Amide-based insulin prodrugs
18 claims: 3 independent, 15 dependent
- 1A鎖とB鎖を有するインスリンプロドラッグであって、 前記インスリンプロドラッグは、一般構造U-Vを有する、アミド結合したジペプチドを有し、 前記A鎖は、配列Z-GIVEQCCX 1 SICSLYQLENX 2 CX 3 (配列番号3)を有し、 前記B鎖は、配列J-X 14 -X 4 LCGX 5 X 6 LVEALX 7 LVCGERGFX 8 (配列番号14)を有し、ここで、 ZおよびJは独立に、Hであるか、あるいは、 前記 ジペプチドであり、 X 14 は、結合であるか、あるいは、FVNQ(配列番号11)、VNQ、NQ、およびQからなる群から選択される1~4個のアミノ酸からなる配列であり、 X 1 は、スレオニンおよびヒスチジンからなる群から選択され、 X 2 は、以下の一般構造を有するアミノ酸であり:[式中、Xは、OH、NH 2 、NHR 10 (ここでR 10 は、一般構造U-Vのジペプチドである)、および、OCH 3 からなる群から選択される]、 X 3 は、アスパラギン、オルニチン、グリシン、アラニン、スレオニン、およびセリンからなる群から選択され、 X 4 は、ヒスチジンおよびスレオニンからなる群から選択され、 X 5 は、アラニン、グリシン、およびセリンからなる群から選択され、 X 6 は、ヒスチジン、アスパラギン酸、グルタミン酸、ホモシステイン酸、およびシステイン酸からなる群から選択され、 X 7 は、以下の一般構造のアミノ酸であり: [式中、X 12 は、OH、NH 2 、NHR 11 (ここでR 11 は、一般構造U-Vのジペプチドである)、およびOCH 3 からなる群から選択される]、 X 8 は、ヒスチジン、アスパラギン、または以下の一般構造のアミノ酸であり: [式中、X 13 は、H、OH、NH 2 、NHR 12 (ここでR 12 は、一般構造U-Vのジペプチドである)、およびOCH 3 からなる群から選択される](ここで、上式中、 Uは、Aib、Gly、Ala、Leu、Met、Asn、Glu、Asp、Gln、His、Lys、Arg、Ser、Cys、Pro、Phe、Tyr、Trp、Ile、Val、Thr、d-Ala、d-Leu、d-Met、d-Asn、d-Glu、d-Asp、d-Gln、d-His、d-Lys、d-Arg、d-Ser、d-Cys、d-Pro、d-Phe、d-Tyr、d-Trp、d-Ile、d-Val、およびd-Thrからなる群から選択されるアミノ酸であり、 Vは、Gly(N-メチル)、Ala(N-メチル)、Leu(N-メチル)、Met(N-メチル)、Asn(N-メチル)、Glu(N-メチル)、Asp(N-メチル)、Gln(N-メチル)、His(N-メチル)、Lys(N-メチル)、Arg(N-メチル)、Ser(N-メチル)、Cys(N-メチル)、Phe(N-メチル)、Tyr(N-メチル)、Trp(N-メチル)、Ile(N-メチル)、Val(N-メチル)、Thr(N-メチル)、d-Ala(N-メチル)、d-Leu(N-メチル)、d-Met(N-メチル)、d-Asn(N-メチル)、d-Glu(N-メチル)、d-Asp(N-メチル)、d-Gln(N-メチル)、d-His(N-メチル)、d-Lys(N-メチル)、d-Arg(N-メチル)、d-Ser(N-メチル)、d-Cys(N-メチル)、d-Phe(N-メチル)、d-Tyr(N-メチル)、d-Trp(N-メチル)、d-Ile(N-メチル)、d-Val(N-メチル)、d-Thr(N-メチル)、Gly(N-ヘキシル)、Ala(N-ヘキシル)、Leu(N-ヘキシル)、Met(N-ヘキシル)、Asn(N-ヘキシル)、Glu(N-ヘキシル)、Asp(N-ヘキシル)、Gln(N-ヘキシル)、His(N-ヘキシル)、Lys(N-ヘキシル)、Arg(N-ヘキシル)、Ser(N-ヘキシル)、Cys(N-ヘキシル)、Phe(N-ヘキシル)、Tyr(N-ヘキシル)、Trp(N-ヘキシル)、Ile(N-ヘキシル)、Val(N-ヘキシル)、Thr(N-ヘキシル)、d-Ala(N-ヘキシル)、d-Leu(N-ヘキシル)、d-Met(N-ヘキシル)、d-Asn(N-ヘキシル)、d-Glu(N-ヘキシル)、d-Asp(N-ヘキシル)、d-Gln(N-ヘキシル)、d-His(N-ヘキシル)、d-Lys(N-ヘキシル)、d-Arg(N-ヘキシル)、d-Ser(N-ヘキシル)、d-Cys(N-ヘキシル)、d-Phe(N-ヘキシル)、d-Tyr(N-ヘキシル)、d-Trp(N-ヘキシル)、d-Ile(N-ヘキシル)、d-Val(N-ヘキシル)、d-Thr(N-ヘキシル)、Tyr(N-C 1 -C 8 アルキル)、Trp(N-C 1 -C 8 アルキル)、Phe(N-C 1 -C 8 アルキル)、およびGly(N-C 1 -C 8 アルキル)からなる群から選択されるアミノ酸である)、 ただし、X、X 12 、X 13 、J、およびZのうちの1つだけが、一般構造U-Vのジペプチドを有することを条件とする、プロドラッグ。
- 2前記一般構造U-Vのジペプチドが、Aib-Gly(N-ヘキシル)、dLys-Gly(N-ヘキシル)、dCys-Gly(N-ヘキシル)、dAla-Gly(N-ヘキシル)、Aib-Gly(N-メチル)、dLys-Gly(N-メチル)、dCys-Gly(N-メチル)、dAla-Gly(N-ヘキシル)、Aib-Phe(N-メチル)、dLys-Phe(N-メチル)、dCys-Phe(N-メチル)、dAla-Gly(N-メチル)、dCys-Gly(N-メチル)、dLys-Gly(N-メチル)、およびAib-Gly(N-メチル)からなる群から選択される、請求項1に記載のプロドラッグ。
- 3Vは、グリシン(N-メチル)、グリシン(N-エチル)、グリシン(N-プロピル)、グリシン(N-ブチル)、グリシン(N-ペンチル)、グリシン(N-ヘキシル)、グリシン(N-ヘプチル)、およびグリシン(N-オクチル)からなる群から選択される、請求項1に記載のプロドラッグ。
- 4Vは、グリシン(N-メチル)である、請求項3に記載のプロドラッグ。
- 5Vは、グリシン(N-ヘキシル)である、請求項3に記載のプロドラッグ。
- 6前記ジペプチドは、前記A鎖または前記B鎖のN末端アミノ酸のαアミノ基とアミド結合を介して結合している、請求項1~5のいずれか1項に記載のプロドラッグ。
- 7前記ジペプチドは、前記A鎖または前記B鎖の側鎖の脂肪族アミノ基とアミド結合を介して結合している、請求項1~5のいずれか1項に記載のプロドラッグ。
- 8X 1 はスレオニンであり、 X 3 はアスパラギンであり、 前記B鎖は、配列FVNQHLCGSHLVEALYLVCGERGFFYTPKT(配列番号8)または配列FVNQHLCGSHLVEALYLVCGERGFFYTKPT(配列番号9)を有する、請求項1~6のいずれか1項に記載のプロドラッグ。
- 9Uは、リジン、システイン、d-リジン、およびd-システインからなる群から選択される、請求項1 または8 に記載のプロドラッグ。
- 10前記プロドラッグに親水体が共有結合している、請求項1~9のいずれか1項に記載のプロドラッグ。
- 11前記親水体はポリエチレングリコールである、請求項10に記載のプロドラッグ。
- 12前記ポリエチレングリコールは、U-Vに共有結合している、請求項11に記載のプロドラッグ。
- 13前記プロドラッグにアシル基またはアルキル基が共有結合している、請求項1~12のいずれか1項に記載のプロドラッグ。
- 14前記アシル基またはアルキル基は、U-Vに共有結合している、請求項13に記載のプロドラッグ。
- 15前記B鎖のカルボキシ末端は、8~12個のアミノ酸からなるペプチドリンカーを介して前記A鎖のアミノ末端に結合し、単鎖インスリン類縁体を形成する、請求項1~14のいずれか1項に記載のプロドラッグ。
- 16前記ペプチドリンカーは、GYGSSSRRAPQT(配列番号23)、GYGSSSX 7 X 8 (配列番号71)、およびGAGSSSRRAPQT(配列番号70)からなる群から選択され、ここでX 7 およびX 8 は独立に、オルニチン、アルギニン、またはリジンである、請求項15に記載のプロドラッグ。
- 17請求項1~16のいずれか1項に記載のプロドラッグを含む、薬学的組成物。
- 18糖尿病の治療剤である、請求項17に記載の薬学的組成物。
Independent claims18
284 paragraphs, as filed
== Cross-reference to related applications == This application claims the priority benefit of US Provisional Patent Application No. 61 / 358,192, filed June 24, 2010. The entire disclosure of this provisional patent application is expressly incorporated herein by reference.
== Incorporation of materials submitted electronically == One computer-readable base / amino acid sequence list (27 kilobytes of ASCII (text format)) with file name "IN216461" created on June 23, 2011 was submitted at the same time as this specification, and the entire contents were submitted. Is incorporated herein by reference.
Insulin is a peptide hormone consisting of a heterodimer derived from a low-activity single-chain proinsulin precursor and biosynthesized by enzyme treatment. Human insulin consists of two peptide chains ("A chain" (SEQ ID NO: 1) and "B chain" (SEQ ID NO: 2)), which are bound to each other by disulfide bonds and have a total of 51 amino acids. The C-terminal region of the B chain and the two terminal regions of the A chain form a three-dimensional structure and gather at the binding site that has a high affinity for the insulin receptor.
Insulin exerts a very rare ability to lower blood sugar levels in almost every form of diabetes. Unfortunately, the pharmacological effects of insulin are not glucose-sensitive, and administration of insulin can lead to excessive effects leading to life-threatening hypoglycemia. Contradictory pharmacological effects, such as the extreme difficulty of normalizing blood glucose without causing hypoglycemia, are characteristic of insulin therapy. Moreover, natural insulin has a short duration of action and needs to be modified before it can be used to control basal glucose. Established methods for delaying the onset of insulin action include reducing solubility and albumin binding.
<p num="0005"> For example, two commercially available insulin derivatives were prepared to obtain longer-acting characteristics. Specifically, insulin derivatives [GlyA21, ArgB31, ArgB32] insulin were prepared to increase the pI of insulin from 5.4 to 6.7. This causes the peptide to precipitate at physiological pH, resulting in slower adsorption and duration of action (see Bolli et al., Diabetologia 1999, 42, 1151-1167). However, this insulin derivative increases the affinity of IGF-1 and increases the proliferative effect, which may lead to tumor formation. Another commercially available insulin derivative is [LysB29-tetradecanoyl, des (B30)] insulin, which LysB29 is C.<sub>14</sub>It is acylated with fatty acids (Mayer et al., Peptide Science, 88, 5, 687-713). The presence of fatty acid chains enhances peptide binding to serum albumin and prolongs plasma half-life. However, this derivative has the drawback of low in vivo activity. Furthermore, both of these insulin derivatives have different manifestations of biological effects from patient to patient.</p><p num="0006"> Prodrug chemistry allows tight control over the onset and duration of action after insulin diffuses from the site of administration and reaches equilibrium in plasma at very narrow concentrations. Compared to existing long-lasting insulin analogs and preparations, the main value of such methods is that insulin accumulates in the blood compartment rather than in the subcutaneous adipose tissue infused. That's what it means. This eliminates the variability in absorption that occurs with prior art insulin derivatives that delay the onset of action. It also enables administration of peptide hormones by routes other than subcutaneous injection.</p><p num="0007"> When insulin binds to a receptor, biological stimulation occurs, but at the same time, insulin-induced pharmacological actions begin to be inactivated by the enzymatic degradation of insulin peptides. The use of insulin prodrug derivatives also has the advantage that this method can prolong the biological half-life of insulin by suppressing the recognition of the prodrug by the corresponding receptor. Despite the advantages described above, the preparation of such prodrugs is complicated and it has not been possible to prepare effective prodrug derivatives of insulin. To build an effective prodrug-hormone, a structural location of the active site is needed that can form the basis for the reversible binding of the prodrug components. This structural location provides two important properties: (1) the possibility of selective chemical modification and (2) the ability to achieve high activity in its natural form when the prodrug component is removed. I need to be able to do it. The insulin prodrugs disclosed herein will be chemically converted to a structure recognizable by the receptor, depending on the rate of this chemical conversion. The timing and duration of the onset of biological action in vivo is determined. The chemistry of prodrugs disclosed in this application utilizes an intramolecular chemical reaction that is independent of additional chemicals, namely enzymes or enzyme inhibitors.</p><p num="0008"> The ideal prodrug should be water soluble under physiological conditions (eg pH 7.2, 37 ° C) and should be stable in powder form for long-term storage. In addition, it must not cause an immune reaction and must be less active than the drug substance. In general, a prodrug is less than 10% active of the drug substance, and in one embodiment, the prodrug is less than 10%, less than 5%, about 1% or less than 1% of the drug substance. In addition, the prodrug must be quantitatively converted to the active drug within a defined time when injected into the body. Applicants are the first to disclose insulin prodrug analogs that meet each of these objectives.</p>
<p num="0009"> Peptide-based drugs are highly effective pharmaceutical agents having the characteristics of having a relatively short duration of action and a variable therapeutic index. The present disclosure relates to insulin prodrugs, the prodrug derivatives being designed to delay the onset of action and prolong the half-life of the drug. Delaying the onset of action has the advantage in that it can be distributed throughout the body before the prodrug is activated. Therefore, administration of the prodrug eliminates the problem caused by maximal activity at the same time as administration, and increases the therapeutic index of the drug substance.</p><p num="0010"> According to one embodiment, a prodrug derivative of insulin is prepared by covalently binding the dipeptide to the active site of the insulin peptide via an amide bond. In one embodiment, the dipeptide covalently binds to the insulin peptide at a position that interferes with the function of insulin interacting with the insulin and IGF-1 receptors. Then, when the dipeptide is removed by an intramolecular reaction, diketopiperazine or diketomorpholine is formed under physiological conditions and in an inactive state by an enzyme, and the complete activity of the polypeptide is restored.</p><p num="0011"> In one embodiment, an insulin prodrug having a general structure UO-insulin is provided. Here, U is an amino acid or hydroxyic acid, and O is an N-alkylated amino acid bound to an insulin peptide by forming an amide bond between UO and the amine of the insulin peptide. In one embodiment, the UO dipeptide binds via an amide bond at the N-terminus or at the side chain of the amino acid corresponding to the A19, B16 or B25 position of each A or B chain. The structure of UO is, in one embodiment, selected so that the chemical reaction cleavage of UO from an insulin peptide is completed at least about 90% within about 1 to about 720 hours under physiological conditions in PBS. Will be done. In one embodiment, the half-life (t) at which UO is chemically cleaved from an insulin peptide.<sub>1/2</sub>), But under physiological conditions in PBS, at least about 1 hour to about 1 week.</p><p num="0012"> In one embodiment, U and O are selected to inhibit the enzymatic cleavage of UO dipeptides from insulin peptides by enzymes contained in mammalian serum. In one embodiment, U and / or O have a half-life in which UO is cleaved from the insulin peptide under physiological conditions in PBS, and UO is cleaved from the insulin peptide in a solution containing the DPP-IV protease. Cleavage of UO from insulin prodrugs occurs at a rate more than twice that of the ideal enzyme-free condition in the presence of DPP-IV protease under physiological conditions. Not) is selected. In one embodiment, the amino acid of the insulin peptide to which U, O or UO is bound is an unencoded amino acid. In one embodiment, U and / or O are amino acids that have a D-type stereoisomer configuration. In some exemplary embodiments, U is an amino acid with a D-type stereoisomer configuration and O is an amino acid with an L-type stereoisomer configuration. In some exemplary embodiments, U is an amino acid with an L-type stereoisomer configuration and O is an amino acid with a D-type stereoisomer configuration. In some exemplary embodiments, U is an amino acid with a D-type stereoisomer configuration and O is an amino acid with a D-type stereoisomer configuration. In one embodiment, O is an N-alkylated amino acid, but not proline.</p><p num="0013"> In one embodiment, the dipeptide prodrug element comprises a compound having the general structure of formula I below.</p><p num="0014"><img id="000002" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or aryl, or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH.</p><p num="0015"> In one embodiment, the dipeptide prodrug element comprises a compound having the general structure of formula I below.</p><p num="0016"><img id="000003" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.</p><p num="0017"> In one embodiment, the dipeptide extension comprises a compound having the following general structure:</p><p num="0018"><img id="000004" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In the formula, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>8</sub>Form a cycloalkyl ring, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH. In one embodiment, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle.</p><p num="0019"> According to one embodiment, an insulin prodrug analog having an A chain and a B chain is provided, where the A chain is sequenced Z-GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>It has (SEQ ID NO: 3) and the B chain is the sequence JX.<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>(SEQ ID NO: 14). The indications of Z and J in the structural formulas of the A and B chains are independently H (forming an N-terminal amine) or a dipeptide having the following general structure.</p><p num="0020"><img id="000005" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>14</sub>X the "J" element<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>(SEQ ID NO: 14) A bond that connects to the N-terminus of the sequence, or X<sub>14</sub>X the "J" element<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>(SEQ ID NO: 14) A sequence consisting of 1 to 4 amino acids selected from the group consisting of FVNQ (SEQ ID NO: 11), VNQ, NQ, and Q connected to the N-terminal of the sequence is shown.</p><p num="0021"> X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000006" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide having the following general structure. ]<img id="000007" he="30" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Is selected from the group consisting of asparagine, ornithine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000008" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>12</sub>Is OH, NH<sub>2</sub>, NHR<sub>11</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>11</sub>Is a dipeptide having the following general structure. ]<img id="000009" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>8</sub>Is a histidine, asparagine or an amino acid having the following general structure,<img id="000010" he="50" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>13</sub>Is H, OH, NH<sub>2</sub>, NHR<sub>12</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>12</sub>Is a dipeptide having the following general structure. ]<img id="000011" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In the formula, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, but X, X<sub>12</sub>, X<sub>13</sub>, J and Z only have dipeptides of the following general structure:</p><p num="0022"><img id="000012" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />In one embodiment, the insulin analog is X<sub>8</sub>Has the sequences just above, except that is histidine or asparagine. In one embodiment, J or Z has a dipeptide of formula I and R<sub>4</sub>And R<sub>3</sub>If, together with the atoms to which they are bonded, form a 4-membered, 5-membered or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.</p><p num="0023"> According to one embodiment, Z, J, R<sub>10</sub>, R<sub>11</sub>Or R<sub>12</sub>Dipeptides present in contain compounds having the general structure of formula I.</p><p num="0024"><img id="000013" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.</p><p num="0025"> According to one embodiment, Z, J, R<sub>10</sub>, R<sub>11</sub>Or R<sub>12</sub>Dipeptides present in contain compounds having the general structure of formula I.</p><p num="0026"><img id="000014" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>8</sub>Independently, H or C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.</p><p num="0027"> According to one embodiment, the A chain of the insulin peptide is sequenced Z-GIVEQCCTSICSLYQLENX.<sub>2</sub>Provided by an insulin analog having CN (SEQ ID NO: 6) and having a sequence selected from the group consisting of HLCCGSHLVEALYLVCGERGFF (SEQ ID NO: 7), FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 8), FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 9). Will be done.</p><p num="0028"> Here, Z is H or is a dipeptide having the following general structure.</p><p num="0029"><img id="000015" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>2</sub>Is an amino acid having the following general structure,<img id="000016" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In the formula, R<sub>10</sub>Is a dipeptide that is H or has the following general structure:</p><p num="0030"><img id="000017" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />However, Z and R<sub>10</sub>Are neither H nor dipeptides having the following general structure.</p><p num="0031"><img id="000018" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> According to one embodiment, a single chain insulin prodrug analog is provided. In this embodiment, the carboxy terminus of the human insulin B chain or its functional analog is covalently attached to the human insulin A chain or the N terminus of its functional analog. In this case, the following general structure<img id="000019" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />The dipeptide prodrug moiety with is covalently attached via an amide bond at the N-terminus of the peptide or at the side chain of the amino acid corresponding to the A19, B16 or B25 position of each A or B chain. In one embodiment, the B chain is attached to the A chain via a peptide linker consisting of 4-12 or 4-8 amino acids.</p><p num="0032"> In another embodiment, the hydrophilics covalently bind to the peptide, increasing the solubility of insulin prodrug analogs. In one embodiment, the hydrophilic body binds to an insulin analog via a linker. In one embodiment, the hydrophilic is the N-terminal α-amine of the B chain (directly or indirectly via the linker) or the amino acid at position 28 of SEQ ID NO: 9 or the amino acid at position 29 of SEQ ID NO: 8. It binds to the side chain. In one embodiment, the hydrophilic body is a polyethylene glycol (PEG) chain selected from the range of molecular weights ranging from about 500 to about 40,000 daltons. In one embodiment, the polyethylene glycol chain is selected from a molecular weight range of about 500 to about 5,000 Dalton. In another embodiment, the polyethylene glycol chain has a molecular weight of about 10,000 to about 20,000 daltons.</p><p num="0033"> Acylation or alkylation can prolong the half-life of insulin peptides in the blood. Acylation or alkylation conveniently delays the onset of action at the insulin receptor and / or prolongs the duration of action upon activation of the prodrug. The insulin analog may be acylated or alkylated at the same amino acid position where the hydrophilic body binds, or may be acylated or alkylated at a different amino acid position.</p><p num="0034"> According to one embodiment, any of the novel insulin prodrug analogs disclosed herein are preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% purity levels. , 97%, 98% or 99%, and comprises a pharmaceutically acceptable diluent, carrier or excipient. Such compositions contain at least 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml of A19 insulin analogs as disclosed herein. , 7mg / ml, 8mg / ml, 9mg / ml, 10mg / ml, 11mg / ml, 12mg / ml, 13mg / ml, 14mg / ml, 15mg / ml, 16mg / ml, 17mg / ml, 18mg / ml, 19mg It may be contained in / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml or higher. In one embodiment, the pharmaceutical composition comprises an aqueous solution that is sterilized and optionally stored and housed in various packaging containers. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be packaged as part of a kit containing a disposable device for administering the composition to a patient. The container or kit may be labeled for storage at room temperature or refrigerator temperature.</p><p num="0035"> According to one embodiment, an improved method of regulating blood glucose levels in insulin-dependent patients is provided. The method comprises administering to the patient an insulin prodrug analog of the present disclosure in a therapeutically effective amount to control diabetes. In one embodiment, the insulin prodrug analog is PEGylated with a PEG chain selected from a molecular weight range of about 5,000 to about 40,000 daltons.</p>
<figref num="1">It is a schematic diagram of a two-step synthetic strategy for preparing human insulin. The details of this procedure will be described in Example 1.</figref><figref num="2">It is a graph which compared the specific binding of an insulin receptor with respect to synthetic human insulin and purified natural insulin. This synthetic insulin was synthesized by the method shown in detail in FIG. As is clear from the data shown in the graph, the two molecules have similar binding activity.</figref><figref num="3">Natural insulin and A19 insulin analogs (insulin (p-NH)<sub>2</sub>-F)<sup>19</sup>) Is a graph comparing the relative binding force to the insulin receptor. As is clear from the data shown in the graph, the two molecules have similar binding activity.</figref><figref num="4">Natural insulin and IGF1 (YL)<sup>B16B17</sup>It is a graph which compared the relative binding force to an insulin receptor of an analog. As is clear from the data shown in the graph, the two molecules have similar binding activity.</figref><figref num="5">IGF1 (Y<sup>B16</sup>L<sup>B17</sup>) (P-NH<sub>2</sub>-F)<sup>A19</sup>FIG. 6 is a schematic diagram of a synthetic scheme used to prepare analogs.</figref><figref num="6">IGF1 (Y<sup>B16</sup>L<sup>B17</sup>) (P-NH<sub>2</sub>-F)<sup>A19</sup>And IGF1 (Y<sup>B16</sup>L<sup>B17</sup>) (P-NH<sub>2</sub>-F)<sup>A19</sup>It is a graph comparing the relative binding force to the insulin receptor of the dipeptide extended form of. Here, the dipeptide AibAla binds at position A19 (ie, IGF1 (Y).<sup>B16</sup>L<sup>B17</sup>)<sup>A19</sup>-Aib Ala).</figref><figref num="7A">The activity of the dimer prepared according to the present disclosure is shown. Figure 7A shows two single-stranded IGFs.<sup>B16B17</sup>Derivative peptide (IGF-1B chain [C<sup>0</sup>H<sup>5</sup>Y<sup>16</sup>L<sup>17</sup>O<sup>22</sup>] -A chain [O<sup>9,14,15</sup>N<sup>18,21</sup>]; SEQ ID NO: 68) shows the structure of the IGF-1 single chain dimer linked by a disulfide bond between the side chains at the amino terminus of the B chain.</figref><figref num="7B">The activity of the dimer prepared according to the present disclosure is shown. Figure 7B shows insulin, IGF-1, single chain IGF<sup>B16B17</sup>Derivative peptide dimer, double-stranded IGF<sup>B16B17</sup>It is a graph which shows the relative binding force to an insulin receptor of a derivative peptide dimer.</figref><figref num="7C">The activity of the dimer prepared according to the present disclosure is shown. Figure 7C shows insulin, IGF-1, double-stranded IGF<sup>B16B17</sup>It is a graph which shows the relative activity for inducing the phosphorylation of an insulin receptor of a derivative peptide dimer.</figref><figref num="8">IGF1A (Ala)<sup>6,7,11,20</sup>Amide IGF<sup>B16B17</sup>Derivative peptide: ((pNH<sub>2</sub>-F)<sup>19</sup>It is a figure which shows the decomposition of the Aib-Pro prodrug form of. The dipeptide was incubated in PBS at pH 7.4 at 37 ° C for a predetermined time. A small amount was taken at 20 minutes (Fig. 8A), 81 minutes (Fig. 8B), and 120 minutes (Fig. 8C) after the start of incubation, the reaction was stopped at 0.1% TFA, and the test was performed by analytical HPLC. Peak a (IGF1A (Ala))<sup>6,7,11,20</sup>(pNH<sub>2</sub>-F)<sup>1</sup>Amide) and peak b (IGF1A (Ala))<sup>6,7,11,20</sup>(Aib-Pro-pNH-F)<sup>19</sup>Amide) was identified by LC-MS and the peak area was integrated and quantified. These data are based on IGF1A (Ala)<sup>6,7,11,20</sup>(Aib-Pro-pNH-F)<sup>19</sup>From amide to IGF1A (Ala)<sup>6,7,11,20</sup>(pNH<sub>2</sub>-F)<sup>1</sup>It shows autonomous and enzyme-independent conversion over time to amides.</figref><figref num="9A">It is a graph which shows the activity in vitro of a prodrug Aib, dPro-IGF1YL (a19 4-aminophenylalanine-bound dipeptide). Figure 9A shows the temporal (0 hours, 2.5 hours, 10.6) of natural insulin (measured at 4 ° C at 1 hour) and A19 IGF prodrug analog (Aib, dPro-IGF1YL) incubated in PBS. Time) It is a graph comparing the relative binding force to an insulin receptor. As evidenced by the data shown in the graph, conversion of the prodrug form to the active IGF1YL peptide restores the active state from the A19 IGF prodrug analog sample.</figref><figref num="9B">It is a graph which shows the activity in vitro of a prodrug Aib, dPro-IGF1YL (a19 4-aminophenylalanine-bound dipeptide). Figure 9B shows the temporal (0 hours, 0 hours) of natural insulin (measured at 4 ° C for 1.5 hours) and A19 IGF prodrug analogs (Aib, dPro-IGF1YL) incubated in 20% plasma / PBS. (1.5 hours, 24.8 hours) It is a graph comparing the relative binding force to the insulin receptor. As evidenced by the data shown in the graph, conversion of the prodrug form to the active IGF1YL peptide restores the active state from the A19 IGF prodrug analog sample.</figref><figref num="10A">It is a graph showing the in vitro activity of the prodrug dK, (N-isobutyl G) -IGF1YL (in this case, the dipeptide dK, (N-isobutyl G) is an insulin analog with A19 4-aminophenylalanine via an amide bond. It is attached to the body). Figure 10A shows natural insulin (measured at 4 ° C at 1 hour) and A19 IGF prodrug analog (IGF1YL: dK, (N-isobutyl G)) incubated in PBS over time (0 hours,). 5 hours, 52 hours) It is a graph comparing the relative binding force to the insulin receptor. As is clear from the data shown in the graph, when the prodrug form is converted to the active IGF1YL peptide, A19 IGF pro Recovers from drug analog samples to a highly active state.</figref><figref num="10B">It is a graph showing the in vitro activity of the prodrug dK, (N-isobutyl G) -IGF1YL (in this case, the dipeptide dK, (N-isobutyl G) is an insulin analog with A19 4-aminophenylalanine via an amide bond. It is attached to the body). Figure 10B shows the temporal insulin (measured at 4 ° C. at 1.5 hours) and A19 IGF prodrug analog (IGF1YL: dK, (N-isobutyl G)) incubated in 20% plasma / PBS. (0 hours, 3.6 hours, 24.8 hours) It is a graph comparing the relative binding force to the insulin receptor. As is clear from the data shown in the graph, when the prodrug form is converted to the active IGF1YL peptide. , A19 IGF Prodrug Recovers from analog samples to a highly active state.</figref><figref num="11A">The prodrug dK (e-acetyl), Sar) -IGF1YL (in this case, the acylated dipeptide dK (e-acetyl), Sar) binds to the insulin analog with A19 4-aminophenylalanine via an amide bond. It is a graph which shows the activity in vitro. Figure 11A shows the temporal (0 hours) of natural insulin (measured at 4 ° C at 1 hour) and A19 IGF prodrug analog (IGF1YL: dK (e-acetyl), Sar) incubated in PBS. , 7.2 hours, 91.6 hours) It is a graph comparing the relative binding force to the insulin receptor. As evidenced by the data shown in the graph, conversion of the prodrug form to the active IGF1YL peptide restores the active state from the A19 IGF prodrug analog sample.</figref><figref num="11B">The prodrug dK (e-acetyl), Sar) -IGF1YL (in this case, the acylated dipeptide dK (e-acetyl), Sar) binds to the insulin analog with A19 4-aminophenylalanine via an amide bond. It is a graph which shows the activity in vitro. Figure 11B shows the time course of natural insulin (measured at 4 ° C for 1.5 hours) and A19 IGF prodrug analog (IGF1YL: dK (e-acetyl), Sar) incubated with 20% plasma / PBS. (0 hour, 9 hours, 95 hours) It is a graph comparing the relative binding force to an insulin receptor. As evidenced by the data shown in the graph, conversion of the prodrug form to the active IGF1YL peptide restores the active state from the A19 IGF prodrug analog sample.</figref><figref num="12">Heteroduplex of natural insulin, A-chain of IGF-1 and heteroduplex of B-chain, single-chain IGF-1 analog (the carboxy end of B-chain is directly attached to the N-terminal of A-chain of IGF-1. It is a graph comparing the relative binding force to the insulin receptor.</figref><figref num="13">Heteroduplex of natural insulin, IGF-1, IGF-1 delta heteroduplex, single chain IGF-1 delta single chain analog (the carboxy end of the B chain is via a peptide linker consisting of the sequence GYGSSSOR (SEQ ID NO: 69). It is a graph comparing the relative binding force to the insulin receptor (which binds to the N-terminal of the A chain of IGF-1). In this case, the IGF-1 delta analogs are amino acid-substituted HA8, OA9, OA14, OA15, QA17, It has a natural IGF-1 sequence, including NA21, YB16, LB17, OB22.</figref><figref num="14">It is a bar graph showing the relative binding activity of a single chain insulin analog to the IGF-1 receptor or an insulin receptor of A or B subtype in vitro, in which case the carboxy terminus of the B chain of natural insulin is IGF. It is bound to the amino end of the A chain of natural insulin via various derivatives of -1 C-peptide or IGF-1 C-peptide. B<sup>0</sup>C<sup>1</sup>A<sup>0</sup>In the insulin analog nomenclature, B<sup>0</sup>And A<sup>0</sup>The symbol indicates the insulin sequences of the A and B chains, whereas the C symbol<sup>1</sup>Indicates the C-peptide of IGF-1. As shown by this data, single-chain insulin analogs that bind the B chain to the A chain via the C-peptide of IGF-1 are potent insulin agonists. In addition, modifying the second position (eg, replacing alanine with natural tyrosine) or deleting the last four amino acids of the C-bridging peptide of IGF-1 results in a highly active insulin-selective single-chain insulin analog. Is formed.</figref><figref num="15">Formula B for the IGF-1 receptor or insulin receptor of A or B subtype<sup>0</sup>C<sup>1</sup>A<sup>0</sup>It is a bar graph which shows the relative binding activity of a single chain insulin analog in vitro. In this case, the natural sequence of the bridging IGF-1 C-peptide is modified by the title amino acid substitutions at the 1st, 2nd, 3rd, 4th or 8th positions. B<sup>0</sup>C<sup>1</sup>A<sup>0</sup>In the insulin analog nomenclature, B<sup>0</sup>And A<sup>0</sup>The symbol indicates the insulin sequences of the A and B chains, whereas the C symbol<sup>1</sup>Indicates the C-peptide of IGF-1.</figref><figref num="16">Single chain B for A subtype insulin receptor<sup>0</sup>C<sup>1</sup>A<sup>0</sup>It is a bar graph which shows the relative binding activity and phosphorylation activity of an insulin analog in vitro. The activity of native IGF-1 C-peptide (010) to various amino acid substitutions or deletions was compared. B<sup>0</sup>C<sup>1</sup>A<sup>0</sup>In the insulin analog nomenclature, B<sup>0</sup>And A<sup>0</sup>The symbol indicates the insulin sequences of the A and B chains, whereas the C symbol<sup>1</sup>Indicates the C-peptide of IGF-1.</figref><figref num="17">It is a graph which shows that the insulin analog having the A chain of IGF-1 is more resistant to the degradation by a specific insulin degrading enzyme (IDE) than the insulin analog having the A chain of insulin.</figref><figref num="18">It is a graph which shows the relative activity of IGF-1, insulin and insulin / IGF chimera which induce cell proliferation in vitro. These results indicate that insulin activity associated with the IGF-1 single-chain insulin analog does not correlate with proliferative activity associated with native IGF-1.</figref><figref num="19A">It shows the activity of the prodrug MIU-29: B1 (Y16, L17, Y25) 29a: A1 (aF19-dLys (Ac), NLeu). Figure 19A shows that when MIU-29 is incubated ex vivo (37 ° C in PBS buffer), in vitro binding to insulin receptors increases over time with a half-life of approximately 4.4 hours. Is shown.</figref><figref num="19B">It shows the activity of the prodrug MIU-29: B1 (Y16, L17, Y25) 29a: A1 (aF19-dLys (Ac), NLeu). Figure 19B shows a prodrug double-stranded insulin analog acylated with a dipeptide prodrug element (MIU-29: [B1 (Y16, L17, Y25) 29a: A1 (aF19-dLys (Ac), NLeu)]. , A graph showing the results of an insulin resistance comparison test conducted in healthy mice in comparison with its parent insulin analog (MIU-27: [B1 (Y16, L17, Y25) 29a: A1 (aF19-NH2)]. The prodrug derivative MIU-29 has a 4-aminophenylalanine substitution at the A19 position. In this case, the dipeptides dLys (Ac), NLeu are covalently attached to the 4-amino position of the A19 residue, and the dipeptide element. The side chains of lysine are acylated with C14 fatty acids. Under physiological conditions, this dipeptide divides autonomously with a half-life of approximately 4.4 hours. Ex ex MIU-29 After incubation in vivo for 24 hours, the resulting compound (labeled "MIU-29c") was administered to mice, which compared their ability to lower blood glucose with that of the parent compound. As shown in FIG. 12, these two compounds acted in much the same way.</figref><figref num="20A">MIU-30a: B1 (Y16, L17, Y25) 29a: A1 (dLys (Ac), Sar-aF19) (acylated dipeptide dLys (Ac), Sar, which is an insulin prodrug analog, is mediated by an amide bond. The results obtained in the insulin resistance comparison test with A19 4-aminophenylalanine bound to insulin analogs are shown. The half-life of prodrugs is estimated to be about 20 hours. The data shown in FIG. 20A reveal that the parent compound has low activity but increased activity after 48 hours of incubation (forming "MIU-30c") in 20% plasma. = Solvent control, = MIU 30a, 90nm / kg; = MIU 30c, 90nm / kg; = MIU 30a, 270nm / kg; = MIU 30c, 270nm / kg.</figref><figref num="20B">MIU-30a: B1 (Y16, L17, Y25) 29a: A1 (dLys (Ac), Sar-aF19) (acylated dipeptide dLys (Ac), Sar, which is an insulin prodrug analog, is mediated by an amide bond. The results obtained in the insulin resistance comparison test with A19 4-aminophenylalanine bound to insulin analogs are shown. The half-life of this prodrug is estimated to be about 20 hours. Similarly, FIG. 20B shows blood glucose AUC after 8 hours in C57 / Blk mice, indicating that in vitro incubation prior to administration increases compound activity over time.</figref><figref num="21">The in vitro activity of the acylated insulin analog MIU 46: B1 (H5,10 Y16, L17, Y25, K29-C14) 28a: A1 (N18,21, aF19NH2) has been acylated. It is a graph which shows the comparison with no corresponding substance (MIU-45) and natural insulin. The acylated insulin has a 4-aminophenylalanine substitution at the A19 position and a lysine substitution at the B29 position, and the side chain of B29 lysine is acylated with a C14 fatty acid. The acylated analog is less active than the parent compound.</figref><figref num="22">Acylated prodrug MIU 42: B1 (Y16, L17, Y25) 29a: A1 (dLys (rE-C14), Sar-aF19) (The amino acid of the dipeptide prodrug element is acylated and the γ-position of the glutamate linker " The in vitro activity of (bound at rE) is shown in 30% ACN / PBS at pH 7.4 and 37 ° C for ex vivo incubation time. As shown by this data, the activity recovers to the parent compound MIU 42 as the incubation time ex vivo increases.</figref><figref num="23A">Data from insulin resistance controlled trials conducted on Detemir and the acylated insulin analog MIU-46 using C57 / Blk mice. The acylated derivative MIU 46: B1 (H5,10 Y16, L17, Y25, K29-C14) 28a: A1 (N18,21, aF19NH2) was acylated with C14 fatty acid via a γ-glutamic acid spacer. It has a lysine substitution at the 29th position. As shown in FIGS. 23A to 23D, the acylated analog MIU-46 is not as potent as Detemir.</figref><figref num="23B">Data from insulin resistance controlled trials conducted on Detemir and the acylated insulin analog MIU-46 using C57 / Blk mice. The acylated derivative MIU 46: B1 (H5,10 Y16, L17, Y25, K29-C14) 28a: A1 (N18,21, aF19NH2) was acylated with C14 fatty acid via a γ-glutamic acid spacer. It has a lysine substitution at the 29th position. As shown in FIGS. 23A to 23D, the acylated analog MIU-46 is not as potent as Detemir.</figref><figref num="23C">Data from insulin resistance controlled trials conducted on Detemir and the acylated insulin analog MIU-46 using C57 / Blk mice. The acylated derivative MIU 46: B1 (H5,10 Y16, L17, Y25, K29-C14) 28a: A1 (N18,21, aF19NH2) was acylated with C14 fatty acid via a γ-glutamic acid spacer. It has a lysine substitution at the 29th position. As shown in FIGS. 23A to 23D, the acylated analog MIU-46 is not as potent as Detemir.</figref><figref num="23D">Data from insulin resistance controlled trials conducted on Detemir and the acylated insulin analog MIU-46 using C57 / Blk mice. The acylated derivative MIU 46: B1 (H5,10 Y16, L17, Y25, K29-C14) 28a: A1 (N18,21, aF19NH2) was acylated with C14 fatty acid via a γ-glutamic acid spacer. It has a lysine substitution at the 29th position. As shown in FIGS. 23A to 23D, the acylated analog MIU-46 is not as potent as Detemir.</figref><figref num="24">The general structure of the PEGylated insulin prodrug embodiment, where 20 kDa PEG binds to the N-terminal α-amine of the B chain via a linker and the a chain has a 4-aminophenylalanine substitution at position A19. .. In this case, the dipeptides (AA1, AA2) are attached to the 4-amino group of 4-aminophenylalanine via an amide bond. In one embodiment, the dipeptides (AA1, AA2) are norleucine, d-lysine (acylated).</figref><figref num="25">FIG. 5 is a schematic diagram of a synthetic scheme used to prepare the general structure shown in FIG. Details of this synthesis will be described in Example 15.</figref><figref num="26">Insulin of various prodrug compounds (having the general structure shown in FIG. 24) for the type A subtype receptor of insulin based on the phosphorylation assay. It is a graph which shows the activity in vitro. The compounds tested are: Natural insulin (), B1Aoa2 (H5, H10, Y16, L17) 25a: A1 (pNH2-F19, N18, N21 (, has aminooxyacetyl (Aoa)) linker, but no PEG, no A19 dipeptide ), B1PEG-Aoa2 (H5, H10, Y16, L17) 25a: A1 (pNH2-F19dLys (rE-C14), Sar-aF19, N18, N21 (, N-terminal 20 kDa PEG, but A19 dipeptide No), B1PEGAoa2 (H5, H10, Y16, L17) 25a: A1 (dLys (rE-C14), βAla-aF19, N18, N21 (, N-terminal 20 kDa PEG, almost uncut dipeptide ( It has dLys (rE-C14), βAla) in A19), B1PEGAoa2 (H5, H10, Y16, L17) 25a: A1 (dLys (rE-C14), Nleu-aF19, N18, N21 (, N-terminal 20kDa) PEG of, self-cleaving dipeptide (dLys (rE-C14), Nleu) administered without incubation step in A19), B1PEGAoa2 (H5, H10, Y16, L17) 25a: A1 (dLys (rE-) C14), Nleu-aF19, N18, N21 (Left black triangle, self-cleaving dipeptide with N-terminal 20 kDa PEG and administered after 78 hours incubation in PBS (dLys (rE-C14), Nleu) The dipeptide dLys (rE-C14), Nleu, under physiological conditions, divides autonomously with a half-life of about 4.4 hours, as mentioned in FIGS. 19A and 19B.</figref><figref num="27">Insulin of various prodrug compounds (having the general structure of FIG. 24) for the B-type subtype receptor of insulin based on the phosphorylation assay. It is a graph which shows the activity in vitro. The compounds tested are: Natural insulin (), B1Aoa2 (H5, H10, Y16, L17) 25a: A1 (pNH2-F19, N18, N21 (, has aminooxyacetyl (Aoa)) linker, but no PEG, no A19 dipeptide ), B1PEG-Aoa2 (H5, H10, Y16, L17) 25a: A1 (pNH2-F19dLys (rE-C14), Sar-aF19, N18, N21 (, N-terminal 20 kDa PEG, but A19 dipeptide No), B1PEGAoa2 (H5, H10, Y16, L17) 25a: A1 (dLys (rE-C14), βAla-aF19, N18, N21 (, N-terminal 20 kDa PEG, almost uncut dipeptide ( It has dLys (rE-C14), βAla) in A19), B1PEGAoa2 (H5, H10, Y16, L17) 25a: A1 (dLys (rE-C14), Nleu-aF19, N18, N21 (, N-terminal 20kDa) PEG of, self-cleaving dipeptide (dLys (rE-C14), Nleu) administered without incubation step in A19), B1PEGAoa2 (H5, H10, Y16, L17) 25a: A1 (dLys (rE-) C14), Nleu-aF19, N18, N21 (Left black triangle, self-cleaving dipeptide with N-terminal 20 kDa PEG and administered after 78 hours incubation in PBS (dLys (rE-C14), Nleu) The dipeptide dLys (rE-C14), Nleu, under physiological conditions, divides autonomously with a half-life of about 4.4 hours, as mentioned in FIGS. 19A and 19B.</figref>
Detailed explanation Definition In describing and claiming the present invention, the following technical terms will be used in accordance with the definitions given below.
As used herein, the term "prodrug" is defined as a compound that is chemically modified before exerting its pharmacological action.
"Bioactive polypeptide" refers to a polypeptide capable of exerting a biological action in vitro and / or in vivo.
As used herein, the term "amino acid" includes molecules that contain both amino and carboxy functional groups, where the amino and carboxylate groups are attached to the same carbon (α carbon). doing. The α-carbon may optionally have one or two other organic substituents. The labeling of amino acids when the isomer type is not specified is intended to include L-type or D-type of amino acids, or racemic mixtures. However, if an amino acid is represented by a three-letter code and contains a superscript number, the D type of amino acid is indicated by including a lowercase d and a superscript number before the three-letter code (for example). , DLys<sup>-1</sup>), Where there is no lowercase d (for example, Lys)<sup>-1</sup>) Is intended to indicate the natural L-type of amino acids. This nomenclature indicates the position of an amino acid in the IGF peptide sequence when it includes the superscript number. In this case, the amino acids located in the IGF sequence are indicated by positive numbers in sequence starting from the N-terminus. Additional amino acids that bind to the IGF peptide at the N-terminus or via the side chain are numbered starting at 0 and increasing in number with negative integer values further away from the IGF sequence. For example, the position of the amino acid in the dipeptide prodrug bound to the N-terminus of IGF is aa.<sup>-1</sup>-aa<sup>0</sup>-IGF is written, but this aa<sup>0</sup>Represents the carboxy-terminal amino acid of the dipeptide, aa<sup>-1</sup>Represents the amino acid at the amino terminus of the dipeptide.
As used herein, the term "hydroxy acid" refers to a modified amino acid that replaces the amino group of the α carbon with a hydroxy group.
As used herein, the expression "unencoded amino acid" refers to the following 20 amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn. , Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, including amino acids that are not L isomers.
A "dipeptide" is a compound formed by the binding of an α-amino acid or α-hydroxy acid to another amino acid via a peptide bond.
As used herein, the expression "cleave by chemical reaction" includes non-enzymatic reactions that lead to the cleavage of chemical covalent bonds, unless otherwise noted.
"Bioactive polypeptide" refers to a polypeptide capable of exerting a biological action in vitro and / or in vivo.
As used herein, general reference to peptides is intended to include peptides with modified amino and carboxy terminus. For example, in the amino acid sequence that selects standard amino acids, not only the standard amino acids at the N-terminal and C-terminal, but also the amide group instead of the corresponding hydroxy acid at the N-terminal and / or the carboxylic acid at the C-terminal. It is intended to include the corresponding C-terminal amino acid modified to include.
As used herein, an "acylated" amino acid is an amino acid that contains an unnatural acyl group relative to a natural amino acid, regardless of the means by which it is made. Examples of methods for producing acylated amino acids and acylated peptides are known in the art, where amino acids are acylated and then included in the peptide, or the peptide is chemically synthesized after peptide synthesis. Includes acylation. In one embodiment, the acyl group attaches to the peptide, (i) prolonging the half-life in blood, (ii) delaying the onset of action, (iii) prolonging the duration of action, (iv) proteases such as DPP-IV. To improve resistance to, (v) enhance one or more of the actions at the insulin peptide receptor.
As used herein, an "alkylated" amino acid is an amino acid that contains an unnatural alkyl group relative to a natural amino acid, regardless of the means by which it is made. Examples of methods for producing alkylated amino acids and alkylated peptides are known in the art, where amino acids can be alkylated and then included in the peptide, or the peptide can be chemically incorporated after peptide synthesis. Includes alkylation. Without being bound by a particular theory, peptide alkylation has similar, if not identical, effects to peptide acylation, such as prolonging the half-life in blood, delaying the onset of action, and prolonging the duration of action. It is believed that this will be achieved by improving resistance to proteases such as DPP-IV and enhancing the action on insulin receptors.
As used herein, the expression "pharmaceutically acceptable carrier" refers to emulsions such as phosphate buffered saline, water, oil-in-water emulsions or water-in-oil emulsions, as well as various types. Includes standard pharmaceutical carriers such as wetting agents. The term also includes drugs approved by US federal regulatory authorities for use in animals, including humans, or drugs listed in the United States Pharmacopeia.
As used herein, the expression "pharmaceutically acceptable salt" is a salt of a compound that retains the biological activity of the parent compound and has no biological or otherwise desirable point. Is shown. Many of the compounds disclosed herein are capable of forming acid and / or basic salts in the presence of amino and / or carboxy groups or similar groups.
Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Examples of the salt derived from the inorganic base include sodium salt, potassium salt, lithium salt, ammonium salt, calcium salt and magnesium salt. Examples of the salt derived from the organic base include, but are not limited to, a salt of a primary amine, a salt of a secondary amine, and a salt of a tertiary amine.
Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Examples of the salt derived from the inorganic acid include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvate, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartrate acid, citric acid, benzoic acid and silicic acid. , Mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid and the like.
As used herein, the expression "treat" refers to the prevention of a particular disorder or condition, or the alleviation of symptoms associated with a particular disorder or condition and / or the prevention or elimination of these symptoms. Including. For example, as used herein, the phrase "treating diabetes" generally indicates maintaining blood glucose levels near normal levels and increasing blood glucose levels depending on the individual situation. It may also include lowering or lowering.
As used herein, the "effective" or "therapeutically effective" amount of prodrug is the amount of prodrug that is non-toxic but sufficient to achieve the desired effect. For example, one desirable effect would be the ability to prevent or treat hyperglycemia. The "effective" amount will vary from person to person to treatment, depending on the individual's age, general condition, administration mode, and so on. Therefore, it is not always possible to specify an accurate "effective amount". However, the appropriate "effective" amount in an individual case can be determined by one of ordinary skill in the art using routine experiments.
The term "parenteral" means not through the gastrointestinal tract, but by some other route, such as intranasal, inhaled, subcutaneous, intramuscular, intraspinal or intravenous.
As used herein, the expression "natural insulin peptide" refers to SEQ ID NOs: 1 and 2 as well as a heterodimer consisting of 51 amino acids having the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2. It is intended to show a single chain insulin analog having. The term "insulin peptide", when used herein without any other descriptive terminology, is a heterodimer consisting of 51 amino acids with the A chain of SEQ ID NO: 1 and the B chain of SEQ ID NO: 2. Not only that, it includes its single-chain insulin analogs, including those disclosed, for example, in WO 96/34882 and US Pat. No. 6,630,348 (the disclosure of which is incorporated herein by reference). It is intended that these also include heterodimers and single chain analogs with modified derivatives of natural A and / or B chains. As an example, modification of the amino acid at position A19, position B16 or position B25 to 4-aminophenylalanine, or A5, A8, A9, A10, A12, A14, A15, A17, A18, A21, B1, B2, One or more amino acid substitutions at positions selected from B3, B4, B5, B9, B10, B13, B14, B17, B20, B21, B22, B23, B26, B27, B28, B29, B30, or Any or all deletions of B1-4 and B26-30 can be mentioned. An "insulin prodrug analog", as used herein, is a position that interferes with insulin activity or the activity of an IGF1 insulin analog (eg, the ability to interact with insulin and IGF-1 receptors). Insulin peptide modified by co-binding of a dipeptide via an amide bond (or an IGF1-based insulin analog as disclosed in Example 9).
As used herein, the term "single-chain insulin analog" includes a group of structurally related proteins in which the A and B chains of insulin are covalently linked.
As used herein, "modification" of an amino acid refers to the substitution, addition or deletion of an amino acid or the induction of an amino acid by adding and / or removing a chemical group from the amino acid to a human protein. It includes substitutions or additions with atypical or unnatural amino acids, as well as the 20 commonly found amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich (Milwaukee, WI), ChemPep Inc. (Miami, FL), and Genzyme Pharmaceuticals (Cambridge, MA). Amino acids that are not typical may be purchased from commercial sources, newly synthesized from natural amino acids, or chemically modified or derivatized.
As used herein, "substitution" of an amino acid means that one amino acid residue is replaced by a different amino acid residue. Throughout this application, any reference to the position of a particular amino acid by letter and number (eg, position A5) is either chain A (SEQ ID NO: 1) or chain B (SEQ ID NO: 2) of natural human insulin. ), Refers to the position of the amino acid at the corresponding position on the A chain (for example, the position of A5) or the B chain (for example, the position of B5), or the position of the corresponding amino acid in any analog thereof. For example, without further detail, the term "B28 position" herein means the corresponding B27 position if it is the B chain of an insulin analog lacking the first amino acid of SEQ ID NO: 2. ..
As used herein, the expression "conservative amino acid substitution" is defined herein as an exchange within one of the following five groups:
I. Aliphatic, non-polar or slightly polar small residues Ala, Ser, Thr, Pro, Gly II. Polarized, negatively charged residues and their amides Asp, Asn, Glu, Gln III. Polar and positively charged residues His, Arg, Lys, Ornithine (Orn) IV. Aliphatic, large non-polar residues Met, Leu, Ile, Val, Cys, Norleucine (Nle), Homocysteine V. Large aromatic residues
Phe, Tyr, Trp, Acetyl Phenylalanine As used herein, the general term "polyethylene glycol chain" or "PEG chain" is the general formula H (OCH).<sub>2</sub>CH<sub>2</sub>)<sub>n</sub>A mixture of a condensed polymer (branched or linear) of ethylene oxide and water, represented by OH (where n is at least 9 in the formula). In the absence of further features, the term is intended to include polymers of ethylene glycol selected from the range of 500-80,000 Dalton average total molecular weight. A "polyethylene glycol chain" or "PEG chain" is used in combination with a numeric suffix to indicate its approximate average molecular weight. For example, PEG-5,000 exhibits a polyethylene glycol chain with an average total molecular weight of about 5,000 daltons.
As used herein, "PEGylated" and similar terms refer to a compound modified from its natural state by binding a polyethylene glycol chain to the compound. A "PEGylated polypeptide" is a polypeptide having a PEG chain covalently attached to the polypeptide.
As used herein , a "linker" is a bond, molecule or group of molecules that binds two separate things together. The linker may provide the optimum spacing between the two, or it may further provide an unstable bond that allows the two to be separated from each other. Unstable bonds include photocleavable groups, acid-sensitive moieties, base-sensitive moieties, and enzyme-cleavable groups.
As used herein, an "insulin dimer" is a complex having two insulin peptides covalently attached to each other via a linker. The term insulin dimer includes both insulin homodimers and insulin heterodimers when used without any other limiting wording. Insulin homodimers contain two identical subunits (each with its own A and B chains), whereas insulin heterodimers are two subunits that are substantially similar to each other but not identical to each other. including.
n can be 1-6, "C<sub>1</sub>~ C<sub>n</sub>The expression "alkyl", as used herein, refers to a branched or linear alkyl group having from one to a specified number of carbon atoms. General C<sub>1</sub>~ C<sub>6</sub>Examples of the alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
n can be 2-6, "C<sub>2</sub>~ C<sub>n</sub>The expression "alkenyl" as used herein represents a branched or linear olefin-based unsaturated group having two to a specified number of carbon atoms and at least one double bond. .. Examples of such groups are 1-propenyl, 2-propenyl (-CH).<sub>2</sub>-CH = CH<sub>2</sub>), 1,3-Butadienyl, (-CH = CHCH = CH<sub>2</sub>), 1-Butenyl (-CH = CHCH)<sub>2</sub>CH<sub>3</sub>), Hexenyl, pentenyl, etc., but are not limited to these.
"C" where n can be 2-6<sub>2</sub>~ C<sub>n</sub>The expression "alkynyl" refers to a branched or linear unsaturated group having 2 to n carbon atoms and at least one triple bond. Examples of such groups include, but are not limited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl and the like.
As used herein, the term "aryl" is a monocyclic having one or two aromatic rings, such as, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, etc. Or a bicyclic carbocyclic system. The size of the aryl ring and the presence of substituents or bonding groups are indicated by the number of carbons present. For example, "(C<sub>1</sub>~ C<sub>3</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>The expression "aryl)" is C<sub>1</sub>~ C<sub>3</sub>C bonded to the parent moiety via the alkyl chain of<sub>5</sub>~ C<sub>10</sub>Indicates the aryl of.
The term "heteroaryl", as used herein, is a monocycle containing one or two aromatic rings, the aromatic ring containing at least one nitrogen, oxygen or sulfur atom. Indicates a cyclic or bicyclic ring system. The size of the heteroaryl ring and the presence of substituents or bonding groups are indicated by the number of carbons present. For example, "(C<sub>1</sub>~ C<sub>n</sub>Alkyl) (C<sub>5</sub>~ C<sub>6</sub>The expression "heteroaryl)" is C bonded to the parent moiety via an alkyl chain consisting of 1 to "n" carbons.<sub>5</sub>~ C<sub>6</sub>Shows heteroaryl.
"C<sub>3</sub>~ C<sub>n</sub>The expression "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic ring having a carbon atom and a hydrogen atom, and the subscript number indicates the number of carbon atoms present. For example, C<sub>3</sub>~ C<sub>8</sub>Cycloalkyl represents the compounds cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl.
"C<sub>3</sub>~ C<sub>n</sub>The expression "heterocycle" refers to a cycloalkyl ring system containing 1 to "n-1" heteroatoms, which heteroatoms are selected from the group consisting of oxygen, sulfur and nitrogen. For example, "5-membered heterocycle" or "C"<sub>5</sub>The phrase "heterocycle" is a 5-membered heterocycle with one heteroatom (eg, thiophene, pyrrole, furan, etc.), a 5-membered ring with two heteroatoms at the 1,2 or 1,3 positions. Heterocycles (eg, oxazole, pyrazole, imidazole, thiazole, purine, etc.) and 5-membered heterocycles with three heteroatoms (eg, triazole, thiazazole, etc.), but are not limited thereto. ..
"C<sub>3</sub>~ C<sub>n</sub>The expression "membered ring" as used herein refers to a saturated or unsaturated hydrocarbon ring structure in which a total of 3 to "n" components combine with each other to form a ring, in this case. The components of the ring are selected from the group consisting of C, O, S and N. This expression is intended to include cycloalkyl, heterocycles, aryls, heteroaryls.
As used herein, the term "halo" refers to one or more components of the group consisting of fluorine, chlorine, bromine, iodine.
As used herein, the term "patient" includes, but is limited to, domesticated warm-blooded vertebrates (eg, domestic animals, horses, cats, dogs, and other pets, unless otherwise specified. Is not intended to include humans).
Embodiment The present disclosure provides insulin prodrug derivatives prepared to improve the therapeutic index of the underlying insulin peptide by delaying the onset of action of the insulin peptide and prolonging its half-life. In the insulin prodrug chemistry disclosed herein, the prodrug can be activated by an enzymatic degradation mechanism. The prodrug chemistry disclosed herein is capable of chemically binding to the amine at the active site to form an amide that returns to the parent amine upon diketopiperazine formation and release of the prodrug element. This novel biologically preferred prodrug chemistry degrades autonomously under physiological conditions (eg, an aqueous environment at pH about 7, 37 ° C) and is independent of enzymatic degradation. The duration of action of the prodrug derivative depends on the choice of dipeptide prodrug sequence, providing flexibility in the prodrug preparation.
In one embodiment, a prodrug is provided that, under physiological conditions, has a half-life (t1 / 2) of enzyme-independent activation of 1-100 hours. The physiological conditions disclosed herein are intended to include a temperature of about 35 to about 40 ° C and a pH of about 7.0 to about 7.4 in an aqueous environment, more generally pH 7.2 to 7.4, Intended to include temperatures of 36-38 ° C. In one embodiment, a dipeptide capable of forming diketopiperazine under physiological conditions is covalently attached to an insulin peptide via an amide bond.
Fortunately, the rate of cleavage and therefore the activation of the prodrug depends on the structure and character isomerism of the dipeptide precursor and also on the strength of the nucleophile. The prodrugs disclosed herein will eventually be chemically converted into recognizable structures by the natural receptors of the drug, depending on the rate of this chemical conversion, in vivo. The timing and duration of biological action is determined. The chemistry of prodrugs disclosed in this application utilizes intramolecular chemical reactions that are independent of additional chemicals or enzymes. The conversion rate is controlled by the chemistry and cleavage of the dipeptide substituent under physiological conditions. Because the physiological pH and temperature are tightly regulated within a very limited range, the rate of prodrug-to-drug conversion is highly reproducible, both in one patient and between patients. ..
As disclosed herein, bioactive polypeptides have a long half-life of at least 1 hour, more generally greater than 20 hours but less than 100 hours, and are enzyme-independent, uniquely chemical. The reaction driven by instability provides a prodrug that is transformed into an active form under physiological conditions. In one embodiment, the enzyme-independent activation of the prodrug, which can be determined by incubating the prodrug at 37 ° C, pH 7.2 in phosphate buffer (PBS, etc.), has a t1 / 2 hour of 1 to 100 hours. More generally, it is 12 to 72 hours, and in one embodiment, t1 / 2 is 24 to 48 hours. In one embodiment, the half-life of the prodrug is about 1 hour, about 8 hours, about 12 hours, about 20 hours, about 24 hours, about 48 hours or about 72 hours. The half-lives of various prodrugs are expressed in equation t<sub>1/2</sub>Calculated using = 0.693 / k (in the equation, "k" is the primary rate constant of the prodrug decomposition). In one embodiment, activation of the prodrug occurs after cleavage of the amide-bound dipeptide, formation of diketopiperazine or diketomorpholine, the active insulin peptide.
Specific dipeptides consisting of natural or synthetic amino acids have been identified that facilitate intramolecular degradation under physiological conditions to release the active insulin peptide. The dipeptide can bind to an amino group present in natural insulin (via an amide bond) or to an amino group introduced into the insulin peptide by modification of the natural insulin peptide. In one embodiment, the dipeptide structure is selected to withstand cleavage by peptidases present in mammalian serum, such as dipeptidyl peptidase IV (DPP-IV). Therefore, in one embodiment, when the reaction is carried out in the presence of serum protease using physiological conditions, it is from a bioactive peptide (eg, insulin peptide (Q)) as compared to the reaction in which no protease is present. The rate of cleavage of the dipeptide protease element cannot be increased substantially (eg, more than double). Thus, the half-life in which the dipeptide prodrug element is cleaved from the insulin peptide (under physiological conditions in PBS) is that the dipeptide prodrug element is cleaved from the insulin peptide in a solution containing the DPP-IV protease. Less than twice, three times, four times or five times the half-life. In one embodiment, the solution containing the DPP-IV protease is serum, especially mammalian serum (including human serum).
According to one embodiment, the dipeptide prodrug element has a structural UO, where U is an amino acid or a hydroxy acid and O is an N-alkylated amino acid. In one embodiment, the amino acid of the insulin peptide to which U, O or UO is bound is an unencoded amino acid. In one embodiment, U and / or O are amino acids that have a D-type stereoisomer configuration. In some exemplary embodiments, U is an amino acid with a D-type stereoisomer configuration and O is an amino acid with an L-type stereoisomer configuration. In some exemplary embodiments, U is an amino acid with an L-type stereoisomer configuration and O is an amino acid with a D-type stereoisomer configuration. In some exemplary embodiments, U is an amino acid with a D-type stereoisomer configuration and O is an amino acid with a D-type stereoisomer configuration. In one embodiment, O is an N-alkylated amino acid, but not proline. In one embodiment, the N-alkylated group of amino acid O is C<sub>1</sub>~ C<sub>18</sub>Alkylated, in one embodiment the N-alkylated group is C<sub>1</sub>~ C<sub>6</sub>It is alkyl. In one embodiment, the UO is a dipeptide having the structure of formula I as defined herein.
In one embodiment, the dipeptide is an amino group selected from the N-terminal amino group of the A or B chain or the side chain amino group of an amino acid present at the active site of the insulin peptide and binds to the insulin peptide. According to one embodiment, the dipeptide extension covalently binds to the insulin peptide via the amine in the side chain of the lysine residue at or near the active site. In one embodiment, the dipeptide extension is attached via a synthetic or modified amino acid, where the synthetic or modified amino acid exhibits a functional group suitable for covalent binding of the dipeptide extension (eg,). , Amino phenylalanine aromatic amines, etc.). According to one embodiment, the dipeptide is an amino group at the N-terminus of the A or B chain, or an aromatic amine (eg, 4-aminophenylalanine residue) present at the A19, B16 or B25 positions. An amino group selected from the amino groups of the side chains such as, which binds to an insulin peptide. In one embodiment, the UO dipeptide binds at position A19 via 4-aminophenylalanine present at position A19.
Dipeptide prodrug elements are designed to autonomously cleave amide bonds to insulin analogs under physiological conditions and in the absence of enzymatic activity. In one embodiment, the N-terminal amino acid of the dipeptide extension has a C-alkylated amino acid (eg, aminoisobutyl acid). In one embodiment, the C-terminal amino acid of the dipeptide has an N-alkylated amino acid (eg, proline or N-methylglycine). In one embodiment, the dipeptide has an N-terminal C-alkylated amino acid sequence, followed by an N-alkylated amino acid.
Applicants have found that the 4-aminophenyl amino acid moiety can be selectively inserted into the 19th natural tyrosine of the A chain without compromising the activity of the insulin peptide (see Figure 3). Subsequent chemical amidation of the amino group at the active site with the dipeptide prodrug moiety disclosed herein dramatically reduces the binding activity of the insulin receptor, thus providing a suitable prodrug of insulin. (See Figure 6; data include insulin (p-NH)<sub>2</sub>-F)<sup>A19</sup>IGF1Y16L17 (p-NH) proved to have activity comparable to<sub>2</sub>-F)<sup>A19</sup>Indicates an analog. See Figure 4). Thus, in one embodiment, the dipeptide prodrug element is attached to the aromatic ring of A19 4-aminophenylalanine via an amide bond. In this case, the C-terminal amino acid of the dipeptide has an N-alkylated amino acid, and the N-terminal amino acid of the dipeptide is any amino acid.
A dipeptide prodrug moiety may be attached to another site of the insulin peptide to prepare an insulin prodrug or depot analog. According to one embodiment, it is selected from the group consisting of an A chain, a B chain, and an amino group at the N-terminal of the A chain or the B chain via an amide bond, or an amino group at the side chain of an amino acid other than the terminal. Insulin prodrug / depot with a dipeptide that is bound to one or more sites (eg, bound to the aromatic amine of a 4-aminophenylalanine residue present at position A19, B16 or B25). An analog is provided. In one embodiment, the insulin peptide has two dipeptide elements. These dipeptide elements are optionally PEGylated, alkylated, acylated, or attached to a depot polymer. In one embodiment, the dipeptide has an N-terminal C-alkylated amino acid, followed by an N-alkylated amino acid.
The A and B chains with insulin prodrug analogs may have the native sequences of the respective peptides (ie, SEQ ID NO: 1 and SEQ ID NO: 2), and SEQ ID NO: 1 and / or SEQ ID NO: 2 It may have a derivative of. This derivative is modified to 4-aminophenylalanine at the position A19, B16 or B25 and / or A5, A8, A9, A10, A14, A15, A17, A18, A19 and A21, B1, B2, B3, B4. , B5, B9, B10, B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 and B30 at a position selected from one or more amino acid substitutions or B1-4 and Includes deletions at any or all positions of B26-30. In one embodiment, the dipeptide prodrug element is attached to the N-terminal amino group of the A or B chain, and the C-terminal amino acid of the dipeptide prodrug element has an N-alkylated amino acid and is a dipeptide prodrug. The N-terminal amino acid of the drug element is any amino acid. However, when the C-terminal amino acid of the dipeptide is proline, the N-terminal amino acid of the dipeptide has a C-alkylated amino acid.
In one embodiment, the dipeptide prodrug element has the general structure of formula I.
<img id="000020" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or aryl, or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, but R<sub>4</sub>And R<sub>3</sub>If, together with the atoms to which they are bonded, form a 4-membered, 5-membered or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither H.
In another embodiment, the dipeptide prodrug element has the following general structure:
<img id="000021" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>8</sub>Independently, H or C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, except that the dipeptide of formula I binds via the amine at the N-terminus of the peptide and is R.<sub>4</sub>And R<sub>3</sub>However, if these are combined with the bonded atoms to form a 5- or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither H. In one embodiment, the first and / or second amino acids of the dipeptide prodrug element are amino acids that have a D-type stereoisomer configuration.
In one embodiment, the prodrug element of Equation I is provided, in the equation, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>8</sub>Form a cycloalkyl ring, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, R<sub>8</sub>Is H. In one embodiment, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>6</sub>Alkyl, CH<sub>2</sub>OH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle. In another embodiment, R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>8</sub>Is H.
In another embodiment, a prodrug element of formula I is provided, in the formula, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>8</sub>Form a cycloalkyl ring, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo, R<sub>8</sub>Is H, but R<sub>4</sub>And R<sub>3</sub>However, if these are combined with the bonded atoms to form a 5- or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither H. In one embodiment, the first and / or second amino acids of the dipeptide prodrug element are unencoded amino acids, and in one embodiment, amino acids that have a D-type stereoisomer configuration.
In other embodiments, the dipeptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>And R<sub>8</sub>Independently, H or C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo, R<sub>8</sub>Is H.
In another embodiment, the dipeptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>18</sub>Alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
In another embodiment, the dipeptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is (CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, but R<sub>1</sub>And R<sub>2</sub>Are both not hydrogen and R<sub>4</sub>Or R<sub>8</sub>At least one of them is hydrogen.
In another embodiment, the peptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is (CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 6-membered heterocycle, R<sub>4</sub>Is hydrogen and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>8</sub>Is hydrogen, R<sub>5</sub>Is NH<sub>2</sub>However, R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
In another embodiment, the dipeptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NH<sub>2</sub>However, R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
In another embodiment, the dipeptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is (CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>4</sub>Is (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>And R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH, R<sub>8</sub>Is hydrogen, but R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
In another embodiment, the dipeptide prodrug element has the structure of formula I, in the formula, R<sub>1</sub>Is hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>2</sub>Is hydrogen, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, but R<sub>1</sub>Is alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>If, then R<sub>1</sub>And R<sub>5</sub>Together with the atoms to which they are bonded form a 4- to 11-membered heterocycle.
According to one embodiment, an insulin prodrug analog having an insulin peptide and an amide-binding dipeptide is provided. In particular, the insulin prodrug analog has an A-chain sequence and a B-chain sequence, and the A-chain has the sequence Z-GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>-R<sub>13</sub>1-9, 1-5 or 1 at a position selected from positions (SEQ ID NO: 3) or A5, A8, A9, A10, A14, A15, A17, A18 (relative to the A chain of natural insulin). ~ 3 amino acid modifications have a sequence different from SEQ ID NO: 3, have an analog thereof, and the B chain sequence is JX<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>The sequence of (SEQ ID NO: 14) or (relative to the B chain of natural insulin; that is, the amino acid X of SEQ ID NO: 14<sub>4</sub>Corresponds to position B5 of natural insulin) at a position selected from the positions B1, B2, B3, B4, B5, B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 and B30. , 1-10, 1-5 or 1-3 amino acid modifications having a sequence different from that of SEQ ID NO: 14 and its analogs. Z and J are independently H or dipeptides having the general structure of formula I below.
<img id="000022" he="26" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>14</sub>X the "J" element<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>(SEQ ID NO: 14) A bond that connects to an array, or X<sub>14</sub>X the "J" element<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>(SEQ ID NO: 14) X connected to the array<sub>9</sub>The sequence consisting of 1 to 4 amino acids selected from the group consisting of VNQ (SEQ ID NO: 21), VNQ, NQ and Q is shown.
X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000023" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, m is an integer selected from 0 to 3, and X is OH, NH.<sub>2</sub>, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide that is H or has the general structure of formula I below. ]<img id="000024" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Is selected from the group consisting of asparagine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000025" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, m is an integer selected from 0 to 3, and X<sub>12</sub>Is OH, NH<sub>2</sub>, NHR<sub>11</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>11</sub>Is a dipeptide that is H or has the general structure of formula I below. ]<img id="000026" he="30" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>8</sub>Is a histidine, arginine or an amino acid having the following general structure,<img id="000027" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, m is an integer selected from 0 to 3, and X<sub>13</sub>Is H, OH, NH<sub>2</sub>, NHR<sub>12</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>12</sub>Is a dipeptide that is H or has the general structure of formula I below. ]<img id="000028" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine, in the formula, R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or aryl, or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, R<sub>13</sub>Is COOH or CONH<sub>2</sub>Is. In one embodiment, X<sub>8</sub>Is histidine, arginine or tyrosine. In another embodiment, X<sub>8</sub>Is an amino acid having the following general structure.
<img id="000029" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In one embodiment, R<sub>13</sub>Is COOH, and the amino acid at the carboxy terminal of the B chain is an amide (CONH) instead of the natural α-carbon carboxy group.<sub>2</sub>). In one embodiment, X, X<sub>12</sub>, X<sub>13</sub>, J, Z, one or more, are dipeptides having the general structure of formula I below.
<img id="000030" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In one embodiment, X, X<sub>12</sub>, X<sub>13</sub>, J, Z contain dipeptides having the general structure of formula I.
<img id="000031" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> According to one embodiment, X, X<sub>12</sub>, X<sub>13</sub>, J, Z are at least one dipeptide having the general structure of formula I below.
<img id="000032" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In one embodiment, X, X<sub>12</sub>, X<sub>13</sub>, J, Z contain only one of the dipeptides having the general structure of formula I.
<img id="000033" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />(That is, only one dipeptide prodrug element binds to the insulin peptide). In addition, the dipeptide prodrug element binds to the N-terminus of the A or B chain (ie, either J or Z has a dipeptide) and R<sub>4</sub>And R<sub>3</sub>If, together with the atoms to which they are bonded, form a 4-membered, 5-membered or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>At least one of them is other than H, and in one embodiment, R<sub>1</sub>And R<sub>2</sub>Are both other than H. In one embodiment, J and Z are both H and X<sub>12</sub>Is OH and X<sub>13</sub>Is H or OH and X is NHR<sub>10</sub>And R<sub>10</sub>Is a dipeptide having the general structure of formula I. In another embodiment, the A chain is sequenced GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>-R<sub>13</sub>It has (SEQ ID NO: 3) and the B chain sequence is X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>It has the sequence of (SEQ ID NO: 14), m is 1 and X<sub>12</sub>Is OH and X<sub>13</sub>Is H or OH and X is NHR<sub>10</sub>And R<sub>10</sub>Is a dipeptide having the structure of the general formula I, R<sub>13</sub>Is COOH, and the amino acid at the carboxy terminal of the B chain is an amide (CONH) instead of the natural α-carbon carboxy group.<sub>2</sub>), And the rest of the symbols are as defined above.
In one embodiment, X, X<sub>12</sub>, X<sub>13</sub>, J, Z are dipeptides having the general structure of Formula I below.
<img id="000034" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, but R<sub>4</sub>And R<sub>3</sub>However, if these are combined with the bonded atoms to form a 5- or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither H. W<sub>1</sub>If is N, it will be obvious to those skilled in the art that the nitrogen atom binds to H under physiological conditions. In one embodiment, J or Z has a dipeptide of formula I and R<sub>4</sub>And R<sub>3</sub>If, together with the atoms to which they are bonded, form a 4-membered, 5-membered or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
In one embodiment, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, R<sub>8</sub>Is H. According to another embodiment, m is 1 and R<sub>8</sub>Is H and R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>4</sub>Alkyl, (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle. In one embodiment, m is 1 and R<sub>8</sub>Is H and R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle. In another embodiment, X<sub>9</sub>Is phenylalanine, a single dipeptide extension attached to the insulin peptide via an amide bond to the N-terminus of the A or B chain. In another embodiment, the insulin peptide has a 4-aminophenylalanine substitution at position 19 of the A chain, and the single dipeptide extension is via an amide bond formed by the aromatic amine of 4-aminophenylalanine. It is bound to the peptide. In one embodiment, the insulin analogs disclosed herein have a C-terminal amide or ester instead of the C-terminal carboxylate of the A and / or B chains.
According to one embodiment, the dipeptide of formula I is further modified to include a large polymer that interferes with the ability of the insulin analog to interact with the insulin receptor or IGF-1 receptor. Subsequent cleavage of the dipeptide releases an insulin analog from the dipeptide complex, which is fully active. According to one embodiment, J has a dipeptide of formula I and comprises a large polymer that interferes with the ability of bound insulin analogs to interact with the insulin receptor or IGF-1 receptor. Further modifies the dipeptide of. According to one embodiment, X, X<sub>12</sub>, X<sub>13</sub>, J, Z comprises a dipeptide having the general structure of formula I.
<img id="000035" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />Here, the dipeptide of formula I is PEGylated, alkylated or acylated. In one embodiment, either J, Z or X has an acylated or PEGylated formula I dipeptide, and in one embodiment J has an acylated or PEGylated formula I dipeptide. Have.
In one embodiment, the dipeptide polymer element is covalently attached to the insulin peptide via the N-terminal amide bond of the A or B chain, or to the amine-bearing side chain of a non-terminal amino acid. The dipeptide further comprises a depot polymer bound to the dipeptide. In one embodiment, the natural amino acid of the insulin peptide is replaced with an amino acid suitable for forming an amide bond with the dipeptide of formula I. In one embodiment, the depot-retaining dipeptide binds at a position selected from A14, A19, B16, B28, B29. In one embodiment, two or more depot polymers are attached to a single dipeptide element. The depot polymer is biocompatible and large enough that the insulin peptide modified by the covalent bond of the dipeptide cannot be anchored at the injection site and / or interact with its corresponding receptor upon administration to the patient. Selected to have. Subsequent cleavage of the dipeptide releases an insulin peptide to interact with the intended target.
According to one embodiment, the depot polymer is selected from biocompatible polymers known to those of skill in the art. Depot polymers generally range in size from about 20,000 to about 120,000 daltons. In one embodiment, the depot polymer ranges in size from about 40,000 to about 100,000 or from about 40,000 to about 80,000 daltons. In one embodiment, the depot polymer is about 40,000, about 50,000, about 60,000, about 70,000 or about 80,000 daltons in size. Suitable depot polymers include dextran, polylactide, polyglycolide, caprolactone polymer, poly (caprolactone), polyacid anhydride, polyamine, polyesteramide, polyorthoester, polydioxanone, polyacetal, polyketal, polycarbonate, polyphosphoester, polyester. , Polybutylene terephthalate, polyorthocarbonate, polyphosphazene, succinate, poly (apple acid), poly (amino acid), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, polysaccharides, chitin, chitosan, hyaluronic acid, copolymers thereof, Examples include, but are not limited to, terpolymers, mixtures and biodegradable polymers and copolymers thereof (such as, but not limited to, caprolactone polymers, polycaprolactones and copolymers containing polybutylene terephthalate). In one embodiment, the depot polymer is selected from the group consisting of polyethylene glycol, dextran, polylactic acid, polyglycolic acid, copolymers of lactic acid and glycolic acid, and in one specific embodiment, the depot polymer is polyethylene glycol. .. In one embodiment, the depot polymer is polyethylene glycol and the total molecular weight of the depot polymer (s) bound to the dipeptide element is from about 40,000 to about 80,000 daltons.
According to one embodiment, the dipeptide element of formula I further comprises a polyethylene oxide, alkyl or acyl group. In one embodiment, one or more polyethylene oxide chains are attached to a dipeptide of formula I and the total molecular weight of the polyethylene oxide chains ranges from about 20,000 to about 80,000 daltons or 40,000 to 80,000 daltons or 40,000 to 60,000 daltons. Is. In one embodiment, the polyethylene oxide is polyethylene glycol. In one embodiment, at least one polyethylene glycol chain having a molecular weight of about 40,000 daltons or about 20,000 daltons is attached to the dipeptide of the formula, either directly or indirectly via a linker / spacer. In another embodiment, the dipeptide of formula I is acylated with a sufficiently large acyl group to bind serum albumin and inactivate insulin analogs upon administration. The acyl group may be linear or branched and, in one embodiment, is a C16-C30 fatty acid. For example, the acyl group may be any of C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid or C30 fatty acid. In one embodiment, the acyl group is a C16-C20 fatty acid, eg, a C18 fatty acid or a C20 fatty acid.
According to one embodiment, an insulin prodrug analog having an A-chain sequence and a B-chain sequence is provided. A chain is the sequence GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>-R<sub>13</sub>It has (SEQ ID NO: 3) and the B chain sequence is X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>It has the sequence of (SEQ ID NO: 14). here, X<sub>14</sub>X the "J" element<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>-R<sub>14</sub>(SEQ ID NO: 14) N-terminal amine, X, connected to the sequence<sub>9</sub>It is selected from the group consisting of VNQ (SEQ ID NO: 21), VNQ, NQ, and Q.
X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000036" he="54" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> Where U is an amino acid or a hydroxy acid and O is an N-alkylated amino acid that is attached via an amide bond. X<sub>3</sub>Is selected from the group consisting of asparagine, ornithine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000037" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, m is an integer selected from 0 to 3, and X<sub>12</sub>Is OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>8</sub>Is an amino acid having the following general structure,<img id="000038" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, m is an integer selected from 0 to 3, and X<sub>13</sub>Is H, OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine, R<sub>13</sub>And R<sub>14</sub>Independently, COOH or CONH<sub>2</sub>Is. In one embodiment, X<sub>7</sub>And X<sub>8</sub>Are both tyrosine and R<sub>13</sub>Is COOH, and the amino acid at the carboxy terminal of the B chain is an amide (CONH) instead of the natural α-carbon carboxy group.<sub>2</sub>).
According to one embodiment, the sequence Z-GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>1-3 amino acid modifications at positions selected from the positions A5, A8, A9, A10, A14, A15, A17, A18 (relative to the A chain sequence of natural insulin) (SEQ ID NO: 3) A compound having an analog thereof having a sequence different from that of SEQ ID NO: 3 is provided. In this embodiment, X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000039" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In the formula, X is OH, NH<sub>2</sub>, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>And Z are independently H or dipeptides with structural UO, where U is an amino acid or hydroxy acid, O is an N-alkylated amino acid, and O is an amide bond. It binds to the peptide of SEQ ID NO: 3 by formation. However, R<sub>10</sub>And Z are not identical, and the amino acid of SEQ ID NO: 3 to which U, O or UO binds is an unencoded amino acid. In one embodiment, under physiological conditions in PBS, cleavage of the dipeptide from SEQ ID NO: 3 by chemical reaction is completed at least about 90% within about 1 to about 720 hours. In another embodiment, the half-life of chemical reaction cleavage of UO from SEQ ID NO: 3 (t).<sub>1/2</sub>) Is at least about 1 hour to about 1 week in PBS under physiological conditions. The compound in one embodiment further comprises the B chain of insulin bound to the A chain of SEQ ID NO: 3 either by intermolecular disulfide bonds or as a recombinant single chain polypeptide.
The choice of binding site for a dipeptide element substituent and a dipeptide prodrug element may affect the rate of cleavage of the dipeptide prodrug element from an insulin peptide by a chemical reaction. In one embodiment, an insulin prodrug having an A-chain sequence and a B-chain sequence is provided. A chain is the sequence GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>It has (SEQ ID NO: 3) and the B chain sequence is X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>It has the sequence of LVEALYLVCGERGFF (SEQ ID NO: 4). here, X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000040" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>3</sub>Is selected from the group consisting of asparagine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>14</sub>Is a join, X<sub>9</sub>Selected from the group consisting of VNQ (SEQ ID NO: 21), VNQ, NQ, Q, X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine, the dipeptide prodrug element has the following structure:<img id="000041" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />It binds to the α-amino group of the N-terminal amino acid of the peptide of SEQ ID NO: 3 or SEQ ID NO: 4, but R<sub>4</sub>And R<sub>3</sub>If, together with the atoms to which they are bonded, form a 4-membered, 5-membered or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen. In this embodiment, t in PBS under physiological conditions.<sub>1/2</sub>A compound is provided in which is about 1 hour.
During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>18</sub>Alkyl or aryl, or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>-Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is an amine.
In other embodiments, it binds at the N-terminus and t<sub>1/2</sub>A prodrug having a prodrug element, where is, for example, about 1 hour, has a dipeptide prodrug element having the following structure.
<img id="000042" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>18</sub>Alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
Or GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>A chain sequence of (SEQ ID NO: 3) and X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>An insulin prodrug with the B chain sequence of (SEQ ID NO: 14) is provided, the dipeptide prodrug element is attached to the α-amino group of the N-terminal amino acid of the peptide of SEQ ID NO: 3 or SEQ ID NO: 14 and into PBS. Under physiological conditions, t<sub>1/2</sub>Is about 6 to about 24 hours. In one embodiment, such a compound has a prodrug element of formula I, in the formula, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Selected from the group consisting of alkyl and aryl, or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>-Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl and aryl, R<sub>5</sub>Is an amine, but R<sub>1</sub>And R<sub>2</sub>Are both not hydrogen and R<sub>4</sub>Or R<sub>8</sub>One of them is hydrogen.
In another embodiment, GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>A chain sequence of (SEQ ID NO: 3) and X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>An insulin prodrug having the B chain sequence of (SEQ ID NO: 14) is provided. The dipeptide prodrug element binds to the α-amino group of the N-terminal amino acid of the peptide of SEQ ID NO: 3 or SEQ ID NO: 14, and under physiological conditions in PBS, t<sub>1/2</sub>Is about 72 to about 168 hours. In one embodiment, such a compound has a prodrug element of formula I and During the ceremony R<sub>1</sub>Is hydrogen, C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl and aryl, R<sub>2</sub>Is H, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is an amine or N-substituted amine or hydroxyl, However, R<sub>1</sub>If is alkyl or aryl, then R<sub>1</sub>And R<sub>5</sub>Together with the atoms to which they are bonded form a 4- to 11-membered heterocycle.
In one embodiment, it has a dipeptide prodrug element attached to the N-terminal α-amino acid of an insulin A-chain peptide or B-chain peptide, t.<sub>1/2</sub>For example, a prodrug that lasts from about 12 to about 72 hours and, in one embodiment, from about 12 to about 48 hours, has a dipeptide prodrug element having the following structure:
<img id="000043" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is (CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, but R<sub>1</sub>And R<sub>2</sub>Are both not hydrogen and R<sub>4</sub>Or R<sub>8</sub>At least one of them is hydrogen.
In one embodiment, it has a dipeptide prodrug element attached to the N-terminal amino acid of an insulin A-chain peptide or B-chain peptide, t.<sub>1/2</sub>For example, a prodrug that lasts from about 12 to about 72 hours and, in one embodiment, from about 12 to about 48 hours, has a dipeptide prodrug element having the following structure:
<img id="000044" he="24" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is (CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 6-membered heterocycle, R<sub>4</sub>Is hydrogen and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>5</sub>Is NH<sub>2</sub>Is. However, R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
In other embodiments, it has a dipeptide prodrug element attached to the N-terminal amino acid of an insulin A-chain peptide or B-chain peptide, t.<sub>1/2</sub>For example, a prodrug that lasts from about 12 to about 72 hours and, in one embodiment, from about 12 to about 48 hours, has a dipeptide prodrug element having the following structure:
<img id="000045" he="26" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl R<sub>4</sub>Is hydrogen, R<sub>5</sub>Is NH<sub>2</sub>Is. However, R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
In one embodiment, it has a dipeptide prodrug element attached to the N-terminal amino acid of an insulin A-chain peptide or B-chain peptide, t.<sub>1/2</sub>For example, a prodrug that lasts from about 12 to about 72 hours and, in one embodiment, from about 12 to about 48 hours, has a dipeptide prodrug element having the following structure:
<img id="000046" he="26" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is (CH<sub>2</sub>)<sub>p</sub>Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl R<sub>4</sub>Is (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>And R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH. However, R<sub>1</sub>And R<sub>2</sub>Are neither hydrogen.
It also has a dipeptide prodrug element bound to the N-terminal α-amino acid of insulin A-chain peptide or B-chain peptide, t<sub>1/2</sub>Prodrugs are provided, for example, for about 72 to about 168 hours, where the dipeptide prodrug element has the following structure:
<img id="000047" he="26" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>Is hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo. However, R<sub>1</sub>Is alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>If, then R<sub>1</sub>And R<sub>5</sub>Together with the atoms to which they are bonded form a 4- to 11-membered heterocycle.
In one embodiment, the dipeptide prodrug element binds to an amine on the side chain of an amino acid other than the terminal of the insulin peptide. In this embodiment, t<sub>1/2</sub>A prodrug, for example, for about 1 hour, has the following structure:
<img id="000048" he="26" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>8</sub>Alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>-Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NH<sub>2</sub>And R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
In addition, t<sub>1/2</sub>For example, for about 6 to about 24 hours, a prodrug having a dipeptide prodrug element bound to a side chain of an amino acid other than the terminal has a dipeptide prodrug element having the following structure.
<img id="000049" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>-Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>And R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, but R<sub>1</sub>And R<sub>2</sub>Are both not hydrogen and R<sub>4</sub>Or R<sub>8</sub>At least one of them is hydrogen.
Also, t<sub>1/2</sub>Is provided, for example, for about 72 to about 168 hours, and a prodrug having a dipeptide prodrug element bound to the side chain of an amino acid other than the terminal of the insulin peptide is provided, wherein the dipeptide prodrug element has the following structure. ..
<img id="000050" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>It is selected from the group consisting of alkyl) OH and halo, but R<sub>1</sub>And R<sub>2</sub>Are both independently alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>If, then R<sub>1</sub>Or R<sub>2</sub>One of (CH<sub>2</sub>)<sub>p</sub>Through R<sub>5</sub>(In the formula, p is 2-9).
In one embodiment, the dipeptide prodrug element binds to an amine in the side chain of an amino acid other than the terminal of the insulin peptide, and the amino acid other than the terminal has the structure of Formula IV below.
<img id="000051" he="42" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony n is an integer selected from 1 to 4. In one embodiment, n is 3 or 4, and in one embodiment, the non-terminal amino acid is lysine. In one embodiment, the dipeptide prodrug element binds to the primary amine on the side chain of the amino acid at position 28 or 29 of the B chain of the insulin peptide.
In one embodiment, an insulin prodrug having an A-chain sequence and a B-chain sequence is provided. A chain is the sequence GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>It has (SEQ ID NO: 3) and the B chain sequence is X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>It has the sequence of (SEQ ID NO: 14). here, X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000052" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide that is H or has the following general structure: ]<img id="000053" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>-Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>And R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo, X<sub>3</sub>Is selected from the group consisting of asparagine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000054" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>12</sub>Is OH, NH<sub>2</sub>, NHR<sub>11</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>11</sub>Is a dipeptide that is H or has the following general structure: ]<img id="000055" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>8</sub>Is an amino acid having the following general structure,<img id="000056" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>13</sub>Is H, OH, NH<sub>2</sub>, NHR<sub>12</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>12</sub>Is a dipeptide that is H or has the following general structure: ]<img id="000057" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>14</sub>Is hydrogen (forming an N-terminal amine), X<sub>9</sub>Selected from the group consisting of VNQ (SEQ ID NO: 21), VNQ, NQ, Q, X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine, provided that R<sub>10</sub>, R<sub>11</sub>And R<sub>12</sub>Only one of them is a dipeptide having the following general structure:
<img id="000058" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> According to one embodiment in which the dipeptide prodrug element binds to the amino substituent of the aryl group of the aromatic amino acid, a prodrug preparation having the desired activation time is provided. For example, the structure of Equation III below<img id="000059" he="62" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> (In the formula, m is an integer of 0 to 3), and under physiological conditions in PBS, an insulin prodrug having t1 / 2 of about 1 hour is provided. In one embodiment where the insulin prodrug has the structure of formula III and has such a half-life, R<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>18</sub>Alkyl or aryl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Selected from the group consisting of alkyl and aryl, R<sub>5</sub>Is an amine or hydroxyl. In one embodiment, m is 1.
In one embodiment, the dipeptide prodrug element binds to the insulin peptide via an amine present at the aryl group of the aromatic amino acid of the insulin peptide.<sub>1/2</sub>The prodrug, for example, for about 1 hour, has the following dipeptide structure:
<img id="000060" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> In the formula, R<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>18</sub>Alkyl or (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>And R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 12-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>5</sub>Is NH<sub>2</sub>Or OH, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
In another embodiment, it has the structure of formula III (where m is an integer of 0 to 3) and under physiological conditions in PBS, t1 / 2 is about 6 to about 24 hours. Is an insulin prodrug. In one embodiment where the insulin prodrug has the structure of formula III and has such a half-life, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Selected from the group consisting of alkyl and aryl, or R<sub>1</sub>And R<sub>2</sub>Is-(CH<sub>2</sub>)<sub>p</sub>-Join via (in the formula, p is 2-9), R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 6-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Selected from the group consisting of alkyl and aryl, R<sub>5</sub>Is an amine or N-substituted amine. In one embodiment, m is 1.
In one embodiment, it has a dipeptide prodrug element linked via an amine present in the aryl group of an aromatic amino acid, t<sub>1/2</sub>Prodrugs are provided, for example, for about 6 to about 24 hours, and the dipeptide has the following structure:
<img id="000061" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 6-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
In another embodiment, it has the structure of formula III (where m is an integer from 0 to 3) and under physiological conditions in PBS, t1 / 2 is about 72 to about 168 hours. Is an insulin prodrug. In one embodiment where the insulin prodrug has the structure of formula III and has such a half-life, R<sub>1</sub>And R<sub>2</sub>Independently, hydrogen, C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl and aryl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 6-membered heterocycle, R<sub>4</sub>And R<sub>8</sub>Are each hydrogen, R<sub>5</sub>Is selected from the group consisting of amines, N-substituted amines, and hydroxyls. In one embodiment, m is 1.
In one embodiment, it has a dipeptide prodrug element linked via an aromatic amino acid, t<sub>1/2</sub>Prodrugs are provided that are, for example, about 72 to about 168 hours. The dipeptide has the following structure.
<img id="000062" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>Is hydrogen, C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>1</sub>And R<sub>5</sub>Together with the atoms to which they are bonded form a 4- to 11-membered heterocycle, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 4- to 6-membered heterocycle, R<sub>4</sub>Is hydrogen or R<sub>3</sub>Form a 4- to 6-membered heterocycle with and R<sub>8</sub>Is hydrogen, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
In one embodiment, the insulin prodrug analog is GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>-R<sub>13</sub>A chain sequence of (SEQ ID NO: 3) and X<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>It has the B chain sequence of (SEQ ID NO: 14). here, X<sub>1</sub>Is selected from the group consisting of threonine, histidine, arginine, lysine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000063" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>3</sub>Are asparagine, glycine, alanine, threonine or serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is tyrosine, X<sub>8</sub>Is tyrosine or phenylalanine.
X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine, X<sub>10</sub>Is an aspartic acid-lysine dipeptide, a lysine-proline dipeptide or a proline-lysine dipeptide, X<sub>11</sub>Is a threonine, alanine or threonine-arginine-arginine tripeptide, and the B chain has a carboxy-terminal extension consisting of 1 to 4 amino acids, and the carboxy-terminal extension is an amino acid having the following structure. Has.
<img id="000064" he="38" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony m is an integer from 0 to 3 n is an integer from 1 to 4 R<sub>12</sub>Is a dipeptide having the following general structure.
<img id="000065" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo, R<sub>13</sub>Is COOH or CONH<sub>2</sub>Is.
In one embodiment, the insulin prodrug analog is the sequence GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>-R<sub>13</sub>A chain sequence containing (SEQ ID NO: 3) and JX<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>(SEQ ID NO: 14) or JX<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>YTX<sub>10</sub> X<sub>11</sub>-R<sub>14</sub>It has a B chain sequence containing the sequence of (SEQ ID NO: 5), where J is an H (forming an N-terminal amine) or a dipeptide having the following general structure:<img id="000066" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>14</sub>Is a join that connects the "J" element to array number 14, or X<sub>14</sub>Indicates a sequence consisting of 1 to 4 amino acids selected from the group consisting of FVNQ (SEQ ID NO: 11), VNQ, NQ, and Q connecting the "J" element to SEQ ID NO: 14. X<sub>1</sub>Is selected from the group consisting of threonine, histidine, arginine, lysine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000067" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>3</sub>Are asparagine, glycine, alanine, threonine or serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000068" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>12</sub>Is OH, OCH<sub>3</sub>, NH<sub>2</sub>, NHR<sub>11</sub>Selected from the group consisting of R<sub>11</sub>Is a dipeptide having the following general structure. ]<img id="000069" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>8</sub>Is an amino acid having the following general structure,<img id="000070" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>13</sub>Is H, OH, OCH<sub>3</sub>, NH<sub>2</sub>, NHR<sub>12</sub>Selected from the group consisting of R<sub>12</sub>Is a dipeptide having the following general structure. ]<img id="000071" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine, X<sub>10</sub>Is an aspartic acid-lysine dipeptide, a lysine-proline dipeptide or a proline-lysine dipeptide, X<sub>11</sub>Is a threonine, alanine or threonine-arginine-arginine tripeptide, In the formula, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, R<sub>13</sub>And R<sub>14</sub>Independently, COOH or CONH<sub>2</sub>However, X<sub>12</sub>, X<sub>13</sub>Or only one of J is a dipeptide having the following general structure:
<img id="000072" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />(That is, only one peptide of the dipeptide prodrug element binds to the insulin peptide). In one embodiment, J is a dipeptide having the following general structure:
<img id="000073" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />X and X<sub>12</sub>Are OH and X<sub>13</sub>Is H, but R<sub>4</sub>And R<sub>3</sub>However, when these are combined with the bonded atoms to form a 5-membered heterocycle, R<sub>1</sub>And R<sub>2</sub>Are both other than H. In another embodiment, X<sub>12</sub>Is NHR<sub>11</sub>And J and X<sub>13</sub>Are each H and X is OH. In another embodiment, X<sub>13</sub>Is NHR<sub>12</sub>And X and X<sub>12</sub>Are OH and J is H, respectively. In one embodiment, the B chain is the sequence JX<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>YTPKT (SEQ ID NO: 15) or JX<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>Has YTKPT (SEQ ID NO: 16), where J, X<sub>4</sub>, X<sub>5</sub>, X<sub>6</sub>, X<sub>7</sub>, X<sub>8</sub>, X<sub>9</sub>Is as defined just above. In another embodiment, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle. In another embodiment, X<sub>4</sub>Is histidine, X<sub>5</sub>Is serine and X<sub>6</sub>Is histidine.
In another embodiment, Z-GIVE QCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>A chain sequence of (SEQ ID NO: 3) and X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>An insulin prodrug analog with a B chain sequence comprising the sequence of LVEALYLVCGERGFF (SEQ ID NO: 4) is provided. here, Z is H or is a dipeptide having the following general structure:<img id="000074" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000075" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide having the following general structure. ]<img id="000076" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Is selected from the group consisting of asparagine, glycine, alanine, threonine or serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, During the ceremony R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, except that if X is OH, then both X and Z are not dipeptides and Z is not H. In one embodiment, Z is the following general structure<img id="000077" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />Is a dipeptide with R<sub>4</sub>And R<sub>3</sub>However, when these are combined with the bonded atoms to form a 5-membered heterocycle, R<sub>1</sub>And R<sub>2</sub>At least one of them is other than H. In one embodiment, the A chain is Z-GIVEQCCX.<sub>1</sub>SICSLYQLENYCX<sub>3</sub>It has the sequence of (SEQ ID NO: 17), and the B chain sequence is the sequence X.<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALYLVCGERGFFYTPKT (SEQ ID NO: 12) or X<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>It has LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13).
In another embodiment, GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>A chain sequence having (SEQ ID NO: 3) and sequence X<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALYLVCGERGFFYTPKT (SEQ ID NO: 12) or X<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>A B chain sequence with LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13) and an insulin prodrug analog with are provided, where X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000078" he="55" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> U is an amino acid or hydroxy acid, O is an N-alkylated amino acid, X<sub>3</sub>Are asparagine, glycine, alanine, threonine or serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>9</sub>Is selected from the group consisting of phenylalanine and desaminophenylalanine. In one embodiment, UO exhibits a dipeptide of the following general structure:
<img id="000079" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH. In another embodiment, X<sub>7</sub>Is tyrosine and X<sub>8</sub>Is phenylalanine and X<sub>9</sub>Is phenylalanine, and in yet another embodiment, X<sub>4</sub>Is histidine, X<sub>5</sub>Is serine and X<sub>6</sub>Is histidine. In another embodiment, UO exhibits a dipeptide of the following general structure:
<img id="000080" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>, R<sub>8</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or R<sub>4</sub>And R<sub>8</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo.
In another embodiment, Z-GIVEQCCTSICSLYQLENX<sub>2</sub>CX<sub>3</sub>-R<sub>13</sub>A chain sequence of (SEQ ID NO: 18) and X<sub>4</sub>LCGSHLVEALYLVCGERGFF-R<sub>14</sub>An insulin prodrug analog having a B chain sequence comprising the sequence of (SEQ ID NO: 19) is provided. here, Z is H or is an amide-binding dipeptide having the following general structure:<img id="000081" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>2</sub>Is an amino acid having the following general structure,<img id="000082" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH and NHR<sub>10</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide having the following general structure. ]<img id="000083" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Is serine, asparagine or glycine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, R<sub>13</sub>And R<sub>14</sub>Independently, COOH or CONH<sub>2</sub>However, if Z is H, then X is not OH, and if X is OH, then Z is not H. In one embodiment, R<sub>13</sub>Is COOH and R<sub>14</sub>Is CONH<sub>2</sub>Is. In another embodiment, X<sub>2</sub>Is an amino acid having the general structure of the following formula III,<img id="000084" he="66" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> Z is H and X<sub>3</sub>Is serine and X<sub>4</sub>Is histidine, R<sub>13</sub>Is COOH and R<sub>14</sub>Is CONH<sub>2</sub>Is. In yet another embodiment R<sub>1</sub>H and C<sub>1</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl R<sub>2</sub>Is H, C<sub>1</sub>~ C<sub>6</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>2</sub>And R<sub>6</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl R<sub>4</sub>H and C<sub>1</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle . In another embodiment, R<sub>3</sub>Is CH<sub>3</sub>And R<sub>4</sub>Is H and R<sub>5</sub>Is NH<sub>2</sub>Or R<sub>5</sub>Is NH<sub>2</sub>And R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle. According to one embodiment, the B chain of the insulin prodrug analog is the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT-R.<sub>14</sub>(SEQ ID NO: 8), FVNQHLCGSHLVEALYLVCGERGFFYTKPT-R<sub>14</sub>(SEQ ID NO: 9) or FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR-R<sub>14</sub>Has (SEQ ID NO: 10), where R<sub>14</sub>Is COOH or CONH<sub>2</sub>And in one embodiment, R<sub>14</sub>Is CONH<sub>2</sub>Is.
In one embodiment, the sequence Z-GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>An insulin prodrug analog with the polypeptide of (SEQ ID NO: 3) is provided. Z is a dipeptide having the following general structure.
<img id="000085" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000086" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>3</sub>Are asparagine, glycine, alanine, threonine or serine, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH. In one embodiment, X<sub>1</sub>Is threonine and X<sub>3</sub>Is asparagine or glycine, in another embodiment R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>4</sub>Alkyl, (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle, but R<sub>4</sub>And R<sub>3</sub>However, if these are combined with the bonded atoms to form a 5- or 6-membered heterocycle, then R<sub>1</sub>And R<sub>2</sub>Are both other than H.
In one embodiment, the array GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>An insulin prodrug analog with the polypeptide of (SEQ ID NO: 3) is provided, wherein X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000087" he="64" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Are asparagine, glycine, alanine, threonine or serine, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>6</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH. In one embodiment, GIVEQCCX as defined just above.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>The A chain of (SEQ ID NO: 3) is sequence X by a disulfide bond or as a single chain polypeptide.<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>It binds to the B chain having (SEQ ID NO: 14), where X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is tyrosine, X<sub>8</sub>Is phenylalanine.
In another embodiment, the dipeptide prodrug element has the structure of formula I and During the ceremony R<sub>1</sub>, R<sub>2</sub>, R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H, C<sub>1</sub>~ C<sub>8</sub>Alkyl or R<sub>6</sub>And R<sub>1</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is hydrogen, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl) OH and halo. In another embodiment, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl and R<sub>4</sub>H and C<sub>1</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl, or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle. In one embodiment, X<sub>1</sub>Is threonine and X<sub>3</sub>Is asparagine or glycine.
In one embodiment, R<sub>1</sub>H and C<sub>1</sub>~ C<sub>6</sub>Selected from the group consisting of alkyl, R<sub>2</sub>Is H, C<sub>1</sub>~ C<sub>6</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) C (O) NH<sub>2</sub>, CH<sub>2</sub>OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), CH<sub>2</sub>(C<sub>6</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>2</sub>And R<sub>6</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>4</sub>Alkyl, (C<sub>3</sub>~ C<sub>6</sub>) Cycloalkyl, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>Selected from the group consisting of, or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH.
According to one embodiment, a single chain insulin prodrug analog is provided in which the carboxy terminus of the human insulin B chain or its functional analog is covalently attached to the N end of the human insulin A chain or its functional analog. In addition, the following general structure<img id="000088" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />The dipeptide prodrug moiety with is covalently linked via an amide bond at the N-terminus of the peptide or on the side chain of the amino acid (eg, corresponding to A19, B16 or B25 of the A or B chain of each native insulin. Including position)). According to one embodiment, the single chain insulin analog comprises a compound of formula BPA, where B is the B chain of human insulin or a functional or prodrug analog of the B chain as disclosed herein. One of the bodies, A is the A chain of human insulin or one of the functional or prodrug analogs of the A chain as disclosed herein, and P is the A chain. Is a linker containing a peptide linker that covalently connects insulin to the B chain. In one embodiment, the linker is a peptide linker consisting of about 5 to about 18 or about 10 to about 14 or about 4 to about 8 or about 6 amino acids. In one embodiment, the B chain is attached to the A chain via a peptide linker consisting of 4-12 or 4-8 amino acids. In one embodiment, the single chain analog peptide linkers are GYGSSSRRAPQT; SEQ ID NO: 23, GYGSSSRR (SEQ ID NO: 66), GYGSSSOR (SEQ ID NO: 69), GYGSSSX.<sub>7</sub>X<sub>8</sub>A sequence selected from the group consisting of (SEQ ID NO: 71) or GAGSSSRRAPQT (SEQ ID NO: 70), or a sequence in which 1-3 amino acid substitutions or 1-2 amino acid substitutions differ from SEQ ID NOs: 89, 90 or 79. And here, X<sub>7</sub>And X<sub>8</sub>Are independently ornithine, arginine or lysine. Optionally, the bridging moiety of the single chain analog can also serve as a binding site for the dipeptide element, and in one embodiment the bridging moiety is the suitable side for binding the dipeptide element of the invention via an amide bond. It is a peptide linker having an amino acid having a chain group.
In one embodiment, the single chain insulin prodrug analog of formula BPA is the sequence GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>A chain with (SEQ ID NO: 3) and JX<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>It has a B chain sequence containing the sequence of (SEQ ID NO: 14), where J is H or is a dipeptide having the following general structure:
<img id="000089" he="27" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> here, X<sub>14</sub>Is a binding or a sequence consisting of 1 to 4 amino acids selected from the group consisting of FVNQ (SEQ ID NO: 11), VNQ, NQ, Q. X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000090" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide that is H or has the following general structure: ]<img id="000091" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Are asparagine, glycine, alanine, threonine or serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000092" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>12</sub>Is OH, OCH<sub>3</sub>, NHR<sub>11</sub>Selected from the group consisting of R<sub>11</sub>Is a dipeptide that is H or has the following general structure: ]<img id="000093" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>8</sub>Is an amino acid having the following general structure,<img id="000094" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>13</sub>Is H, OH, OCH<sub>3</sub>, NHR<sub>12</sub>Selected from the group consisting of R<sub>12</sub>Is a dipeptide that is H or has the following general structure: ]<img id="000095" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>H and C<sub>1</sub>~ C<sub>8</sub>Selected from the group consisting of alkyl R<sub>2</sub>And R<sub>4</sub>Independently, H, C<sub>1</sub>~ C<sub>8</sub>Alkyl, C<sub>2</sub>~ C<sub>8</sub>Alkenyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>5</sub>~ C<sub>9</sub>Selected from the group consisting of heteroaryl) R<sub>3</sub>Is C<sub>1</sub>~ C<sub>8</sub>Alkyl, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>3</sub>~ C<sub>6</sub>) Selected from the group consisting of cycloalkyl or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>5</sub>Is NHR<sub>6</sub>Or OH, R<sub>6</sub>Is H or R<sub>6</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH, but Z, J, R<sub>10</sub>, R<sub>11</sub>Or R<sub>12</sub>Only one of them is a dipeptide having the following general structure:
<img id="000096" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />(That is, only one of the dipeptide prodrug elements binds to the insulin peptide).
In one embodiment, J is a dipeptide having the following general structure:
<img id="000097" he="29" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X and X<sub>12</sub>Are OH and X<sub>13</sub>Is H, but R<sub>4</sub>And R<sub>3</sub>However, when these are combined with the bonded atoms to form a 5-membered heterocycle, R<sub>1</sub>And R<sub>2</sub>Are both other than H. In another embodiment, X<sub>12</sub>Has a dipeptide of formula I, J and X<sub>13</sub>Are each H and X is OH. In another embodiment, X<sub>13</sub>Has a dipeptide of formula I, X and X<sub>12</sub>Are OH and J is H, respectively. In another embodiment, X has a dipeptide of formula I, J and X.<sub>13</sub>Are each H and X<sub>12</sub>Is OH. In one embodiment, the B chain is the sequence JX<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>YTPKT (SEQ ID NO: 15) or JX<sub>9</sub>VNQX<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCGERGFX<sub>8</sub>Has YTKPT (SEQ ID NO: 16), J, X<sub>4</sub>, X<sub>5</sub>, X<sub>6</sub>, X<sub>7</sub>, X<sub>8</sub>, X<sub>9</sub>Is as defined just above. In another embodiment, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>6</sub>Alkyl and R<sub>4</sub>Is H, C<sub>1</sub>~ C<sub>4</sub>Selected from the group consisting of alkyl or R<sub>3</sub>And R<sub>4</sub>Together with the atoms to which they are bonded form a 5-membered heterocycle. In another embodiment, X<sub>4</sub>Is histidine, X<sub>5</sub>Is serine and X<sub>6</sub>Is histidine.
In one embodiment, the single chain insulin analog has a compound of formula BPA, where. B is X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>A B-chain sequence having the sequence of LVEALYLVCG ERGFF (SEQ ID NO: 4) or a functional analog thereof. A is GIVEQCCX<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>It is an A chain sequence having the sequence of (SEQ ID NO: 3) or a functional analog thereof, and "P" is a linker such as a linker consisting of 8 to 12 amino acids, for example. X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000098" he="66" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> X<sub>3</sub>Is asparagine or glycine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid.
P is a linker such as a peptide linker that covalently connects the amino terminus of the A chain to the carboxy terminus of the B chain. In another embodiment, the single chain insulin analog comprises a compound of formula APB, where A is the A chain of human insulin or a functional analog thereof and B is the B chain of human insulin or Its functional analog, P, is a linker, such as a peptide linker, that covalently links the amino terminus of the B chain to the carboxy terminus of the A chain. In one embodiment, the peptide linker has 4-8 amino acids.
According to one embodiment, the peptide linker is 5-18 amino acids in length, Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 22), Gly-Tyr-Gly-Ser-Ser. -Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 23), Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO: 32), Gly-Gly-Gly-Gly-Gly-Lys -Arg (SEQ ID NO: 24), Arg-Arg-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 25), Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 26), Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 27), Gly-Gly-Tyr-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 28), Arg-Arg- Tyr-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 29), Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 30), Arg-Arg-His-Pro- It has a sequence selected from the group consisting of Gly-Asp-Val-Gly-Gly (SEQ ID NO: 31). In one embodiment, the peptide linker is 7-12 amino acids in length and is sequenced Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 22) or Gly-Tyr-Gly-Ser-Ser- It has Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 23).
In another embodiment, the peptide linkers are AGRGSGK (SEQ ID NO: 35), AGLGSGK (SEQ ID NO: 36), AGMGSGK (SEQ ID NO: 37), ASMGSGK (SEQ ID NO: 38), TGLGSGQ (SEQ ID NO: 39), TGLGRGK (SEQ ID NO: 40). ), TGLGSGK (SEQ ID NO: 41), HGLYSGK (SEQ ID NO: 42), KGLGSGQ (SEQ ID NO: 43), VGLMSGK (SEQ ID NO: 44), VGLSSGQ (SEQ ID NO: 45), VGLYSGK (SEQ ID NO: 46), VGLSSGK (SEQ ID NO: 47) , VGMSSGK (SEQ ID NO: 48), VWSSSGK (SEQ ID NO: 49), VGSSSGK (SEQ ID NO: 50), VGMSSGK (SEQ ID NO: 51), TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO: 54), Has a sequence selected from the group consisting of VKLSSGQ (SEQ ID NO: 55), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57) SRVSRRSR (SEQ ID NO: 65), GYGSSSRRAPQT (SEQ ID NO: 23), VGLSKGQ (SEQ ID NO: 58). .. In one embodiment, the linker has GSSSRRAP (SEQ ID NO: 67) or SRVSRRSR (SEQ ID NO: 65).
In one embodiment, the single chain insulin analog is the amino acid sequence Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val- Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu- Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn (SEQ ID NO: 33) or Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu- Ala-Leu -Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr- Thr-Pro-Lys-Thr-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu- Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn (SEQ ID NO: 34), Xaa is an amino acid having the following general structure.
<img id="000099" he="66" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> The insulin peptides disclosed herein may be part of a dimer, trimmer or even higher order multimer comprising at least two, three or four or more peptides bound via a linker. , At least one or both peptides have a dipeptide prodrug element attached to the insulin peptide. The dimer may be a homodimer or a heterodimer and is selected from the group consisting of natural insulin, natural IGF-1, natural IGF-II and insulin analog peptides as disclosed herein. Has a peptide. In one embodiment, the linker is selected from the group consisting of bifunctional thiol crosslinkers and bifunctional amine crosslinkers. In certain embodiments, the linker is PEG, eg, 5 kDa PEG, 20 kDa PEG. In some embodiments, the linker is a disulfide bond.
For example, each monomer of the dimer may contain a Cys residue (eg, a terminal or non-terminal Cys), and the sulfur atom of each Cys residue is involved in the formation of disulfide bonds. Each monomer of the dimer represents an A-chain and a B-chain heterodimer. The A and B chains are linked via disulfide bonds or prepared as single chain peptides. In some aspects of the invention, via terminal amino acids (such as N-terminal or C-terminal), non-terminal amino acids, or terminal amino acids of at least one monomer and non-terminal amino acids of at least one other monomer. Then, the monomers are bonded to each other. In a specific embodiment, the monomer does not bind via the N-terminal amino acid. In some embodiments, the multimer monomers are attached to each other in a "tail-to-tail" orientation, where the C-terminal amino acids of each monomer are attached to each other. The conjugate moiety may be covalently linked to any insulin peptide described herein, including dimers, trimmers or higher-order multimers.
Further modifications of the prodrugs disclosed herein may be used to improve the solubility of the peptide in aqueous solution at physiological pH, while preventing renal excretion of the peptide and prolonging the shelf life of the peptide. Good. Peptides are easily excreted due to their relatively small molecular size compared to plasma proteins. When the molecular weight of the peptide is greater than 40 kDa, it exceeds the renal threshold and stays in plasma for a significantly longer period of time. Thus, in one embodiment, the peptide prodrug is further modified to include covalently bound hydrophilics.
In one embodiment, the hydrophilic body is the plasma protein polyethylene oxide chain or the Fc portion of an immunoglobulin. Thus, in one embodiment, the prodrugs disclosed herein are further modified to include one or more hydrophilic groups covalently attached to the side chain of an amino acid.
According to one embodiment, the hydrophilic is placed at the N-terminal amino acid of the B chain or the carboxy-terminal of the B chain (eg, at position 28 of SEQ ID NO: 9 / SEQ ID NO: 13 or position 29 of SEQ ID NO: 8 / SEQ ID NO: 12). By binding to any of the side chains of the lysine amino acid located at, etc.), the insulin prodrug disclosed herein is further modified. In one embodiment, a single chain insulin prodrug analog is provided in which one of the amino acids of the peptide linker is modified by binding the hydrophilic to the side chain of the peptide linker. In one embodiment, the modified amino acid is cysteine, lysine or acetylphenylalanine. In one embodiment, the peptide linkers are TGLGSGQ (SEQ ID NO: 39), VGLSSGQ (SEQ ID NO: 45), VGLSSGK (SEQ ID NO: 47), TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO: 54). , VKLSSGQ (SEQ ID NO: 55), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57), VGLSKGQ (SEQ ID NO: 58). Tie to the chain.
In another embodiment, a modified amino acid is added to the carboxy terminus of the B chain of the insulin prodrug to further modify the insulin prodrug analog disclosed herein. In this case, the amino acid added to the C-terminus is modified to have a hydrophilic body that binds to this amino acid. In one embodiment, the amino acid added to the C-terminus is modified cysteine, lysine or acetylphenylalanine. In one embodiment, the hydrophilic is selected from the group consisting of plasma proteins, polyethylene oxide chains, Fc moieties of immunoglobulins.
In one embodiment, the hydrophilic group is a polyethylene oxide chain, and in one embodiment, two or more polyethylene oxide chains are covalently attached to two or more amino acid side chains of an insulin prodrug analog. To do. According to one embodiment, the hydrophilic is a position selected from the group consisting of the C-terminus or N-terminus of the A9, A14, A15, B22, B28, B29, B chain, the insulin prodrug disclosed herein. Covalently binds to the side chain of an amino acid in an analog. For insulin prodrug analogs with multiple polyethylene oxide chains, the polyethylene oxide chain is an amino acid at the N-terminal amino acid of the B chain or at the side chain of the lysine amino acid at the carboxy terminus of the B chain, or at the C-terminal of the peptide. Can be attached by adding one, where the added amino acid has a polyethylene oxide chain attached to its side chain. According to one embodiment, a polyethylene oxide chain or other hydrophilic body having a dipeptide prodrug element is attached to the side chain of one of the two amino acids. In one embodiment, the dipeptide prodrug element has lysine (in a D-type or L-type configuration) with the polyethylene oxide chain bound to the amine side chain of lysine.
Bonding of hydrophilic bodies Covalent binding of a hydrophilic body to a peptide enhances the solubility of the insulin analogs disclosed herein in another embodiment. The hydrophilic is capable of binding to the insulin analog under any suitable conditions used to react the protein with the activated polymer molecule. The reactive group of the target compound (eg, aldehyde group, amino group, ester group, thiol group, α-haloacetyl group, maleimide group or hydrazino group) has a PEG moiety (eg, aldehyde group, amino group, ester group, thiol). Including acylation, reductive alkylation, Michael addition, thiol alkylation or other chemically selective binding / ligation methods that attach via reactive groups of groups (such as groups, α-haloacetyl groups, maleimide groups or hydrazino groups). Any means known in the art may be used. Sulfones, maleimides, sulfhydryls, thiols, triflate, tresilates, aziridines, oxylans, 5-pyridyls, without limitation, as activating groups that can be used to attach water-soluble polymers to one or more proteins. Can be given. When attached to a peptide by reductive alkylation, the polymer selected must have a single reactive aldehyde so that the degree of polymerization can be controlled. For example, Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002); Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002); Zalipsky et al., Adv. See Drug Delivery Rev. 16: 157-182 (1995).
Suitable hydrophilics include polyethylene glycol (PEG), polypropylene glycol, polyoxyethylated polyol (POG, etc.), polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), polyoxyalkylene, Polyethylene glycol propionaldehyde, ethylene glycol / propylene glycol copolymer, monomethoxy-polyethylene glycol, mono- (C1-C10) alkoxy- or aryloxy-polyethylene glycol, carboxymethyl cellulose, polyacetal, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly -1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, poly (β-amino acid) (either homopolymer or random copolymer), poly (n-vinylpyrrolidone) polyethylene glycol , Polypropylene glycol homopolymer (PPG) and other polyalkylene oxides, polypropylene oxide / ethylene oxide copolymers, coronic acid or other polysaccharide polymers, Ficoll or dextran and mixtures thereof.
Hydrophilic bodies such as polyethylene glycol chains according to one embodiment are selected from a molecular weight in the range of about 500 to about 40,000 daltons. In one embodiment, hydrophilics such as PEG are selected with a molecular weight ranging from about 500 to about 5,000 Dalton or about 1,000 to about 5,000 Dalton. In another embodiment, hydrophilics such as PEG have a molecular weight of about 10,000 to about 20,000 daltons. In yet another exemplary embodiment, hydrophilics such as PEG have a molecular weight of about 20,000 to about 40,000 daltons.
In one embodiment, dextran is used as the hydrophilic. Dextran is a polysaccharide polymer of glucose subunits that preferentially binds by α1-6 bonds. Dextran can be as many as about 1 kDa to about 100 kDa, or about 5, about 10, about 15 or about 20 kDa to about 20, about 30, about 40, about 50, about 60, about 70, about 80 or about 90 kDa. Available in the molecular weight range.
Linear or branched polymers are being considered. The resulting conjugate preparation may be essentially monodisperse or polydisperse, with approximately 0.5, 0.7, 1, 1.2, 1.5 or 2 polymers per peptide. It may have a portion.
According to one embodiment, the insulin prodrug analogs disclosed herein are further modified by amino acid substitutions. In this case, the substituting amino acid has a side chain suitable for cross-linking with a hydrophilic body, for example polyethylene glycol. In one embodiment, in order to introduce or facilitate binding, the amino acid at the position where the hydrophilic of the insulin prodrug analog binds is replaced with a natural or synthetic amino acid (or this is C). It is added to the end). For example, in one embodiment, A5, A8, A9, A10, A12, A14, A15, A17, A18, B1, B2, B3, B4, B5, B13, B14, B17, B21, B22, B26, B27, B28. , B29, B30, replace the natural amino acid with a lysine residue, cysteine residue or acetylphenylalanine residue (or add a lysine residue, cysteine residue or acetylphenylalanine residue to the C-terminus) ) Allows co-bonding of cysteine glycol chains.
In one embodiment, the insulin prodrug analog has a single cysteine residue added to the carboxy terminus of the B chain, or the insulin prodrug analog is replaced with at least one cysteine residue. The side chain of the cysteine residue in this case is further modified with a thiol-reactive reagent such as maleimide, vinyl sulfone, 2-pyridylthio, haloalkyl, haloacyl and the like. These thiol-reactive reagents may contain not only a carboxy group, a keto group, a hydroxy group, an ether group, but also other hydrophilic substances such as polyethylene glycol units. In another embodiment, the insulin prodrug analog has a single lysine residue added to the carboxy terminus of the B chain, or the insulin prodrug analog has a lysine substituted and substituted lysine residue. The side chains are further modified with amine-reactive reagents such as hydrophilic aldehydes such as polyethylene glycol or active esters of carboxylic acids (succinimide, anhydride, etc.).
In embodiments where the insulin prodrug analog has a polyethylene glycol chain, the polyethylene glycol chain may be linear or branched. According to one embodiment, polyethylene glycol chains are selected from an average molecular weight in the range of about 20,000 to about 60,000 daltons. It is possible to bind multiple polyethylene glycol chains to insulin prodrug analogs to provide insulin prodrug analogs with optimized solubility and blood clearance properties. In one embodiment, the insulin prodrug analog binds to a single polyethylene glycol chain with an average molecular weight selected from the range of about 20,000 to about 60,000 daltons. In another embodiment, the insulin prodrug analog is attached to two polyethylene glycol chains and the total average molecular weight of the two chains is selected from the range of about 40,000 to about 80,000 daltons. In one embodiment, a single polyethylene glycol chain with an average molecular weight of 20,000 or 60,000 daltons is attached to the insulin prodrug analog. In another embodiment, a single polyethylene glycol chain is attached to an insulin prodrug analog, the average molecular weight of which is selected from the range of about 40,000 to about 50,000 daltons. In one embodiment, two polyethylene glycol chains are attached to an insulin prodrug analog, each of the first and second polyethylene glycol chains having an average molecular weight of 20,000 daltons. In another embodiment, the two polyethylene glycol chains are attached to the insulin prodrug analog, and the first and second polyethylene glycol chains each have an average molecular weight of 40,000 daltons.
In another embodiment, an insulin prodrug analog having two or more polyethylene glycol chains covalently attached to the peptide is provided. In this case, the total molecular weight of the polyethylene glycol chain is from about 40,000 to about 60,000 daltons. In one embodiment, the PEGylated insulin prodrug analog is a polyethylene attached to one or more amino acids at the N-terminus of the B chain and / or at position 28 of SEQ ID NO: 9 or position 29 of SEQ ID NO: 8. It has a glycol chain and the total molecular weight of the PEG chain (s) is about 40,000 to about 80,000 daltons.
According to one embodiment, the insulin peptide or prodrug / depot derivative thereof is subjected to chemical PEG (eg, set) as described in International Patent Application Publication WO2009 / 023270 and US Patent Application Publication US2008 / 0286808. It fuses with an auxiliary peptide capable of forming an elongated structure similar to (eg, PEG (rPEG) molecule). The rPEG molecule is not polyethylene glycol. The rPEG molecule in some embodiments is a polypeptide having one or more of glycine, serine, glutamic acid, aspartic acid, alanine or proline. In some embodiments, the rPEG is a homopolymer such as polyglycine, polyserine, polyglutamic acid, polyaspartic acid, polyalanine or polyproline. In other embodiments, rPEG is poly (Gly-Ser), poly (Gly-Glu), poly (Gly-Ala), poly (Gly-Asp), poly (Gly-Pro), poly (Ser-Glu). Includes a repeating structure of two amino acids, such as. In some embodiments, rPEG comprises three different amino acids, such as poly (Gly-Ser-Glu). In a specific embodiment, rPEG prolongs the half-life of insulin peptides. In some embodiments, the rPEG has a positive charge as a whole or a negative charge as a whole. RPEG in some embodiments lacks secondary structure. In one embodiment, the rPEG is 10 or more amino acids in length, and in one embodiment, it is about 40 to about 50 amino acids in length. The co-peptide in some embodiments is fused to the N-terminus or C-terminus of the peptide of the invention via a peptide bond or proteinase cleavage site or inserted into the loop of the peptide of the invention. In some embodiments, rPEG has an affinity tag or binds to PEG greater than 5 kDa. In one embodiment, rPEG increases the hydrodynamic radius of the peptides of the invention, prolongs serum half-life, and increases protease resistance.
According to one embodiment, an insulin prodrug analog in which a plasma protein is covalently attached to the amino acid side chain of a peptide is provided to improve the solubility, stability and / or pharmacokinetics of the insulin prodrug analog. Will be done. For example, it is possible to covalently bind serum albumin to the insulin prodrug analogs presented herein. In one embodiment, the plasma protein is covalently attached to the N-terminus of the B chain and / or the amino acid corresponding to position 28 of SEQ ID NO: 9 or position 29 of SEQ ID NO: 8.
According to one embodiment, a linear amino acid sequence representing the Fc portion of an immunoglobulin molecule is disclosed herein to improve the solubility, stability and / or pharmacokinetics of insulin prodrug analogs. An insulin prodrug analog is provided that is covalently attached to the amino acid side chain of the insulin prodrug analog. For example, the amino acid sequence representing the Fc portion of an immunoglobulin molecule can be covalently attached to the N-terminus of the B chain or the C-terminus of the A or B chain with an extended end. For example, the amino acid sequence representing the Fc portion of the immunoglobulin molecule can be covalently attached to the C-terminus of the B chain, for example, binding to the amino acid corresponding to the 28th of SEQ ID NO: 9 or the 29th of SEQ ID NO: 8. can give. The Fc moiety is generally isolated from IgG, but any immunoglobulin-derived Fc peptide fragment should function equally.
In a specific embodiment of the invention, the insulin prodrug analog has an alkyl or acyl group by direct alkylation or acylation of the amine, hydroxyl or thiol side chain of the amino acid of the insulin prodrug analog. Modify the body. In one embodiment, the insulin prodrug analog is directly acylated with an amine, hydroxyl or thiol in the side chain of the amino acid. In one embodiment, acylation is performed at one or more of the positions selected from A9, A14, A15, B22, B28 or B29. In this regard, the acylated insulin prodrug analog may have the amino acid sequence of A chain of SEQ ID NO: 3 and the B chain of SEQ ID NO: 5, or A9, A14, A15, At least one of the amino acids at position B22, B28 or B29 has the modified amino acid sequence of SEQ ID NO: 3 and / or SEQ ID NO: 5, modified with an amino acid having a side chain amine, hydroxyl or thiol. May be. In some specific embodiments, the direct acylation of the insulin prodrug analog is done via the amine, hydroxyl or thiol of the side chain of the amino acid at the position of B28 or B29. In another embodiment, the insulin prodrug analog has an acyl group of a carboxylic acid attached to the ε-amino group of Lys with 1 to 24 carbon atoms at positions B28 or B29. In one embodiment, one of the amino acids of the peptide linker is modified to have an acyl group by direct acylation of an amine, hydroxyl or thiol on the side chain of the amino acid of the peptide linker, a single chain insulin prodrug. An analog is provided. According to one embodiment, the single chain insulin analog peptide linkers are AGRGSGK (SEQ ID NO: 35), AGLGSGK (SEQ ID NO: 36), AGMGSGK (SEQ ID NO: 37), ASMGSGK (SEQ ID NO: 38), TGLGSGQ (SEQ ID NO: 39). ), TGL GRGK (SEQ ID NO: 40), TGLGSGK (SEQ ID NO: 41), HGLYSGK (SEQ ID NO: 42), KGLGSGQ (SEQ ID NO: 43), VGLMSGK (SEQ ID NO: 44), VGLSSGQ (SEQ ID NO: 45), VGLYSGK (SEQ ID NO: 46), VGLSSGK ( SEQ ID NO: 47), VGMSSGK (SEQ ID NO: 48), VWSSSGK (SEQ ID NO: 49), VGSSSGK (SEQ ID NO: 50), VGMSSGK (SEQ ID NO: 51), TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO: 53) At least one of the A-chain, B-chain or bridging peptides selected from the group consisting of No. 54), VKLSSGQ (SEQ ID NO: 55), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57), VGLSKGQ (SEQ ID NO: 58). One lysine residue is chemically modified by acylation. In one embodiment, the acylating group has 1-5, 10-12 or 12-24 carbon chains.
According to one embodiment, the insulin prodrug analog as disclosed herein is further modified to bind another compound to the dipeptide portion of the analog prodrug. In one embodiment, the side chain of an amino acid having a dipeptide prodrug element is PEGylated, acylated, or alkylated. In one embodiment, the dipeptide is acylated with a group having 1-5, 10-12 or 12-24 carbon chains. In one embodiment, the dipeptide is PEGylated with a polyethylene glycol chain of 40-80 KDa. In one embodiment, the dipeptide prodrug element is PEGylated and the insulin peptide bound to the dipeptide is acylated. This includes, for example, acylation at the C-terminal lysine of the B chain. According to one embodiment, the hydrophilic or trapping macromolecule is R of a dipeptide having the following general structure:<sub>2</sub>Covalently bond to the side chain.
<img id="000100" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>2</sub>Is (C<sub>1</sub>~ C<sub>4</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) SH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Selected from the group consisting of. In one embodiment, R<sub>2</sub>Is (C<sub>3</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>Is. Capturing macromolecules are known to those of skill in the art and include dextran and high molecular weight polyethylene glycol (ie, 80 KDa and above). Binding of the captive macromolecule to the dipeptide moiety keeps the prodrug anchored while the active insulin peptide is slowly released, based on the cleavage kinetics of the amide bond of the dipeptide.
The present disclosure also includes other conjugates in which the insulin prodrug analogs of the invention are optionally attached to the conjugate via a covalent bond and optionally via a linker. Bonds are carried out by physical forces such as chemical covalent bonds, electrostatic interactions, hydrogen-hydrogen interactions, ionic interactions, van der Waals interactions or hydrophobic interactions or hydrophilic interactions. Just do it. Biobiosin-avidin, ligands / receptors, enzymes / substrates, nucleic acid / nucleic acid binding proteins, lipids / lipid binding proteins, cell adhesion molecule binding partners, or any binding partner or fragments thereof that have an affinity for each other. A wide variety of non-covalent bonding systems are available.
Examples of conjugates are heterologous peptides or polypeptides (including plasma proteins, etc.), drugs for targeting, immunoglobulins or parts thereof (such as variable regions, CDR or Fc regions), radioactive isotopes, fluorophores or enzymes. Diagnostic labels such as labels, polymers such as water-soluble polymers or other therapeutic or diagnostic agents include, but are not limited to. In one embodiment, a conjugate comprising the insulin prodrug analog of the present disclosure and a plasma protein is provided, wherein the plasma protein is selected from the group consisting of albumin, transferrin, fibrinogen. In one embodiment, the plasma protein portion of the conjugate is albumin or transferrin. In one embodiment, the linker is 1 to about 60 or 1 to 30 atoms long or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms or 10 to 20 atoms. It has an atomic chain of several lengths. In one embodiment, all atoms in the chain are carbon atoms. In some embodiments, the atoms of the chain in the backbone of the linker are selected from the group consisting of C, O, N, S. The atom of the chain and the linker can be selected according to the expected solubility (hydrophilicity) so as to provide a more soluble conjugate. In one embodiment, the linker provides a functional group that is cleaved by an enzyme or other catalyst or hydrolyzed state found in the target tissue or organ or cell. In one embodiment, the length of the linker is long enough to reduce the possibility of steric hindrance. If the linker is a covalent or peptidyl bond and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such a peptidyl linker may be of any length. Illustrative linkers are about 1 to about 50 amino acids, 5 to 50, 3 to 5, 5 to 10, 5 to 15 or 10 to 30 amino acids in length. Alternatively, such fusion proteins may be produced by recombinant genetic engineering methods known to those of skill in the art.
Couplings and fusions The present disclosure also includes other conjugates in which the insulin analogs disclosed herein are attached to the conjugate moiety, optionally via a covalent bond and optionally via a linker. Bonds are carried out by physical forces such as chemical covalent bonds, electrostatic interactions, hydrogen-hydrogen interactions, ionic interactions, van der Waals interactions or hydrophobic interactions or hydrophilic interactions. Just do it. Biobiosin-avidin, ligands / receptors, enzymes / substrates, nucleic acid / nucleic acid binding proteins, lipids / lipid binding proteins, cell adhesion molecule binding partners, or any binding partner or fragments thereof that have an affinity for each other. A wide variety of non-covalent bonding systems are available.
Direct by reacting the targeted amino acid residues of the peptide with selected side chains of these targeted amino acids or organic derivatizing agents capable of reacting with N-terminal or C-terminal residues. The peptide may be attached to the conjugate moiety by a covalent bond. Reactive groups of conjugates or peptides include aldehyde groups, amino groups, ester groups, thiol groups, α-haloacetyl groups, maleimide groups or hydrazino groups. Derivatizers include maleimide benzoyl succinimide ester (linked via a cysteine residue), N-hydroxysuccinimide (via a lysine residue), glutaraldehyde, succinic anhydride or other agents known in the art. can give. Alternatively, intermediate carriers such as polysaccharide carriers or polypeptide carriers can be used to indirectly bind the conjugate moiety to the peptide. An example of a polysaccharide carrier is aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids and other amino acids, such as serine, to give the bound carriers the desired dissolving properties. can give.
The cystenyl residue most commonly reacts with α-haloacetic acids (and corresponding amines) such as chloroacetic acid or chloroacetamide to produce carboxymethyl or carboxamide methyl derivatives. Cistenyl residues are also bromotrifluoroacetone, α-bromo-β- (5-imide zoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p- Derivatized by reaction with chloromercury benzoic acid, 2-chloromercury-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
Since histidyl residues are relatively specific to the histidyl side chain, they are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0. p-Bromophenacyl bromide is also useful. This reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0.
Ridinyl and amino-terminal residues react with succinic acid or other carboxylic acid anhydrides. Derivatization using these agents has the effect of reversing the charge of lysinyl residues. Other suitable reagents for derivatizing α-amino-containing residues include imide esters such as methylpicolin imidazole, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzene sulfonic acid, O-methylisourea, 2, Transaminase-catalyzed reaction with 4-pentandione and glyoxylic acid can be mentioned.
Arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butandione, 1,2-cyclohexanedione, ninhydrin. Guanidine functional group pK<sub>a</sub>Therefore, it is necessary to carry out the reaction under alkaline conditions to derivatize the arginine residue. In addition, these reagents may react with the lysine group as well as the ε-amino group of arginine.
Unique modifications can be made to tyrosyl residues, and of particular interest is the introduction of spectral labeling into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to produce O-acetyltyrosyl species and 3-nitro derivatives, respectively.
The carboxyl side group (aspartyl or glutamyl) is selectively modified by reaction with carbodiimide (RN = C = N-R'). In the formula, R and R'are different alkyl groups such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide. Is. In addition, the reaction with ammonium ions converts aspartyl and glutamyl residues to asparaginyl and glutamyl residues.
Other modifications include phosphorylation of proline and lysine, phosphorylation of hydroxyl groups on ceryl or threonyl residues, methylation of α-amino groups on lysine, arginine, and histidine side chains (TE Creighton, Proteins). : Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), Deamidation of asparagine or glutamine, acetylation and / or amidation of N-terminal amines or C-terminal carboxylic acids Esterylation of the group can be mentioned.
Another type of covalent modification is to chemically or enzymatically bind the glycoside to the peptide. Of (a) arginine and histidine, (b) free carboxy groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxy groups such as those of serine, threonine or hydroxyproline, (e) tyrosine or tryptophan Sugars (s) may be attached to aromatic residues such as those or the amide group of (f) glutamine. These methods are described in WO87 / 05330, published on September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981). ..
Examples of conjugate moieties that can bind to the insulin analogs described herein are heterologous peptides or polypeptides (including plasma proteins, etc.), agents for targeting, immunoglobulins or parts thereof (variable regions, CDRs). (Or Fc region, etc.), diagnostic labels such as radioisotopes, fluorophores or enzyme labels, polymers such as water-soluble polymers or other therapeutic or diagnostic agents, but not limited to these. .. In one embodiment, a conjugate comprising the insulin analogs disclosed herein and a plasma protein is provided, wherein the plasma protein is selected from the group consisting of albumin, transferrin, fibrinogen, globulin.
In one embodiment, the linker is 1 to about 60 or 1 to 30 atoms long or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms or 10 to 20 atoms. It has an atomic chain of several lengths. In one embodiment, all atoms in the chain are carbon atoms. In some embodiments, the atoms of the chain in the backbone of the linker are selected from the group consisting of C, O, N, S. The atom of the chain and the linker can be selected according to the expected solubility (hydrophilicity) so as to provide a more soluble conjugate. In one embodiment, the linker provides a functional group that is cleaved by an enzyme or other catalyst or hydrolyzed state found in the target tissue or organ or cell. In one embodiment, the length of the linker is long enough to reduce the possibility of steric hindrance. If the linker is a covalent or peptidyl bond and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such a peptidyl linker may be of any length. Illustrative linkers are about 1 to about 50 amino acids, 5 to 50, 3 to 5, 5 to 10, 5 to 15 or 10 to 30 amino acids in length. Alternatively, such fusion proteins may be produced by recombinant genetic engineering methods known to those of skill in the art.
As mentioned above, in one embodiment, the insulin analog binds (for example, fuses) to an immunoglobulin or part thereof (such as a variable region, CDR or Fc region). Well-known types of immunoglobulins (Igs) include IgG, IgA, IgE, IgD or IgM. The Fc region is the C-terminal region of the Ig heavy chain and has actions such as reuse (prolonging the half-life), antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cellular cytotoxicity (CDC). It is responsible for binding to the Fc receptor.
For example, according to some definitions, the heavy chain Fc region of human IgG ranges from the 226th cysteine of the heavy chain to the C-terminus. The "hinge region" usually ranges from the 216th glutamic acid to the 230th proline of human IgG1 (for other IgG isotype hinge regions, by aligning the cysteines involved in cysteine binding with the IgG1 sequence. Can be aligned). The Fc region of IgG has two constant domains, CH2 and CH3. The CH2 domain of the Fc region of human IgG usually ranges from amino acid 231 to amino acid 341. The CH3 domain of the Fc region of human IgG usually ranges from amino acids 342 to 447. Kabat et al. 1991, Sequences of Proteins of Immunological Interest, US Based on the Department of Public Health, Bethesda, Md. In a related embodiment, the Fc region, in addition to CH1, is one or more natural constant regions or modified constant regions of immunoglobulin heavy chains (CH2 and CH3 regions of IgG and IgA, or IgE. It may have a CH3 region and a CH4 region).
Suitable conjugate moieties have a portion of the immunoglobulin sequence that contains the FcRn binding site. The salvage receptor FcRn is responsible for reusing immunoglobulins and returning them to blood circulation. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372: 379). The main contact area between Fc and FcRn is near the junction of CH2, main and CH3 domains. Both Fc and FcRn contacts are within a single Ig heavy chain. The major contact sites are the 248th, 250th to 257th, 272nd, 285th, 288th, 290th to 291st, 308th to 311th, 314th amino acid residues of the CH2 domain and 385th of the CH3 domain. Contains amino acid residues from the 387th to the 387th, the 428th, and the 433rd to the 436th.
Some conjugate moieties may or may not have FcγR binding sites (s). FcγR is responsible for ADCC and CDC. Examples of positions in the Fc region that are in direct contact with FcγR are amino acids 234 to 239 (lower side in the hinge region), amino acids 265 to 269 (B / C loop), and amino acids 297 to 299. (C'/ E loop), the 327th to 332nd amino acid (F / G) loop (Sondermann et al., Nature 406: 267-273, 2000). The underside of IgE within the hinge region is also involved in FcRI binding (Henry, et al., Biochemistry 36, 15568-15578, 1997). Residues involved in binding to the IgA receptor are described in Lewis et al., (J Immunol. 175: 6964-701, 2005). Amino acid residues involved in binding to the IgE receptor are described in Sayers et al. (J Biol Chem. 279 (34): 35320-5, 2004).
Amino acid modification may be performed on the Fc region of immunoglobulin. Such mutated Fc regions are at least one amino acid modification in the CH3 domain of the Fc region (residues 342 to 447) and / or in the CH2 domain of the Fc region (residues 231 to 341). Has at least one amino acid modification of. Mutations that are thought to increase affinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem. 276: 6591). Other mutations may reduce the binding of the Fc region to FcγRI, FcγRIIA, FcγRIIB and / or FcγRIIIA without significantly reducing affinity for FcRn. For example, substituting Asn at position 297 of the Fc region with Ala or another amino acid eliminates the highly conserved N-glycosylation site and reduces immunogenicity with an extended half-life of the Fc region, resulting in FcγR. May reduce binding to (Routledge et al. 1995, Transplantation 60: 847; Friend et al. 1999, Transplantation 68: 1632; Shields et al. 1995, J. Biol. Chem. 276: 6591). The 233rd to 236th amino acid modifications of IgG1 that reduce the binding to FcγR have been made (Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armor et al. 1999, Eur. J. Immunol. 29:2613). Examples of several amino acid substitutions are described in US Pat. Nos. 7,355,008 and 7,381,408, each of which is incorporated herein by reference in its entirety.
Acylation and alkylation According to one embodiment, the insulin analogs disclosed herein are modified to include acyl or alkyl groups. Acylation or alkylation can prolong the half-life of insulin analogs in the blood. Conveniently, acylation or alkylation delays onset of action and / or prolongs the duration of action at the insulin and / or IGF-1 receptors and / or improves resistance to proteases such as DPP-IV. And / or improve solubility. Insulin analogs may be acylated or alkylated at the same amino acid positions to which the hydrophilics bind, or they may be acylated or alkylated at different amino acid positions.
In one embodiment, the invention comprises an acyl or alkyl group covalently attached to an amino acid at the position corresponding to A10, B28, B29 of natural insulin or at the C- or N-terminus of the A or B chain. It provides an insulin analog modified to. The insulin analog may further have a spacer between the amino acid of the insulin analog and the acyl or alkyl group. In one embodiment, the acyl group is a fatty acid or bile acid or a salt thereof, eg, C4-C30 fatty acid, C8-C24 fatty acid, cholic acid, C4-C30 alkyl, C8-C24 alkyl or a steroid moiety of a bile acid. It is an alkyl having. The spacer may be any moiety as long as it has a suitable reactive group for attaching an acyl group or an alkyl group. In an exemplary embodiment, the spacer may be an amino acid, dipeptide, tripeptide or hydrophilic bifunctional spacer. In one embodiment, the spacers are Trp, Glu, Asp, Cys, and NH.<sub>2</sub>(CH<sub>2</sub>CH<sub>2</sub>O) n (CH<sub>2</sub>) Selected from the group consisting of spacers with mCOOH, in the equation m is any integer from 1 to 6 and n is any integer from 2 to 12. Such acylated or alkylated insulin peptides may further comprise a hydrophilic, optionally polyethylene glycol. Any of the above insulin analogs may have two acyl groups or two alkyl groups or a combination thereof.
Acylation may occur at any position on the insulin analog as long as the insulin agonist activity of the insulin analog is retained. The acyl group may be one that directly covalently binds to the amino acid of the insulin analog, or may be one that indirectly covalently binds to the amino acid of the insulin analog via a spacer. Here, the spacer is located between the amino acid and the acyl group of the insulin peptide. In a specific aspect of the invention, the insulin analog is modified to include an acyl group by directly acylating an amine, hydroxyl or thiol in the side chain of the amino acid of the insulin peptide. In one embodiment, the insulin analog is directly acylated via an amine, hydroxyl or thiol in the side chain of the amino acid. In one embodiment, the acylation is at the position corresponding to A10, B28, B29 of the natural insulin or at the C- or N-terminus of the A or B chain. In this regard, the acylated insulin analog may have the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10, the positions corresponding to A10, B28, B29 of the natural insulin, or the A chain or A modified amino acid with one or more amino acid modifications in which at least one of the amino acids at the C-terminal or N-terminal position of the B chain is modified with an amino acid having an amine, hydroxyl or thiol in the side chain. It may be an array. In some specific embodiments, the direct acylation of the insulin peptide is done at the position corresponding to A10, B28, B29 of the native insulin via the amine, hydroxyl or thiol of the side chain of the amino acid. According to one embodiment, one of the amino acid side chains of the dipeptide element is acylated.
In one embodiment, the amino acid acylated is an amino acid of formula IV below.
<img id="000101" he="34" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />In some embodiments by way of example, the amino acid of formula IV is an amino acid of n 4 (Lys) or n 3 (Orn).
In another embodiment, the amino acid having a hydroxyl group in the side chain is the amino acid of formula V below.
<img id="000102" he="33" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />In some embodiments by way of example, the amino acid of formula V is an amino acid with n of 1 (Ser).
In yet another embodiment, the amino acid having a thiol in the side chain is the amino acid of formula VI below.
<img id="000103" he="33" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />In some embodiments by way of example, the amino acid of formula VI is an amino acid with n of 1 (Cys).
In some embodiments by way of example, the insulin analog is modified to include an acyl group by acylation of the amine, hydroxyl or thiol of the spacer, which spacer is A10 (by amino acid number of wild-type insulin). , B28 or B29, is attached to the side chain of the amino acid. The amino acid to which the spacer is bound may be any amino acid as long as it has a portion that enables binding to the spacer. For example, on the side chain, NH<sub>2</sub>, Amino acids with -OH or -COOH (eg Lys, Orn, Ser, Asp or Glu) are suitable. In one embodiment, the spacer is an amino acid having a side chain amine, hydroxyl or thiol, or a dipeptide or tripeptide comprising an amino acid having a side chain amine, hydroxyl or thiol.
If acylation occurs via the amine group of the spacer, this acylation can occur via the amino acid α-amine or the side chain amine. In the example where the α-amine is acylated, the amino acid of the spacer may be any amino acid. For example, the amino acid of the spacer may be a hydrophobic amino acid such as Gly, Ala, Val, Leu, Ile, Trp, Met, Phe, Tyr. Alternatively, the amino acids in the spacer may be acidic residues such as Asp and Glu. In an example where the side chain amine of the spacer amino acid is acylated, the spacer amino acid is an amino acid having a side chain amine, such as an amino acid of formula I (such as Lys or Orn). In this case, both the α-amine of the spacer amino acid and the amine of the side chain can be acylated so that the insulin peptide is diacylated. The disclosure also contemplates diacylated insulin analogs.
If acylation occurs via the hydroxy group of the spacer, one of the amino acids, or dipeptides or tripeptides, may be an amino acid of formula II. In a specific embodiment as an example, the amino acid is Ser.
If acylation occurs via the thiol group of the spacer, one of the amino acids or dipeptide or tripeptide amino acids may be an amino acid of formula III. In a specific embodiment as an example, the amino acid is Cys.
In one embodiment, the spacer may be a hydrophilic bifunctional spacer. In a specific embodiment, the spacer has an aminopoly (alkyloxy) carboxylate. In this regard, spacers are, for example, NH<sub>2</sub>(CH<sub>2</sub>CH<sub>2</sub>O)<sub>n</sub>(CH<sub>2</sub>)<sub>m</sub>It may contain COOH, where m is any integer from 1 to 6 and n is any integer from 2 to 12. One example is 8-amino-3,6-dioxaoctanoic acid, which is commercially available from Peptides International, Inc. (Louisville, KY).
Suitable methods for acylating peptides with amines, hydroxyls and thiols are known in the art. For example, Miller, Biochem Biophys Res Commun 218: 377-382 (1996); Shimohigashi and Stammer, Int J Pept Protein Res 19: 54-62 (1982); and Previero et al., Biochim Biophys Acta 263: 7-13 ( 1972) (Acylation with hydroxyl); and San and Silvius, J Pept Res 66: 169-180 (2005) (Acylation with thiol); Bioconjugate Chem. "Chemical Modifications of Proteins: History and Applications" pages 1, 2-12 (1990); Hashimoto et al., Pharmacuetical Res. See "Synthesis of Palmitoyl Derivatives of Insulin and their Biological Activity" Vol. 6, No: 2 pp.171-176 (1989).
The acyl group of the acylated insulin peptide may have any size, such as any carbon chain length, and may be linear or branched. In some specific embodiments of the invention, the acyl group is a C4-C30 fatty acid. For example, the acyl group can be any of C4 fatty acid, C6 fatty acid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fatty acid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid, C26 fatty acid, C28 fatty acid or C30 fatty acid. There may be. In one embodiment, the acyl group is a C8-C20 fatty acid, such as a C14 or C16 fatty acid.
In another embodiment, the acyl group is a bile acid. The bile acid may be any suitable bile acid. Examples include, but are not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, cholesterol acid and the like.
In a specific embodiment, the insulin analog comprises an alkylated desaminocysteine spacer, i.e., a cholesterol acid bound to the Lys residue of the insulin analog via an alkylated 3-mercaptopropionic acid spacer. The alkylated desaminocysteine spacer is, for example, a desaminocysteine spacer having a dodecaethylene glycol moiety. In one embodiment, the insulin analog has the following structure:
<img id="000104" he="97" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> The acylated insulin analogs described herein may be further modified to have hydrophilics. In some specific embodiments, the hydrophilic body may have a polyethylene glycol (PEG) chain. Uptake of hydrophilics can be achieved by any suitable means, such as any of the methods described herein.
Alternatively, the acylated insulin peptide may have a spacer that is acylated and modified to contain a hydrophilic substance. Non-limiting examples of suitable spacers include spacers having one or more amino acids selected from the group consisting of Cys, Lys, Orn, homocysteine, Ac-Phe.
According to one embodiment, the insulin analog is an ester for the purpose of prolonging the blood half-life and / or delaying the onset of action and / or prolonging the duration of action and / or improving resistance to proteases such as DPP-IV. , Ethers, thioethers, amides or alkylamines modified to contain alkyl groups attached to the analogs via an alkylamine bond.
The alkyl group of the alkylated insulin peptide may have any size, such as any carbon chain length, and may be linear or branched. In one embodiment of the invention, the alkyl group is a C1-C30 alkyl. For example, the alkyl groups are C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl. , C28 alkyl or C30 alkyl. In one embodiment, the alkyl group is C8 to C20 alkyl, for example C14 alkyl or C16 alkyl.
In some specific embodiments, the alkyl group has a steroid moiety of a bile acid such as cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid, taurocholic acid, glycocholic acid, cholesterol acid.
According to some embodiments, the dipeptide prodrug element may be further modified to have a hydrophilic body. In some embodiments, the hydrophilic body is a polyethylene glycol chain. According to some embodiments, a 40k or larger polyethylene glycol chain is covalently attached to the side chain of the amino acid A or B of the dipeptide prodrug element. In another embodiment, the dipeptide prodrug element is in addition to or in place of the above, eg, of C4-C30 fatty acid, C8-C24 fatty acid, cholic acid, C4-C30 alkyl, C8-C24 alkyl or bile acid. It is acylated or alkylated with a fatty acid such as alkyl having a steroid moiety or a bile acid or a salt thereof. The amino acid "A" of the dipeptide prodrug element is covalently shared with the PEG molecule via, for example, d-lysine, which is covalently attached to an acyl or alkyl group via its side chain amino group, or its side chain sulfhydryl group. It may have a bound d-cysteine. The dipeptide prodrug element may be directly capable of binding to a hydrophilic, acyl or alkyl group or to a hydrophilic, acyl or alkyl group via a spacer as described herein. It may be possible. Alternatively, the dipeptide prodrug element is a depot such as dextran or a large PEG molecule (80,000 daltons or larger) that serves to retain the prodrug to the injection site until the active insulin peptide (Q) is released by cleavage of the dipeptide. It can bind to proteins.
Effect of structure of dipeptide prodrug element on cleavage rate As described herein above, the rate at which the dipeptide prodrug element AB is cleaved from a bioactive peptide (eg, insulin peptide (Q)), thereby activating the prodrug, is determined by the rate of activation of the dipeptide prodrug element amino acid. Depends on structure (including N-alkylation, number of substituents, length or bulk) and steric isomerism. The rate of cleavage of the dipeptide prodrug element AB from (eg, insulin peptide (Q)) also depends on the stability, nucleophilicity, and steric hindrance of the leaving group of Q during diketopiperazine formation. Some of these structural features are described below in Category I, Category II, and Category III, which form part of the present invention. Explicitly excluded from these categories is, to the extent that it is fully contained and / or overlapped with any part of any of the subcategories described herein, and is novel to the subject matter claimed. As well as the peptide sequences disclosed in the international patent application PCT / US2009 / 68745 (filed December 18, 2009) or its sequence table, as well as the international patent application PCT / US2009 / 68745 (2009). It is a subcategory of (1) dipeptide prodrug elements, (2) amino acids A and / or (3) amino acids B disclosed in (filed December 18).
According to one embodiment, the autonomously cleaved dipeptide element (AB) covalently binds to the insulin peptide (Q) via an amide bond between the aliphatic amino groups of AB and Q. For example, the aliphatic amino group AB may be attached to the α-amino group of the N-terminal amino acid of the A chain or the B chain. Alternatively, the aliphatic amino group AB may be attached to the aliphatic amino group in the side chain of Q. In one embodiment, AB binds to the amine in the side chain of lysine. For example, the position of B29 in the b-chain sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 8) or the position of B28 in the B-chain sequence FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 9) can be mentioned.
In one embodiment, Q is an insulin peptide having an A chain and a B chain, wherein the A chain is the sequence GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>It has (SEQ ID NO: 3), and the B chain is the sequence X.<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>Has (SEQ ID NO: 14) X<sub>14</sub>Is a binding or a sequence consisting of 1 to 4 amino acids selected from the group consisting of FVNQ (SEQ ID NO: 11), VNQ, NQ, Q. X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000105" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, OCH<sub>3</sub>Selected from the group consisting of. ] X<sub>3</sub>Is selected from the group consisting of asparagine, ornithine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is tyrosine, X<sub>8</sub>Is histidine, asparagine or tyrosine, and AB binds to Q via an amide bond between AB and the aliphatic amino group of Q.
In one embodiment, Q is an insulin peptide having an A chain and a B chain, Q is an insulin peptide having an A chain and a B chain, and the A chain is the sequence GIVEQCCX.<sub>1</sub>SICSLYQLENX<sub>2</sub>CX<sub>3</sub>It has (SEQ ID NO: 3), and the B chain is the sequence X.<sub>14</sub>-X<sub>4</sub>LCGX<sub>5</sub>X<sub>6</sub>LVEALX<sub>7</sub>LVCG ERGFX<sub>8</sub>Has (SEQ ID NO: 14) X<sub>14</sub>Is a binding or a sequence consisting of 1 to 4 amino acids selected from the group consisting of FVNQ (SEQ ID NO: 11), VNQ, NQ, Q. X<sub>1</sub>Is selected from the group consisting of threonine and histidine, X<sub>2</sub>Is an amino acid having the following general structure,<img id="000106" he="49" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X is OH, NH<sub>2</sub>, NHR<sub>10</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>10</sub>Is a dipeptide of general structure AB. ] X<sub>3</sub>Is selected from the group consisting of asparagine, ornithine, glycine, alanine, threonine, serine, X<sub>4</sub>Is selected from the group consisting of histidine and threonine, X<sub>5</sub>Is selected from the group consisting of alanine, glycine, serine, X<sub>6</sub>Is selected from the group consisting of histidine, aspartic acid, glutamic acid, homocysteine acid, cysteic acid, X<sub>7</sub>Is an amino acid having the following general structure,<img id="000107" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>12</sub>Is OH, NH<sub>2</sub>, NHR<sub>11</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>11</sub>Is a dipeptide of general structure AB. ] X<sub>8</sub>Is a histidine, asparagine or an amino acid having the following general structure:
<img id="000108" he="47" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> [In the formula, X<sub>13</sub>Is H, OH, NH<sub>2</sub>, NHR<sub>12</sub>, OCH<sub>3</sub>Selected from the group consisting of R<sub>12</sub>Is a dipeptide of general structure AB, which binds to Q via an amide bond between AB and the aromatic amino group of the amino acid side chain of Q. ] Category I: Composition of amino acid B of dipeptide prodrug element In some embodiments, the half-life of the prodrug, which is at least about 1 hour to about 1 week under physiological conditions in PBS, eg, the half-life in which Q to AB are chemically cleaved (t).<sub>1/2</sub>) Depends on the presence and length of the N-alkyl substituent on the amino acid B. For example, a prodrug with a short N-alkyl substituent on the amino acid B (eg Gly (N-methyl)) has a longer prodrug with a long N-alkyl substituent on the amino acid B (eg Gly (N-hexyl)). ) Etc.), the cutting speed of AB is slower and the half-life is longer.
In some embodiments, the half-life of the prodrug depends on the degree of substitution of the dipeptide prodrug element B at the amino acid position.
In some embodiments, the half-life of the prodrug depends on the presence or absence of the alkyl side chain and the degree of substitution of the alkyl side chain in the amino acid B of the dipeptide prodrug element at the β-position. For example, a prodrug with an amino acid of N-alkylated B that is disubstituted at the β-position (for example, N-alkylated isoleucine) is N-alkylated that is mono-substituted at the β-position. It cleaves AB more slowly and has a longer half-life than prodrugs with the amino acid of B (eg, N-alkylated isoleucine). In addition, prodrugs with amino acids of N-alkylated B that are monosubstituted at the β-position (eg, N-alkylated leucine) are N-alkylated B that are unsubstituted at the β-position. AB cleaves slower and has a longer half-life than prodrugs with the amino acids (eg, N-alkylated alanine). In addition, prodrugs with the β-unsubstituted N-alkylated B amino acid (eg, N-alkylated alanine) are prodrugs with glycine or N-alkylated glycine as the B amino acid. AB is cleaved more slowly and has a longer half-life.
In some embodiments, the half-life of the prodrug depends on the bulkiness of the side chain of the amino acid B. For example, a prodrug with a bulkier side chain on the amino acid B (eg, N-alkylated phenylalanine) is a prodrug with a lower side chain on the amino acid B (eg, N-). It cleaves AB more slowly and has a longer half-life than alkylated alanine, etc.). The rate of cleavage of a dipeptide may also depend on the amine of the drug to which it binds (such as insulin). In particular, when the same dipeptide binds to an aromatic amine instead of an N-terminal amine, it is cleaved even faster, and the dipeptide bound to the N-terminal amine is more than if the dipeptide was bound to the amine on the side chain of the lysine residue. Is also disconnected quickly.
The composition of the amino acid B of the dipeptide prodrug element can be classified into subcategories IA, IB, and IC below. Usually, the dipeptide prodrug element of subcategory IA is cleaved fastest and the dipeptide prodrug element of subcategory IC is cleaved the slowest.
Subcategory IA: Glycine in which the amino acid B of the dipeptide prodrug element is N-alkylated. In some embodiments, the prodrug has a structural ABQ. Here, Q is a bioactive peptide (insulin peptide, etc.), and AB has the following structure.
<img id="000109" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>And R<sub>8</sub>Are each H R<sub>5</sub>Is NHR<sub>6</sub>And R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>4</sub>Alkyl or R<sub>5</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>7</sub>Is selected from the group consisting of H and OH.
In some embodiments, the amino acids B are glycine (N-methyl), glycine (N-ethyl), glycine (N-propyl), glycine (N-butyl), glycine (N-pentyl), glycine (N). -Selected from the group consisting of hexyl), glycine (N-heptyl), and glycine (N-octyl). For example, the amino acid of B may be glycine (N-methyl) or glycine (N-hexyl).
In some embodiments, R<sub>1</sub>And R<sub>2</sub>If both are hydrogen, then R<sub>3</sub>Is C<sub>1</sub>~ C<sub>4</sub>It is alkyl. In some embodiments, R<sub>1</sub>Or R<sub>2</sub>If one of them is other than hydrogen, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>4</sub>It is alkyl.
Subcategory IB: Amino acid B of the dipeptide prodrug element is unsubstituted or mono-substituted at the β-position In some embodiments, the prodrug has a structural ABQ. Here, Q is a bioactive peptide (insulin peptide, etc.), and AB has the following structure.
<img id="000110" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>Is CH<sub>3</sub>, CH<sub>2</sub>(C<sub>1</sub>~ C<sub>10</sub>Alkyl), CH<sub>2</sub>(C<sub>2</sub>~ C<sub>10</sub>Alkenyl), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>10</sub>Alkyl) OH, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>10</sub>Alkyl) SH, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) CONH<sub>2</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) COOH, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) NH<sub>2</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>1</sub>~ C<sub>3</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>12</sub>Alkyl) (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>4</sub>And R<sub>3</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>8</sub>Is H R<sub>5</sub>Is NHR<sub>6</sub>Or R<sub>5</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>4</sub>Alkyl R<sub>7</sub>Is selected from the group consisting of H and OH.
In some embodiments, R<sub>4</sub>Is CH<sub>3</sub>, CH<sub>2</sub>(C<sub>1</sub>~ C<sub>4</sub>Alkyl), CH<sub>2</sub>(C<sub>1</sub>~ C<sub>4</sub>) Alkenyl, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>4</sub>Alkyl) OH, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>4</sub>Alkyl) SH, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) CONH<sub>2</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) COOH, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>Selected from the group consisting of.
A non-limiting example of the amino acid B in these embodiments is alanine (NC).<sub>1</sub>~ C<sub>10</sub>Alkyl), leucine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), methionine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), asparagine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), glutamic acid (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), aspartic acid (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), glutamine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), histidine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), lysine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), arginine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), serine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), Cysteine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl) can be mentioned.
In some embodiments, the amino acid of B is alanine (NC).<sub>1</sub>~ C<sub>6</sub>Alkyl), leucine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), methionine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), asparagine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), glutamic acid (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), aspartic acid (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), glutamine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), histidine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), lysine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), arginine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), serine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), Cysteine (NC)<sub>1</sub>~ C<sub>6</sub>Selected from the group consisting of alkyl).
For example, the amino acids B are alanine (N-methyl), leucine (N-methyl), methionine (N-methyl), asparagine (N-methyl), glutamine (N-methyl), aspartic acid (N-methyl), It may contain glutamine (N-methyl), histidine (N-methyl), lysine (N-methyl), arginine (N-methyl), serine (N-methyl), cysteine (N-methyl).
In some embodiments, R<sub>4</sub>Is CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>3</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, CH<sub>2</sub>(C<sub>1</sub>~ C<sub>3</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), CH<sub>2</sub>(C<sub>0</sub>~ C<sub>12</sub>Alkyl) (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, and O, and R<sub>7</sub>Is selected from the group consisting of H and OH.
A non-limiting example of the amino acid B in these embodiments is phenylalanine (NC).<sub>1</sub>~ C<sub>10</sub>Alkyl), tyrosine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), tryptophan (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl) Phenylalanine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), tyrosine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), tryptophan (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl) can be mentioned. In some embodiments, the amino acid of B is phenylalanine (NC).<sub>1</sub>~ C<sub>6</sub>Alkyl), tyrosine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), tryptophan (NC)<sub>1</sub>~ C<sub>6</sub>Selected from the group consisting of alkyl). For example, the amino acid B may include phenylalanine (N-methyl), tyrosine (N-methyl), tryptophan (N-methyl).
In some embodiments, the amino acid of B is proline. In some embodiments, proline is excluded from subcategory IB.
Subcategory IC: Amino acid B of dipeptide prodrug element disubstituted at β-position In some embodiments, the prodrug has a structural ABQ. Here, Q is a bioactive peptide (insulin peptide, etc.), and AB has the following structure.
<img id="000111" he="28" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /> During the ceremony R<sub>1</sub>And R<sub>2</sub>Independently, H, C<sub>1</sub>~ C<sub>18</sub>Alkyl, C<sub>2</sub>~ C<sub>18</sub>Alkenyl, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>18</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl, R<sub>3</sub>Is C<sub>1</sub>~ C<sub>18</sub>Alkyl R<sub>4</sub>Independently, CH (C<sub>1</sub>~ C<sub>8</sub>Alkyl)<sub>2</sub>, CH (C<sub>2</sub>~ C<sub>8</sub>Alkenyl)<sub>2</sub>, CH (C<sub>1</sub>~ C<sub>8</sub>Alkyl) (OH), CH (C)<sub>1</sub>~ C<sub>8</sub>Alkyl) ((C<sub>1</sub>~ C<sub>8</sub>Alkyl) SH), CH (C)<sub>1</sub>~ C<sub>3</sub>Alkyl) ((C<sub>1</sub>~ C<sub>8</sub>Alkyl) (NH<sub>2</sub>)) Selected from the group R<sub>8</sub>Is H, R<sub>5</sub>Is NHR<sub>6</sub>Or R<sub>5</sub>And R<sub>2</sub>Together with the atoms to which they are bonded form a 4-membered, 5- or 6-membered heterocycle, R<sub>6</sub>Is H or C<sub>1</sub>~ C<sub>4</sub>Alkyl R<sub>7</sub>Is selected from the group consisting of H and OH.
In some embodiments, R<sub>4</sub>Is CH (C<sub>1</sub>~ C<sub>8</sub>Alkyl)<sub>2</sub>Or CH (C<sub>1</sub>~ C<sub>8</sub>Alkyl) OH. As a non-limiting example of the amino acid B, isoleucine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), valine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl), threonine (NC)<sub>1</sub>~ C<sub>10</sub>Alkyl) can be mentioned. In some embodiments, the amino acid of B is isoleucine (NC).<sub>1</sub>~ C<sub>6</sub>Alkyl), valine (NC)<sub>1</sub>~ C<sub>6</sub>Alkyl), threonine (NC)<sub>1</sub>~ C<sub>6</sub>Selected from the group consisting of alkyl). For example, the amino acid B may include isoleucine (N-methyl), valine (N-methyl), threonine (N-methyl).
Category II: Composition of amino acid A of dipeptide prodrug element In some embodiments, the half-life of the prodrug depends on the number of substituents at the α-position of the amino acid A. For example, a prodrug with an amino acid of A, which is an α-monosubstituted amino acid (such as Ala), is more cleaved than a prodrug having an amino acid of A, which is an α, α-disubstituted amino acid (such as Aib). It is slow and has a long half-life.
In some embodiments, the half-life of the prodrug depends on the degree of alkylation of the amino acid A with the α-amino group. Generally, the greater the degree of alkylation, the slower the cleavage rate and the longer the half-life of the prodrug. For example, a dipeptide prodrug element with N-alkylated Ala cleaves later than Ala and has a longer half-life.
The composition of the amino acid A of the dipeptide prodrug element can be classified below into subcategories IIA and IIB. Usually, the dipeptide prodrug element of subcategory IIA is cleaved faster than the dipeptide prodrug element of subcategory IIB.
Subcategory IIA: Amino acid A in the dipeptide prodrug element is disubstituted at the α-position In some embodiments, the amino acid A in the dipeptide prodrug element is disubstituted at the α-position. In these embodiments, R of the structures described in subcategories IA, IB, IC<sub>1</sub>And R<sub>2</sub>Independently, C<sub>1</sub>~ C<sub>10</sub>Alkyl, C<sub>2</sub>~ C<sub>10</sub>Alkenyl, (C<sub>1</sub>~ C<sub>10</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>10</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Forming cycloalkyl and R<sub>7</sub>Is selected from the group consisting of H and OH.
For example, the amino acid of A may include aminoisobutyric acid (Aib).
Subcategory IIB: Amino acid A of the dipeptide prodrug element is unsubstituted or mono-substituted at the α-position. In some embodiments, the amino acid A in the dipeptide prodrug element is unsubstituted or mono-substituted at the α-position. In these embodiments, R of the structures described in subcategories IA, IB, IC<sub>1</sub>Is H and R of the structure described in the subcategories IA, IB, IC<sub>2</sub>Is H, C<sub>1</sub>~ C<sub>10</sub>Alkyl, C<sub>2</sub>~ C<sub>10</sub>Alkenyl, (C<sub>1</sub>~ C<sub>10</sub>Alkyl) OH, (C<sub>1</sub>~ C<sub>10</sub>Alkyl) SH, (C<sub>2</sub>~ C<sub>3</sub>Alkyl) SCH<sub>3</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) CONH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) COOH, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NH<sub>2</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) NHC (NH<sub>2</sub><sup>+</sup>) NH<sub>2</sub>, (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>6</sub>Cycloalkyl), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>2</sub>~ C<sub>5</sub>Heterocycle), (C<sub>0</sub>~ C<sub>4</sub>Alkyl) (C<sub>6</sub>~ C<sub>10</sub>Aryl) R<sub>7</sub>, (C<sub>1</sub>~ C<sub>4</sub>Alkyl) (C<sub>3</sub>~ C<sub>9</sub>Heteroaryl), C<sub>1</sub>~ C<sub>12</sub>Alkyl (W<sub>1</sub>) C<sub>1</sub>~ C<sub>12</sub>Selected from the group consisting of alkyl, R<sub>7</sub>Is selected from the group consisting of H and OH, W<sub>1</sub>Is a heteroatom selected from the group consisting of N, S, O, or R<sub>1</sub>And R<sub>2</sub>Together with the atoms to which they are bonded, C<sub>3</sub>~ C<sub>12</sub>Form cycloalkyl or R<sub>2</sub>And R<sub>5</sub>Together with the atoms to which they are bonded form a 4-membered, 5-membered or 6-membered heterocycle.
In some embodiments, the amino acid A of the dipeptide prodrug element has a "d" character isomer. Non-limiting examples of the amino acid A in these embodiments include lysine, cysteine, alanine. For example, d-lysine, d-cysteine, d-alanine. In some embodiments, d-characteristics may result in reduced proteolytic degradation of the prodrug peptide and a longer half-life.
In some embodiments, the amino acid of A is Ala (NC).<sub>1</sub>~ C<sub>4</sub>Alkyl), Lys (NC)<sub>1</sub>~ C<sub>4</sub>Alkyl), Cys (NC)<sub>1</sub>~ C<sub>4</sub>Alkylated) with groups having 1 to 4 carbon atoms. For example, the amino acid of A may be Ala (N-methyl), Lys (N-methyl), Cys (N-methyl). N-alkylation of the amino acid A reduces the rate of cleavage of the dipeptide prodrug element from Q and increases its half-life.
Category III: Binding site of dipeptide prodrug element (AB) to peptide drug (Q) In some embodiments, the half-life of the prodrug depends on the steric hindrance, nucleophilicity, and stability of the leaving group of Q during diketopiperazine formation. The half-life of the prodrug is shortened as the steric hindrance of the leaving group decreases, the nucleophilicity of the leaving group decreases, or the stability of the leaving group after cleavage increases. The type of leaving group for Q can be determined by the type of bond between the amino group of AB and Q, as described below in subcategories IIIA and IIIB. In general, the dipeptide prodrug element of subcategory IIIB cleaves from Q faster and has a shorter half-life than the dipeptide prodrug element of subcategory IIIA.
AB bound to the aliphatic amino group of subcategory IIIA: Q In some embodiments, as described herein above, the half-life (t) in which Q to AB are chemically cleaved in PBS under physiological conditions.<sub>1/2</sub>) Is attached to Q via an amide bond between the aliphatic amino group of AB and Q so that a prodrug can be obtained that is at least about 1 hour to about 1 week.
In some embodiments, AB binds to Q via an amide bond between AB and the α-amino group of the N-terminal amino acid of Q. For example, in PBS, under physiological conditions, the half-life (t) in which Q to AB are chemically cleaved.<sub>1/2</sub>Amino acids in any of subcategories IA, IB, IC and A in subcategories IIA and IIB so that a prodrug can be obtained in which) is at least about 1 hour to about 1 week. The dipeptide prodrug element having may be bound to the N-terminal amino acid of Q.
In some embodiments, AB binds to Q via an amide bond between the aliphatic amino group of the side chain of the amino acids AB and Q. For example, in PBS, under physiological conditions, the half-life (t) in which Q to AB are chemically cleaved.<sub>1/2</sub>Amino acids in any of subcategories IA, IB, IC and A in subcategories IIA and IIB so that prodrugs can be obtained for at least about 1 hour to about 1 week. The dipeptide prodrug element having may be attached to the aliphatic amino group of the side chain of the amino acid of Q.
In some embodiments, if AB is attached to Q via an amide bond between AB and the aliphatic amino group of Q, then A must be an α, α-disubstituted amino acid (sub). Either category IIA) or B must be N-alkylated (either subcategory IA, IB or IC) or both. For example, if A is an α-monosubstituted amino acid (such as Ala), B is not N-alkylated, and AB is attached to Q via the aliphatic amino group of Q, then AB is significantly It will not be disconnected.
In another embodiment, AB is an amino acid (such as glycine) that binds to Q via an amide bond between AB and the aliphatic amino group of Q and A is not substituted at the α-position. If B is a subcategory IA amino acid (N-alkylated glycine), the N-alkyl substituents on the B amino acid are at least 5 carbon atoms long (eg NC).<sub>5</sub>~ C<sub>8</sub>Alkyl).
In yet another embodiment, AB is attached to Q via an amide bond between AB and the aliphatic amino group of Q, and the amino acid of A is unsubstituted or mono-substituted at the α-position. If yes (subcategory IIB), the amino acid in B is not proline.
Subcategory IIIB: AB attached to the aromatic amino group of Q In some embodiments, as described herein above, the half-life (t) in which Q to AB are chemically cleaved in PBS under physiological conditions.<sub>1/2</sub>) Is attached to Q via an amide bond between the aromatic amino group of the side chain of the amino acid of AB and Q so that a prodrug can be obtained that is at least about 1 hour to about 1 week. For example, in PBS, under physiological conditions, the half-life (t) in which Q to AB are chemically cleaved.<sub>1/2</sub>Amino acids in any of subcategories IA, IB, IC and A in subcategories IIA and IIB so that a prodrug can be obtained in which) is at least about 1 hour to about 1 week. The dipeptide prodrug element having may be attached to the aromatic amino group of the side chain of the amino acid of Q.
It is possible to combine any of the amino acids B defined in Category I with any of the amino acids A defined in Category II to form a dipeptide prodrug element. The dipeptide prodrug element may be attached to any of the positions described in Category III. For the half-life of prodrugs, (i) Number of substitution groups at the α-position of the amino acid of A (ii) Degree of N-alkylation of amino acids A and B (iii) Number of β-substituted cardinal numbers of amino acid of B (iv) The bulkiness of the side chain of the amino acid of B (iii) Steric hindrance, nucleophilicity, and stability of the leaving group of Q during diketopiperazine formation It can be adjusted by selecting.
Modification of dipeptide prodrug element AB As already described herein, it is also possible to further modify the dipeptide prodrug element described above to have a hydrophilic, acyl or alkyl group. In some embodiments, the dipeptide prodrug element has a lysine attached to an acyl or alkyl group via an amino group in its side chain. In some embodiments, the dipeptide prodrug element has a cysteine attached to a hydrophilic body (eg, 40 kD PEG) via a side chain sulfhydryl group. For hydrophilics, acyl groups or alkyl groups, they may be attached directly to the dipeptide prodrug element or via spacers. In some embodiments by way of example, a hydrophilic group, an alkyl group and / or an acyl group is attached to the amino acid A of the dipeptide prodrug element.
In some embodiments, the following dipeptide prodrug elements are PEGylated: dCys-Gly (N-hexyl) dCys-Gly (N-methyl), dCys-Phe (N-methyl). In some embodiments, the following dipeptide prodrug elements have an acyl group: dLys-Gly (N-hexyl), dLys-Gly (N-methyl), dLys-Phe (N-methyl). In some embodiments, the following dipeptide prodrug elements have an alkyl group: dLys-Gly (N-hexyl), dLys-Gly (N-methyl), dLys-Phe (N-methyl).
Embodiment as an example The dipeptide prodrug element of the present invention may have a combination of any of the B amino acids of Category I and any of the A amino acids of Category II. A non-limiting example of amino acids suitable for the amino acids A and B of the dipeptide prodrug element is given in the table below.
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In some embodiments, the dipeptide prodrug element has a combination of any one of A1 to A77 and any one of B1 to B113. For example, combinations of amino acids A and B in the dipeptide prodrug element include: A1-B1; A1-B2; A1-B3; A1-B4; A1-B5; A1-B6; A1-B7; A1-B8; A1-B9; A1-B10; A1-B11; A1-B12; A1-B13; A1-B14; A1-B15; A1-B16; A1-B17; A1-B18; A1- B19; A1-B20; A1-B21; A1-B22; A1-B23; A1-B24; A1-B25; A1-B26; A1-B27; A1-B28; A1-B29; A1-B30; A1-B31; A1-B32; A1-B33; A1-B34; A1-B35; A1-B36; A1-B37; A1-B38; A1-B39; A1-B40; A1-B41; A1-B42; A1-B43; A1- B44; A1-B45; A1-B46; A1-B47; A1-B48; A1-B49; A1-B50; A1-B51; A1-B52; A1-B53; A1-B54; A1-B55; A1-B56; A1-B57; A1-B58; A1-B59; A1-B60; A1-B61; A1-B62; A1-B63; A1-B64; A1-B65; A1-B66; A1-B67; A1-B68; A1- B69; A1-B70; A1-B71; A1-B72; A1-B73; A1-B74; A1-B75; A1-B76; A1-B77; A1-B78; A1-B79; A1-B80; A1-B81; A1-B82; A1-B83; A1-B84; A1-B85; A1-B86; A1-B87; A1-B88; A1-B89; A1-B90; A1-B91; A1-B92; A1-B93; A1- B94; A1-B95; A1-B96; A1-B97; A1-B98; A1-B99; A1-B100; A1-B101; A1-B102; A1-B103; A1-B104; In some embodiments, the dipeptide prodrug element has a combination of any one of A1-A154 and any one of B1-B113. For example, combinations of the A and B amino acids of the dipeptide prodrug element include: A1-B1; A1-B2; A1-B3; A1-B4; A1-B5; A1-B6; A1-B7; A1-B8; A1-B9; A1-B10; A1-B11; A1-B12; A1-B13; A1-B14; A1-B15; A1-B16; A1-B17; A1-B18; A1- B19; A1-B20; A1-B21; A1-B22; A1-B23; A1-B24; A1-B25; A1-B26; A1-B27; A1-B28; A1-B29; A1-B30; A1-B31; A1-B32; A1-B33; A1-B34; A1-B35; A1-B36; A1-B37; A1-B38; A1-B39; A1-B40; A1-B41; A1-B42; A1-B43; A1- B44; A1-B45; A1-B46; A1-B47; A1-B48; A1-B49; A1-B50; A1-B51; A1-B52; A1-B53; A1-B54; A1-B55; A1-B56; A1-B57; A1-B58; A1-B59; A1-B60; A1-B61; A1-B62; A1-B63; A1-B64; A1-B65; A1-B66; A1-B67; A1-B68; A1- B69; A1-B70; A1-B71; A1-B72; A1-B73; A1-B74; A1-B75; A1-B76; A1-B77; A1-B78; A1-B79; A1-B80; A1-B81; A1-B82; A1-B83; A1-B84; A1-B85; A1-B86; A1-B87; A1-B88; A1-B89; A1-B90; A1-B91; A1-B92; A1-B93; A1- B94; A1-B95; A1-B96; A1-B97; A1-B98; A1-B99; A1-B100; A1-B101; A1-B102; A1-B103; A1-B104; A2-B1; A2-B2; A2-B3; A2-B4; A2-B5; A2-B6; A2-B7; A2-B8; A2-B9; A2-B10; A2-B11; A2-B12; A2- B13; A2-B14; A2-B15; A2-B16; A2-B17; A2-B18; A2-B19; A2-B20; A2-B21; A2-B22; A2-B23; A2-B24; A2-B25; A2-B26; A2-B27; A2-B28; A2-B29; A2-B30; A2-B31; A2-B32; A2-B33; A2-B34; A2-B35; A2-B36; A2-B37; A2- B38; A2-B39; A2-B40; A2-B41; A2-B42; A2-B43; A2-B44; A2-B45; A2-B46; A2-B47; A2-B48; A2-B49; A2-B50; A2-B51; A2-B52; A2-B53; A2-B54; A2-B55; A2-B56; A2-B57; A2-B58; A2-B59; A2-B60; A2-B61; A2-B62; A2- B63; A2-B64; A2-B65; A2-B66; A2-B67; A2-B68; A2-B69; A2-B70; A2-B71; A2-B72; A2-B73; A2-B74; A2-B75; A2-B76; A2-B77; A2-B78; A2-B79; A2-B80; A2-B81; A2-B82; A2-B83; A2-B84; A2-B85; A2-B86; A2-B87; A2- B88; A2-B89; A2-B90; A2-B91; A2-B92; A2-B93; A2-B94; A2-B95; A2-B96; A2-B97; A2-B98; A2-B99; A2-B100; A2-B101; A2-B102; A2-B103; A2-B104; A2-B105; A2-B106; A2-B107; A2-B108; A2-B109; A2-B110; A2-B111; A2-B112; A2- B113; A3-B1; A3-B2; A3-B3; A3-B4; A3-B5; A3-B6; A3-B7; A3-B8; A3-B9; A3-B10; A3-B11; A3-B12; A3- B13; A3-B14; A3-B15; A3-B16; A3-B17; A3-B18; A3-B19; A3-B20; A3-B21; A3-B22; A3-B23; A3-B24; A3-B25; A3-B26; A3-B27; A3-B28; A3-B29; A3-B30; A3-B31; A3-B32; A3-B33; A3-B34; A3-B35; A3-B36; A3-B37; A3- B38; A3-B39; A3-B40; A3-B41; A3-B42; A3-B43; A3-B44; A3-B45; A3-B46; A3-B47; A3-B48; A3-B49; A3-B50; A3-B51; A3-B52; A3-B53; A3-B54; A3-B55; A3-B56; A3-B57; A3-B58; A3-B59; A3-B60; A3-B61; A3-B62; A3- B63; A3-B64; A3-B65; A3-B66; A3-B67; A3-B68; A3-B69; A3-B70; A3-B71; A3-B72; A3-B73; A3-B74; A3-B75; A3-B76; A3-B77; A3-B78; A3-B79; A3-B80; A3-B81; A3-B82; A3-B83; A3-B84; A3-B85; A3-B86; A3-B87; A3- B88; A3-B89; A3-B90; A3-B91; A3-B92; A3-B93; A3-B94; A3-B95; A3-B96; A3-B97; A3-B98; A3-B99; A3-B100; A3-B101; A3-B102; A3-B103; A3-B104; A3-B105; A3-B106; A3-B107; A3-B108; A3-B109; A3-B110; A3-B111; A3-B112; A3- B113; A4-B1; A4-B2; A4-B3; A4-B4; A4-B5; A4-B6; A4-B7; A4-B8; A4-B9; A4-B10; A4-B11; A4-B12; A4- B13; A4-B14; A4-B15; A4-B16; A4-B17; A4-B18; A4-B19; A4-B20; A4-B21; A4-B22; A4-B23; A4-B24; A4-B25; A4-B26; A4-B27; A4-B28; A4-B29; A4-B30; A4-B31; A4-B32; A4-B33; A4-B34; A4-B35; A4-B36; A4-B37; A4- B38; A4-B39; A4-B40; A4-B41; A4-B42; A4-B43; A4-B44; A4-B45; A4-B46; A4-B47; A4-B48; A4-B49; A4-B50; A4-B51; A4-B52; A4-B53; A4-B54; A4-B55; A4-B56; A4-B57; A4-B58; A4-B59; A4-B60; A4-B61; A4-B62; A4- B63; A4-B64; A4-B65; A4-B66; A4-B67; A4-B68; A4-B69; A4-B70; A4-B71; A4-B72; A4-B73; A4-B74; A4-B75; A4-B76; A4-B77; A4-B78; A4-B79; A4-B80; A4-B81; A4-B82; A4-B83; A4-B84; A4-B85; A4-B86; A4-B87; A4- B88; A4-B89; A4-B90; A4-B91; A4-B92; A4-B93; A4-B94; A4-B95; A4-B96; A4-B97; A4-B98; A4-B99; A4-B100; A4-B101; A4-B102; A4-B103; A4-B104; A4-B105; A4-B106; A4-B107; A4-B108; A4-B109; A4-B110; A4-B111; A4-B112; A4- B113; A5-B1; A5-B2; A5-B3; A5-B4; A5-B5; A5-B6; A5-B7; A5-B8; A5-B9; A5-B10; A5-B11; A5-B12; A5- B13; A5-B14; A5-B15; A5-B16; A5-B17; A5-B18; A5-B19; A5-B20; A5-B21; A5-B22; A5-B23; A5-B24; A5-B25; A5-B26; A5-B27; A5-B28; A5-B29; A5-B30; A5-B31; A5-B32; A5-B33; A5-B34; A5-B35; A5-B36; A5-B37; A5- B38; A5-B39; A5-B40; A5-B41; A5-B42; A5-B43; A5-B44; A5-B45; A5-B46; A5-B47; A5-B48; A5-B49; A5-B50; A5-B51; A5-B52; A5-B53; A5-B54; A5-B55; A5-B56; A5-B57; A5-B58; A5-B59; A5-B60; A5-B61; A5-B62; A5- B63; A5-B64; A5-B65; A5-B66; A5-B67; A5-B68; A5-B69; A5-B70; A5-B71; A5-B72; A5-B73; A5-B74; A5-B75; A5-B76; A5-B77; A5-B78; A5-B79; A5-B80; A5-B81; A5-B82; A5-B83; A5-B84; A5-B85; A5-B86; A5-B87; A5- B88; A5-B89; A5-B90; A5-B91; A5-B92; A5-B93; A5-B94; A5-B95; A5-B96; A5-B97; A5-B98; A5-B99; A5-B100; A5-B101; A5-B102; A5-B103; A5-B104; A5-B105; A5-B106; A5-B107; A5-B108; A5-B109; A5-B110; A5-B111; A5-B112; A5- B113; A6-B1; A6-B2; A6-B3; A6-B4; A6-B5; A6-B6; A6-B7; A6-B8; A6-B9; A6-B10; A6-B11; A6-B12; A6- B13; A6-B14; A6-B15; A6-B16; A6-B17; A6-B18; A6-B19; A6-B20; A6-B21; A6-B22; A6-B23; A6-B24; A6-B25; A6-B26; A6-B27; A6-B28; A6-B29; A6-B30; A6-B31; A6-B32; A6-B33; A6-B34; A6-B35; A6-B36; A6-B37; A6- B38; A6-B39; A6-B40; A6-B41; A6-B42; A6-B43; A6-B44; A6-B45; A6-B46; A6-B47; A6-B48; A6-B49; A6-B50; A6-B51; A6-B52; A6-B53; A6-B54; A6-B55; A6-B56; A6-B57; A6-B58; A6-B59; A6-B60; A6-B61; A6-B62; A6- B63; A6-B64; A6-B65; A6-B66; A6-B67; A6-B68; A6-B69; A6-B70; A6-B71; A6-B72; A6-B73; A6-B74; A6-B75; A6-B76; A6-B77; A6-B78; A6-B79; A6-B80; A6-B81; A6-B82; A6-B83; A6-B84; A6-B85; A6-B86; A6-B87; A6- B88; A6-B89; A6-B90; A6-B91; A6-B92; A6-B93; A6-B94; A6-B95; A6-B96; A6-B97; A6-B98; A6-B99; A6-B100; A6-B101; A6-B102; A6-B103; A6-B104; A6-B105; A6-B106; A6-B107; A6-B108; A6-B109; A6-B110; A6-B111; A6-B112; A6- B113; A7-B1; A7-B2; A7-B3; A7-B4; A7-B5; A7-B6; A7-B7; A7-B8; A7-B9; A7-B10; A7-B11; A7-B12; A7- B13; A7-B14; A7-B15; A7-B16; A7-B17; A7-B18; A7-B19; A7-B20; A7-B21; A7-B22; A7-B23; A7-B24; A7-B25; A7-B26; A7-B27; A7-B28; A7-B29; A7-B30; A7-B31; A7-B32; A7-B33; A7-B34; A7-B35; A7-B36; A7-B37; A7- B38; A7-B39; A7-B40; A7-B41; A7-B42; A7-B43; A7-B44; A7-B45; A7-B46; A7-B47; A7-B48; A7-B49; A7-B50; A7-B51; A7-B52; A7-B53; A7-B54; A7-B55; A7-B56; A7-B57; A7-B58; A7-B59; A7-B60; A7-B61; A7-B62; A7- B63; A7-B64; A7-B65; A7-B66; A7-B67; A7-B68; A7-B69; A7-B70; A7-B71; A7-B72; A7-B73; A7-B74; A7-B75; A7-B76; A7-B77; A7-B78; A7-B79; A7-B80; A7-B81; A7-B82; A7-B83; A7-B84; A7-B85; A7-B86; A7-B87; A7- B88; A7-B89; A7-B90; A7-B91; A7-B92; A7-B93; A7-B94; A7-B95; A7-B96; A7-B97; A7-B98; A7-B99; A7-B100; A7-B101; A7-B102; A7-B103; A7-B104; A7-B105; A7-B106; A7-B107; A7-B108; A7-B109; A7-B110; A7-B111; A7-B112; A7- B113; A8-B1; A8-B2; A8-B3; A8-B4; A8-B5; A8-B6; A8-B7; A8-B8; A8-B9; A8-B10; A8-B11; A8-B12; A8- B13; A8-B14; A8-B15; A8-B16; A8-B17; A8-B18; A8-B19; A8-B20; A8-B21; A8-B22; A8-B23; A8-B24; A8-B25; A8-B26; A8-B27; A8-B28; A8-B29; A8-B30; A8-B31; A8-B32; A8-B33; A8-B34; A8-B35; A8-B36; A8-B37; A8- B38; A8-B39; A8-B40; A8-B41; A8-B42; A8-B43; A8-B44; A8-B45; A8-B46; A8-B47; A8-B48; A8-B49; A8-B50; A8-B51; A8-B52; A8-B53; A8-B54; A8-B55; A8-B56; A8-B57; A8-B58; A8-B59; A8-B60; A8-B61; A8-B62; A8- B63; A8-B64; A8-B65; A8-B66; A8-B67; A8-B68; A8-B69; A8-B70; A8-B71; A8-B72; A8-B73; A8-B74; A8-B75; A8-B76; A8-B77; A8-B78; A8-B79; A8-B80; A8-B81; A8-B82; A8-B83; A8-B84; A8-B85; A8-B86; A8-B87; A8- B88; A8-B89; A8-B90; A8-B91; A8-B92; A8-B93; A8-B94; A8-B95; A8-B96; A8-B97; A8-B98; A8-B99; A8-B100; A8-B101; A8-B102; A8-B103; A8-B104; A8-B105; A8-B106; A8-B107; A8-B108; A8-B109; A8-B110; A8-B111; A8-B112; A8- B113; A9-B1; A9-B2; A9-B3; A9-B4; A9-B5; A9-B6; A9-B7; A9-B8; A9-B9; A9-B10; A9-B11; A9-B12; A9- B13; A9-B14; A9-B15; A9-B16; A9-B17; A9-B18; A9-B19; A9-B20; A9-B21; A9-B22; A9-B23; A9-B24; A9-B25; A9-B26; A9-B27; A9-B28; A9-B29; A9-B30; A9-B31; A9-B32; A9-B33; A9-B34; A9-B35; A9-B36; A9-B37; A9- B38; A9-B39; A9-B40; A9-B41; A9-B42; A9-B43; A9-B44; A9-B45; A9-B46; A9-B47; A9-B48; A9-B49; A9-B50; A9-B51; A9-B52; A9-B53; A9-B54; A9-B55; A9-B56; A9-B57; A9-B58; A9-B59; A9-B60; A9-B61; A9-B62; A9- B63; A9-B64; A9-B65; A9-B66; A9-B67; A9-B68; A9-B69; A9-B70; A9-B71; A9-B72; A9-B73; A9-B74; A9-B75; A9-B76; A9-B77; A9-B78; A9-B79; A9-B80; A9-B81; A9-B82; A9-B83; A9-B84; A9-B85; A9-B86; A9-B87; A9- B88; A9-B89; A9-B90; A9-B91; A9-B92; A9-B93; A9-B94; A9-B95; A9-B96; A9-B97; A9-B98; A9-B99; A9-B100; A9-B101; A9-B102; A9-B103; A9-B104; A9-B105; A9-B106; A9-B107; A9-B108; A9-B109; A9-B110; A9-B111; A9-B112; A9- B113; A10-B1; A10-B2; A10-B3; A10-B4; A10-B5; A10-B6; A10-B7; A10-B8; A10-B9; A10-B10; A10-B11; A10-B12; A10- B13; A10-B14; A10-B15; A10-B16; A10-B17; A10-B18; A10-B19; A10-B20; A10-B21; A10-B22; A10-B23; A10-B24; A10-B25; A10-B26; A10-B27; A10-B28; A10-B29; A10-B30; A10-B31; A10-B32; A10-B33; A10-B34; A10-B35; A10-B36; A10-B37; A10- B38; A10-B39; A10-B40; A10-B41; A10-B42; A10-B43; A10-B44; A10-B45; A10-B46; A10-B47; A10-B48; A10-B49; A10-B50; A10-B51; A10-B52; A10-B53; A10-B54; A10-B55; A10-B56; A10-B57; A10-B58; A10-B59; A10-B60; A10-B61; A10-B62; A10- B63; A10-B64; A10-B65; A10-B66; A10-B67; A10-B68; A10-B69; A10-B70; A10-B71; A10-B72; A10-B73; A10-B74; A10-B75; A10-B76; A10-B77; A10-B78; A10-B79; A10-B80; A10-B81; A10-B82; A10-B83; A10-B84; A10-B85; A10-B86; A10-B87; A10- B88; A10-B89; A10-B90; A10-B91; A10-B92; A10-B93; A10-B94; A10-B95; A10-B96; A10-B97; A10-B98; A10-B99; A10-B100; A10-B101; A10-B102; A10-B103; A10-B104; A10-B105; A10-B106; A10-B107;A10-B108; A10-B109; A10-B110; A10-B111; A10-B112; A10-B113; A11-B1; A11-B2; A11-B3; A11-B4; A11-B5; A11-B6; A11-B7; A11-B8; A11-B9; A11-B10; A11-B11; A11-B12; A11- B13; A11-B14; A11-B15; A11-B16; A11-B17; A11-B18; A11-B19; A11-B20; A11-B21; A11-B22; A11-B23; A11-B24; A11-B25; A11-B26; A11-B27; A11-B28; A11-B29; A11-B30; A11-B31; A11-B32; A11-B33; A11-B34; A11-B35; A11-B36; A11-B37; A11- B38; A11-B39; A11-B40; A11-B41; A11-B42; A11-B43; A11-B44; A11-B45; A11-B46; A11-B47; A11-B48; A11-B49; A11-B50; A11-B51; A11-B52; A11-B53; A11-B54; A11-B55; A11-B56; A11-B57; A11-B58; A11-B59; A11-B60; A11-B61; A11-B62; A11- B63; A11-B64; A11-B65; A11-B66; A11-B67; A11-B68; A11-B69; A11-B70; A11-B71; A11-B72; A11-B73; A11-B74; A11-B75; A11-B76; A11-B77; A11-B78; A11-B79; A11-B80; A11-B81; A11-B82; A11-B83; A11-B84; A11-B85; A11-B86; A11-B87; A11- B88; A11-B89; A11-B90; A11-B91; A11-B92; A11-B93; A11-B94; A11-B95; A11-B96; A11-B97; A11-B98; A11-B99; A11-B100; A11-B101; A11-B102; A11-B103; A11-B104; A11-B105; A11-B106; A11-B107;A11-B108; A11-B109; A11-B110; A11-B111; A11-B112; A11-B113; A12-B1; A12-B2; A12-B3; A12-B4; A12-B5; A12-B6; A12-B7; A12-B8; A12-B9; A12-B10; A12-B11; A12-B12; A12- B13; A12-B14; A12-B15; A12-B16; A12-B17; A12-B18; A12-B19; A12-B20; A12-B21; A12-B22; A12-B23; A12-B24; A12-B25; A12-B26; A12-B27; A12-B28; A12-B29; A12-B30; A12-B31; A12-B32; A12-B33; A12-B34; A12-B35; A12-B36; A12-B37; A12- B38; A12-B39; A12-B40; A12-B41; A12-B42; A12-B43; A12-B44; A12-B45; A12-B46; A12-B47; A12-B48; A12-B49; A12-B50; A12-B51; A12-B52; A12-B53; A12-B54; A12-B55; A12-B56; A12-B57; A12-B58; A12-B59; A12-B60; A12-B61; A12-B62; A12- B63; A12-B64; A12-B65; A12-B66; A12-B67; A12-B68; A12-B69; A12-B70; A12-B71; A12-B72; A12-B73; A12-B74; A12-B75; A12-B76; A12-B77; A12-B78; A12-B79; A12-B80; A12-B81; A12-B82; A12-B83; A12-B84; A12-B85; A12-B86; A12-B87; A12- B88; A12-B89; A12-B90; A12-B91; A12-B92; A12-B93; A12-B94; A12-B95; A12-B96; A12-B97; A12-B98; A12-B99; A12-B100; A12-B101; A12-B102; A12-B103; A12-B104; A12-B105; A12-B106; A12-B107;A12-B108; A12-B109; A12-B110; A12-B111; A12-B112; A12-B113; A13-B1; A13-B2; A13-B3; A13-B4; A13-B5; A13-B6; A13-B7; A13-B8; A13-B9; A13-B10; A13-B11; A13-B12; A13- B13; A13-B14; A13-B15; A13-B16; A13-B17; A13-B18; A13-B19; A13-B20; A13-B21; A13-B22; A13-B23; A13-B24; A13-B25; A13-B26; A13-B27; A13-B28; A13-B29; A13-B30; A13-B31; A13-B32; A13-B33; A13-B34; A13-B35; A13-B36; A13-B37; A13- B38; A13-B39; A13-B40; A13-B41; A13-B42; A13-B43; A13-B44; A13-B45; A13-B46; A13-B47; A13-B48; A13-B49; A13-B50; A13-B51; A13-B52; A13-B53; A13-B54; A13-B55; A13-B56; A13-B57; A13-B58; A13-B59; A13-B60; A13-B61; A13-B62; A13- B63; A13-B64; A13-B65; A13-B66; A13-B67; A13-B68; A13-B69; A13-B70; A13-B71; A13-B72; A13-B73; A13-B74; A13-B75; A13-B76; A13-B77; A13-B78; A13-B79; A13-B80; A13-B81; A13-B82; A13-B83; A13-B84; A13-B85; A13-B86; A13-B87; A13- B88; A13-B89; A13-B90; A13-B91; A13-B92; A13-B93; A13-B94; A13-B95; A13-B96; A13-B97; A13-B98; A13-B99; A13-B100; A13-B101; A13-B102; A13-B103; A13-B104; A13-B105; A13-B106; A13-B107;A13-B108; A13-B109; A13-B110; A13-B111; A13-B112; A13-B113; A14-B1; A14-B2; A14-B3; A14-B4; A14-B5; A14-B6; A14-B7; A14-B8; A14-B9; A14-B10; A14-B11; A14-B12; A14- B13; A14-B14; A14-B15; A14-B16; A14-B17; A14-B18; A14-B19; A14-B20; A14-B21; A14-B22; A14-B23; A14-B24; A14-B25; A14-B26; A14-B27; A14-B28; A14-B29; A14-B30; A14-B31; A14-B32; A14-B33; A14-B34; A14-B35; A14-B36; A14-B37; A14- B38; A14-B39; A14-B40; A14-B41; A14-B42; A14-B43; A14-B44; A14-B45; A14-B46; A14-B47; A14-B48; A14-B49; A14-B50; A14-B51; A14-B52; A14-B53; A14-B54; A14-B55; A14-B56; A14-B57; A14-B58; A14-B59; A14-B60; A14-B61; A14-B62; A14- B63; A14-B64; A14-B65; A14-B66; A14-B67; A14-B68; A14-B69; A14-B70; A14-B71; A14-B72; A14-B73; A14-B74; A14-B75; A14-B76; A14-B77; A14-B78; A14-B79; A14-B80; A14-B81; A14-B82; A14-B83; A14-B84; A14-B85; A14-B86; A14-B87; A14- B88; A14-B89; A14-B90; A14-B91; A14-B92; A14-B93; A14-B94; A14-B95; A14-B96; A14-B97; A14-B98; A14-B99; A14-B100; A14-B101; A14-B102; A14-B103; A14-B104; A14-B105; A14-B106; A14-B107;A14-B108; A14-B109; A14-B110; A14-B111; A14-B112; A14-B113; A15-B1; A15-B2; A15-B3; A15-B4; A15-B5; A15-B6; A15-B7; A15-B8; A15-B9; A15-B10; A15-B11; A15-B12; A15- B13; A15-B14; A15-B15; A15-B16; A15-B17; A15-B18; A15-B19; A15-B20; A15-B21; A15-B22; A15-B23; A15-B24; A15-B25; A15-B26; A15-B27; A15-B28; A15-B29; A15-B30; A15-B31; A15-B32; A15-B33; A15-B34; A15-B35; A15-B36; A15-B37; A15- B38; A15-B39; A15-B40; A15-B41; A15-B42; A15-B43; A15-B44; A15-B45; A15-B46; A15-B47; A15-B48; A15-B49; A15-B50; A15-B51; A15-B52; A15-B53; A15-B54; A15-B55; A15-B56; A15-B57; A15-B58; A15-B59; A15-B60; A15-B61; A15-B62; A15- B63; A15-B64; A15-B65; A15-B66; A15-B67; A15-B68; A15-B69; A15-B70; A15-B71; A15-B72; A15-B73; A15-B74; A15-B75; A15-B76; A15-B77; A15-B78; A15-B79; A15-B80; A15-B81; A15-B82; A15-B83; A15-B84; A15-B85; A15-B86; A15-B87; A15- B88; A15-B89; A15-B90; A15-B91; A15-B92; A15-B93; A15-B94; A15-B95; A15-B96; A15-B97; A15-B98; A15-B99; A15-B100; A15-B101; A15-B102; A15-B103; A15-B104; A15-B105; A15-B106; A15-B107;A15-B108; A15-B109; A15-B110; A15-B111; A15-B112; A15-B113; A16-B1; A16-B2; A16-B3; A16-B4; A16-B5; A16-B6; A16-B7; A16-B8; A16-B9; A16-B10; A16-B11; A16-B12; A16- B13; A16-B14; A16-B15; A16-B16; A16-B17; A16-B18; A16-B19; A16-B20; A16-B21; A16-B22; A16-B23; A16-B24; A16-B25; A16-B26; A16-B27; A16-B28; A16-B29; A16-B30; A16-B31; A16-B32; A16-B33; A16-B34; A16-B35; A16-B36; A16-B37; A16- B38; A16-B39; A16-B40; A16-B41; A16-B42; A16-B43; A16-B44; A16-B45; A16-B46; A16-B47; A16-B48; A16-B49; A16-B50; A16-B51; A16-B52; A16-B53; A16-B54; A16-B55; A16-B56; A16-B57; A16-B58; A16-B59; A16-B60; A16-B61; A16-B62; A16- B63; A16-B64; A16-B65; A16-B66; A16-B67; A16-B68; A16-B69; A16-B70; A16-B71; A16-B72; A16-B73; A16-B74; A16-B75; A16-B76; A16-B77; A16-B78; A16-B79; A16-B80; A16-B81; A16-B82; A16-B83; A16-B84; A16-B85; A16-B86; A16-B87; A16- B88; A16-B89; A16-B90; A16-B91; A16-B92; A16-B93; A16-B94; A16-B95; A16-B96; A16-B97; A16-B98; A16-B99; A16-B100; A16-B101; A16-B102; A16-B103; A16-B104; A16-B105; A16-B106; A16-B107;A16-B108; A16-B109; A16-B110; A16-B111; A16-B112; A16-B113; A17-B1; A17-B2; A17-B3; A17-B4; A17-B5; A17-B6; A17-B7; A17-B8; A17-B9; A17-B10; A17-B11; A17-B12; A17- B13; A17-B14; A17-B15; A17-B16; A17-B17; A17-B18; A17-B19; A17-B20; A17-B21; A17-B22; A17-B23; A17-B24; A17-B25; A17-B26; A17-B27; A17-B28; A17-B29; A17-B30; A17-B31; A17-B32; A17-B33; A17-B34; A17-B35; A17-B36; A17-B37; A17- B38; A17-B39; A17-B40; A17-B41; A17-B42; A17-B43; A17-B44; A17-B45; A17-B46; A17-B47; A17-B48; A17-B49; A17-B50; A17-B51; A17-B52; A17-B53; A17-B54; A17-B55; A17-B56; A17-B57; A17-B58; A17-B59; A17-B60; A17-B61; A17-B62; A17- B63; A17-B64; A17-B65; A17-B66; A17-B67; A17-B68; A17-B69; A17-B70; A17-B71; A17-B72; A17-B73; A17-B74; A17-B75; A17-B76; A17-B77; A17-B78; A17-B79; A17-B80; A17-B81; A17-B82; A17-B83; A17-B84; A17-B85; A17-B86; A17-B87; A17- B88; A17-B89; A17-B90; A17-B91; A17-B92; A17-B93; A17-B94; A17-B95; A17-B96; A17-B97; A17-B98; A17-B99; A17-B100; A17-B101; A17-B102; A17-B103; A17-B104; A17-B105; A17-B106; A17-B107;A17-B108; A17-B109; A17-B110; A17-B111; A17-B112; A17-B113; A18-B1; A18-B2; A18-B3; A18-B4; A18-B5; A18-B6; A18-B7; A18-B8; A18-B9; A18-B10; A18-B11; A18-B12; A18- B13; A18-B14; A18-B15; A18-B16; A18-B17; A18-B18; A18-B19; A18-B20; A18-B21; A18-B22; A18-B23; A18-B24; A18-B25; A18-B26; A18-B27; A18-B28; A18-B29; A18-B30; A18-B31; A18-B32; A18-B33; A18-B34; A18-B35; A18-B36; A18-B37; A18- B38; A18-B39; A18-B40; A18-B41; A18-B42; A18-B43; A18-B44; A18-B45; A18-B46; A18-B47; A18-B48; A18-B49; A18-B50; A18-B51; A18-B52; A18-B53; A18-B54; A18-B55; A18-B56; A18-B57; A18-B58; A18-B59; A18-B60; A18-B61; A18-B62; A18- B63; A18-B64; A18-B65; A18-B66; A18-B67; A18-B68; A18-B69; A18-B70; A18-B71; A18-B72; A18-B73; A18-B74; A18-B75; A18-B76; A18-B77; A18-B78; A18-B79; A18-B80; A18-B81; A18-B82; A18-B83; A18-B84; A18-B85; A18-B86; A18-B87; A18- B88; A18-B89; A18-B90; A18-B91; A18-B92; A18-B93; A18-B94; A18-B95; A18-B96; A18-B97; A18-B98; A18-B99; A18-B100; A18-B101; A18-B102; A18-B103; A18-B104; A18-B105; A18-B106; A18-B107;A18-B108; A18-B109; A18-B110; A18-B111; A18-B112; A18-B113; A19-B1; A19-B2; A19-B3; A19-B4; A19-B5; A19-B6; A19-B7; A19-B8; A19-B9; A19-B10; A19-B11; A19-B12; A19- B13; A19-B14; A19-B15; A19-B16; A19-B17; A19-B18; A19-B19; A19-B20; A19-B21; A19-B22; A19-B23; A19-B24; A19-B25; A19-B26; A19-B27; A19-B28; A19-B29; A19-B30; A19-B31; A19-B32; A19-B33; A19-B34; A19-B35; A19-B36; A19-B37; A19- B38; A19-B39; A19-B40; A19-B41; A19-B42; A19-B43; A19-B44; A19-B45; A19-B46; A19-B47; A19-B48; A19-B49; A19-B50; A19-B51; A19-B52; A19-B53; A19-B54; A19-B55; A19-B56; A19-B57; A19-B58; A19-B59; A19-B60; A19-B61; A19-B62; A19- B63; A19-B64; A19-B65; A19-B66; A19-B67; A19-B68; A19-B69; A19-B70; A19-B71; A19-B72; A19-B73; A19-B74; A19-B75; A19-B76; A19-B77; A19-B78; A19-B79; A19-B80; A19-B81; A19-B82; A19-B83; A19-B84; A19-B85; A19-B86; A19-B87; A19- B88; A19-B89; A19-B90; A19-B91; A19-B92; A19-B93; A19-B94; A19-B95; A19-B96; A19-B97; A19-B98; A19-B99; A19-B100; A19-B101; A19-B102; A19-B103; A19-B104; A19-B105; A19-B106; A19-B107;A19-B108; A19-B109; A19-B110; A19-B111; A19-B112; A19-B113; A20-B1; A20-B2; A20-B3; A20-B4; A20-B5; A20-B6; A20-B7; A20-B8; A20-B9; A20-B10; A20-B11; A20-B12; A20- B13; A20-B14; A20-B15; A20-B16; A20-B17; A20-B18; A20-B19; A20-B20; A20-B21; A20-B22; A20-B23; A20-B24; A20-B25; A20-B26; A20-B27; A20-B28; A20-B29; A20-B30; A20-B31; A20-B32; A20-B33; A20-B34; A20-B35; A20-B36; A20-B37; A20- B38; A20-B39; A20-B40; A20-B41; A20-B42; A20-B43; A20-B44; A20-B45; A20-B46; A20-B47; A20-B48; A20-B49; A20-B50; A20-B51; A20-B52; A20-B53; A20-B54; A20-B55; A20-B56; A20-B57; A20-B58; A20-B59; A20-B60; A20-B61; A20-B62; A20- B63; A20-B64; A20-B65; A20-B66; A20-B67; A20-B68; A20-B69; A20-B70; A20-B71; A20-B72; A20-B73; A20-B74; A20-B75; A20-B76; A20-B77; A20-B78; A20-B79; A20-B80; A20-B81; A20-B82; A20-B83; A20-B84; A20-B85; A20-B86; A20-B87; A20- B88; A20-B89; A20-B90; A20-B91; A20-B92; A20-B93; A20-B94; A20-B95; A20-B96; A20-B97; A20-B98; A20-B99; A20-B100; A20-B101; A20-B102; A20-B103; A20-B104; A20-B105; A20-B106; A20-B107;A20-B108; A20-B109; A20-B110; A20-B111; A20-B112; A20-B113; A21-B1; A21-B2; A21-B3; A21-B4; A21-B5; A21-B6; A21-B7; A21-B8; A21-B9; A21-B10; A21-B11; A21-B12; A21- B13; A21-B14; A21-B15; A21-B16; A21-B17; A21-B18; A21-B19; A21-B20; A21-B21; A21-B22; A21-B23; A21-B24; A21-B25; A21-B26; A21-B27; A21-B28; A21-B29; A21-B30; A21-B31; A21-B32; A21-B33; A21-B34; A21-B35; A21-B36; A21-B37; A21- B38; A21-B39; A21-B40; A21-B41; A21-B42; A21-B43; A21-B44; A21-B45; A21-B46; A21-B47; A21-B48; A21-B49; A21-B50; A21-B51; A21-B52; A21-B53; A21-B54; A21-B55; A21-B56; A21-B57; A21-B58; A21-B59; A21-B60; A21-B61; A21-B62; A21- B63; A21-B64; A21-B65; A21-B66; A21-B67; A21-B68; A21-B69; A21-B70; A21-B71; A21-B72; A21-B73; A21-B74; A21-B75; A21-B76; A21-B77; A21-B78; A21-B79; A21-B80; A21-B81; A21-B82; A21-B83; A21-B84; A21-B85; A21-B86; A21-B87; A21- B88; A21-B89; A21-B90; A21-B91; A21-B92; A21-B93; A21-B94; A21-B95; A21-B96; A21-B97; A21-B98; A21-B99; A21-B100; A21-B101; A21-B102; A21-B103; A21-B104; A21-B105; A21-B106; A21-B107;A21-B108; A21-B109; A21-B110; A21-B111; A21-B112; A21-B113; A22-B1; A22-B2; A22-B3; A22-B4; A22-B5; A22-B6; A22-B7; A22-B8; A22-B9; A22-B10; A22-B11; A22-B12; A22- B13; A22-B14; A22-B15; A22-B16; A22-B17; A22-B18; A22-B19; A22-B20; A22-B21; A22-B22; A22-B23; A22-B24; A22-B25; A22-B26; A22-B27; A22-B28; A22-B29; A22-B30; A22-B31; A22-B32; A22-B33; A22-B34; A22-B35; A22-B36; A22-B37; A22- B38; A22-B39; A22-B40; A22-B41; A22-B42; A22-B43; A22-B44; A22-B45; A22-B46; A22-B47; A22-B48; A22-B49; A22-B50; A22-B51; A22-B52; A22-B53; A22-B54; A22-B55; A22-B56; A22-B57; A22-B58; A22-B59; A22-B60; A22-B61; A22-B62; A22- B63; A22-B64; A22-B65; A22-B66; A22-B67; A22-B68; A22-B69; A22-B70; A22-B71; A22-B72; A22-B73; A22-B74; A22-B75; A22-B76; A22-B77; A22-B78; A22-B79; A22-B80; A22-B81; A22-B82; A22-B83; A22-B84; A22-B85; A22-B86; A22-B87; A22- B88; A22-B89; A22-B90; A22-B91; A22-B92; A22-B93; A22-B94; A22-B95; A22-B96; A22-B97; A22-B98; A22-B99; A22-B100; A22-B101; A22-B102; A22-B103; A22-B104; A22-B105; A22-B106; A22-B107;A22-B108; A22-B109; A22-B110; A22-B111; A22-B112; A22-B113; A23-B1; A23-B2; A23-B3; A23-B4; A23-B5; A23-B6; A23-B7; A23-B8; A23-B9; A23-B10; A23-B11; A23-B12; A23- B13; A23-B14; A23-B15; A23-B16; A23-B17; A23-B18; A23-B19; A23-B20; A23-B21; A23-B22; A23-B23; A23-B24; A23-B25; A23-B26; A23-B27; A23-B28; A23-B29; A23-B30; A23-B31; A23-B32; A23-B33; A23-B34; A23-B35; A23-B36; A23-B37; A23- B38; A23-B39; A23-B40; A23-B41; A23-B42; A23-B43; A23-B44; A23-B45; A23-B46; A23-B47; A23-B48; A23-B49; A23-B50; A23-B51; A23-B52; A23-B53; A23-B54; A23-B55; A23-B56; A23-B57; A23-B58; A23-B59; A23-B60; A23-B61; A23-B62; A23- B63; A23-B64; A23-B65; A23-B66; A23-B67; A23-B68; A23-B69; A23-B70; A23-B71; A23-B72; A23-B73; A23-B74; A23-B75; A23-B76; A23-B77; A23-B78; A23-B79; A23-B80; A23-B81; A23-B82; A23-B83; A23-B84; A23-B85; A23-B86; A23-B87; A23- B88; A23-B89; A23-B90; A23-B91; A23-B92; A23-B93; A23-B94; A23-B95; A23-B96; A23-B97; A23-B98; A23-B99; A23-B100; A23-B101; A23-B102; A23-B103; A23-B104; A23-B105; A23-B106; A23-B107;A23-B108; A23-B109; A23-B110; A23-B111; A23-B112; A23-B113; A24-B1; A24-B2; A24-B3; A24-B4; A24-B5; A24-B6; A24-B7; A24-B8; A24-B9; A24-B10; A24-B11; A24-B12; A24- B13; A24-B14; A24-B15; A24-B16; A24-B17; A24-B18; A24-B19; A24-B20; A24-B21; A24-B22; A24-B23; A24-B24; A24-B25; A24-B26; A24-B27; A24-B28; A24-B29; A24-B30; A24-B31; A24-B32; A24-B33; A24-B34; A24-B35; A24-B36; A24-B37; A24- B38; A24-B39; A24-B40; A24-B41; A24-B42; A24-B43; A24-B44; A24-B45; A24-B46; A24-B47; A24-B48; A24-B49; A24-B50; A24-B51; A24-B52; A24-B53; A24-B54; A24-B55; A24-B56; A24-B57; A24-B58; A24-B59; A24-B60; A24-B61; A24-B62; A24- B63; A24-B64; A24-B65; A24-B66; A24-B67; A24-B68; A24-B69; A24-B70; A24-B71; A24-B72; A24-B73; A24-B74; A24-B75; A24-B76; A24-B77; A24-B78; A24-B79; A24-B80; A24-B81; A24-B82; A24-B83; A24-B84; A24-B85; A24-B86; A24-B87; A24- B88; A24-B89; A24-B90; A24-B91; A24-B92; A24-B93; A24-B94; A24-B95; A24-B96; A24-B97; A24-B98; A24-B99; A24-B100; A24-B101; A24-B102; A24-B103; A24-B104; A24-B105; A24-B106; A24-B107;A24-B108; A24-B109; A24-B110; A24-B111; A24-B112; A24-B113; A25-B1; A25-B2; A25-B3; A25-B4; A25-B5; A25-B6; A25-B7; A25-B8; A25-B9; A25-B10; A25-B11; A25-B12; A25- B13; A25-B14; A25-B15; A25-B16; A25-B17; A25-B18; A25-B19; A25-B20; A25-B21; A25-B22; A25-B23; A25-B24; A25-B25; A25-B26; A25-B27; A25-B28; A25-B29; A25-B30; A25-B31; A25-B32; A25-B33; A25-B34; A25-B35; A25-B36; A25-B37; A25- B38; A25-B39; A25-B40; A25-B41; A25-B42; A25-B43; A25-B44; A25-B45; A25-B46; A25-B47; A25-B48; A25-B49; A25-B50; A25-B51; A25-B52; A25-B53; A25-B54; A25-B55; A25-B56; A25-B57; A25-B58; A25-B59; A25-B60; A25-B61; A25-B62; A25- B63; A25-B64; A25-B65; A25-B66; A25-B67; A25-B68; A25-B69; A25-B70; A25-B71; A25-B72; A25-B73; A25-B74; A25-B75; A25-B76; A25-B77; A25-B78; A25-B79; A25-B80; A25-B81; A25-B82; A25-B83; A25-B84; A25-B85; A25-B86; A25-B87; A25- B88; A25-B89; A25-B90; A25-B91; A25-B92; A25-B93; A25-B94; A25-B95; A25-B96; A25-B97; A25-B98; A25-B99; A25-B100; A25-B101; A25-B102; A25-B103; A25-B104; A25-B105; A25-B106; A25-B107;A25-B108; A25-B109; A25-B110; A25-B111; A25-B112; A25-B113; A26-B1; A26-B2; A26-B3; A26-B4; A26-B5; A26-B6; A26-B7; A26-B8; A26-B9; A26-B10; A26-B11; A26-B12; A26- B13; A26-B14; A26-B15; A26-B16; A26-B17; A26-B18; A26-B19; A26-B20; A26-B21; A26-B22; A26-B23; A26-B24; A26-B25; A26-B26; A26-B27; A26-B28; A26-B29; A26-B30; A26-B31; A26-B32; A26-B33; A26-B34; A26-B35; A26-B36; A26-B37; A26- B38; A26-B39; A26-B40; A26-B41; A26-B42; A26-B43; A26-B44; A26-B45; A26-B46; A26-B47; A26-B48; A26-B49; A26-B50; A26-B51; A26-B52; A26-B53; A26-B54; A26-B55; A26-B56; A26-B57; A26-B58; A26-B59; A26-B60; A26-B61; A26-B62; A26- B63; A26-B64; A26-B65; A26-B66; A26-B67; A26-B68; A26-B69; A26-B70; A26-B71; A26-B72; A26-B73; A26-B74; A26-B75; A26-B76; A26-B77; A26-B78; A26-B79; A26-B80; A26-B81; A26-B82; A26-B83; A26-B84; A26-B85; A26-B86; A26-B87; A26- B88; A26-B89; A26-B90; A26-B91; A26-B92; A26-B93; A26-B94; A26-B95; A26-B96; A26-B97; A26-B98; A26-B99; A26-B100; A26-B101; A26-B102; A26-B103; A26-B104; A26-B105; A26-B106; A26-B107;A26-B108; A26-B109; A26-B110; A26-B111; A26-B112; A26-B113; A27-B1; A27-B2; A27-B3; A27-B4; A27-B5; A27-B6; A27-B7; A27-B8; A27-B9; A27-B10; A27-B11; A27-B12; A27- B13; A27-B14; A27-B15; A27-B16; A27-B17; A27-B18; A27-B19; A27-B20; A27-B21; A27-B22; A27-B23; A27-B24; A27-B25; A27-B26; A27-B27; A27-B28; A27-B29; A27-B30; A27-B31; A27-B32; A27-B33; A27-B34; A27-B35; A27-B36; A27-B37; A27- B38; A27-B39; A27-B40; A27-B41; A27-B42; A27-B43; A27-B44; A27-B45; A27-B46; A27-B47; A27-B48; A27-B49; A27-B50; A27-B51; A27-B52; A27-B53; A27-B54; A27-B55; A27-B56; A27-B57; A27-B58; A27-B59; A27-B60; A27-B61; A27-B62; A27- B63; A27-B64; A27-B65; A27-B66; A27-B67; A27-B68; A27-B69; A27-B70; A27-B71; A27-B72; A27-B73; A27-B74; A27-B75; A27-B76; A27-B77; A27-B78; A27-B79; A27-B80; A27-B81; A27-B82; A27-B83; A27-B84; A27-B85; A27-B86; A27-B87; A27- B88; A27-B89; A27-B90; A27-B91; A27-B92; A27-B93; A27-B94; A27-B95; A27-B96; A27-B97; A27-B98; A27-B99; A27-B100; A27-B101; A27-B102; A27-B103; A27-B104; A27-B105; A27-B106; A27-B107;A27-B108; A27-B109; A27-B110; A27-B111; A27-B112; A27-B113; A28-B1; A28-B2; A28-B3; A28-B4; A28-B5; A28-B6; A28-B7; A28-B8; A28-B9; A28-B10; A28-B11; A28-B12; A28- B13; A28-B14; A28-B15; A28-B16; A28-B17; A28-B18; A28-B19; A28-B20; A28-B21; A28-B22; A28-B23; A28-B24; A28-B25; A28-B26; A28-B27; A28-B28; A28-B29; A28-B30; A28-B31; A28-B32; A28-B33; A28-B34; A28-B35; A28-B36; A28-B37; A28- B38; A28-B39; A28-B40; A28-B41; A28-B42; A28-B43; A28-B44; A28-B45; A28-B46; A28-B47; A28-B48; A28-B49; A28-B50; A28-B51; A28-B52; A28-B53; A28-B54; A28-B55; A28-B56; A28-B57; A28-B58; A28-B59; A28-B60; A28-B61; A28-B62; A28- B63; A28-B64; A28-B65; A28-B66; A28-B67; A28-B68; A28-B69; A28-B70; A28-B71; A28-B72; A28-B73; A28-B74; A28-B75; A28-B76; A28-B77; A28-B78; A28-B79; A28-B80; A28-B81; A28-B82; A28-B83; A28-B84; A28-B85; A28-B86; A28-B87; A28- B88; A28-B89; A28-B90; A28-B91; A28-B92; A28-B93; A28-B94; A28-B95; A28-B96; A28-B97; A28-B98; A28-B99; A28-B100; A28-B101; A28-B102; A28-B103; A28-B104; A28-B105; A28-B106; A28-B107;A28-B108; A28-B109; A28-B110; A28-B111; A28-B112; A28-B113; A29-B1; A29-B2; A29-B3; A29-B4; A29-B5; A29-B6; A29-B7; A29-B8; A29-B9; A29-B10; A29-B11; A29-B12; A29- B13; A29-B14; A29-B15; A29-B16; A29-B17; A29-B18; A29-B19; A29-B20; A29-B21; A29-B22; A29-B23; A29-B24; A29-B25; A29-B26; A29-B27; A29-B28; A29-B29; A29-B30; A29-B31; A29-B32; A29-B33; A29-B34; A29-B35; A29-B36; A29-B37; A29- B38; A29-B39; A29-B40; A29-B41; A29-B42; A29-B43; A29-B44; A29-B45; A29-B46; A29-B47; A29-B48; A29-B49; A29-B50; A29-B51; A29-B52; A29-B53; A29-B54; A29-B55; A29-B56; A29-B57; A29-B58; A29-B59; A29-B60; A29-B61; A29-B62; A29- B63; A29-B64; A29-B65; A29-B66; A29-B67; A29-B68; A29-B69; A29-B70; A29-B71; A29-B72; A29-B73; A29-B74; A29-B75; A29-B76; A29-B77; A29-B78; A29-B79; A29-B80; A29-B81; A29-B82; A29-B83; A29-B84; A29-B85; A29-B86; A29-B87; A29- B88; A29-B89; A29-B90; A29-B91; A29-B92; A29-B93; A29-B94; A29-B95; A29-B96; A29-B97; A29-B98; A29-B99; A29-B100; A29-B101; A29-B102; A29-B103; A29-B104; A29-B105; A29-B106; A29-B107;A29-B108; A29-B109; A29-B110; A29-B111; A29-B112; A29-B113; A30-B1; A30-B2; A30-B3; A30-B4; A30-B5; A30-B6; A30-B7; A30-B8; A30-B9; A30-B10; A30-B11; A30-B12; A30- B13; A30-B14; A30-B15; A30-B16; A30-B17; A30-B18; A30-B19; A30-B20; A30-B21; A30-B22; A30-B23; A30-B24; A30-B25; A30-B26; A30-B27; A30-B28; A30-B29; A30-B30; A30-B31; A30-B32; A30-B33; A30-B34; A30-B35; A30-B36; A30-B37; A30- B38; A30-B39; A30-B40; A30-B41; A30-B42; A30-B43; A30-B44; A30-B45; A30-B46; A30-B47; A30-B48; A30-B49; A30-B50; A30-B51; A30-B52; A30-B53; A30-B54; A30-B55; A30-B56; A30-B57; A30-B58; A30-B59; A30-B60; A30-B61; A30-B62; A30- B63; A30-B64; A30-B65; A30-B66; A30-B67; A30-B68; A30-B69; A30-B70; A30-B71; A30-B72; A30-B73; A30-B74; A30-B75; A30-B76; A30-B77; A30-B78; A30-B79; A30-B80; A30-B81; A30-B82; A30-B83; A30-B84; A30-B85; A30-B86; A30-B87; A30- B88; A30-B89; A30-B90; A30-B91; A30-B92; A30-B93; A30-B94; A30-B95; A30-B96; A30-B97; A30-B98; A30-B99; A30-B100; A30-B101; A30-B102; A30-B103; A30-B104; A30-B105; A30-B106; A30-B107;A30-B108; A30-B109; A30-B110; A30-B111; A30-B112; A30-B113; A31-B1; A31-B2; A31-B3; A31-B4; A31-B5; A31-B6; A31-B7; A31-B8; A31-B9; A31-B10; A31-B11; A31-B12; A31- B13; A31-B14; A31-B15; A31-B16; A31-B17; A31-B18; A31-B19; A31-B20; A31-B21; A31-B22; A31-B23; A31-B24; A31-B25; A31-B26; A31-B27; A31-B28; A31-B29; A31-B30; A31-B31; A31-B32; A31-B33; A31-B34; A31-B35; A31-B36; A31-B37; A31- B38; A31-B39; A31-B40; A31-B41; A31-B42; A31-B43; A31-B44; A31-B45; A31-B46; A31-B47; A31-B48; A31-B49; A31-B50; A31-B51; A31-B52; A31-B53; A31-B54; A31-B55; A31-B56; A31-B57; A31-B58; A31-B59; A31-B60; A31-B61; A31-B62; A31- B63; A31-B64; A31-B65; A31-B66; A31-B67; A31-B68; A31-B69; A31-B70; A31-B71; A31-B72; A31-B73; A31-B74; A31-B75; A31-B76; A31-B77; A31-B78; A31-B79; A31-B80; A31-B81; A31-B82; A31-B83; A31-B84; A31-B85; A31-B86; A31-B87; A31- B88; A31-B89; A31-B90; A31-B91; A31-B92; A31-B93; A31-B94; A31-B95; A31-B96; A31-B97; A31-B98; A31-B99; A31-B100; A31-B101; A31-B102; A31-B103; A31-B104; A31-B105; A31-B106; A31-B107;A31-B108; A31-B109; A31-B110; A31-B111; A31-B112; A31-B113; A32-B1; A32-B2; A32-B3; A32-B4; A32-B5; A32-B6; A32-B7; A32-B8; A32-B9; A32-B10; A32-B11; A32-B12; A32- B13; A32-B14; A32-B15; A32-B16; A32-B17; A32-B18; A32-B19; A32-B20; A32-B21; A32-B22; A32-B23; A32-B24; A32-B25; A32-B26; A32-B27; A32-B28; A32-B29; A32-B30; A32-B31; A32-B32; A32-B33; A32-B34; A32-B35; A32-B36; A32-B37; A32- B38; A32-B39; A32-B40; A32-B41; A32-B42; A32-B43; A32-B44; A32-B45; A32-B46; A32-B47; A32-B48; A32-B49; A32-B50; A32-B51; A32-B52; A32-B53; A32-B54; A32-B55; A32-B56; A32-B57; A32-B58; A32-B59; A32-B60; A32-B61; A32-B62; A32- B63; A32-B64; A32-B65; A32-B66; A32-B67; A32-B68; A32-B69; A32-B70; A32-B71; A32-B72; A32-B73; A32-B74; A32-B75; A32-B76; A32-B77; A32-B78; A32-B79; A32-B80; A32-B81; A32-B82; A32-B83; A32-B84; A32-B85; A32-B86; A32-B87; A32- B88; A32-B89; A32-B90; A32-B91; A32-B92; A32-B93; A32-B94; A32-B95; A32-B96; A32-B97; A32-B98; A32-B99; A32-B100; A32-B101; A32-B102; A32-B103; A32-B104; A32-B105; A32-B106; A32-B107;A32-B108; A32-B109; A32-B110; A32-B111; A32-B112; A32-B113; A33-B1; A33-B2; A33-B3; A33-B4; A33-B5; A33-B6; A33-B7; A33-B8; A33-B9; A33-B10; A33-B11; A33-B12; A33- B13; A33-B14; A33-B15; A33-B16; A33-B17; A33-B18; A33-B19; A33-B20; A33-B21; A33-B22; A33-B23; A33-B24; A33-B25; A33-B26; A33-B27; A33-B28; A33-B29; A33-B30; A33-B31; A33-B32; A33-B33; A33-B34; A33-B35; A33-B36; A33-B37; A33- B38; A33-B39; A33-B40; A33-B41; A33-B42; A33-B43; A33-B44; A33-B45; A33-B46; A33-B47; A33-B48; A33-B49; A33-B50; A33-B51; A33-B52; A33-B53; A33-B54; A33-B55; A33-B56; A33-B57; A33-B58; A33-B59; A33-B60; A33-B61; A33-B62; A33- B63; A33-B64; A33-B65; A33-B66; A33-B67; A33-B68; A33-B69; A33-B70; A33-B71; A33-B72; A33-B73; A33-B74; A33-B75; A33-B76; A33-B77; A33-B78; A33-B79; A33-B80; A33-B81; A33-B82; A33-B83; A33-B84; A33-B85; A33-B86; A33-B87; A33- B88; A33-B89; A33-B90; A33-B91; A33-B92; A33-B93; A33-B94; A33-B95; A33-B96; A33-B97; A33-B98; A33-B99; A33-B100; A33-B101; A33-B102; A33-B103; A33-B104; A33-B105; A33-B106; A33-B107;A33-B108; A33-B109; A33-B110; A33-B111; A33-B112; A33-B113; A34-B1; A34-B2; A34-B3; A34-B4; A34-B5; A34-B6; A34-B7; A34-B8; A34-B9; A34-B10; A34-B11; A34-B12; A34-B13; A34-B14; A34-B15; A34-B16; A34-B17; A34-B18; A34- B19; A34-B20; A34-B21; A34-B22; A34-B23; A34-B24; A34-B25; A34-B26; A34-B27; A34-B28; A34-B29; A34-B30; A34-B31; A34-B32; A34-B33; A34-B34; A34-B35; A34-B36; A34-B37; A34-B38; A34-B39; A34-B40; A34-B41; A34-B42; A34-B43; A34- B44; A34-B45; A34-B46; A34-B47; A34-B48; A34-B49; A34-B50; A34-B51; A34-B52; A34-B53; A34-B54; A34-B55; A34-B56; A34-B57; A34-B58; A34-B59; A34-B60; A34-B61; A34-B62; A34-B63; A34-B64; A34-B65; A34-B66; A34-B67; A34-B68; A34- B69; A34-B70; A34-B71; A34-B72; A34-B73; A34-B74; A34-B75; A34-B76; A34-B77; A34-B78; A34-B79; A34-B80; A34-B81; A34-B82; A34-B83; A34-B84; A34-B85; A34-B86; A34-B87; A34-B88; A34-B89; A34-B90; A34-B91; A34-B92; A34-B93; A34- B94; A34-B95; A34-B96; A34-B97; A34-B98; A34-B99; A34-B100; A34-B101; A34-B102; A34-B103; A34-B104; A34-B105; A34-B106; A34-B107; A34-B108; A34-B109; A34-B110; A34-B111; A34-B112;A34-B113; A35-B1; A35-B2; A35-B3; A35-B4; A35-B5; A35-B6; A35-B7; A35-B8; A35-B9; A35-B10; A35-B11; A35-B12; A35- B13; A35-B14; A35-B15; A35-B16; A35-B17; A35-B18; A35-B19; A35-B20; A35-B21; A35-B22; A35-B23; A35-B24; A35-B25; A35-B26; A35-B27; A35-B28; A35-B29; A35-B30; A35-B31; A35-B32; A35-B33; A35-B34; A35-B35; A35-B36; A35-B37; A35- B38; A35-B39; A35-B40; A35-B41; A35-B42; A35-B43; A35-B44; A35-B45; A35-B46; A35-B47; A35-B48; A35-B49; A35-B50; A35-B51; A35-B52; A35-B53; A35-B54; A35-B55; A35-B56; A35-B57; A35-B58; A35-B59; A35-B60; A35-B61; A35-B62; A35- B63; A35-B64; A35-B65; A35-B66; A35-B67; A35-B68; A35-B69; A35-B70; A35-B71; A35-B72; A35-B73; A35-B74; A35-B75; A35-B76; A35-B77; A35-B78; A35-B79; A35-B80; A35-B81; A35-B82; A35-B83; A35-B84; A35-B85; A35-B86; A35-B87; A35- B88; A35-B89; A35-B90; A35-B91; A35-B92; A35-B93; A35-B94; A35-B95; A35-B96; A35-B97; A35-B98; A35-B99; A35-B100; A35-B101; A35-B102; A35-B103; A35-B104; A35-B105; A35-B106; A35-B107;A35-B108; A35-B109; A35-B110; A35-B111; A35-B112; A35-B113; A36-B1; A36-B2; A36-B3; A36-B4; A36-B5; A36-B6; A36-B7; A36-B8; A36-B9; A36-B10; A36-B11; A36-B12; A36- B13; A36-B14; A36-B15; A36-B16; A36-B17; A36-B18; A36-B19; A36-B20; A36-B21; A36-B22; A36-B23; A36-B24; A36-B25; A36-B26; A36-B27; A36-B28; A36-B29; A36-B30; A36-B31; A36-B32; A36-B33; A36-B34; A36-B35; A36-B36; A36-B37; A36- B38; A36-B39; A36-B40; A36-B41; A36-B42; A36-B43; A36-B44; A36-B45; A36-B46; A36-B47; A36-B48; A36-B49; A36-B50; A36-B51; A36-B52; A36-B53; A36-B54; A36-B55; A36-B56; A36-B57; A36-B58; A36-B59; A36-B60; A36-B61; A36-B62; A36- B63; A36-B64; A36-B65; A36-B66; A36-B67; A36-B68; A36-B69; A36-B70; A36-B71; A36-B72; A36-B73; A36-B74; A36-B75; A36-B76; A36-B77; A36-B78; A36-B79; A36-B80; A36-B81; A36-B82; A36-B83; A36-B84; A36-B85; A36-B86; A36-B87; A36- B88; A36-B89; A36-B90; A36-B91; A36-B92; A36-B93; A36-B94; A36-B95; A36-B96; A36-B97; A36-B98; A36-B99; A36-B100; A36-B101; A36-B102; A36-B103; A36-B104; A36-B105; A36-B106; A36-B107;A36-B108; A36-B109; A36-B110; A36-B111; A36-B112; A36-B113; A37-B1; A37-B2; A37-B3; A37-B4; A37-B5; A37-B6; A37-B7; A37-B8; A37-B9; A37-B10; A37-B11; A37-B12; A37- B13; A37-B14; A37-B15; A37-B16; A37-B17; A37-B18; A37-B19; A37-B20; A37-B21; A37-B22; A37-B23; A37-B24; A37-B25; A37-B26; A37-B27; A37-B28; A37-B29; A37-B30; A37-B31; A37-B32; A37-B33; A37-B34; A37-B35; A37-B36; A37-B37; A37- B38; A37-B39; A37-B40; A37-B41; A37-B42; A37-B43; A37-B44; A37-B45; A37-B46; A37-B47; A37-B48; A37-B49; A37-B50; A37-B51; A37-B52; A37-B53; A37-B54; A37-B55; A37-B56; A37-B57; A37-B58; A37-B59; A37-B60; A37-B61; A37-B62; A37- B63; A37-B64; A37-B65; A37-B66; A37-B67; A37-B68; A37-B69; A37-B70; A37-B71; A37-B72; A37-B73; A37-B74; A37-B75; A37-B76; A37-B77; A37-B78; A37-B79; A37-B80; A37-B81; A37-B82; A37-B83; A37-B84; A37-B85; A37-B86; A37-B87; A37- B88; A37-B89; A37-B90; A37-B91; A37-B92; A37-B93; A37-B94; A37-B95; A37-B96; A37-B97; A37-B98; A37-B99; A37-B100; A37-B101; A37-B102; A37-B103; A37-B104; A37-B105; A37-B106; A37-B107;A37-B108; A37-B109; A37-B110; A37-B111; A37-B112; A37-B113; A38-B1; A38-B2; A38-B3; A38-B4; A38-B5; A38-B6; A38-B7; A38-B8; A38-B9; A38-B10; A38-B11; A38-B12; A38- B13; A38-B14; A38-B15; A38-B16; A38-B17; A38-B18; A38-B19; A38-B20; A38-B21; A38-B22; A38-B23; A38-B24; A38-B25; A38-B26; A38-B27; A38-B28; A38-B29; A38-B30; A38-B31; A38-B32; A38-B33; A38-B34; A38-B35; A38-B36; A38-B37; A38- B38; A38-B39; A38-B40; A38-B41; A38-B42; A38-B43; A38-B44; A38-B45; A38-B46; A38-B47; A38-B48; A38-B49; A38-B50; A38-B51; A38-B52; A38-B53; A38-B54; A38-B55; A38-B56; A38-B57; A38-B58; A38-B59; A38-B60; A38-B61; A38-B62; A38- B63; A38-B64; A38-B65; A38-B66; A38-B67; A38-B68; A38-B69; A38-B70; A38-B71; A38-B72; A38-B73; A38-B74; A38-B75; A38-B76; A38-B77; A38-B78; A38-B79; A38-B80; A38-B81; A38-B82; A38-B83; A38-B84; A38-B85; A38-B86; A38-B87; A38- B88; A38-B89; A38-B90; A38-B91; A38-B92; A38-B93; A38-B94; A38-B95; A38-B96; A38-B97; A38-B98; A38-B99; A38-B100; A38-B101; A38-B102; A38-B103; A38-B104; A38-B105; A38-B106; A38-B107;A38-B108; A38-B109; A38-B110; A38-B111; A38-B112; A38-B113; A39-B1; A39-B2; A39-B3; A39-B4; A39-B5; A39-B6; A39-B7; A39-B8; A39-B9; A39-B10; A39-B11; A39-B12; A39- B13; A39-B14; A39-B15; A39-B16; A39-B17; A39-B18; A39-B19; A39-B20; A39-B21; A39-B22; A39-B23; A39-B24; A39-B25; A39-B26; A39-B27; A39-B28; A39-B29; A39-B30; A39-B31; A39-B32; A39-B33; A39-B34; A39-B35; A39-B36; A39-B37; A39- B38; A39-B39; A39-B40; A39-B41; A39-B42; A39-B43; A39-B44; A39-B45; A39-B46; A39-B47; A39-B48; A39-B49; A39-B50; A39-B51; A39-B52; A39-B53; A39-B54; A39-B55; A39-B56; A39-B57; A39-B58; A39-B59; A39-B60; A39-B61; A39-B62; A39- B63; A39-B64; A39-B65; A39-B66; A39-B67; A39-B68; A39-B69; A39-B70; A39-B71; A39-B72; A39-B73; A39-B74; A39-B75; A39-B76; A39-B77; A39-B78; A39-B79; A39-B80; A39-B81; A39-B82; A39-B83; A39-B84; A39-B85; A39-B86; A39-B87; A39- B88; A39-B89; A39-B90; A39-B91; A39-B92; A39-B93; A39-B94; A39-B95; A39-B96; A39-B97; A39-B98; A39-B99; A39-B100; A39-B101; A39-B102; A39-B103; A39-B104; A39-B105; A39-B106; A39-B107;A39-B108; A39-B109; A39-B110; A39-B111; A39-B112; A39-B113; A40-B1; A40-B2; A40-B3; A40-B4; A40-B5; A40-B6; A40-B7; A40-B8; A40-B9; A40-B10; A40-B11; A40-B12; A40- B13; A40-B14; A40-B15; A40-B16; A40-B17; A40-B18; A40-B19; A40-B20; A40-B21; A40-B22; A40-B23; A40-B24; A40-B25; A40-B26; A40-B27; A40-B28; A40-B29; A40-B30; A40-B31; A40-B32; A40-B33; A40-B34; A40-B35; A40-B36; A40-B37; A40- B38; A40-B39; A40-B40; A40-B41; A40-B42; A40-B43; A40-B44; A40-B45; A40-B46; A40-B47; A40-B48; A40-B49; A40-B50; A40-B51; A40-B52; A40-B53; A40-B54; A40-B55; A40-B56; A40-B57; A40-B58; A40-B59; A40-B60; A40-B61; A40-B62; A40- B63; A40-B64; A40-B65; A40-B66; A40-B67; A40-B68; A40-B69; A40-B70; A40-B71; A40-B72; A40-B73; A40-B74; A40-B75; A40-B76; A40-B77; A40-B78; A40-B79; A40-B80; A40-B81; A40-B82; A40-B83; A40-B84; A40-B85; A40-B86; A40-B87; A40- B88; A40-B89; A40-B90; A40-B91; A40-B92; A40-B93; A40-B94; A40-B95; A40-B96; A40-B97; A40-B98; A40-B99; A40-B100; A40-B101; A40-B102; A40-B103; A40-B104; A40-B105; A40-B106; A40-B107;A40-B108; A40-B109; A40-B110; A40-B111; A40-B112; A40-B113; A41-B1; A41-B2; A41-B3; A41-B4; A41-B5; A41-B6; A41-B7; A41-B8; A41-B9; A41-B10; A41-B11; A41-B12; A41- B13; A41-B14; A41-B15; A41-B16; A41-B17; A41-B18; A41-B19; A41-B20; A41-B21; A41-B22; A41-B23; A41-B24; A41-B25; A41-B26; A41-B27; A41-B28; A41-B29; A41-B30; A41-B31; A41-B32; A41-B33; A41-B34; A41-B35; A41-B36; A41-B37; A41- B38; A41-B39; A41-B40; A41-B41; A41-B42; A41-B43; A41-B44; A41-B45; A41-B46; A41-B47; A41-B48; A41-B49; A41-B50; A41-B51; A41-B52; A41-B53; A41-B54; A41-B55; A41-B56; A41-B57; A41-B58; A41-B59; A41-B60; A41-B61; A41-B62; A41- B63; A41-B64; A41-B65; A41-B66; A41-B67; A41-B68; A41-B69; A41-B70; A41-B71; A41-B72; A41-B73; A41-B74; A41-B75; A41-B76; A41-B77; A41-B78; A41-B79; A41-B80; A41-B81; A41-B82; A41-B83; A41-B84; A41-B85; A41-B86; A41-B87; A41- B88; A41-B89; A41-B90; A41-B91; A41-B92; A41-B93; A41-B94; A41-B95; A41-B96; A41-B97; A41-B98; A41-B99; A41-B100; A41-B101; A41-B102; A41-B103; A41-B104; A41-B105; A41-B106; A41-B107;A41-B108; A41-B109; A41-B110; A41-B111; A41-B112; A41-B113; A42-B1; A42-B2; A42-B3; A42-B4; A42-B5; A42-B6; A42-B7; A42-B8; A42-B9; A42-B10; A42-B11; A42-B12; A42- B13; A42-B14; A42-B15; A42-B16; A42-B17; A42-B18; A42-B19; A42-B20; A42-B21; A42-B22; A42-B23; A42-B24; A42-B25; A42-B26; A42-B27; A42-B28; A42-B29; A42-B30; A42-B31; A42-B32; A42-B33; A42-B34; A42-B35; A42-B36; A42-B37; A42- B38; A42-B39; A42-B40; A42-B41; A42-B42; A42-B43; A42-B44; A42-B45; A42-B46; A42-B47; A42-B48; A42-B49; A42-B50; A42-B51; A42-B52; A42-B53; A42-B54; A42-B55; A42-B56; A42-B57; A42-B58; A42-B59; A42-B60; A42-B61; A42-B62; A42- B63; A42-B64; A42-B65; A42-B66; A42-B67; A42-B68; A42-B69; A42-B70; A42-B71; A42-B72; A42-B73; A42-B74; A42-B75; A42-B76; A42-B77; A42-B78; A42-B79; A42-B80; A42-B81; A42-B82; A42-B83; A42-B84; A42-B85; A42-B86; A42-B87; A42- B88; A42-B89; A42-B90; A42-B91; A42-B92; A42-B93; A42-B94; A42-B95; A42-B96; A42-B97; A42-B98; A42-B99; A42-B100; A42-B101; A42-B102; A42-B103; A42-B104; A42-B105; A42-B106; A42-B107;A42-B108; A42-B109; A42-B110; A42-B111; A42-B112; A42-B113; A43-B1; A43-B2; A43-B3; A43-B4; A43-B5; A43-B6; A43-B7; A43-B8; A43-B9; A43-B10; A43-B11; A43-B12; A43- B13; A43-B14; A43-B15; A43-B16; A43-B17; A43-B18; A43-B19; A43-B20; A43-B21; A43-B22; A43-B23; A43-B24; A43-B25; A43-B26; A43-B27; A43-B28; A43-B29; A43-B30; A43-B31; A43-B32; A43-B33; A43-B34; A43-B35; A43-B36; A43-B37; A43- B38; A43-B39; A43-B40; A43-B41; A43-B42; A43-B43; A43-B44; A43-B45; A43-B46; A43-B47; A43-B48; A43-B49; A43-B50; A43-B51; A43-B52; A43-B53; A43-B54; A43-B55; A43-B56; A43-B57; A43-B58; A43-B59; A43-B60; A43-B61; A43-B62; A43- B63; A43-B64; A43-B65; A43-B66; A43-B67; A43-B68; A43-B69; A43-B70; A43-B71; A43-B72; A43-B73; A43-B74; A43-B75; A43-B76; A43-B77; A43-B78; A43-B79; A43-B80; A43-B81; A43-B82; A43-B83; A43-B84; A43-B85; A43-B86; A43-B87; A43- B88; A43-B89; A43-B90; A43-B91; A43-B92; A43-B93; A43-B94; A43-B95; A43-B96; A43-B97; A43-B98; A43-B99; A43-B100; A43-B101; A43-B102; A43-B103; A43-B104; A43-B105; A43-B106; A43-B107;A43-B108; A43-B109; A43-B110; A43-B111; A43-B112; A43-B113; A44-B1; A44-B2; A44-B3; A44-B4; A44-B5; A44-B6; A44-B7; A44-B8; A44-B9; A44-B10; A44-B11; A44-B12; A44- B13; A44-B14; A44-B15; A44-B16; A44-B17; A44-B18; A44-B19; A44-B20; A44-B21; A44-B22; A44-B23; A44-B24; A44-B25; A44-B26; A44-B27; A44-B28; A44-B29; A44-B30; A44-B31; A44-B32; A44-B33; A44-B34; A44-B35; A44-B36; A44-B37; A44- B38; A44-B39; A44-B40; A44-B41; A44-B42; A44-B43; A44-B44; A44-B45; A44-B46; A44-B47; A44-B48; A44-B49; A44-B50; A44-B51; A44-B52; A44-B53; A44-B54; A44-B55; A44-B56; A44-B57; A44-B58; A44-B59; A44-B60; A44-B61; A44-B62; A44- B63; A44-B64; A44-B65; A44-B66; A44-B67; A44-B68; A44-B69; A44-B70; A44-B71; A44-B72; A44-B73; A44-B74; A44-B75; A44-B76; A44-B77; A44-B78; A44-B79; A44-B80; A44-B81; A44-B82; A44-B83; A44-B84; A44-B85; A44-B86; A44-B87; A44- B88; A44-B89; A44-B90; A44-B91; A44-B92; A44-B93; A44-B94; A44-B95; A44-B96; A44-B97; A44-B98; A44-B99; A44-B100; A44-B101; A44-B102; A44-B103; A44-B104; A44-B105; A44-B106; A44-B107;A44-B108; A44-B109; A44-B110; A44-B111; A44-B112; A44-B113; A45-B1; A45-B2; A45-B3; A45-B4; A45-B5; A45-B6; A45-B7; A45-B8; A45-B9; A45-B10; A45-B11; A45-B12; A45- B13; A45-B14; A45-B15; A45-B16; A45-B17; A45-B18; A45-B19; A45-B20; A45-B21; A45-B22; A45-B23; A45-B24; A45-B25; A45-B26; A45-B27; A45-B28; A45-B29; A45-B30; A45-B31; A45-B32; A45-B33; A45-B34; A45-B35; A45-B36; A45-B37; A45- B38; A45-B39; A45-B40; A45-B41; A45-B42; A45-B43; A45-B44; A45-B45; A45-B46; A45-B47; A45-B48; A45-B49; A45-B50; A45-B51; A45-B52; A45-B53; A45-B54; A45-B55; A45-B56; A45-B57; A45-B58; A45-B59; A45-B60; A45-B61; A45-B62; A45- B63; A45-B64; A45-B65; A45-B66; A45-B67; A45-B68; A45-B69; A45-B70; A45-B71; A45-B72; A45-B73; A45-B74; A45-B75; A45-B76; A45-B77; A45-B78; A45-B79; A45-B80; A45-B81; A45-B82; A45-B83; A45-B84; A45-B85; A45-B86; A45-B87; A45- B88; A45-B89; A45-B90; A45-B91; A45-B92; A45-B93; A45-B94; A45-B95; A45-B96; A45-B97; A45-B98; A45-B99; A45-B100; A45-B101; A45-B102; A45-B103; A45-B104; A45-B105; A45-B106; A45-B107;A45-B108; A45-B109; A45-B110; A45-B111; A45-B112; A45-B113; A46-B1; A46-B2; A46-B3; A46-B4; A46-B5; A46-B6; A46-B7; A46-B8; A46-B9; A46-B10; A46-B11; A46-B12; A46- B13; A46-B14; A46-B15; A46-B16; A46-B17; A46-B18; A46-B19; A46-B20; A46-B21; A46-B22; A46-B23; A46-B24; A46-B25; A46-B26; A46-B27; A46-B28; A46-B29; A46-B30; A46-B31; A46-B32; A46-B33; A46-B34; A46-B35; A46-B36; A46-B37; A46- B38; A46-B39; A46-B40; A46-B41; A46-B42; A46-B43; A46-B44; A46-B45; A46-B46; A46-B47; A46-B48; A46-B49; A46-B50; A46-B51; A46-B52; A46-B53; A46-B54; A46-B55; A46-B56; A46-B57; A46-B58; A46-B59; A46-B60; A46-B61; A46-B62; A46- B63; A46-B64; A46-B65; A46-B66; A46-B67; A46-B68; A46-B69; A46-B70; A46-B71; A46-B72; A46-B73; A46-B74; A46-B75; A46-B76; A46-B77; A46-B78; A46-B79; A46-B80; A46-B81; A46-B82; A46-B83; A46-B84; A46-B85; A46-B86; A46-B87; A46- B88; A46-B89; A46-B90; A46-B91; A46-B92; A46-B93; A46-B94; A46-B95; A46-B96; A46-B97; A46-B98; A46-B99; A46-B100; A46-B101; A46-B102; A46-B103; A46-B104; A46-B105; A46-B106; A46-B107;A46-B108; A46-B109; A46-B110; A46-B111; A46-B112; A46-B113; A47-B1; A47-B2; A47-B3; A47-B4; A47-B5; A47-B6; A47-B7; A47-B8; A47-B9; A47-B10; A47-B11; A47-B12; A47- B13; A47-B14; A47-B15; A47-B16; A47-B17; A47-B18; A47-B19; A47-B20; A47-B21; A47-B22; A47-B23; A47-B24; A47-B25; A47-B26; A47-B27; A47-B28; A47-B29; A47-B30; A47-B31; A47-B32; A47-B33; A47-B34; A47-B35; A47-B36; A47-B37; A47- B38; A47-B39; A47-B40; A47-B41; A47-B42; A47-B43; A47-B44; A47-B45; A47-B46; A47-B47; A47-B48; A47-B49; A47-B50; A47-B51; A47-B52; A47-B53; A47-B54; A47-B55; A47-B56; A47-B57; A47-B58; A47-B59; A47-B60; A47-B61; A47-B62; A47- B63; A47-B64; A47-B65; A47-B66; A47-B67; A47-B68; A47-B69; A47-B70; A47-B71; A47-B72; A47-B73; A47-B74; A47-B75; A47-B76; A47-B77; A47-B78; A47-B79; A47-B80; A47-B81; A47-B82; A47-B83; A47-B84; A47-B85; A47-B86; A47-B87; A47- B88; A47-B89; A47-B90; A47-B91; A47-B92; A47-B93; A47-B94; A47-B95; A47-B96; A47-B97; A47-B98; A47-B99; A47-B100; A47-B101; A47-B102; A47-B103; A47-B104; A47-B105; A47-B106; A47-B107;A47-B108; A47-B109; A47-B110; A47-B111; A47-B112; A47-B113; A48-B1; A48-B2; A48-B3; A48-B4; A48-B5; A48-B6; A48-B7; A48-B8; A48-B9; A48-B10; A48-B11; A48-B12; A48- B13; A48-B14; A48-B15; A48-B16; A48-B17; A48-B18; A48-B19; A48-B20; A48-B21; A48-B22; A48-B23; A48-B24; A48-B25; A48-B26; A48-B27; A48-B28; A48-B29; A48-B30; A48-B31; A48-B32; A48-B33; A48-B34; A48-B35; A48-B36; A48-B37; A48- B38; A48-B39; A48-B40; A48-B41; A48-B42; A48-B43; A48-B44; A48-B45; A48-B46; A48-B47; A48-B48; A48-B49; A48-B50; A48-B51; A48-B52; A48-B53; A48-B54; A48-B55; A48-B56; A48-B57; A48-B58; A48-B59; A48-B60; A48-B61; A48-B62; A48- B63; A48-B64; A48-B65; A48-B66; A48-B67; A48-B68; A48-B69; A48-B70; A48-B71; A48-B72; A48-B73; A48-B74; A48-B75; A48-B76; A48-B77; A48-B78; A48-B79; A48-B80; A48-B81; A48-B82; A48-B83; A48-B84; A48-B85; A48-B86; A48-B87; A48- B88; A48-B89; A48-B90; A48-B91; A48-B92; A48-B93; A48-B94; A48-B95; A48-B96; A48-B97; A48-B98; A48-B99; A48-B100; A48-B101; A48-B102; A48-B103; A48-B104; A48-B105; A48-B106; A48-B107;A48-B108; A48-B109; A48-B110; A48-B111; A48-B112; A48-B113; A49-B1; A49-B2; A49-B3; A49-B4; A49-B5; A49-B6; A49-B7; A49-B8; A49-B9; A49-B10; A49-B11; A49-B12; A49- B13; A49-B14; A49-B15; A49-B16; A49-B17; A49-B18; A49-B19; A49-B20; A49-B21; A49-B22; A49-B23; A49-B24; A49-B25; A49-B26; A49-B27; A49-B28; A49-B29; A49-B30; A49-B31; A49-B32; A49-B33; A49-B34; A49-B35; A49-B36; A49-B37; A49- B38; A49-B39; A49-B40; A49-B41; A49-B42; A49-B43; A49-B44; A49-B45; A49-B46; A49-B47; A49-B48; A49-B49; A49-B50; A49-B51; A49-B52; A49-B53; A49-B54; A49-B55; A49-B56; A49-B57; A49-B58; A49-B59; A49-B60; A49-B61; A49-B62; A49- B63; A49-B64; A49-B65; A49-B66; A49-B67; A49-B68; A49-B69; A49-B70; A49-B71; A49-B72; A49-B73; A49-B74; A49-B75; A49-B76; A49-B77; A49-B78; A49-B79; A49-B80; A49-B81; A49-B82; A49-B83; A49-B84; A49-B85; A49-B86; A49-B87; A49- B88; A49-B89; A49-B90; A49-B91; A49-B92; A49-B93; A49-B94; A49-B95; A49-B96; A49-B97; A49-B98; A49-B99; A49-B100; A49-B101; A49-B102; A49-B103; A49-B104; A49-B105; A49-B106; A49-B107;A49-B108; A49-B109; A49-B110; A49-B111; A49-B112; A49-B113; A50-B1; A50-B2; A50-B3; A50-B4; A50-B5; A50-B6; A50-B7; A50-B8; A50-B9; A50-B10; A50-B11; A50-B12; A50- B13; A50-B14; A50-B15; A50-B16; A50-B17; A50-B18; A50-B19; A50-B20; A50-B21; A50-B22; A50-B23; A50-B24; A50-B25; A50-B26; A50-B27; A50-B28; A50-B29; A50-B30; A50-B31; A50-B32; A50-B33; A50-B34; A50-B35; A50-B36; A50-B37; A50- B38; A50-B39; A50-B40; A50-B41; A50-B42; A50-B43; A50-B44; A50-B45; A50-B46; A50-B47; A50-B48; A50-B49; A50-B50; A50-B51; A50-B52; A50-B53; A50-B54; A50-B55; A50-B56; A50-B57; A50-B58; A50-B59; A50-B60; A50-B61; A50-B62; A50- B63; A50-B64; A50-B65; A50-B66; A50-B67; A50-B68; A50-B69; A50-B70; A50-B71; A50-B72; A50-B73; A50-B74; A50-B75; A50-B76; A50-B77; A50-B78; A50-B79; A50-B80; A50-B81; A50-B82; A50-B83; A50-B84; A50-B85; A50-B86; A50-B87; A50- B88; A50-B89; A50-B90; A50-B91; A50-B92; A50-B93; A50-B94; A50-B95; A50-B96; A50-B97; A50-B98; A50-B99; A50-B100; A50-B101; A50-B102; A50-B103; A50-B104; A50-B105; A50-B106; A50-B107;A50-B108; A50-B109; A50-B110; A50-B111; A50-B112; A50-B113; A51-B1; A51-B2; A51-B3; A51-B4; A51-B5; A51-B6; A51-B7; A51-B8; A51-B9; A51-B10; A51-B11; A51-B12; A51- B13; A51-B14; A51-B15; A51-B16; A51-B17; A51-B18; A51-B19; A51-B20; A51-B21; A51-B22; A51-B23; A51-B24; A51-B25; A51-B26; A51-B27; A51-B28; A51-B29; A51-B30; A51-B31; A51-B32; A51-B33; A51-B34; A51-B35; A51-B36; A51-B37; A51- B38; A51-B39; A51-B40; A51-B41; A51-B42; A51-B43; A51-B44; A51-B45; A51-B46; A51-B47; A51-B48; A51-B49; A51-B50; A51-B51; A51-B52; A51-B53; A51-B54; A51-B55; A51-B56; A51-B57; A51-B58; A51-B59; A51-B60; A51-B61; A51-B62; A51- B63; A51-B64; A51-B65; A51-B66; A51-B67; A51-B68; A51-B69; A51-B70; A51-B71; A51-B72; A51-B73; A51-B74; A51-B75; A51-B76; A51-B77; A51-B78; A51-B79; A51-B80; A51-B81; A51-B82; A51-B83; A51-B84; A51-B85; A51-B86; A51-B87; A51- B88; A51-B89; A51-B90; A51-B91; A51-B92; A51-B93; A51-B94; A51-B95; A51-B96; A51-B97; A51-B98; A51-B99; A51-B100; A51-B101; A51-B102; A51-B103; A51-B104; A51-B105; A51-B106; A51-B107;A51-B108; A51-B109; A51-B110; A51-B111; A51-B112; A51-B113; A52-B1; A52-B2; A52-B3; A52-B4; A52-B5; A52-B6; A52-B7; A52-B8; A52-B9; A52-B10; A52-B11; A52-B12; A52- B13; A52-B14; A52-B15; A52-B16; A52-B17; A52-B18; A52-B19; A52-B20; A52-B21; A52-B22; A52-B23; A52-B24; A52-B25; A52-B26; A52-B27; A52-B28; A52-B29; A52-B30; A52-B31; A52-B32; A52-B33; A52-B34; A52-B35; A52-B36; A52-B37; A52- B38; A52-B39; A52-B40; A52-B41; A52-B42; A52-B43; A52-B44; A52-B45; A52-B46; A52-B47; A52-B48; A52-B49; A52-B50; A52-B51; A52-B52; A52-B53; A52-B54; A52-B55; A52-B56; A52-B57; A52-B58; A52-B59; A52-B60; A52-B61; A52-B62; A52- B63; A52-B64; A52-B65; A52-B66; A52-B67; A52-B68; A52-B69; A52-B70; A52-B71; A52-B72; A52-B73; A52-B74; A52-B75; A52-B76; A52-B77; A52-B78; A52-B79; A52-B80; A52-B81; A52-B82; A52-B83; A52-B84; A52-B85; A52-B86; A52-B87; A52- B88; A52-B89; A52-B90; A52-B91; A52-B92; A52-B93; A52-B94; A52-B95; A52-B96; A52-B97; A52-B98; A52-B99; A52-B100; A52-B101; A52-B102; A52-B103; A52-B104; A52-B105; A52-B106; A52-B107;A52-B108; A52-B109; A52-B110; A52-B111; A52-B112; A52-B113; A53-B1; A53-B2; A53-B3; A53-B4; A53-B5; A53-B6; A53-B7; A53-B8; A53-B9; A53-B10; A53-B11; A53-B12; A53- B13; A53-B14; A53-B15; A53-B16; A53-B17; A53-B18; A53-B19; A53-B20; A53-B21; A53-B22; A53-B23; A53-B24; A53-B25; A53-B26; A53-B27; A53-B28; A53-B29; A53-B30; A53-B31; A53-B32; A53-B33; A53-B34; A53-B35; A53-B36; A53-B37; A53- B38; A53-B39; A53-B40; A53-B41; A53-B42; A53-B43; A53-B44; A53-B45; A53-B46; A53-B47; A53-B48; A53-B49; A53-B50; A53-B51; A53-B52; A53-B53; A53-B54; A53-B55; A53-B56; A53-B57; A53-B58; A53-B59; A53-B60; A53-B61; A53-B62; A53- B63; A53-B64; A53-B65; A53-B66; A53-B67; A53-B68; A53-B69; A53-B70; A53-B71; A53-B72; A53-B73; A53-B74; A53-B75; A53-B76; A53-B77; A53-B78; A53-B79; A53-B80; A53-B81; A53-B82; A53-B83; A53-B84; A53-B85; A53-B86; A53-B87; A53- B88; A53-B89; A53-B90; A53-B91; A53-B92; A53-B93; A53-B94; A53-B95; A53-B96; A53-B97; A53-B98; A53-B99; A53-B100; A53-B101; A53-B102; A53-B103; A53-B104; A53-B105; A53-B106; A53-B107;A53-B108; A53-B109; A53-B110; A53-B111; A53-B112; A53-B113; A54-B1; A54-B2; A54-B3; A54-B4; A54-B5; A54-B6; A54-B7; A54-B8; A54-B9; A54-B10; A54-B11; A54-B12; A54- B13; A54-B14; A54-B15; A54-B16; A54-B17; A54-B18; A54-B19; A54-B20; A54-B21; A54-B22; A54-B23; A54-B24; A54-B25; A54-B26; A54-B27; A54-B28; A54-B29; A54-B30; A54-B31; A54-B32; A54-B33; A54-B34; A54-B35; A54-B36; A54-B37; A54- B38; A54-B39; A54-B40; A54-B41; A54-B42; A54-B43; A54-B44; A54-B45; A54-B46; A54-B47; A54-B48; A54-B49; A54-B50; A54-B51; A54-B52; A54-B53; A54-B54; A54-B55; A54-B56; A54-B57; A54-B58; A54-B59; A54-B60; A54-B61; A54-B62; A54- B63; A54-B64; A54-B65; A54-B66; A54-B67; A54-B68; A54-B69; A54-B70; A54-B71; A54-B72; A54-B73; A54-B74; A54-B75; A54-B76; A54-B77; A54-B78; A54-B79; A54-B80; A54-B81; A54-B82; A54-B83; A54-B84; A54-B85; A54-B86; A54-B87; A54- B88; A54-B89; A54-B90; A54-B91; A54-B92; A54-B93; A54-B94; A54-B95; A54-B96; A54-B97; A54-B98; A54-B99; A54-B100; A54-B101; A54-B102; A54-B103; A54-B104; A54-B105; A54-B106; A54-B107;A54-B108; A54-B109; A54-B110; A54-B111; A54-B112; A54-B113; A55-B1; A55-B2; A55-B3; A55-B4; A55-B5; A55-B6; A55-B7; A55-B8; A55-B9; A55-B10; A55-B11; A55-B12; A55- B13; A55-B14; A55-B15; A55-B16; A55-B17; A55-B18; A55-B19; A55-B20; A55-B21; A55-B22; A55-B23; A55-B24; A55-B25; A55-B26; A55-B27; A55-B28; A55-B29; A55-B30; A55-B31; A55-B32; A55-B33; A55-B34; A55-B35; A55-B36; A55-B37; A55- B38; A55-B39; A55-B40; A55-B41; A55-B42; A55-B43; A55-B44; A55-B45; A55-B46; A55-B47; A55-B48; A55-B49; A55-B50; A55-B51; A55-B52; A55-B53; A55-B54; A55-B55; A55-B56; A55-B57; A55-B58; A55-B59; A55-B60; A55-B61; A55-B62; A55- B63; A55-B64; A55-B65; A55-B66; A55-B67; A55-B68; A55-B69; A55-B70; A55-B71; A55-B72; A55-B73; A55-B74; A55-B75; A55-B76; A55-B77; A55-B78; A55-B79; A55-B80; A55-B81; A55-B82; A55-B83; A55-B84; A55-B85; A55-B86; A55-B87; A55- B88; A55-B89; A55-B90; A55-B91; A55-B92; A55-B93; A55-B94; A55-B95; A55-B96; A55-B97; A55-B98; A55-B99; A55-B100; A55-B101; A55-B102; A55-B103; A55-B104; A55-B105; A55-B106; A55-B107;A55-B108; A55-B109; A55-B110; A55-B111; A55-B112; A55-B113; A56-B1; A56-B2; A56-B3; A56-B4; A56-B5; A56-B6; A56-B7; A56-B8; A56-B9; A56-B10; A56-B11; A56-B12; A56- B13; A56-B14; A56-B15; A56-B16; A56-B17; A56-B18; A56-B19; A56-B20; A56-B21; A56-B22; A56-B23; A56-B24; A56-B25; A56-B26; A56-B27; A56-B28; A56-B29; A56-B30; A56-B31; A56-B32; A56-B33; A56-B34; A56-B35; A56-B36; A56-B37; A56- B38; A56-B39; A56-B40; A56-B41; A56-B42; A56-B43; A56-B44; A56-B45; A56-B46; A56-B47; A56-B48; A56-B49; A56-B50; A56-B51; A56-B52; A56-B53; A56-B54; A56-B55; A56-B56; A56-B57; A56-B58; A56-B59; A56-B60; A56-B61; A56-B62; A56- B63; A56-B64; A56-B65; A56-B66; A56-B67; A56-B68; A56-B69; A56-B70; A56-B71; A56-B72; A56-B73; A56-B74; A56-B75; A56-B76; A56-B77; A56-B78; A56-B79; A56-B80; A56-B81; A56-B82; A56-B83; A56-B84; A56-B85; A56-B86; A56-B87; A56- B88; A56-B89; A56-B90; A56-B91; A56-B92; A56-B93; A56-B94; A56-B95; A56-B96; A56-B97; A56-B98; A56-B99; A56-B100; A56-B101; A56-B102; A56-B103; A56-B104; A56-B105; A56-B106; A56-B107;A56-B108; A56-B109; A56-B110; A56-B111; A56-B112; A56-B113; A57-B1; A57-B2; A57-B3; A57-B4; A57-B5; A57-B6; A57-B7; A57-B8; A57-B9; A57-B10; A57-B11; A57-B12; A57- B13; A57-B14; A57-B15; A57-B16; A57-B17; A57-B18; A57-B19; A57-B20; A57-B21; A57-B22; A57-B23; A57-B24; A57-B25; A57-B26; A57-B27; A57-B28; A57-B29; A57-B30; A57-B31; A57-B32; A57-B33; A57-B34; A57-B35; A57-B36; A57-B37; A57- B38; A57-B39; A57-B40; A57-B41; A57-B42; A57-B43; A57-B44; A57-B45; A57-B46; A57-B47; A57-B48; A57-B49; A57-B50; A57-B51; A57-B52; A57-B53; A57-B54; A57-B55; A57-B56; A57-B57; A57-B58; A57-B59; A57-B60; A57-B61; A57-B62; A57- B63; A57-B64; A57-B65; A57-B66; A57-B67; A57-B68; A57-B69; A57-B70; A57-B71; A57-B72; A57-B73; A57-B74; A57-B75; A57-B76; A57-B77; A57-B78; A57-B79; A57-B80; A57-B81; A57-B82; A57-B83; A57-B84; A57-B85; A57-B86; A57-B87; A57- B88; A57-B89; A57-B90; A57-B91; A57-B92; A57-B93; A57-B94; A57-B95; A57-B96; A57-B97; A57-B98; A57-B99; A57-B100; A57-B101; A57-B102; A57-B103; A57-B104; A57-B105; A57-B106; A57-B107;A57-B108; A57-B109; A57-B110; A57-B111; A57-B112; A57-B113; A58-B1; A58-B2; A58-B3; A58-B4; A58-B5; A58-B6; A58-B7; A58-B8; A58-B9; A58-B10; A58-B11; A58-B12; A58- B13; A58-B14; A58-B15; A58-B16; A58-B17; A58-B18; A58-B19; A58-B20; A58-B21; A58-B22; A58-B23; A58-B24; A58-B25; A58-B26; A58-B27; A58-B28; A58-B29; A58-B30; A58-B31; A58-B32; A58-B33; A58-B34; A58-B35; A58-B36; A58-B37; A58- B38; A58-B39; A58-B40; A58-B41; A58-B42; A58-B43; A58-B44; A58-B45; A58-B46; A58-B47; A58-B48; A58-B49; A58-B50; A58-B51; A58-B52; A58-B53; A58-B54; A58-B55; A58-B56; A58-B57; A58-B58; A58-B59; A58-B60; A58-B61; A58-B62; A58- B63; A58-B64; A58-B65; A58-B66; A58-B67; A58-B68; A58-B69; A58-B70; A58-B71; A58-B72; A58-B73; A58-B74; A58-B75; A58-B76; A58-B77; A58-B78; A58-B79; A58-B80; A58-B81; A58-B82; A58-B83; A58-B84; A58-B85; A58-B86; A58-B87; A58- B88; A58-B89; A58-B90; A58-B91; A58-B92; A58-B93; A58-B94; A58-B95; A58-B96; A58-B97; A58-B98; A58-B99; A58-B100; A58-B101; A58-B102; A58-B103; A58-B104; A58-B105; A58-B106; A58-B107;A58-B108; A58-B109; A58-B110; A58-B111; A58-B112; A58-B113; A59-B1; A59-B2; A59-B3; A59-B4; A59-B5; A59-B6; A59-B7; A59-B8; A59-B9; A59-B10; A59-B11; A59-B12; A59- B13; A59-B14; A59-B15; A59-B16; A59-B17; A59-B18; A59-B19; A59-B20; A59-B21; A59-B22; A59-B23; A59-B24; A59-B25; A59-B26; A59-B27; A59-B28; A59-B29; A59-B30; A59-B31; A59-B32; A59-B33; A59-B34; A59-B35; A59-B36; A59-B37; A59- B38; A59-B39; A59-B40; A59-B41; A59-B42; A59-B43; A59-B44; A59-B45; A59-B46; A59-B47; A59-B48; A59-B49; A59-B50; A59-B51; A59-B52; A59-B53; A59-B54; A59-B55; A59-B56; A59-B57; A59-B58; A59-B59; A59-B60; A59-B61; A59-B62; A59- B63; A59-B64; A59-B65; A59-B66; A59-B67; A59-B68; A59-B69; A59-B70; A59-B71; A59-B72; A59-B73; A59-B74; A59-B75; A59-B76; A59-B77; A59-B78; A59-B79; A59-B80; A59-B81; A59-B82; A59-B83; A59-B84; A59-B85; A59-B86; A59-B87; A59- B88; A59-B89; A59-B90; A59-B91; A59-B92; A59-B93; A59-B94; A59-B95; A59-B96; A59-B97; A59-B98; 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A77- B63; A77-B64; A77-B65; A77-B66; A77-B67; A77-B68; A77-B69; A77-B70; A77-B71; A77-B72; A77-B73; A77-B74; A77-B75; A77-B76; A77-B77; A77-B78; A77-B79; A77-B80; A77-B81; A77-B82; A77-B83; A77-B84; A77-B85; A77-B86; A77-B87; A77- B88; A77-B89; A77-B90; A77-B91; A77-B92; A77-B93; A77-B94; A77-B95; A77-B96; A77-B97; A77-B98; A77-B99; A77-B100; A77-B101; A77-B102; A77-B103; A77-B104; A77-B105; A77-B106; A77-B107;A77-B108; A77-B109; A77-B110; A77-B111; A77-B112; A77-B113; A78-B1; A78-B2; A78-B3; A78-B4; A78-B5; A78-B6; A78-B7; A78-B8; A78-B9; A78-B10; A78-B11; A78-B12; A78- B13; A78-B14; A78-B15; A78-B16; A78-B17; A78-B18; A78-B19; A78-B20; A78-B21; A78-B22; A78-B23; A78-B24; A78-B25; A78-B26; A78-B27; A78-B28; A78-B29; A78-B30; A78-B31; A78-B32; A78-B33; A78-B34; A78-B35; A78-B36; A78-B37; A78- B38; A78-B39; A78-B40; A78-B41; A78-B42; A78-B43; A78-B44; A78-B45; A78-B46; A78-B47; A78-B48; A78-B49; A78-B50; A78-B51; A78-B52; A78-B53; A78-B54; A78-B55; A78-B56; A78-B57; A78-B58; A78-B59; A78-B60; A78-B61; A78-B62; A78- B63; A78-B64; A78-B65; A78-B66; A78-B67; A78-B68; A78-B69; A78-B70; A78-B71; A78-B72; A78-B73; A78-B74; A78-B75; A78-B76; A78-B77; A78-B78; A78-B79; A78-B80; A78-B81; A78-B82; A78-B83; A78-B84; A78-B85; A78-B86; A78-B87; A78- B88; A78-B89; A78-B90; A78-B91; A78-B92; A78-B93; A78-B94; A78-B95; A78-B96; A78-B97; A78-B98; A78-B99; A78-B100; A78-B101; A78-B102; A78-B103; A78-B104; A78-B105; A78-B106; A78-B107;A78-B108; A78-B109; A78-B110; A78-B111; A78-B112; A78-B113; A79-B1; A79-B2; A79-B3; A79-B4; A79-B5; A79-B6; A79-B7; A79-B8; A79-B9; A79-B10; A79-B11; A79-B12; A79- B13; A79-B14; A79-B15; A79-B16; A79-B17; A79-B18; A79-B19; A79-B20; A79-B21; A79-B22; A79-B23; A79-B24; A79-B25; A79-B26; A79-B27; A79-B28; A79-B29; A79-B30; A79-B31; A79-B32; A79-B33; A79-B34; A79-B35; A79-B36; A79-B37; A79- B38; A79-B39; A79-B40; A79-B41; A79-B42; A79-B43; A79-B44; A79-B45; A79-B46; A79-B47; A79-B48; A79-B49; A79-B50; A79-B51; A79-B52; A79-B53; A79-B54; A79-B55; A79-B56; A79-B57; A79-B58; A79-B59; A79-B60; A79-B61; A79-B62; A79- B63; A79-B64; A79-B65; A79-B66; A79-B67; A79-B68; A79-B69; A79-B70; A79-B71; A79-B72; A79-B73; A79-B74; A79-B75; A79-B76; A79-B77; A79-B78; A79-B79; A79-B80; A79-B81; A79-B82; A79-B83; A79-B84; A79-B85; A79-B86; A79-B87; A79- B88; A79-B89; A79-B90; A79-B91; A79-B92; A79-B93; A79-B94; A79-B95; A79-B96; A79-B97; A79-B98; A79-B99; A79-B100; A79-B101; A79-B102; A79-B103; A79-B104; A79-B105; A79-B106; A79-B107;A79-B108; A79-B109; A79-B110; A79-B111; A79-B112; A79-B113; A80-B1; A80-B2; A80-B3; A80-B4; A80-B5; A80-B6; A80-B7; A80-B8; A80-B9; A80-B10; A80-B11; A80-B12; A80- B13; A80-B14; A80-B15; A80-B16; A80-B17; A80-B18; A80-B19; A80-B20; A80-B21; A80-B22; A80-B23; A80-B24; A80-B25; A80-B26; A80-B27; A80-B28; A80-B29; A80-B30; A80-B31; A80-B32; A80-B33; A80-B34; A80-B35; A80-B36; A80-B37; A80- B38; A80-B39; A80-B40; A80-B41; A80-B42; A80-B43; A80-B44; A80-B45; A80-B46; A80-B47; A80-B48; A80-B49; A80-B50; A80-B51; A80-B52; A80-B53; A80-B54; A80-B55; A80-B56; A80-B57; A80-B58; A80-B59; A80-B60; A80-B61; A80-B62; A80- B63; A80-B64; A80-B65; A80-B66; A80-B67; A80-B68; A80-B69; A80-B70; A80-B71; A80-B72; A80-B73; A80-B74; A80-B75; A80-B76; A80-B77; A80-B78; A80-B79; A80-B80; A80-B81; A80-B82; A80-B83; A80-B84; A80-B85; A80-B86; A80-B87; A80- B88; A80-B89; A80-B90; A80-B91; A80-B92; A80-B93; A80-B94; A80-B95; A80-B96; A80-B97; A80-B98; A80-B99; A80-B100; A80-B101; A80-B102; A80-B103; A80-B104; A80-B105; A80-B106; A80-B107;A80-B108; A80-B109; A80-B110; A80-B111; A80-B112; A80-B113; A81-B1; A81-B2; A81-B3; A81-B4; A81-B5; A81-B6; A81-B7; A81-B8; A81-B9; A81-B10; A81-B11; A81-B12; A81- B13; A81-B14; A81-B15; A81-B16; A81-B17; A81-B18; A81-B19; A81-B20; A81-B21; A81-B22; A81-B23; A81-B24; A81-B25; A81-B26; A81-B27; A81-B28; A81-B29; A81-B30; A81-B31; A81-B32; A81-B33; A81-B34; A81-B35; A81-B36; A81-B37; A81- B38; A81-B39; A81-B40; A81-B41; A81-B42; A81-B43; A81-B44; A81-B45; A81-B46; A81-B47; A81-B48; A81-B49; A81-B50; A81-B51; A81-B52; A81-B53; A81-B54; A81-B55; A81-B56; A81-B57; A81-B58; A81-B59; A81-B60; A81-B61; A81-B62; A81- B63; A81-B64; A81-B65; A81-B66; A81-B67; A81-B68; A81-B69; A81-B70; A81-B71; A81-B72; A81-B73; A81-B74; A81-B75; A81-B76; A81-B77; A81-B78; A81-B79; A81-B80; A81-B81; A81-B82; A81-B83; A81-B84; A81-B85; A81-B86; A81-B87; A81- B88; A81-B89; A81-B90; A81-B91; A81-B92; A81-B93; A81-B94; A81-B95; A81-B96; A81-B97; A81-B98; A81-B99; A81-B100; A81-B101; A81-B102; A81-B103; A81-B104; A81-B105; A81-B106; A81-B107;A81-B108; A81-B109; A81-B110; A81-B111; A81-B112; A81-B113; A82-B1; A82-B2; A82-B3; A82-B4; A82-B5; A82-B6; A82-B7; A82-B8; A82-B9; A82-B10; A82-B11; A82-B12; A82- B13; A82-B14; A82-B15; A82-B16; A82-B17; A82-B18; A82-B19; A82-B20; A82-B21; A82-B22; A82-B23; A82-B24; A82-B25; A82-B26; A82-B27; A82-B28; A82-B29; A82-B30; A82-B31; A82-B32; A82-B33; A82-B34; A82-B35; A82-B36; A82-B37; A82- B38; A82-B39; A82-B40; A82-B41; A82-B42; A82-B43; A82-B44; A82-B45; A82-B46; A82-B47; A82-B48; A82-B49; A82-B50; A82-B51; A82-B52; A82-B53; A82-B54; A82-B55; A82-B56; A82-B57; A82-B58; A82-B59; A82-B60; A82-B61; A82-B62; A82- B63; A82-B64; A82-B65; A82-B66; A82-B67; A82-B68; A82-B69; A82-B70; A82-B71; A82-B72; A82-B73; A82-B74; A82-B75; A82-B76; A82-B77; A82-B78; A82-B79; A82-B80; A82-B81; A82-B82; A82-B83; A82-B84; A82-B85; A82-B86; A82-B87; A82- B88; A82-B89; A82-B90; A82-B91; A82-B92; A82-B93; A82-B94; A82-B95; A82-B96; A82-B97; A82-B98; A82-B99; A82-B100; A82-B101; A82-B102; A82-B103; A82-B104; A82-B105; A82-B106; A82-B107;A82-B108; A82-B109; A82-B110; A82-B111; A82-B112; A82-B113; A83-B1; A83-B2; A83-B3; A83-B4; A83-B5; A83-B6; A83-B7; A83-B8; A83-B9; A83-B10; A83-B11; A83-B12; A83- B13; A83-B14; A83-B15; A83-B16; A83-B17; A83-B18; A83-B19; A83-B20; A83-B21; A83-B22; A83-B23; A83-B24; A83-B25; A83-B26; A83-B27; A83-B28; A83-B29; A83-B30; A83-B31; A83-B32; A83-B33; A83-B34; A83-B35; A83-B36; A83-B37; A83- B38; A83-B39; A83-B40; A83-B41; A83-B42; A83-B43; A83-B44; A83-B45; A83-B46; A83-B47; A83-B48; A83-B49; A83-B50; A83-B51; A83-B52; A83-B53; A83-B54; A83-B55; A83-B56; A83-B57; A83-B58; A83-B59; A83-B60; A83-B61; A83-B62; A83- B63; A83-B64; A83-B65; A83-B66; A83-B67; A83-B68; A83-B69; A83-B70; A83-B71; A83-B72; A83-B73; A83-B74; A83-B75; A83-B76; A83-B77; A83-B78; A83-B79; A83-B80; A83-B81; A83-B82; A83-B83; A83-B84; A83-B85; A83-B86; A83-B87; A83- B88; A83-B89; A83-B90; A83-B91; A83-B92; A83-B93; A83-B94; A83-B95; A83-B96; A83-B97; A83-B98; A83-B99; A83-B100; A83-B101; A83-B102; A83-B103; A83-B104; A83-B105; A83-B106; A83-B107;A83-B108; A83-B109; A83-B110; A83-B111; A83-B112; A83-B113; A84-B1; A84-B2; A84-B3; A84-B4; A84-B5; A84-B6; A84-B7; A84-B8; A84-B9; A84-B10; A84-B11; A84-B12; A84- B13; A84-B14; A84-B15; A84-B16; A84-B17; A84-B18; A84-B19; A84-B20; A84-B21; A84-B22; A84-B23; A84-B24; A84-B25; A84-B26; A84-B27; A84-B28; A84-B29; A84-B30; A84-B31; A84-B32; A84-B33; A84-B34; A84-B35; A84-B36; A84-B37; A84- B38; A84-B39; A84-B40; A84-B41; A84-B42; A84-B43; A84-B44; A84-B45; A84-B46; A84-B47; A84-B48; A84-B49; A84-B50; A84-B51; A84-B52; A84-B53; A84-B54; A84-B55; A84-B56; A84-B57; A84-B58; A84-B59; A84-B60; A84-B61; A84-B62; A84- B63; A84-B64; A84-B65; A84-B66; A84-B67; A84-B68; A84-B69; A84-B70; A84-B71; A84-B72; A84-B73; A84-B74; A84-B75; A84-B76; A84-B77; A84-B78; A84-B79; A84-B80; A84-B81; A84-B82; A84-B83; A84-B84; A84-B85; A84-B86; A84-B87; A84- B88; A84-B89; A84-B90; A84-B91; A84-B92; A84-B93; A84-B94; A84-B95; A84-B96; A84-B97; A84-B98; A84-B99; A84-B100; A84-B101; A84-B102; A84-B103; A84-B104; A84-B105; A84-B106; A84-B107;A84-B108; A84-B109; A84-B110; A84-B111; A84-B112; A84-B113; A85-B1; A85-B2; A85-B3; A85-B4; A85-B5; A85-B6; A85-B7; A85-B8; A85-B9; A85-B10; A85-B11; A85-B12; A85- B13; A85-B14; A85-B15; A85-B16; A85-B17; A85-B18; A85-B19; A85-B20; A85-B21; A85-B22; A85-B23; A85-B24; A85-B25; A85-B26; A85-B27; A85-B28; A85-B29; A85-B30; A85-B31; A85-B32; A85-B33; A85-B34; A85-B35; A85-B36; A85-B37; A85- B38; A85-B39; A85-B40; A85-B41; A85-B42; A85-B43; A85-B44; A85-B45; A85-B46; A85-B47; A85-B48; A85-B49; A85-B50; A85-B51; A85-B52; A85-B53; A85-B54; A85-B55; A85-B56; A85-B57; A85-B58; A85-B59; A85-B60; A85-B61; A85-B62; A85- B63; A85-B64; A85-B65; A85-B66; A85-B67; A85-B68; A85-B69; A85-B70; A85-B71; A85-B72; A85-B73; A85-B74; A85-B75; A85-B76; A85-B77; A85-B78; A85-B79; A85-B80; A85-B81; A85-B82; A85-B83; A85-B84; A85-B85; A85-B86; A85-B87; A85- B88; A85-B89; A85-B90; A85-B91; A85-B92; A85-B93; A85-B94; A85-B95; A85-B96; A85-B97; A85-B98; A85-B99; A85-B100; A85-B101; A85-B102; A85-B103; A85-B104; A85-B105; A85-B106; A85-B107;A85-B108; A85-B109; A85-B110; A85-B111; A85-B112; A85-B113; A86-B1; A86-B2; A86-B3; A86-B4; A86-B5; A86-B6; A86-B7; A86-B8; A86-B9; A86-B10; A86-B11; A86-B12; A86- B13; A86-B14; A86-B15; A86-B16; A86-B17; A86-B18; A86-B19; A86-B20; A86-B21; A86-B22; A86-B23; A86-B24; A86-B25; A86-B26; A86-B27; A86-B28; A86-B29; A86-B30; A86-B31; A86-B32; A86-B33; A86-B34; A86-B35; A86-B36; A86-B37; A86- B38; A86-B39; A86-B40; A86-B41; A86-B42; A86-B43; A86-B44; A86-B45; A86-B46; A86-B47; A86-B48; A86-B49; A86-B50; A86-B51; A86-B52; A86-B53; A86-B54; A86-B55; A86-B56; A86-B57; A86-B58; A86-B59; A86-B60; A86-B61; A86-B62; A86- B63; A86-B64; A86-B65; A86-B66; A86-B67; A86-B68; A86-B69; A86-B70; A86-B71; A86-B72; A86-B73; A86-B74; A86-B75; A86-B76; A86-B77; A86-B78; A86-B79; A86-B80; A86-B81; A86-B82; A86-B83; A86-B84; A86-B85; A86-B86; A86-B87; A86- B88; A86-B89; A86-B90; A86-B91; A86-B92; A86-B93; A86-B94; A86-B95; A86-B96; A86-B97; A86-B98; A86-B99; A86-B100; A86-B101; A86-B102; A86-B103; A86-B104; A86-B105; A86-B106; A86-B107;A86-B108; A86-B109; A86-B110; A86-B111; A86-B112; A86-B113; A87-B1; A87-B2; A87-B3; A87-B4; A87-B5; A87-B6; A87-B7; A87-B8; A87-B9; A87-B10; A87-B11; A87-B12; A87- B13; A87-B14; A87-B15; A87-B16; A87-B17; A87-B18; A87-B19; A87-B20; A87-B21; A87-B22; A87-B23; A87-B24; A87-B25; A87-B26; A87-B27; A87-B28; A87-B29; A87-B30; A87-B31; A87-B32; A87-B33; A87-B34; A87-B35; A87-B36; A87-B37; A87- B38; A87-B39; A87-B40; A87-B41; A87-B42; A87-B43; A87-B44; A87-B45; A87-B46; A87-B47; A87-B48; A87-B49; A87-B50; A87-B51; A87-B52; A87-B53; A87-B54; A87-B55; A87-B56; A87-B57; A87-B58; A87-B59; A87-B60; A87-B61; A87-B62; A87- B63; A87-B64; A87-B65; A87-B66; A87-B67; A87-B68; A87-B69; A87-B70; A87-B71; A87-B72; A87-B73; A87-B74; A87-B75; A87-B76; A87-B77; A87-B78; A87-B79; A87-B80; A87-B81; A87-B82; A87-B83; A87-B84; A87-B85; A87-B86; A87-B87; A87- B88; A87-B89; A87-B90; A87-B91; A87-B92; A87-B93; A87-B94; A87-B95; A87-B96; A87-B97; A87-B98; A87-B99; A87-B100; A87-B101; A87-B102; A87-B103; A87-B104; A87-B105; A87-B106; A87-B107;A87-B108; A87-B109; A87-B110; A87-B111; A87-B112; A87-B113; A88-B1; A88-B2; A88-B3; A88-B4; A88-B5; A88-B6; A88-B7; A88-B8; A88-B9; A88-B10; A88-B11; A88-B12; A88- B13; A88-B14; A88-B15; A88-B16; A88-B17; A88-B18; A88-B19; A88-B20; A88-B21; A88-B22; A88-B23; A88-B24; A88-B25; A88-B26; A88-B27; A88-B28; A88-B29; A88-B30; A88-B31; A88-B32; A88-B33; A88-B34; A88-B35; A88-B36; A88-B37; A88- B38; A88-B39; A88-B40; A88-B41; A88-B42; A88-B43; A88-B44; A88-B45; A88-B46; A88-B47; A88-B48; A88-B49; A88-B50; A88-B51; A88-B52; A88-B53; A88-B54; A88-B55; A88-B56; A88-B57; A88-B58; A88-B59; A88-B60; A88-B61; A88-B62; A88- B63; A88-B64; A88-B65; A88-B66; A88-B67; A88-B68; A88-B69; A88-B70; A88-B71; A88-B72; A88-B73; A88-B74; A88-B75; A88-B76; A88-B77; A88-B78; A88-B79; A88-B80; A88-B81; A88-B82; A88-B83; A88-B84; A88-B85; A88-B86; A88-B87; A88- B88; A88-B89; A88-B90; A88-B91; A88-B92; A88-B93; A88-B94; A88-B95; A88-B96; A88-B97; A88-B98; A88-B99; A88-B100; A88-B101; A88-B102; A88-B103; A88-B104; A88-B105; A88-B106; A88-B107;A88-B108; A88-B109; A88-B110; A88-B111; A88-B112; A88-B113; A89-B1; A89-B2; A89-B3; A89-B4; A89-B5; A89-B6; A89-B7; A89-B8; A89-B9; A89-B10; A89-B11; A89-B12; A89- B13; A89-B14; A89-B15; A89-B16; A89-B17; A89-B18; A89-B19; A89-B20; A89-B21; A89-B22; A89-B23; A89-B24; A89-B25; A89-B26; A89-B27; A89-B28; A89-B29; A89-B30; A89-B31; A89-B32; A89-B33; A89-B34; A89-B35; A89-B36; A89-B37; A89- B38; 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A97-B23; A97-B24; A97-B25; A97-B26; A97-B27; A97-B28; A97-B29; A97-B30; A97-B31; A97-B32; A97-B33; A97-B34; A97-B35; A97-B36; A97-B37; A97- B38; A97-B39; A97-B40; A97-B41; A97-B42; A97-B43; A97-B44; A97-B45; A97-B46; A97-B47; A97-B48; A97-B49; A97-B50; A97-B51; A97-B52; A97-B53; A97-B54; A97-B55; A97-B56; A97-B57; A97-B58; A97-B59; A97-B60; A97-B61; A97-B62; A97- B63; A97-B64; A97-B65; A97-B66; A97-B67; A97-B68; A97-B69; A97-B70; A97-B71; A97-B72; A97-B73; A97-B74; A97-B75; A97-B76; A97-B77; A97-B78; A97-B79; A97-B80; A97-B81; A97-B82; A97-B83; A97-B84; A97-B85; A97-B86; A97-B87; A97- B88; A97-B89; A97-B90; A97-B91; A97-B92; A97-B93; A97-B94; A97-B95; A97-B96; A97-B97; A97-B98; A97-B99; A97-B100; A97-B101; A97-B102; A97-B103; A97-B104; A97-B105; A97-B106; A97-B107;A97-B108; A97-B109; A97-B110; A97-B111; A97-B112; A97-B113; A98-B1; A98-B2; A98-B3; A98-B4; A98-B5; A98-B6; A98-B7; A98-B8; A98-B9; A98-B10; A98-B11; A98-B12; A98- B13; A98-B14; A98-B15; A98-B16; A98-B17; A98-B18; A98-B19; A98-B20; A98-B21; A98-B22; A98-B23; A98-B24; A98-B25; A98-B26; A98-B27; A98-B28; A98-B29; A98-B30; A98-B31; A98-B32; A98-B33; A98-B34; A98-B35; A98-B36; A98-B37; A98- B38; A98-B39; A98-B40; A98-B41; A98-B42; A98-B43; A98-B44; A98-B45; A98-B46; A98-B47; A98-B48; A98-B49; A98-B50; A98-B51; A98-B52; A98-B53; A98-B54; A98-B55; A98-B56; A98-B57; A98-B58; A98-B59; A98-B60; A98-B61; A98-B62; A98- B63; A98-B64; A98-B65; A98-B66; A98-B67; A98-B68; A98-B69; A98-B70; A98-B71; A98-B72; A98-B73; A98-B74; A98-B75; A98-B76; A98-B77; A98-B78; A98-B79; A98-B80; A98-B81; A98-B82; A98-B83; A98-B84; A98-B85; A98-B86; A98-B87; A98- B88; A98-B89; A98-B90; A98-B91; A98-B92; A98-B93; A98-B94; A98-B95; A98-B96; A98-B97; A98-B98; A98-B99; A98-B100; A98-B101; A98-B102; A98-B103; A98-B104; A98-B105; A98-B106; A98-B107;A98-B108; A98-B109; A98-B110; A98-B111; A98-B112; A98-B113; A99-B1; A99-B2; A99-B3; A99-B4; A99-B5; A99-B6; A99-B7; A99-B8; A99-B9; A99-B10; A99-B11; A99-B12; A99- B13; A99-B14; A99-B15; A99-B16; A99-B17; A99-B18; A99-B19; A99-B20; A99-B21; A99-B22; A99-B23; A99-B24; A99-B25; A99-B26; A99-B27; A99-B28; A99-B29; A99-B30; A99-B31; A99-B32; A99-B33; A99-B34; A99-B35; A99-B36; A99-B37; A99- B38; A99-B39; A99-B40; A99-B41; A99-B42; A99-B43; A99-B44; A99-B45; A99-B46; A99-B47; A99-B48; A99-B49; A99-B50; A99-B51; A99-B52; A99-B53; A99-B54; A99-B55; A99-B56; A99-B57; A99-B58; A99-B59; A99-B60; A99-B61; A99-B62; A99- B63; A99-B64; A99-B65; A99-B66; A99-B67; A99-B68; A99-B69; A99-B70; A99-B71; A99-B72; A99-B73; A99-B74; A99-B75; A99-B76; A99-B77; A99-B78; A99-B79; A99-B80; A99-B81; A99-B82; A99-B83; A99-B84; A99-B85; A99-B86; A99-B87; A99- B88; A99-B89; A99-B90; A99-B91; A99-B92; A99-B93; A99-B94; A99-B95; A99-B96; A99-B97; A99-B98; A99-B99; A99-B100; A99-B101; A99-B102; A99-B103; A99-B104; A99-B105; A99-B106; A99-B107;A99-B108; A99-B109; A99-B110; A99-B111; A99-B112; A99-B113; A100-B1; A100-B2; A100-B3; A100-B4; A100-B5; A100-B6; A100-B7; A100-B8; A100-B9; A100-B10; A100-B11; A100-B12; A100- B13; A100-B14; A100-B15; A100-B16; A100-B17; A100-B18; A100-B19; A100-B20; A100-B21; A100-B22; A100-B23; A100-B24; A100-B25; A100-B26; A100-B27; A100-B28; A100-B29; A100-B30; A100-B31; A100-B32; A100-B33; A100-B34; A100-B35; A100-B36; A100-B37; A100- B38; A100-B39; A100-B40; A100-B41; A100-B42; A100-B43; A100-B44; A100-B45; A100-B46; A100-B47; A100-B48; A100-B49; A100-B50; A100-B51; A100-B52; A100-B53; A100-B54; A100-B55; A100-B56; A100-B57; A100-B58; A100-B59; A100-B60; A100-B61; A100-B62; A100- B63; A100-B64; A100-B65; A100-B66; A100-B67; A100-B68; A100-B69; A100-B70; A100-B71; A100-B72; A100-B73; A100-B74; A100-B75; A100-B76; A100-B77; A100-B78; A100-B79; A100-B80; A100-B81; A100-B82; A100-B83; A100-B84; A100-B85; A100-B86; A100-B87; A100- B88; A100-B89; A100-B90; A100-B91; A100-B92; A100-B93; A100-B94; A100-B95; A100-B96;A100-B97; A100-B98; A100-B99; A100-B100; A100-B101; A100-B102; A100-B103; A100-B104; A100-B105; A100-B106; A100-B107; A100-B108; A100- B109; A100-B110; A100-B111; A100-B112; A100-B113; A101-B1; A101-B2; A101-B3; A101-B4; A101-B5; A101-B6; A101-B7; A101-B8; A101-B9; A101-B10; A101-B11; A101-B12; A101- B13; A101-B14; A101-B15; A101-B16; A101-B17; A101-B18; A101-B19; A101-B20; A101-B21; A101-B22; A101-B23; A101-B24; A101-B25; A101-B26; A101-B27; A101-B28; A101-B29; A101-B30; A101-B31; A101-B32; A101-B33; A101-B34; A101-B35; A101-B36; A101-B37; A101- B38; A101-B39; A101-B40; A101-B41; A101-B42; A101-B43; A101-B44; A101-B45; A101-B46; A101-B47; A101-B48; A101-B49; A101-B50; A101-B51; A101-B52; A101-B53; A101-B54; A101-B55; A101-B56; A101-B57; A101-B58; A101-B59; A101-B60; A101-B61; A101-B62; A101- B63; A101-B64; A101-B65; A101-B66; A101-B67; A101-B68; A101-B69; A101-B70; A101-B71; A101-B72; A101-B73; A101-B74; A101-B75; A101-B76; A101-B77; A101-B78; A101-B79; A101-B80; A101-B81; A101-B82; A101-B83; A101-B84; A101-B85; A101-B86; A101-B87; A101- B88; A101-B89; A101-B90; A101-B91; A101-B92; A101-B93; A101-B94; A101-B95; A101-B96;A101-B97; A101-B98; A101-B99; A101-B100; A101-B101; A101-B102; A101-B103; A101-B104; A101-B105; A101-B106; A101-B107; A101-B108; A101- B109; A101-B110; A101-B111; A101-B112; A101-B113; A102-B1; A102-B2; A102-B3; A102-B4; A102-B5; A102-B6; A102-B7; A102-B8; A102-B9; A102-B10; A102-B11; A102-B12; A102- B13; A102-B14; A102-B15; A102-B16; A102-B17; A102-B18; A102-B19; A102-B20; A102-B21; A102-B22; A102-B23; A102-B24; A102-B25; A102-B26; A102-B27; A102-B28; A102-B29; A102-B30; A102-B31; A102-B32; A102-B33; A102-B34; A102-B35; A102-B36; A102-B37; A102- B38; A102-B39; A102-B40; A102-B41; A102-B42; A102-B43; A102-B44; A102-B45; A102-B46; A102-B47; A102-B48; A102-B49; A102-B50; A102-B51; A102-B52; A102-B53; A102-B54; A102-B55; A102-B56; A102-B57; A102-B58; A102-B59; A102-B60; A102-B61; A102-B62; A102- B63; A102-B64; A102-B65; A102-B66; A102-B67; A102-B68; A102-B69; A102-B70; A102-B71; A102-B72; A102-B73; A102-B74; A102-B75; A102-B76; A102-B77; A102-B78; A102-B79; A102-B80; A102-B81; A102-B82; A102-B83; A102-B84; A102-B85; A102-B86; A102-B87; A102- B88; A102-B89; A102-B90; A102-B91; A102-B92; A102-B93; A102-B94; A102-B95; A102-B96;A102-B97; A102-B98; A102-B99; A102-B100; A102-B101; A102-B102; A102-B103; A102-B104; A102-B105; A102-B106; A102-B107; A102-B108; A102- B109; A102-B110; A102-B111; A102-B112; A102-B113; A103-B1; A103-B2; A103-B3; A103-B4; A103-B5; A103-B6; A103-B7; A103-B8; A103-B9; A103-B10; A103-B11; A103-B12; A103- B13; A103-B14; A103-B15; A103-B16; A103-B17; A103-B18; A103-B19; A103-B20; A103-B21; A103-B22; A103-B23; A103-B24; A103-B25; A103-B26; A103-B27; A103-B28; A103-B29; A103-B30; A103-B31; A103-B32; A103-B33; A103-B34; A103-B35; A103-B36; A103-B37; A103- B38; A103-B39; A103-B40; A103-B41; A103-B42; A103-B43; A103-B44; A103-B45; A103-B46; A103-B47; A103-B48; A103-B49; A103-B50; A103-B51; A103-B52; A103-B53; A103-B54; A103-B55; A103-B56; A103-B57; A103-B58; A103-B59; A103-B60; A103-B61; A103-B62; A103- B63; A103-B64; A103-B65; A103-B66; A103-B67; A103-B68; A103-B69; A103-B70; A103-B71; A103-B72; A103-B73; A103-B74; A103-B75; A103-B76; A103-B77; A103-B78; A103-B79; A103-B80; A103-B81; A103-B82; A103-B83; A103-B84; A103-B85; A103-B86; A103-B87; A103- B88; A103-B89; A103-B90; A103-B91; A103-B92; A103-B93; A103-B94; A103-B95; A103-B96;A103-B97; A103-B98; A103-B99; A103-B100; A103-B101; A103-B102; A103-B103; A103-B104; A103-B105; A103-B106; A103-B107; A103-B108; A103- B109; A103-B110; A103-B111; A103-B112; A103-B113; A104-B1; A104-B2; A104-B3; A104-B4; A104-B5; A104-B6; A104-B7; A104-B8; A104-B9; A104-B10; A104-B11; A104-B12; A104- B13; A104-B14; A104-B15; A104-B16; A104-B17; A104-B18; A104-B19; A104-B20; A104-B21; A104-B22; A104-B23; A104-B24; A104-B25; A104-B26; A104-B27; A104-B28; A104-B29; A104-B30; A104-B31; A104-B32; A104-B33; A104-B34; A104-B35; A104-B36; A104-B37; A104- B38; A104-B39; A104-B40; A104-B41; A104-B42; A104-B43; A104-B44; A104-B45; A104-B46; A104-B47; A104-B48; A104-B49; A104-B50; A104-B51; A104-B52; A104-B53; A104-B54; A104-B55; A104-B56; A104-B57; A104-B58; A104-B59; A104-B60; A104-B61; A104-B62; A104- B63; A104-B64; A104-B65; A104-B66; A104-B67; A104-B68; A104-B69; A104-B70; A104-B71; A104-B72; A104-B73; A104-B74; A104-B75; A104-B76; A104-B77; A104-B78; A104-B79; A104-B80; A104-B81; A104-B82; A104-B83; A104-B84; A104-B85; A104-B86; A104-B87; A104- B88; A104-B89; A104-B90; A104-B91; A104-B92; A104-B93; A104-B94; A104-B95; A104-B96;A104-B97; A104-B98; A104-B99; A104-B100; A104-B101; A104-B102; A104-B103; A104-B104; A104-B105; A104-B106; A104-B107; A104-B108; A104- B109; A104-B110; A104-B111; A104-B112; A104-B113; A105-B1; A105-B2; A105-B3; A105-B4; A105-B5; A105-B6; A105-B7; A105-B8; A105-B9; A105-B10; A105-B11; A105-B12; A105- B13; A105-B14; A105-B15; A105-B16; A105-B17; A105-B18; A105-B19; A105-B20; A105-B21; A105-B22; A105-B23; A105-B24; A105-B25; A105-B26; A105-B27; A105-B28; A105-B29; A105-B30; A105-B31; A105-B32; A105-B33; A105-B34; A105-B35; A105-B36; A105-B37; A105-B38; A105-B39; A105-B40; A105-B41; A105-B42; A105- B43; A105-B44; A105-B45; A105-B46; A105-B47; A105-B48; A105-B49; A105-B50; A105-B51; A105-B52; A105-B53; A105-B54; A105-B55; A105-B56; A105-B57; A105-B58; A105-B59; A105-B60; A105-B61; A105-B62; A105-B63; A105-B64; A105-B65; A105-B66; A105-B67; A105- B68; A105-B69; A105-B70; A105-B71; A105-B72; A105-B73; A105-B74; A105-B75; A105-B76; A105-B77; A105-B78; A105-B79; A105-B80; A105-B81; A105-B82; A105-B83; A105-B84; A105-B85; A105-B86; A105-B87; A105-B88; A105-B89; A105-B90; A105-B91; A105-B92; A105- B93; A105-B94; A105-B95; A105-B96; A105-B97; A105-B98; A105-B99; A105-B100; A105-B101; A105-B102; A105-B103; A105-B104; A105-B105; A105-B106; A105-B107; A105-B108; A105-B109; A105-B110; A105-B111; A105-B112; A105-B113; A106-B1; A106-B2; A106-B3; A106-B4; A106-B5; A106-B6; A106-B7; A106-B8; A106-B9; A106-B10; A106-B11; A106-B12; A106- B13; A106-B14; A106-B15; A106-B16; A106-B17; A106-B18; A106-B19; A106-B20; A106-B21; A106-B22; A106-B23; A106-B24; A106-B25; A106-B26; A106-B27; A106-B28; A106-B29; A106-B30; A106-B31; A106-B32; A106-B33; A106-B34; A106-B35; A106-B36; A106-B37; A106- B38; A106-B39; A106-B40; A106-B41; A106-B42; A106-B43; A106-B44; A106-B45; A106-B46; A106-B47; A106-B48; A106-B49; A106-B50; A106-B51; A106-B52; A106-B53; A106-B54; A106-B55; A106-B56; A106-B57; A106-B58; A106-B59; A106-B60; A106-B61; A106-B62; A106- B63; A106-B64; A106-B65; A106-B66; A106-B67; A106-B68; A106-B69; A106-B70; A106-B71; A106-B72; A106-B73; A106-B74; A106-B75; A106-B76; A106-B77; A106-B78; A106-B79; A106-B80; A106-B81; A106-B82; A106-B83; A106-B84; A106-B85; A106-B86; A106-B87; A106- B88; A106-B89; A106-B90; A106-B91; A106-B92; A106-B93; A106-B94; A106-B95; A106-B96;A106-B97; A106-B98; A106-B99; A106-B100; A106-B101; A106-B102; A106-B103; A106-B104; A106-B105; A106-B106; A106-B107; A106-B108; A106- B109; A106-B110; A106-B111; A106-B112; A106-B113; A107-B1; A107-B2; A107-B3; A107-B4; A107-B5; A107-B6; A107-B7; A107-B8; A107-B9; A107-B10; A107-B11; A107-B12; A107- B13; A107-B14; A107-B15; A107-B16; A107-B17; A107-B18; A107-B19; A107-B20; A107-B21; A107-B22; A107-B23; A107-B24; A107-B25; A107-B26; A107-B27; A107-B28; A107-B29; A107-B30; A107-B31; A107-B32; A107-B33; A107-B34; A107-B35; A107-B36; A107-B37; A107- B38; A107-B39; A107-B40; A107-B41; A107-B42; A107-B43; A107-B44; A107-B45; A107-B46; A107-B47; A107-B48; A107-B49; A107-B50; A107-B51; A107-B52; A107-B53; A107-B54; A107-B55; A107-B56; A107-B57; A107-B58; A107-B59; A107-B60; A107-B61; A107-B62; A107- B63; A107-B64; A107-B65; A107-B66; A107-B67; A107-B68; A107-B69; A107-B70; A107-B71; A107-B72; A107-B73; A107-B74; A107-B75; A107-B76; A107-B77; A107-B78; A107-B79; A107-B80; A107-B81; A107-B82; A107-B83; A107-B84; A107-B85; A107-B86; A107-B87; A107- B88; A107-B89; A107-B90; A107-B91; A107-B92; A107-B93; A107-B94; A107-B95; A107-B96;A107-B97; A107-B98; A107-B99; A107-B100; A107-B101; A107-B102; A107-B103; A107-B104; A107-B105; A107-B106; A107-B107; A107-B108; A107- B109; A107-B110; A107-B111; A107-B112; A107-B113; A108-B1; A108-B2; A108-B3; A108-B4; A108-B5; A108-B6; A108-B7; A108-B8; A108-B9; A108-B10; A108-B11; A108-B12; A108- B13; A108-B14; A108-B15; A108-B16; A108-B17; A108-B18; A108-B19; A108-B20; A108-B21; A108-B22; A108-B23; A108-B24; A108-B25; A108-B26; A108-B27; A108-B28; A108-B29; A108-B30; A108-B31; A108-B32; A108-B33; A108-B34; A108-B35; A108-B36; A108-B37; A108- B38; A108-B39; A108-B40; A108-B41; A108-B42; A108-B43; A108-B44; A108-B45; A108-B46; A108-B47; A108-B48; A108-B49; A108-B50; A108-B51; A108-B52; A108-B53; A108-B54; A108-B55; A108-B56; A108-B57; A108-B58; A108-B59; A108-B60; A108-B61; A108-B62; A108- B63; A108-B64; A108-B65; A108-B66; A108-B67; A108-B68; A108-B69; A108-B70; A108-B71; A108-B72; A108-B73; A108-B74; A108-B75; A108-B76; A108-B77; A108-B78; A108-B79; A108-B80; A108-B81; A108-B82; A108-B83; A108-B84; A108-B85; A108-B86; A108-B87; A108- B88; A108-B89; A108-B90; A108-B91; A108-B92; A108-B93; A108-B94; A108-B95; A108-B96;A108-B97; A108-B98; A108-B99; A108-B100; A108-B101; A108-B102; A108-B103; A108-B104; A108-B105; A108-B106; A108-B107; A108-B108; A108- B109; A108-B110; A108-B111; A108-B112; A108-B113; A109-B1; A109-B2; A109-B3; A109-B4; A109-B5; A109-B6; A109-B7; A109-B8; A109-B9; A109-B10; A109-B11; A109-B12; A109- B13; A109-B14; A109-B15; A109-B16; A109-B17; A109-B18; A109-B19; A109-B20; A109-B21; A109-B22; A109-B23; A109-B24; A109-B25; A109-B26; A109-B27; A109-B28; A109-B29; A109-B30; A109-B31; A109-B32; A109-B33; A109-B34; A109-B35; A109-B36; A109-B37; A109- B38; A109-B39; A109-B40; A109-B41; A109-B42; A109-B43; A109-B44; A109-B45; A109-B46; A109-B47; A109-B48; A109-B49; A109-B50; A109-B51; A109-B52; A109-B53; A109-B54; A109-B55; A109-B56; A109-B57; A109-B58; A109-B59; A109-B60; A109-B61; A109-B62; A109- B63; A109-B64; A109-B65; A109-B66; A109-B67; A109-B68; A109-B69; A109-B70; A109-B71; A109-B72; A109-B73; A109-B74; A109-B75; A109-B76; A109-B77; A109-B78; A109-B79; A109-B80; A109-B81; A109-B82; A109-B83; A109-B84; A109-B85; A109-B86; A109-B87; A109- B88; A109-B89; A109-B90; A109-B91; A109-B92; A109-B93; A109-B94; A109-B95; A109-B96;A109-B97; A109-B98; A109-B99; A109-B100; A109-B101; A109-B102; A109-B103; A109-B104; A109-B105; A109-B106; A109-B107; A109-B108; A109- B109; A109-B110; A109-B111; A109-B112; A109-B113; A110-B1; A110-B2; A110-B3; A110-B4; A110-B5; A110-B6; A110-B7; A110-B8; A110-B9; A110-B10; A110-B11; A110-B12; A110- B13; A110-B14; A110-B15; A110-B16; A110-B17; A110-B18; A110-B19; A110-B20; A110-B21; A110-B22; A110-B23; A110-B24; A110-B25; A110-B26; A110-B27; A110-B28; A110-B29; A110-B30; A110-B31; A110-B32; A110-B33; A110-B34; A110-B35; A110-B36; A110-B37; A110- B38; A110-B39; A110-B40; A110-B41; A110-B42; A110-B43; A110-B44; A110-B45; A110-B46; A110-B47; A110-B48; A110-B49; A110-B50; A110-B51; A110-B52; A110-B53; A110-B54; A110-B55; A110-B56; A110-B57; A110-B58; A110-B59; A110-B60; A110-B61; A110-B62; A110- B63; A110-B64; A110-B65; A110-B66; A110-B67; A110-B68; A110-B69; A110-B70; A110-B71; A110-B72; A110-B73; A110-B74; A110-B75; A110-B76; A110-B77; A110-B78; A110-B79; A110-B80; A110-B81; A110-B82; A110-B83; A110-B84; A110-B85; A110-B86; A110-B87; A110- B88; A110-B89; A110-B90; A110-B91; A110-B92; A110-B93; A110-B94; A110-B95; A110-B96;A110-B97; A110-B98; A110-B99; A110-B100; A110-B101; A110-B102; A110-B103; A110-B104; A110-B105; A110-B106; A110-B107; A110-B108; A110- B109; A110-B110; A110-B111; A110-B112; A110-B113; A111-B1; A111-B2; A111-B3; A111-B4; A111-B5; A111-B6; A111-B7; A111-B8; A111-B9; A111-B10; A111-B11; A111-B12; A111- B13; A111-B14; A111-B15; A111-B16; A111-B17; A111-B18; A111-B19; A111-B20; A111-B21; A111-B22; A111-B23; A111-B24; A111-B25; A111-B26; A111-B27; A111-B28; A111-B29; A111-B30; A111-B31; A111-B32; A111-B33; A111-B34; A111-B35; A111-B36; A111-B37; A111- B38; A111-B39; A111-B40; A111-B41; A111-B42; A111-B43; A111-B44; A111-B45; A111-B46; A111-B47; A111-B48; A111-B49; A111-B50; A111-B51; A111-B52; A111-B53; A111-B54; A111-B55; A111-B56; A111-B57; A111-B58; A111-B59; A111-B60; A111-B61; A111-B62; A111- B63; A111-B64; A111-B65; A111-B66; A111-B67; A111-B68; A111-B69; A111-B70; A111-B71; A111-B72; A111-B73; A111-B74; A111-B75; A111-B76; A111-B77; A111-B78; A111-B79; A111-B80; A111-B81; A111-B82; A111-B83; A111-B84; A111-B85; A111-B86; A111-B87; A111- B88; A111-B89; A111-B90; A111-B91; A111-B92; A111-B93; A111-B94; A111-B95; A111-B96;A111-B97; A111-B98; A111-B99; A111-B100; A111-B101; A111-B102; A111-B103; A111-B104; A111-B105; A111-B106; A111-B107; A111-B108; A111- B109; A111-B110; A111-B111; A111-B112; A111-B113; A112-B1; A112-B2; A112-B3; A112-B4; A112-B5; A112-B6; A112-B7; A112-B8; A112-B9; A112-B10; A112-B11; A112-B12; A112- B13; A112-B14; A112-B15; A112-B16; A112-B17; A112-B18; A112-B19; A112-B20; A112-B21; A112-B22; A112-B23; A112-B24; A112-B25; A112-B26; A112-B27; A112-B28; A112-B29; A112-B30; A112-B31; A112-B32; A112-B33; A112-B34; A112-B35; A112-B36; A112-B37; A112- B38; A112-B39; A112-B40; A112-B41; A112-B42; A112-B43; A112-B44; A112-B45; A112-B46; A112-B47; A112-B48; A112-B49; A112-B50; A112-B51; A112-B52; A112-B53; A112-B54; A112-B55; A112-B56; A112-B57; A112-B58; A112-B59; A112-B60; A112-B61; A112-B62; A112- B63; A112-B64; A112-B65; A112-B66; A112-B67; A112-B68; A112-B69; A112-B70; A112-B71; A112-B72; A112-B73; A112-B74; A112-B75; A112-B76; A112-B77; A112-B78; A112-B79; A112-B80; A112-B81; A112-B82; A112-B83; A112-B84; A112-B85; A112-B86; A112-B87; A112- B88; A112-B89; A112-B90; A112-B91; A112-B92; A112-B93; A112-B94; A112-B95; A112-B96;A112-B97; A112-B98; A112-B99; A112-B100; A112-B101; A112-B102; A112-B103; A112-B104; A112-B105; A112-B106; A112-B107; A112-B108; A112- B109; A112-B110; A112-B111; A112-B112; A112-B113; A113-B1; A113-B2; A113-B3; A113-B4; A113-B5; A113-B6; A113-B7; A113-B8; A113-B9; A113-B10; A113-B11; A113-B12; A113- B13; A113-B14; A113-B15; A113-B16; A113-B17; A113-B18; A113-B19; A113-B20; A113-B21; A113-B22; A113-B23; A113-B24; A113-B25; A113-B26; A113-B27; A113-B28; A113-B29; A113-B30; A113-B31; A113-B32; A113-B33; A113-B34; A113-B35; A113-B36; A113-B37; A113- B38; A113-B39; A113-B40; A113-B41; A113-B42; A113-B43; A113-B44; A113-B45; A113-B46; A113-B47; A113-B48; A113-B49; A113-B50; A113-B51; A113-B52; A113-B53; A113-B54; A113-B55; A113-B56; A113-B57; A113-B58; A113-B59; A113-B60; A113-B61; A113-B62; A113- B63; A113-B64; A113-B65; A113-B66; A113-B67; A113-B68; A113-B69; A113-B70; A113-B71; A113-B72; A113-B73; A113-B74; A113-B75; A113-B76; A113-B77; A113-B78; A113-B79; A113-B80; A113-B81; A113-B82; A113-B83; A113-B84; A113-B85; A113-B86; A113-B87; A113- B88; A113-B89; A113-B90; A113-B91; A113-B92; A113-B93; A113-B94; A113-B95; A113-B96;A113-B97; A113-B98; A113-B99; A113-B100; A113-B101; A113-B102; A113-B103; A113-B104; A113-B105; A113-B106; A113-B107; A113-B108; A113- B109; A113-B110; A113-B111; A113-B112; A113-B113; A114-B1; A114-B2; A114-B3; A114-B4; A114-B5; A114-B6; A114-B7; A114-B8; A114-B9; A114-B10; A114-B11; A114-B12; A114- B13; A114-B14; A114-B15; A114-B16; A114-B17; A114-B18; A114-B19; A114-B20; A114-B21; A114-B22; A114-B23; A114-B24; A114-B25; A114-B26; A114-B27; A114-B28; A114-B29; A114-B30; A114-B31; A114-B32; A114-B33; A114-B34; A114-B35; A114-B36; A114-B37; A114- B38; A114-B39; A114-B40; A114-B41; A114-B42; A114-B43; A114-B44; A114-B45; A114-B46; A114-B47; A114-B48; A114-B49; A114-B50; A114-B51; A114-B52; A114-B53; A114-B54; A114-B55; A114-B56; A114-B57; A114-B58; A114-B59; A114-B60; A114-B61; A114-B62; A114- B63; A114-B64; A114-B65; A114-B66; A114-B67; A114-B68; A114-B69; A114-B70; A114-B71; A114-B72; A114-B73; A114-B74; A114-B75; A114-B76; A114-B77; A114-B78; A114-B79; A114-B80; A114-B81; A114-B82; A114-B83; A114-B84; A114-B85; A114-B86; A114-B87; A114- B88; A114-B89; A114-B90; A114-B91; A114-B92; A114-B93; A114-B94; A114-B95; A114-B96;A114-B97; A114-B98; A114-B99; A114-B100; A114-B101; A114-B102; A114-B103; A114-B104; A114-B105; A114-B106; A114-B107; A114-B108; A114- B109; A114-B110; A114-B111; A114-B112; A114-B113; A115-B1; A115-B2; A115-B3; A115-B4; A115-B5; A115-B6; A115-B7; A115-B8; A115-B9; A115-B10; A115-B11; A115-B12; A115- B13; A115-B14; A115-B15; A115-B16; A115-B17; A115-B18; A115-B19; A115-B20; A115-B21; A115-B22; A115-B23; A115-B24; A115-B25; A115-B26; A115-B27; A115-B28; A115-B29; A115-B30; A115-B31; A115-B32; A115-B33; A115-B34; A115-B35; A115-B36; A115-B37; A115- B38; A115-B39; A115-B40; A115-B41; A115-B42; A115-B43; A115-B44; A115-B45; A115-B46; A115-B47; A115-B48; A115-B49; A115-B50; A115-B51; A115-B52; A115-B53; A115-B54; A115-B55; A115-B56; A115-B57; A115-B58; A115-B59; A115-B60; A115-B61; A115-B62; A115- B63; A115-B64; A115-B65; A115-B66; A115-B67; A115-B68; A115-B69; A115-B70; A115-B71; A115-B72; A115-B73; A115-B74; A115-B75; A115-B76; A115-B77; A115-B78; A115-B79; A115-B80; A115-B81; A115-B82; A115-B83; A115-B84; A115-B85; A115-B86; A115-B87; A115- B88; A115-B89; A115-B90; A115-B91; A115-B92; A115-B93; A115-B94; A115-B95; A115-B96;A115-B97; A115-B98; A115-B99; A115-B100; A115-B101; A115-B102; A115-B103; A115-B104; A115-B105; A115-B106; A115-B107; A115-B108; A115- B109; A115-B110; A115-B111; A115-B112; A115-B113; A116-B1; A116-B2; A116-B3; A116-B4; A116-B5; A116-B6; A116-B7; A116-B8; A116-B9; A116-B10; A116-B11; A116-B12; A116- B13; A116-B14; A116-B15; A116-B16; A116-B17; A116-B18; A116-B19; A116-B20; A116-B21; A116-B22; A116-B23; A116-B24; A116-B25; A116-B26; A116-B27; A116-B28; A116-B29; A116-B30; A116-B31; A116-B32; A116-B33; A116-B34; A116-B35; A116-B36; A116-B37; A116- B38; A116-B39; A116-B40; A116-B41; A116-B42; A116-B43; A116-B44; A116-B45; A116-B46; A116-B47; A116-B48; A116-B49; A116-B50; A116-B51; A116-B52; A116-B53; A116-B54; A116-B55; A116-B56; A116-B57; A116-B58; A116-B59; A116-B60; A116-B61; A116-B62; A116- B63; A116-B64; A116-B65; A116-B66; A116-B67; A116-B68; A116-B69; A116-B70; A116-B71; A116-B72; A116-B73; A116-B74; A116-B75; A116-B76; A116-B77; A116-B78; A116-B79; A116-B80; A116-B81; A116-B82; A116-B83; A116-B84; A116-B85; A116-B86; A116-B87; A116- B88; A116-B89; A116-B90; A116-B91; A116-B92; A116-B93; A116-B94; A116-B95; A116-B96;A116-B97; A116-B98; A116-B99; A116-B100; A116-B101; A116-B102; A116-B103; A116-B104; A116-B105; A116-B106; A116-B107; A116-B108; A116- B109; A116-B110; A116-B111; A116-B112; A116-B113; A117-B1; A117-B2; A117-B3; A117-B4; A117-B5; A117-B6; A117-B7; A117-B8; A117-B9; A117-B10; A117-B11; A117-B12; A117- B13; A117-B14; A117-B15; A117-B16; A117-B17; A117-B18; A117-B19; A117-B20; A117-B21; A117-B22; A117-B23; A117-B24; A117-B25; A117-B26; A117-B27; A117-B28; A117-B29; A117-B30; A117-B31; A117-B32; A117-B33; A117-B34; A117-B35; A117-B36; A117-B37; A117- B38; A117-B39; A117-B40; A117-B41; A117-B42; A117-B43; A117-B44; A117-B45; A117-B46; A117-B47; A117-B48; A117-B49; A117-B50; A117-B51; A117-B52; A117-B53; A117-B54; A117-B55; A117-B56; A117-B57; A117-B58; A117-B59; A117-B60; A117-B61; A117-B62; A117- B63; A117-B64; A117-B65; A117-B66; A117-B67; A117-B68; A117-B69; A117-B70; A117-B71; A117-B72; A117-B73; A117-B74; A117-B75; A117-B76; A117-B77; A117-B78; A117-B79; A117-B80; A117-B81; A117-B82; A117-B83; A117-B84; A117-B85; A117-B86; A117-B87; A117- B88; A117-B89; A117-B90; A117-B91; A117-B92; A117-B93; A117-B94; A117-B95; A117-B96;A117-B97; A117-B98; A117-B99; A117-B100; A117-B101; A117-B102; A117-B103; A117-B104; A117-B105; A117-B106; A117-B107; A117-B108; A117- B109; A117-B110; A117-B111; A117-B112; A117-B113; A118-B1; A118-B2; A118-B3; A118-B4; A118-B5; A118-B6; A118-B7; A118-B8; A118-B9; A118-B10; A118-B11; A118-B12; A118- B13; A118-B14; A118-B15; A118-B16; A118-B17; A118-B18; A118-B19; A118-B20; A118-B21; A118-B22; A118-B23; A118-B24; A118-B25; A118-B26; A118-B27; A118-B28; A118-B29; A118-B30; A118-B31; A118-B32; A118-B33; A118-B34; A118-B35; A118-B36; A118-B37; A118- B38; A118-B39; A118-B40; A118-B41; A118-B42; A118-B43; A118-B44; A118-B45; A118-B46; A118-B47; A118-B48; A118-B49; A118-B50; A118-B51; A118-B52; A118-B53; A118-B54; A118-B55; A118-B56; A118-B57; A118-B58; A118-B59; A118-B60; A118-B61; A118-B62; A118- B63; A118-B64; A118-B65; A118-B66; A118-B67; A118-B68; A118-B69; A118-B70; A118-B71; A118-B72; A118-B73; A118-B74; A118-B75; A118-B76; A118-B77; A118-B78; A118-B79; A118-B80; A118-B81; A118-B82; A118-B83; A118-B84; A118-B85; A118-B86; A118-B87; A118- B88; A118-B89; A118-B90; A118-B91; A118-B92; A118-B93; A118-B94; A118-B95; A118-B96;A118-B97; A118-B98; A118-B99; A118-B100; A118-B101; A118-B102; A118-B103; A118-B104; A118-B105; A118-B106; A118-B107; A118-B108; A118- B109; A118-B110; A118-B111; A118-B112; A118-B113; A119-B1; A119-B2; A119-B3; A119-B4; A119-B5; A119-B6; A119-B7; A119-B8; A119-B9; A119-B10; A119-B11; A119-B12; A119- B13; A119-B14; A119-B15; A119-B16; A119-B17; A119-B18; A119-B19; A119-B20; A119-B21; A119-B22; A119-B23; A119-B24; A119-B25; A119-B26; A119-B27; A119-B28; A119-B29; A119-B30; A119-B31; A119-B32; A119-B33; A119-B34; A119-B35; A119-B36; A119-B37; A119- B38; A119-B39; A119-B40; A119-B41; A119-B42; A119-B43; A119-B44; A119-B45; A119-B46; A119-B47; A119-B48; A119-B49; A119-B50; A119-B51; A119-B52; A119-B53; A119-B54; A119-B55; A119-B56; A119-B57; A119-B58; A119-B59; A119-B60; A119-B61; A119-B62; A119- B63; A119-B64; A119-B65; A119-B66; A119-B67; A119-B68; A119-B69; A119-B70; A119-B71; A119-B72; A119-B73; A119-B74; A119-B75; A119-B76; A119-B77; A119-B78; A119-B79; A119-B80; A119-B81; A119-B82; A119-B83; A119-B84; A119-B85; A119-B86; A119-B87; A119- B88; A119-B89; A119-B90; A119-B91; A119-B92; A119-B93; A119-B94; A119-B95; A119-B96;A119-B97; A119-B98; A119-B99; A119-B100; A119-B101; A119-B102; A119-B103; A119-B104; A119-B105; A119-B106; A119-B107; A119-B108; A119- B109; A119-B110; A119-B111; A119-B112; A119-B113; A120-B1; A120-B2; A120-B3; A120-B4; A120-B5; A120-B6; A120-B7; A120-B8; A120-B9; A120-B10; A120-B11; A120-B12; A120- B13; A120-B14; A120-B15; A120-B16; A120-B17; A120-B18; A120-B19; A120-B20; A120-B21; A120-B22; A120-B23; A120-B24; A120-B25; A120-B26; A120-B27; A120-B28; A120-B29; A120-B30; A120-B31; A120-B32; A120-B33; A120-B34; A120-B35; A120-B36; A120-B37; A120- B38; A120-B39; A120-B40; A120-B41; A120-B42; A120-B43; A120-B44; A120-B45; A120-B46; A120-B47; A120-B48; A120-B49; A120-B50; A120-B51; A120-B52; A120-B53; A120-B54; A120-B55; A120-B56; A120-B57; A120-B58; A120-B59; A120-B60; A120-B61; A120-B62; A120- B63; A120-B64; A120-B65; A120-B66; A120-B67; A120-B68; A120-B69; A120-B70; A120-B71; A120-B72; A120-B73; A120-B74; A120-B75; A120-B76; A120-B77; A120-B78; A120-B79; A120-B80; A120-B81; A120-B82; A120-B83; A120-B84; A120-B85; A120-B86; A120-B87; A120- B88; A120-B89; A120-B90; A120-B91; A120-B92; A120-B93; A120-B94; A120-B95; A120-B96;A120-B97; A120-B98; A120-B99; A120-B100; A120-B101; A120-B102; A120-B103; A120-B104; A120-B105; A120-B106; A120-B107; A120-B108; A120- B109; A120-B110; A120-B111; A120-B112; A120-B113; A121-B1; A121-B2; A121-B3; A121-B4; A121-B5; A121-B6; A121-B7; A121-B8; A121-B9; A121-B10; A121-B11; A121-B12; A121- B13; A121-B14; A121-B15; A121-B16; A121-B17; A121-B18; A121-B19; A121-B20; A121-B21; A121-B22; A121-B23; A121-B24; A121-B25; A121-B26; A121-B27; A121-B28; A121-B29; A121-B30; A121-B31; A121-B32; A121-B33; A121-B34; A121-B35; A121-B36; A121-B37; A121- B38; A121-B39; A121-B40; A121-B41; A121-B42; A121-B43; A121-B44; A121-B45; A121-B46; A121-B47; A121-B48; A121-B49; A121-B50; A121-B51; A121-B52; A121-B53; A121-B54; A121-B55; A121-B56; A121-B57; A121-B58; A121-B59; A121-B60; A121-B61; A121-B62; A121- B63; A121-B64; A121-B65; A121-B66; A121-B67; A121-B68; A121-B69; A121-B70; A121-B71; A121-B72; A121-B73; A121-B74; A121-B75; A121-B76; A121-B77; A121-B78; A121-B79; A121-B80; A121-B81; A121-B82; A121-B83; A121-B84; A121-B85; A121-B86; A121-B87; A121- B88; A121-B89; A121-B90; A121-B91; A121-B92; A121-B93; A121-B94; A121-B95; A121-B96;A121-B97; A121-B98; A121-B99; A121-B100; A121-B101; A121-B102; A121-B103; A121-B104; A121-B105; A121-B106; A121-B107; A121-B108; A121- B109; A121-B110; A121-B111; A121-B112; A121-B113; A122-B1; A122-B2; A122-B3; A122-B4; A122-B5; A122-B6; A122-B7; A122-B8; A122-B9; A122-B10; A122-B11; A122-B12; A122- B13; A122-B14; A122-B15; A122-B16; A122-B17; A122-B18; A122-B19; A122-B20; A122-B21; A122-B22; A122-B23; A122-B24; A122-B25; A122-B26; A122-B27; A122-B28; A122-B29; A122-B30; A122-B31; A122-B32; A122-B33; A122-B34; A122-B35; A122-B36; A122-B37; A122- B38; A122-B39; A122-B40; A122-B41; A122-B42; A122-B43; A122-B44; A122-B45; A122-B46; A122-B47; A122-B48; A122-B49; A122-B50; A122-B51; A122-B52; A122-B53; A122-B54; A122-B55; A122-B56; A122-B57; A122-B58; A122-B59; A122-B60; A122-B61; A122-B62; A122- B63; A122-B64; A122-B65; A122-B66; A122-B67; A122-B68; A122-B69; A122-B70; A122-B71; A122-B72; A122-B73; A122-B74; A122-B75; A122-B76; A122-B77; A122-B78; A122-B79; A122-B80; A122-B81; A122-B82; A122-B83; A122-B84; A122-B85; A122-B86; A122-B87; A122- B88; A122-B89; A122-B90; A122-B91; A122-B92; A122-B93; A122-B94; A122-B95; A122-B96;A122-B97; A122-B98; A122-B99; A122-B100; A122-B101; A122-B102; A122-B103; A122-B104; A122-B105; A122-B106; A122-B107; A122-B108; A122- B109; A122-B110; A122-B111; A122-B112; A122-B113; A123-B1; A123-B2; A123-B3; A123-B4; A123-B5; A123-B6; A123-B7; A123-B8; A123-B9; A123-B10; A123-B11; A123-B12; A123- B13; A123-B14; A123-B15; A123-B16; A123-B17; A123-B18; A123-B19; A123-B20; A123-B21; A123-B22; A123-B23; A123-B24; A123-B25; A123-B26; A123-B27; A123-B28; A123-B29; A123-B30; A123-B31; A123-B32; A123-B33; A123-B34; A123-B35; A123-B36; A123-B37; A123- B38; A123-B39; A123-B40; A123-B41; A123-B42; A123-B43; A123-B44; A123-B45; A123-B46; A123-B47; A123-B48; A123-B49; A123-B50; A123-B51; A123-B52; A123-B53; A123-B54; A123-B55; A123-B56; A123-B57; A123-B58; A123-B59; A123-B60; A123-B61; A123-B62; A123- B63; A123-B64; A123-B65; A123-B66; A123-B67; A123-B68; A123-B69; A123-B70; A123-B71; A123-B72; A123-B73; A123-B74; A123-B75; A123-B76; A123-B77; A123-B78; A123-B79; A123-B80; A123-B81; A123-B82; A123-B83; A123-B84; A123-B85; A123-B86; A123-B87; A123- B88; A123-B89; A123-B90; A123-B91; A123-B92; A123-B93; A123-B94; A123-B95; A123-B96;A123-B97; A123-B98; A123-B99; A123-B100; A123-B101; A123-B102; A123-B103; A123-B104; A123-B105; A123-B106; A123-B107; A123-B108; A123- B109; A123-B110; A123-B111; A123-B112; A123-B113; A124-B1; A124-B2; A124-B3; A124-B4; A124-B5; A124-B6; A124-B7; A124-B8; A124-B9; A124-B10; A124-B11; A124-B12; A124- B13; A124-B14; A124-B15; A124-B16; A124-B17; A124-B18; A124-B19; A124-B20; A124-B21; A124-B22; A124-B23; A124-B24; A124-B25; A124-B26; A124-B27; A124-B28; A124-B29; A124-B30; A124-B31; A124-B32; A124-B33; A124-B34; A124-B35; A124-B36; A124-B37; A124- B38; A124-B39; A124-B40; A124-B41; A124-B42; A124-B43; A124-B44; A124-B45; A124-B46; A124-B47; A124-B48; A124-B49; A124-B50; A124-B51; A124-B52; A124-B53; A124-B54; A124-B55; A124-B56; A124-B57; A124-B58; A124-B59; A124-B60; A124-B61; A124-B62; A124- B63; A124-B64; A124-B65; A124-B66; A124-B67; A124-B68; A124-B69; A124-B70; A124-B71; A124-B72; A124-B73; A124-B74; A124-B75; A124-B76; A124-B77; A124-B78; A124-B79; A124-B80; A124-B81; A124-B82; A124-B83; A124-B84; A124-B85; A124-B86; A124-B87; A124- B88; A124-B89; A124-B90; A124-B91; A124-B92; A124-B93; A124-B94; A124-B95; A124-B96;A124-B97; A124-B98; A124-B99; A124-B100; A124-B101; A124-B102; A124-B103; A124-B104; A124-B105; A124-B106; A124-B107; A124-B108; A124- B109; A124-B110; A124-B111; A124-B112; A124-B113; A125-B1; A125-B2; A125-B3; A125-B4; A125-B5; A125-B6; A125-B7; A125-B8; A125-B9; A125-B10; A125-B11; A125-B12; A125- B13; A125-B14; A125-B15; A125-B16; A125-B17; A125-B18; A125-B19; A125-B20; A125-B21; A125-B22; A125-B23; A125-B24; A125-B25; A125-B26; A125-B27; A125-B28; A125-B29; A125-B30; A125-B31; A125-B32; A125-B33; A125-B34; A125-B35; A125-B36; A125-B37; A125- B38; A125-B39; A125-B40; A125-B41; A125-B42; A125-B43; A125-B44; A125-B45; A125-B46; A125-B47; A125-B48; A125-B49; A125-B50; A125-B51; A125-B52; A125-B53; A125-B54; A125-B55; A125-B56; A125-B57; A125-B58; A125-B59; A125-B60; A125-B61; A125-B62; A125- B63; A125-B64; A125-B65; A125-B66; A125-B67; A125-B68; A125-B69; A125-B70; A125-B71; A125-B72; A125-B73; A125-B74; A125-B75; A125-B76; A125-B77; A125-B78; A125-B79; A125-B80; A125-B81; A125-B82; A125-B83; A125-B84; A125-B85; A125-B86; A125-B87; A125- B88; A125-B89; A125-B90; A125-B91; A125-B92; A125-B93; A125-B94; A125-B95; A125-B96;A125-B97; A125-B98; A125-B99; A125-B100; A125-B101; A125-B102; A125-B103; A125-B104; A125-B105; A125-B106; A125-B107; A125-B108; A125- B109; A125-B110; A125-B111; A125-B112; A125-B113; A126-B1; A126-B2; A126-B3; A126-B4; A126-B5; A126-B6; A126-B7; A126-B8; A126-B9; A126-B10; A126-B11; A126-B12; A126- B13; A126-B14; A126-B15; A126-B16; A126-B17; A126-B18; A126-B19; A126-B20; A126-B21; A126-B22; A126-B23; A126-B24; A126-B25; A126-B26; A126-B27; A126-B28; A126-B29; A126-B30; A126-B31; A126-B32; A126-B33; A126-B34; A126-B35; A126-B36; A126-B37; A126- B38; A126-B39; A126-B40; A126-B41; A126-B42; A126-B43; A126-B44; A126-B45; A126-B46; A126-B47; A126-B48; A126-B49; A126-B50; A126-B51; A126-B52; A126-B53; A126-B54; A126-B55; A126-B56; A126-B57; A126-B58; A126-B59; A126-B60; A126-B61; A126-B62; A126- B63; A126-B64; A126-B65; A126-B66; A126-B67; A126-B68; A126-B69; A126-B70; A126-B71; A126-B72; A126-B73; A126-B74; A126-B75; A126-B76; A126-B77; A126-B78; A126-B79; A126-B80; A126-B81; A126-B82; A126-B83; A126-B84; A126-B85; A126-B86; A126-B87; A126- B88; A126-B89; A126-B90; A126-B91; A126-B92; A126-B93; A126-B94; A126-B95; A126-B96;A126-B97; A126-B98; A126-B99; A126-B100; A126-B101; A126-B102; A126-B103; A126-B104; A126-B105; A126-B106; A126-B107; A126-B108; A126- B109; A126-B110; A126-B111; A126-B112; A126-B113; A127-B1; A127-B2; A127-B3; A127-B4; A127-B5; A127-B6; A127-B7; A127-B8; A127-B9; A127-B10; A127-B11; A127-B12; A127- B13; A127-B14; A127-B15; A127-B16; A127-B17; A127-B18; A127-B19; A127-B20; A127-B21; A127-B22; A127-B23; A127-B24; A127-B25; A127-B26; A127-B27; A127-B28; A127-B29; A127-B30; A127-B31; A127-B32; A127-B33; A127-B34; A127-B35; A127-B36; A127-B37; A127- B38; A127-B39; A127-B40; A127-B41; A127-B42; A127-B43; A127-B44; A127-B45; A127-B46; A127-B47; A127-B48; A127-B49; A127-B50; A127-B51; A127-B52; A127-B53; A127-B54; A127-B55; A127-B56; A127-B57; A127-B58; A127-B59; A127-B60; A127-B61; A127-B62; A127- B63; A127-B64; A127-B65; A127-B66; A127-B67; A127-B68; A127-B69; A127-B70; A127-B71; A127-B72; A127-B73; A127-B74; A127-B75; A127-B76; A127-B77; A127-B78; A127-B79; A127-B80; A127-B81; A127-B82; A127-B83; A127-B84; A127-B85; A127-B86; A127-B87; A127- B88; A127-B89; A127-B90; A127-B91; A127-B92; A127-B93; A127-B94; A127-B95; A127-B96;A127-B97; A127-B98; A127-B99; A127-B100; A127-B101; A127-B102; A127-B103; A127-B104; A127-B105; A127-B106; A127-B107; A127-B108; A127- B109; A127-B110; A127-B111; A127-B112; A127-B113; A128-B1; A128-B2; A128-B3; A128-B4; A128-B5; A128-B6; A128-B7; A128-B8; A128-B9; A128-B10; A128-B11; A128-B12; A128- B13; A128-B14; A128-B15; A128-B16; A128-B17; A128-B18; A128-B19; A128-B20; A128-B21; A128-B22; A128-B23; A128-B24; A128-B25; A128-B26; A128-B27; A128-B28; A128-B29; A128-B30; A128-B31; A128-B32; A128-B33; A128-B34; A128-B35; A128-B36; A128-B37; A128- B38; A128-B39; A128-B40; A128-B41; A128-B42; A128-B43; A128-B44; A128-B45; A128-B46; A128-B47; A128-B48; A128-B49; A128-B50; A128-B51; A128-B52; A128-B53; A128-B54; A128-B55; A128-B56; A128-B57; A128-B58; A128-B59; A128-B60; A128-B61; A128-B62; A128- B63; A128-B64; A128-B65; A128-B66; A128-B67; A128-B68; A128-B69; A128-B70; A128-B71; A128-B72; A128-B73; A128-B74; A128-B75; A128-B76; A128-B77; A128-B78; A128-B79; A128-B80; A128-B81; A128-B82; A128-B83; A128-B84; A128-B85; A128-B86; A128-B87; A128- B88; A128-B89; A128-B90; A128-B91; A128-B92; A128-B93; A128-B94; A128-B95; A128-B96;A128-B97; A128-B98; A128-B99; A128-B100; A128-B101; A128-B102; A128-B103; A128-B104; A128-B105; A128-B106; A128-B107; A128-B108; A128- B109; A128-B110; A128-B111; A128-B112; A128-B113; A129-B1; A129-B2; A129-B3; A129-B4; A129-B5; A129-B6; A129-B7; A129-B8; A129-B9; A129-B10; A129-B11; A129-B12; A129- B13; A129-B14; A129-B15; A129-B16; A129-B17; A129-B18; A129-B19; A129-B20; A129-B21; A129-B22; A129-B23; A129-B24; A129-B25; A129-B26; A129-B27; A129-B28; A129-B29; A129-B30; A129-B31; A129-B32; A129-B33; A129-B34; A129-B35; A129-B36; A129-B37; A129- B38; A129-B39; A129-B40; A129-B41; A129-B42; A129-B43; A129-B44; A129-B45; A129-B46; A129-B47; A129-B48; A129-B49; A129-B50; A129-B51; A129-B52; A129-B53; A129-B54; A129-B55; A129-B56; A129-B57; A129-B58; A129-B59; A129-B60; A129-B61; A129-B62; A129- B63; A129-B64; A129-B65; A129-B66; A129-B67; A129-B68; A129-B69; A129-B70; A129-B71; A129-B72; A129-B73; A129-B74; A129-B75; A129-B76; A129-B77; A129-B78; A129-B79; A129-B80; A129-B81; A129-B82; A129-B83; A129-B84; A129-B85; A129-B86; A129-B87; A129- B88; A129-B89; A129-B90; A129-B91; A129-B92; A129-B93; A129-B94; A129-B95; A129-B96;A129-B97; A129-B98; A129-B99; A129-B100; A129-B101; A129-B102; A129-B103; A129-B104; A129-B105; A129-B106; A129-B107; A129-B108; A129- B109; A129-B110; A129-B111; A129-B112; A129-B113; A130-B1; A130-B2; A130-B3; A130-B4; A130-B5; A130-B6; A130-B7; A130-B8; A130-B9; A130-B10; A130-B11; A130-B12; A130- B13; A130-B14; A130-B15; A130-B16; A130-B17; A130-B18; A130-B19; A130-B20; A130-B21; A130-B22; A130-B23; A130-B24; A130-B25; A130-B26; A130-B27; A130-B28; A130-B29; A130-B30; A130-B31; A130-B32; A130-B33; A130-B34; A130-B35; A130-B36; A130-B37; A130- B38; A130-B39; A130-B40; A130-B41; A130-B42; A130-B43; A130-B44; A130-B45; A130-B46; A130-B47; A130-B48; A130-B49; A130-B50; A130-B51; A130-B52; A130-B53; A130-B54; A130-B55; A130-B56; A130-B57; A130-B58; A130-B59; A130-B60; A130-B61; A130-B62; A130- B63; A130-B64; A130-B65; A130-B66; A130-B67; A130-B68; A130-B69; A130-B70; A130-B71; A130-B72; A130-B73; A130-B74; A130-B75; A130-B76; A130-B77; A130-B78; A130-B79; A130-B80; A130-B81; A130-B82; A130-B83; A130-B84; A130-B85; A130-B86; A130-B87; A130- B88; A130-B89; A130-B90; A130-B91; A130-B92; A130-B93; A130-B94; A130-B95; A130-B96;A130-B97; A130-B98; A130-B99; A130-B100; A130-B101; A130-B102; A130-B103; A130-B104; A130-B105; A130-B106; A130-B107; A130-B108; A130- B109; A130-B110; A130-B111; A130-B112; A130-B113; A131-B1; A131-B2; A131-B3; A131-B4; A131-B5; A131-B6; A131-B7; A131-B8; A131-B9; A131-B10; A131-B11; A131-B12; A131- B13; A131-B14; A131-B15; A131-B16; A131-B17; A131-B18; A131-B19; A131-B20; A131-B21; A131-B22; A131-B23; A131-B24; A131-B25; A131-B26; A131-B27; A131-B28; A131-B29; A131-B30; A131-B31; A131-B32; A131-B33; A131-B34; A131-B35; A131-B36; A131-B37; A131- B38; A131-B39; A131-B40; A131-B41; A131-B42; A131-B43; A131-B44; A131-B45; A131-B46; A131-B47; A131-B48; A131-B49; A131-B50; A131-B51; A131-B52; A131-B53; A131-B54; A131-B55; A131-B56; A131-B57; A131-B58; A131-B59; A131-B60; A131-B61; A131-B62; A131- B63; A131-B64; A131-B65; A131-B66; A131-B67; A131-B68; A131-B69; A131-B70; A131-B71; A131-B72; A131-B73; A131-B74; A131-B75; A131-B76; A131-B77; A131-B78; A131-B79; A131-B80; A131-B81; A131-B82; A131-B83; A131-B84; A131-B85; A131-B86; A131-B87; A131- B88; A131-B89; A131-B90; A131-B91; A131-B92; A131-B93; A131-B94; A131-B95; A131-B96;A131-B97; A131-B98; A131-B99; A131-B100; A131-B101; A131-B102; A131-B103; A131-B104; A131-B105; A131-B106; A131-B107; A131-B108; A131- B109; A131-B110; A131-B111; A131-B112; A131-B113; A132-B1; A132-B2; A132-B3; A132-B4; A132-B5; A132-B6; A132-B7; A132-B8; A132-B9; A132-B10; A132-B11; A132-B12; A132- B13; A132-B14; A132-B15; A132-B16; A132-B17; A132-B18; A132-B19; A132-B20; A132-B21; A132-B22; A132-B23; A132-B24; A132-B25; A132-B26; A132-B27; A132-B28; A132-B29; A132-B30; A132-B31; A132-B32; A132-B33; A132-B34; A132-B35; A132-B36; A132-B37; A132- B38; A132-B39; A132-B40; A132-B41; A132-B42; A132-B43; A132-B44; A132-B45; A132-B46; A132-B47; A132-B48; A132-B49; A132-B50; A132-B51; A132-B52; A132-B53; A132-B54; A132-B55; A132-B56; A132-B57; A132-B58; A132-B59; A132-B60; A132-B61; A132-B62; A132- B63; A132-B64; A132-B65; A132-B66; A132-B67; A132-B68; A132-B69; A132-B70; A132-B71; A132-B72; A132-B73; A132-B74; A132-B75; A132-B76; A132-B77; A132-B78; A132-B79; A132-B80; A132-B81; A132-B82; A132-B83; A132-B84; A132-B85; A132-B86; A132-B87; A132- B88; A132-B89; A132-B90; A132-B91; A132-B92; A132-B93; A132-B94; A132-B95; A132-B96;A132-B97; A132-B98; A132-B99; A132-B100; A132-B101; A132-B102; A132-B103; A132-B104; A132-B105; A132-B106; A132-B107; A132-B108; A132- B109; A132-B110; A132-B111; A132-B112; A132-B113; A133-B1; A133-B2; A133-B3; A133-B4; A133-B5; A133-B6; A133-B7; A133-B8; A133-B9; A133-B10; A133-B11; A133-B12; A133- B13; A133-B14; A133-B15; A133-B16; A133-B17; A133-B18; A133-B19; A133-B20; A133-B21; A133-B22; A133-B23; A133-B24; A133-B25; A133-B26; A133-B27; A133-B28; A133-B29; A133-B30; A133-B31; A133-B32; A133-B33; A133-B34; A133-B35; A133-B36; A133-B37; A133- B38; A133-B39; A133-B40; A133-B41; A133-B42; A133-B43; A133-B44; A133-B45; A133-B46; A133-B47; A133-B48; A133-B49; A133-B50; A133-B51; A133-B52; A133-B53; A133-B54; A133-B55; A133-B56; A133-B57; A133-B58; A133-B59; A133-B60; A133-B61; A133-B62; A133- B63; A133-B64; A133-B65; A133-B66; A133-B67; A133-B68; A133-B69; A133-B70; A133-B71; A133-B72; A133-B73; A133-B74; A133-B75; A133-B76; A133-B77; A133-B78; A133-B79; A133-B80; A133-B81; A133-B82; A133-B83; A133-B84; A133-B85; A133-B86; A133-B87; A133- B88; A133-B89; A133-B90; A133-B91; A133-B92; A133-B93; A133-B94; A133-B95; A133-B96;A133-B97; A133-B98; A133-B99; A133-B100; A133-B101; A133-B102; A133-B103; A133-B104; A133-B105; A133-B106; A133-B107; A133-B108; A133- B109; A133-B110; A133-B111; A133-B112; A133-B113; A134-B1; A134-B2; A134-B3; A134-B4; A134-B5; A134-B6; A134-B7; A134-B8; A134-B9; A134-B10; A134-B11; A134-B12; A134- B13; A134-B14; A134-B15; A134-B16; A134-B17; A134-B18; A134-B19; A134-B20; A134-B21; A134-B22; A134-B23; A134-B24; A134-B25; A134-B26; A134-B27; A134-B28; A134-B29; A134-B30; A134-B31; A134-B32; A134-B33; A134-B34; A134-B35; A134-B36; A134-B37; A134- B38; A134-B39; A134-B40; A134-B41; A134-B42; A134-B43; A134-B44; A134-B45; A134-B46; A134-B47; A134-B48; A134-B49; A134-B50; A134-B51; A134-B52; A134-B53; A134-B54; A134-B55; A134-B56; A134-B57; A134-B58; A134-B59; A134-B60; A134-B61; A134-B62; A134- B63; A134-B64; A134-B65; A134-B66; A134-B67; A134-B68; A134-B69; A134-B70; A134-B71; A134-B72; A134-B73; A134-B74; A134-B75; A134-B76; A134-B77; A134-B78; A134-B79; A134-B80; A134-B81; A134-B82; A134-B83; A134-B84; A134-B85; A134-B86; A134-B87; A134- B88; A134-B89; A134-B90; A134-B91; A134-B92; A134-B93; A134-B94; A134-B95; A134-B96;A134-B97; A134-B98; A134-B99; A134-B100; A134-B101; A134-B102; A134-B103; A134-B104; A134-B105; A134-B106; A134-B107; A134-B108; A134- B109; A134-B110; A134-B111; A134-B112; A134-B113; A135-B1; A135-B2; A135-B3; A135-B4; A135-B5; A135-B6; A135-B7; A135-B8; A135-B9; A135-B10; A135-B11; A135-B12; A135- B13; A135-B14; A135-B15; A135-B16; A135-B17; A135-B18; A135-B19; A135-B20; A135-B21; A135-B22; A135-B23; A135-B24; A135-B25; A135-B26; A135-B27; A135-B28; A135-B29; A135-B30; A135-B31; A135-B32; A135-B33; A135-B34; A135-B35; A135-B36; A135-B37; A135- B38; A135-B39; A135-B40; A135-B41; A135-B42; A135-B43; A135-B44; A135-B45; A135-B46; A135-B47; A135-B48; A135-B49; A135-B50; A135-B51; A135-B52; A135-B53; A135-B54; A135-B55; A135-B56; A135-B57; A135-B58; A135-B59; A135-B60; A135-B61; A135-B62; A135- B63; A135-B64; A135-B65; A135-B66; A135-B67; A135-B68; A135-B69; A135-B70; A135-B71; A135-B72; A135-B73; A135-B74; A135-B75; A135-B76; A135-B77; A135-B78; A135-B79; A135-B80; A135-B81; A135-B82; A135-B83; A135-B84; A135-B85; A135-B86; A135-B87; A135- B88; A135-B89; A135-B90; A135-B91; A135-B92; A135-B93; A135-B94; A135-B95; A135-B96;A135-B97; A135-B98; A135-B99; A135-B100; A135-B101; A135-B102; A135-B103; A135-B104; A135-B105; A135-B106; A135-B107; A135-B108; A135- B109; A135-B110; A135-B111; A135-B112; A135-B113; A136-B1; A136-B2; A136-B3; A136-B4; A136-B5; A136-B6; A136-B7; A136-B8; A136-B9; A136-B10; A136-B11; A136-B12; A136- B13; A136-B14; A136-B15; A136-B16; A136-B17; A136-B18; A136-B19; A136-B20; A136-B21; A136-B22; A136-B23; A136-B24; A136-B25; A136-B26; A136-B27; A136-B28; A136-B29; A136-B30; A136-B31; A136-B32; A136-B33; A136-B34; A136-B35; A136-B36; A136-B37; A136- B38; A136-B39; A136-B40; A136-B41; A136-B42; A136-B43; A136-B44; A136-B45; A136-B46; A136-B47; A136-B48; A136-B49; A136-B50; A136-B51; A136-B52; A136-B53; A136-B54; A136-B55; A136-B56; A136-B57; A136-B58; A136-B59; A136-B60; A136-B61; A136-B62; A136- B63; A136-B64; A136-B65; A136-B66; A136-B67; A136-B68; A136-B69; A136-B70; A136-B71; A136-B72; A136-B73; A136-B74; A136-B75; A136-B76; A136-B77; A136-B78; A136-B79; A136-B80; A136-B81; A136-B82; A136-B83; A136-B84; A136-B85; A136-B86; A136-B87; A136- B88; A136-B89; A136-B90; A136-B91; A136-B92; A136-B93; A136-B94; A136-B95; A136-B96;A136-B97; A136-B98; A136-B99; A136-B100; A136-B101; A136-B102; A136-B103; A136-B104; A136-B105; A136-B106; A136-B107; A136-B108; A136- B109; A136-B110; A136-B111; A136-B112; A136-B113; A137-B1; A137-B2; A137-B3; A137-B4; A137-B5; A137-B6; A137-B7; A137-B8; A137-B9; A137-B10; A137-B11; A137-B12; A137- B13; A137-B14; A137-B15; A137-B16; A137-B17; A137-B18; A137-B19; A137-B20; A137-B21; A137-B22; A137-B23; A137-B24; A137-B25; A137-B26; A137-B27; A137-B28; A137-B29; A137-B30; A137-B31; A137-B32; A137-B33; A137-B34; A137-B35; A137-B36; A137-B37; A137- B38; A137-B39; A137-B40; A137-B41; A137-B42; A137-B43; A137-B44; A137-B45; A137-B46; A137-B47; A137-B48; A137-B49; A137-B50; A137-B51; A137-B52; A137-B53; A137-B54; A137-B55; A137-B56; A137-B57; A137-B58; A137-B59; A137-B60; A137-B61; A137-B62; A137- B63; A137-B64; A137-B65; A137-B66; A137-B67; A137-B68; A137-B69; A137-B70; A137-B71; A137-B72; A137-B73; A137-B74; A137-B75; A137-B76; A137-B77; A137-B78; A137-B79; A137-B80; A137-B81; A137-B82; A137-B83; A137-B84; A137-B85; A137-B86; A137-B87; A137- B88; A137-B89; A137-B90; A137-B91; A137-B92; A137-B93; A137-B94; A137-B95; A137-B96;A137-B97; A137-B98; A137-B99; A137-B100; A137-B101; A137-B102; A137-B103; A137-B104; A137-B105; A137-B106; A137-B107; A137-B108; A137- B109; A137-B110; A137-B111; A137-B112; A137-B113; A138-B1; A138-B2; A138-B3; A138-B4; A138-B5; A138-B6; A138-B7; A138-B8; A138-B9; A138-B10; A138-B11; A138-B12; A138- B13; A138-B14; A138-B15; A138-B16; A138-B17; A138-B18; A138-B19; A138-B20; A138-B21; A138-B22; A138-B23; A138-B24; A138-B25; A138-B26; A138-B27; A138-B28; A138-B29; A138-B30; A138-B31; A138-B32; A138-B33; A138-B34; A138-B35; A138-B36; A138-B37; A138- B38; A138-B39; A138-B40; A138-B41; A138-B42; A138-B43; A138-B44; A138-B45; A138-B46; A138-B47; A138-B48; A138-B49; A138-B50; A138-B51; A138-B52; A138-B53; A138-B54; A138-B55; A138-B56; A138-B57; A138-B58; A138-B59; A138-B60; A138-B61; A138-B62; A138- B63; A138-B64; A138-B65; A138-B66; A138-B67; A138-B68; A138-B69; A138-B70; A138-B71; A138-B72; A138-B73; A138-B74; A138-B75; A138-B76; A138-B77; A138-B78; A138-B79; A138-B80; A138-B81; A138-B82; A138-B83; A138-B84; A138-B85; A138-B86; A138-B87; A138- B88; A138-B89; A138-B90; A138-B91; A138-B92; A138-B93; A138-B94; A138-B95; A138-B96;A138-B97; A138-B98; A138-B99; A138-B100; A138-B101; A138-B102; A138-B103; A138-B104; A138-B105; A138-B106; A138-B107; A138-B108; A138- B109; A138-B110; A138-B111; A138-B112; A138-B113; A139-B1; A139-B2; A139-B3; A139-B4; A139-B5; A139-B6; A139-B7; A139-B8; A139-B9; A139-B10; A139-B11; A139-B12; A139- B13; A139-B14; A139-B15; A139-B16; A139-B17; A139-B18; A139-B19; A139-B20; A139-B21; A139-B22; A139-B23; A139-B24; A139-B25; A139-B26; A139-B27; A139-B28; A139-B29; A139-B30; A139-B31; A139-B32; A139-B33; A139-B34; A139-B35; A139-B36; A139-B37; A139- B38; A139-B39; A139-B40; A139-B41; A139-B42; A139-B43; A139-B44; A139-B45; A139-B46; A139-B47; A139-B48; A139-B49; A139-B50; A139-B51; A139-B52; A139-B53; A139-B54; A139-B55; A139-B56; A139-B57; A139-B58; A139-B59; A139-B60; A139-B61; A139-B62; A139- B63; A139-B64; A139-B65; A139-B66; A139-B67; A139-B68; A139-B69; A139-B70; A139-B71; A139-B72; A139-B73; A139-B74; A139-B75; A139-B76; A139-B77; A139-B78; A139-B79; A139-B80; A139-B81; A139-B82; A139-B83; A139-B84; A139-B85; A139-B86; A139-B87; A139- B88; A139-B89; A139-B90; A139-B91; A139-B92; A139-B93; A139-B94; A139-B95; A139-B96;A139-B97; A139-B98; A139-B99; A139-B100; A139-B101; A139-B102; A139-B103; A139-B104; A139-B105; A139-B106; A139-B107; A139-B108; A139- B109; A139-B110; A139-B111; A139-B112; A139-B113; A140-B1; A140-B2; A140-B3; A140-B4; A140-B5; A140-B6; A140-B7; A140-B8; A140-B9; A140-B10; A140-B11; A140-B12; A140- B13; A140-B14; A140-B15; A140-B16; A140-B17; A140-B18; A140-B19; A140-B20; A140-B21; A140-B22; A140-B23; A140-B24; A140-B25; A140-B26; A140-B27; A140-B28; A140-B29; A140-B30; A140-B31; A140-B32; A140-B33; A140-B34; A140-B35; A140-B36; A140-B37; A140- B38; A140-B39; A140-B40; A140-B41; A140-B42; A140-B43; A140-B44; A140-B45; A140-B46; A140-B47; A140-B48; A140-B49; A140-B50; A140-B51; A140-B52; A140-B53; A140-B54; A140-B55; A140-B56; A140-B57; A140-B58; A140-B59; A140-B60; A140-B61; A140-B62; A140- B63; A140-B64; A140-B65; A140-B66; A140-B67; A140-B68; A140-B69; A140-B70; A140-B71; A140-B72; A140-B73; A140-B74; A140-B75; A140-B76; A140-B77; A140-B78; A140-B79; A140-B80; A140-B81; A140-B82; A140-B83; A140-B84; A140-B85; A140-B86; A140-B87; A140- B88; A140-B89; A140-B90; A140-B91; A140-B92; A140-B93; A140-B94; A140-B95; A140-B96;A140-B97; A140-B98; A140-B99; A140-B100; A140-B101; A140-B102; A140-B103; A140-B104; A140-B105; A140-B106; A140-B107; A140-B108; A140- B109; A140-B110; A140-B111; A140-B112; A140-B113; A141-B1; A141-B2; A141-B3; A141-B4; A141-B5; A141-B6; A141-B7; A141-B8; A141-B9; A141-B10; A141-B11; A141-B12; A141- B13; A141-B14; A141-B15; A141-B16; A141-B17; A141-B18; A141-B19; A141-B20; A141-B21; A141-B22; A141-B23; A141-B24; A141-B25; A141-B26; A141-B27; A141-B28; A141-B29; A141-B30; A141-B31; A141-B32; A141-B33; A141-B34; A141-B35; A141-B36; A141-B37; A141- B38; A141-B39; A141-B40; A141-B41; A141-B42; A141-B43; A141-B44; A141-B45; A141-B46; A141-B47; A141-B48; A141-B49; A141-B50; A141-B51; A141-B52; A141-B53; A141-B54; A141-B55; A141-B56; A141-B57; A141-B58; A141-B59; A141-B60; A141-B61; A141-B62; A141- B63; A141-B64; A141-B65; A141-B66; A141-B67; A141-B68; A141-B69; A141-B70; A141-B71; A141-B72; A141-B73; A141-B74; A141-B75; A141-B76; A141-B77; A141-B78; A141-B79; A141-B80; A141-B81; A141-B82; A141-B83; A141-B84; A141-B85; A141-B86; A141-B87; A141- B88; A141-B89; A141-B90; A141-B91; A141-B92; A141-B93; A141-B94; A141-B95; A141-B96;A141-B97; A141-B98; A141-B99; A141-B100; A141-B101; A141-B102; A141-B103; A141-B104; A141-B105; A141-B106; A141-B107; A141-B108; A141- B109; A141-B110; A141-B111; A141-B112; A141-B113; A142-B1; A142-B2; A142-B3; A142-B4; A142-B5; A142-B6; A142-B7; A142-B8; A142-B9; A142-B10; A142-B11; A142-B12; A142- B13; A142-B14; A142-B15; A142-B16; A142-B17; A142-B18; A142-B19; A142-B20; A142-B21; A142-B22; A142-B23; A142-B24; A142-B25; A142-B26; A142-B27; A142-B28; A142-B29; A142-B30; A142-B31; A142-B32; A142-B33; A142-B34; A142-B35; A142-B36; A142-B37; A142- B38; A142-B39; A142-B40; A142-B41; A142-B42; A142-B43; A142-B44; A142-B45; A142-B46; A142-B47; A142-B48; A142-B49; A142-B50; A142-B51; A142-B52; A142-B53; A142-B54; A142-B55; A142-B56; A142-B57; A142-B58; A142-B59; A142-B60; A142-B61; A142-B62; A142- B63; A142-B64; A142-B65; A142-B66; A142-B67; A142-B68; A142-B69; A142-B70; A142-B71; A142-B72; A142-B73; A142-B74; A142-B75; A142-B76; A142-B77; A142-B78; A142-B79; A142-B80; A142-B81; A142-B82; A142-B83; A142-B84; A142-B85; A142-B86; A142-B87; A142- B88; A142-B89; A142-B90; A142-B91; A142-B92; A142-B93; A142-B94; A142-B95; A142-B96;A142-B97; A142-B98; A142-B99; A142-B100; A142-B101; A142-B102; A142-B103; A142-B104; A142-B105; A142-B106; A142-B107; A142-B108; A142- B109; A142-B110; A142-B111; A142-B112; A142-B113; A143-B1; A143-B2; A143-B3; A143-B4; A143-B5; A143-B6; A143-B7; A143-B8; A143-B9; A143-B10; A143-B11; A143-B12; A143- B13; A143-B14; A143-B15; A143-B16; A143-B17; A143-B18; A143-B19; A143-B20; A143-B21; A143-B22; A143-B23; A143-B24; A143-B25; A143-B26; A143-B27; A143-B28; A143-B29; A143-B30; A143-B31; A143-B32; A143-B33; A143-B34; A143-B35; A143-B36; A143-B37; A143- B38; A143-B39; A143-B40; A143-B41; A143-B42; A143-B43; A143-B44; A143-B45; A143-B46; A143-B47; A143-B48; A143-B49; A143-B50; A143-B51; A143-B52; A143-B53; A143-B54; A143-B55; A143-B56; A143-B57; A143-B58; A143-B59; A143-B60; A143-B61; A143-B62; A143- B63; A143-B64; A143-B65; A143-B66; A143-B67; A143-B68; A143-B69; A143-B70; A143-B71; A143-B72; A143-B73; A143-B74; A143-B75; A143-B76; A143-B77; A143-B78; A143-B79; A143-B80; A143-B81; A143-B82; A143-B83; A143-B84; A143-B85; A143-B86; A143-B87; A143- B88; A143-B89; A143-B90; A143-B91; A143-B92; A143-B93; A143-B94; A143-B95; A143-B96;A143-B97; A143-B98; A143-B99; A143-B100; A143-B101; A143-B102; A143-B103; A143-B104; A143-B105; A143-B106; A143-B107; A143-B108; A143- B109; A143-B110; A143-B111; A143-B112; A143-B113; A144-B1; A144-B2; A144-B3; A144-B4; A144-B5; A144-B6; A144-B7; A144-B8; A144-B9; A144-B10; A144-B11; A144-B12; A144- B13; A144-B14; A144-B15; A144-B16; A144-B17; A144-B18; A144-B19; A144-B20; A144-B21; A144-B22; A144-B23; A144-B24; A144-B25; A144-B26; A144-B27; A144-B28; A144-B29; A144-B30; A144-B31; A144-B32; A144-B33; A144-B34; A144-B35; A144-B36; A144-B37; A144- B38; A144-B39; A144-B40; A144-B41; A144-B42; A144-B43; A144-B44; A144-B45; A144-B46; A144-B47; A144-B48; A144-B49; A144-B50; A144-B51; A144-B52; A144-B53; A144-B54; A144-B55; A144-B56; A144-B57; A144-B58; A144-B59; A144-B60; A144-B61; A144-B62; A144- B63; A144-B64; A144-B65; A144-B66; A144-B67; A144-B68; A144-B69; A144-B70; A144-B71; A144-B72; A144-B73; A144-B74; A144-B75; A144-B76; A144-B77; A144-B78; A144-B79; A144-B80; A144-B81; A144-B82; A144-B83; A144-B84; A144-B85; A144-B86; A144-B87; A144- B88; A144-B89; A144-B90; A144-B91; A144-B92; A144-B93; A144-B94; A144-B95; A144-B96;A144-B97; A144-B98; A144-B99; A144-B100; A144-B101; A144-B102; A144-B103; A144-B104; A144-B105; A144-B106; A144-B107; A144-B108; A144- B109; A144-B110; A144-B111; A144-B112; A144-B113; A145-B1; A145-B2; A145-B3; A145-B4; A145-B5; A145-B6; A145-B7; A145-B8; A145-B9; A145-B10; A145-B11; A145-B12; A145- B13; A145-B14; A145-B15; A145-B16; A145-B17; A145-B18; A145-B19; A145-B20; A145-B21; A145-B22; A145-B23; A145-B24; A145-B25; A145-B26; A145-B27; A145-B28; A145-B29; A145-B30; A145-B31; A145-B32; A145-B33; A145-B34; A145-B35; A145-B36; A145-B37; A145- B38; A145-B39; A145-B40; A145-B41; A145-B42; A145-B43; A145-B44; A145-B45; A145-B46; A145-B47; A145-B48; A145-B49; A145-B50; A145-B51; A145-B52; A145-B53; A145-B54; A145-B55; A145-B56; A145-B57; A145-B58; A145-B59; A145-B60; A145-B61; A145-B62; A145- B63; A145-B64; A145-B65; A145-B66; A145-B67; A145-B68; A145-B69; A145-B70; A145-B71; A145-B72; A145-B73; A145-B74; A145-B75; A145-B76; A145-B77; A145-B78; A145-B79; A145-B80; A145-B81; A145-B82; A145-B83; A145-B84; A145-B85; A145-B86; A145-B87; A145- B88; A145-B89; A145-B90; A145-B91; A145-B92; A145-B93; A145-B94; A145-B95; A145-B96;A145-B97; A145-B98; A145-B99; A145-B100; A145-B101; A145-B102; A145-B103; A145-B104; A145-B105; A145-B106; A145-B107; A145-B108; A145- B109; A145-B110; A145-B111; A145-B112; A145-B113; A146-B1; A146-B2; A146-B3; A146-B4; A146-B5; A146-B6; A146-B7; A146-B8; A146-B9; A146-B10; A146-B11; A146-B12; A146- B13; A146-B14; A146-B15; A146-B16; A146-B17; A146-B18; A146-B19; A146-B20; A146-B21; A146-B22; A146-B23; A146-B24; A146-B25; A146-B26; A146-B27; A146-B28; A146-B29; A146-B30; A146-B31; A146-B32; A146-B33; A146-B34; A146-B35; A146-B36; A146-B37; A146- B38; A146-B39; A146-B40; A146-B41; A146-B42; A146-B43; A146-B44; A146-B45; A146-B46; A146-B47; A146-B48; A146-B49; A146-B50; A146-B51; A146-B52; A146-B53; A146-B54; A146-B55; A146-B56; A146-B57; A146-B58; A146-B59; A146-B60; A146-B61; A146-B62; A146- B63; A146-B64; A146-B65; A146-B66; A146-B67; A146-B68; A146-B69; A146-B70; A146-B71; A146-B72; A146-B73; A146-B74; A146-B75; A146-B76; A146-B77; A146-B78; A146-B79; A146-B80; A146-B81; A146-B82; A146-B83; A146-B84; A146-B85; A146-B86; A146-B87; A146- B88; A146-B89; A146-B90; A146-B91; A146-B92; A146-B93; A146-B94; A146-B95; A146-B96;A146-B97; A146-B98; A146-B99; A146-B100; A146-B101; A146-B102; A146-B103; A146-B104; A146-B105; A146-B106; A146-B107; A146-B108; A146- B109; A146-B110; A146-B111; A146-B112; A146-B113; A147-B1; A147-B2; A147-B3; A147-B4; A147-B5; A147-B6; A147-B7; A147-B8; A147-B9; A147-B10; A147-B11; A147-B12; A147- B13; A147-B14; A147-B15; A147-B16; A147-B17; A147-B18; A147-B19; A147-B20; A147-B21; A147-B22; A147-B23; A147-B24; A147-B25; A147-B26; A147-B27; A147-B28; A147-B29; A147-B30; A147-B31; A147-B32; A147-B33; A147-B34; A147-B35; A147-B36; A147-B37; A147- B38; A147-B39; A147-B40; A147-B41; A147-B42; A147-B43; A147-B44; A147-B45; A147-B46; A147-B47; A147-B48; A147-B49; A147-B50; A147-B51; A147-B52; A147-B53; A147-B54; A147-B55; A147-B56; A147-B57; A147-B58; A147-B59; A147-B60; A147-B61; A147-B62; A147- B63; A147-B64; A147-B65; A147-B66; A147-B67; A147-B68; A147-B69; A147-B70; A147-B71; A147-B72; A147-B73; A147-B74; A147-B75; A147-B76; A147-B77; A147-B78; A147-B79; A147-B80; A147-B81; A147-B82; A147-B83; A147-B84; A147-B85; A147-B86; A147-B87; A147- B88; A147-B89; A147-B90; A147-B91; A147-B92; A147-B93; A147-B94; A147-B95; A147-B96;A147-B97; A147-B98; A147-B99; A147-B100; A147-B101; A147-B102; A147-B103; A147-B104; A147-B105; A147-B106; A147-B107; A147-B108; A147- B109; A147-B110; A147-B111; A147-B112; A147-B113; A148-B1; A148-B2; A148-B3; A148-B4; A148-B5; A148-B6; A148-B7; A148-B8; A148-B9; A148-B10; A148-B11; A148-B12; A148- B13; A148-B14; A148-B15; A148-B16; A148-B17; A148-B18; A148-B19; A148-B20; A148-B21; A148-B22; A148-B23; A148-B24; A148-B25; A148-B26; A148-B27; A148-B28; A148-B29; A148-B30; A148-B31; A148-B32; A148-B33; A148-B34; A148-B35; A148-B36; A148-B37; A148- B38; A148-B39; A148-B40; A148-B41; A148-B42; A148-B43; A148-B44; A148-B45; A148-B46; A148-B47; A148-B48; A148-B49; A148-B50; A148-B51; A148-B52; A148-B53; A148-B54; A148-B55; A148-B56; A148-B57; A148-B58; A148-B59; A148-B60; A148-B61; A148-B62; A148- B63; A148-B64; A148-B65; A148-B66; A148-B67; A148-B68; A148-B69; A148-B70; A148-B71; A148-B72; A148-B73; A148-B74; A148-B75; A148-B76; A148-B77; A148-B78; A148-B79; A148-B80; A148-B81; A148-B82; A148-B83; A148-B84; A148-B85; A148-B86; A148-B87; A148- B88; A148-B89; A148-B90; A148-B91; A148-B92; A148-B93; A148-B94; A148-B95; A148-B96;A148-B97; A148-B98; A148-B99; A148-B100; A148-B101; A148-B102; A148-B103; A148-B104; A148-B105; A148-B106; A148-B107; A148-B108; A148- B109; A148-B110; A148-B111; A148-B112; A148-B113; A149-B1; A149-B2; A149-B3; A149-B4; A149-B5; A149-B6; A149-B7; A149-B8; A149-B9; A149-B10; A149-B11; A149-B12; A149- B13; A149-B14; A149-B15; A149-B16; A149-B17; A149-B18; A149-B19; A149-B20; A149-B21; A149-B22; A149-B23; A149-B24; A149-B25; A149-B26; A149-B27; A149-B28; A149-B29; A149-B30; A149-B31; A149-B32; A149-B33; A149-B34; A149-B35; A149-B36; A149-B37; A149- B38; A149-B39; A149-B40; A149-B41; A149-B42; A149-B43; A149-B44; A149-B45; A149-B46; A149-B47; A149-B48; A149-B49; A149-B50; A149-B51; A149-B52; A149-B53; A149-B54; A149-B55; A149-B56; A149-B57; A149-B58; A149-B59; A149-B60; A149-B61; A149-B62; A149- B63; A149-B64; A149-B65; A149-B66; A149-B67; A149-B68; A149-B69; A149-B70; A149-B71; A149-B72; A149-B73; A149-B74; A149-B75; A149-B76; A149-B77; A149-B78; A149-B79; A149-B80; A149-B81; A149-B82; A149-B83; A149-B84; A149-B85; A149-B86; A149-B87; A149- B88; A149-B89; A149-B90; A149-B91; A149-B92; A149-B93; A149-B94; A149-B95; A149-B96;A149-B97; A149-B98; A149-B99; A149-B100; A149-B101; A149-B102; A149-B103; A149-B104; A149-B105; A149-B106; A149-B107; A149-B108; A149- B109; A149-B110; A149-B111; A149-B112; A149-B113; A150-B1; A150-B2; A150-B3; A150-B4; A150-B5; A150-B6; A150-B7; A150-B8; A150-B9; A150-B10; A150-B11; A150-B12; A150- B13; A150-B14; A150-B15; A150-B16; A150-B17; A150-B18; A150-B19; A150-B20; A150-B21; A150-B22; A150-B23; A150-B24; A150-B25; A150-B26; A150-B27; A150-B28; A150-B29; A150-B30; A150-B31; A150-B32; A150-B33; A150-B34; A150-B35; A150-B36; A150-B37; A150- B38; A150-B39; A150-B40; A150-B41; A150-B42; A150-B43; A150-B44; A150-B45; A150-B46; A150-B47; A150-B48; A150-B49; A150-B50; A150-B51; A150-B52; A150-B53; A150-B54; A150-B55; A150-B56; A150-B57; A150-B58; A150-B59; A150-B60; A150-B61; A150-B62; A150- B63; A150-B64; A150-B65; A150-B66; A150-B67; A150-B68; A150-B69; A150-B70; A150-B71; A150-B72; A150-B73; A150-B74; A150-B75; A150-B76; A150-B77; A150-B78; A150-B79; A150-B80; A150-B81; A150-B82; A150-B83; A150-B84; A150-B85; A150-B86; A150-B87; A150- B88; A150-B89; A150-B90; A150-B91; A150-B92; A150-B93; A150-B94; A150-B95; A150-B96;A150-B97; A150-B98; A150-B99; A150-B100; A150-B101; A150-B102; A150-B103; A150-B104; A150-B105; A150-B106; A150-B107; A150-B108; A150- B109; A150-B110; A150-B111; A150-B112; A150-B113; A151-B1; A151-B2; A151-B3; A151-B4; A151-B5; A151-B6; A151-B7; A151-B8; A151-B9; A151-B10; A151-B11; A151-B12; A151- B13; A151-B14; A151-B15; A151-B16; A151-B17; A151-B18; A151-B19; A151-B20; A151-B21; A151-B22; A151-B23; A151-B24; A151-B25; A151-B26; A151-B27; A151-B28; A151-B29; A151-B30; A151-B31; A151-B32; A151-B33; A151-B34; A151-B35; A151-B36; A151-B37; A151- B38; A151-B39; A151-B40; A151-B41; A151-B42; A151-B43; A151-B44; A151-B45; A151-B46; A151-B47; A151-B48; A151-B49; A151-B50; A151-B51; A151-B52; A151-B53; A151-B54; A151-B55; A151-B56; A151-B57; A151-B58; A151-B59; A151-B60; A151-B61; A151-B62; A151- B63; A151-B64; A151-B65; A151-B66; A151-B67; A151-B68; A151-B69; A151-B70; A151-B71; A151-B72; A151-B73; A151-B74; A151-B75; A151-B76; A151-B77; A151-B78; A151-B79; A151-B80; A151-B81; A151-B82; A151-B83; A151-B84; A151-B85; A151-B86; A151-B87; A151- B88; A151-B89; A151-B90; A151-B91; A151-B92; A151-B93; A151-B94; A151-B95; A151-B96;A151-B97; A151-B98; A151-B99; A151-B100; A151-B101; A151-B102; A151-B103; A151-B104; A151-B105; A151-B106; A151-B107; A151-B108; A151- B109; A151-B110; A151-B111; A151-B112; A151-B113; A152-B1; A152-B2; A152-B3; A152-B4; A152-B5; A152-B6; A152-B7; A152-B8; A152-B9; A152-B10; A152-B11; A152-B12; A152- B13; A152-B14; A152-B15; A152-B16; A152-B17; A152-B18; A152-B19; A152-B20; A152-B21; A152-B22; A152-B23; A152-B24; A152-B25; A152-B26; A152-B27; A152-B28; A152-B29; A152-B30; A152-B31; A152-B32; A152-B33; A152-B34; A152-B35; A152-B36; A152-B37; A152- B38; A152-B39; A152-B40; A152-B41; A152-B42; A152-B43; A152-B44; A152-B45; A152-B46; A152-B47; A152-B48; A152-B49; A152-B50; A152-B51; A152-B52; A152-B53; A152-B54; A152-B55; A152-B56; A152-B57; A152-B58; A152-B59; A152-B60; A152-B61; A152-B62; A152- B63; A152-B64; A152-B65; A152-B66; A152-B67; A152-B68; A152-B69; A152-B70; A152-B71; A152-B72; A152-B73; A152-B74; A152-B75; A152-B76; A152-B77; A152-B78; A152-B79; A152-B80; A152-B81; A152-B82; A152-B83; A152-B84; A152-B85; A152-B86; A152-B87; A152- B88; A152-B89; A152-B90; A152-B91; A152-B92; A152-B93; A152-B94; A152-B95; A152-B96;A152-B97; A152-B98; A152-B99; A152-B100; A152-B101; A152-B102; A152-B103; A152-B104; A152-B105; A152-B106; A152-B107; A152-B108; A152- B109; A152-B110; A152-B111; A152-B112; A152-B113; A153-B1; A153-B2; A153-B3; A153-B4; A153-B5; A153-B6; A153-B7; A153-B8; A153-B9; A153-B10; A153-B11; A153-B12; A153- B13; A153-B14; A153-B15; A153-B16; A153-B17; A153-B18; A153-B19; A153-B20; A153-B21; A153-B22; A153-B23; A153-B24; A153-B25; A153-B26; A153-B27; A153-B28; A153-B29; A153-B30; A153-B31; A153-B32; A153-B33; A153-B34; A153-B35; A153-B36; A153-B37; A153- B38; A153-B39; A153-B40; A153-B41; A153-B42; A153-B43; A153-B44; A153-B45; A153-B46; A153-B47; A153-B48; A153-B49; A153-B50; A153-B51; A153-B52; A153-B53; A153-B54; A153-B55; A153-B56; A153-B57; A153-B58; A153-B59; A153-B60; A153-B61; A153-B62; A153- B63; A153-B64; A153-B65; A153-B66; A153-B67; A153-B68; A153-B69; A153-B70; A153-B71; A153-B72; A153-B73; A153-B74; A153-B75; A153-B76; A153-B77; A153-B78; A153-B79; A153-B80; A153-B81; A153-B82; A153-B83; A153-B84; A153-B85; A153-B86; A153-B87; A153- B88; A153-B89; A153-B90; A153-B91; A153-B92; A153-B93; A153-B94; A153-B95; A153-B96;A153-B97; A153-B98; A153-B99; A153-B100; A153-B101; A153-B102; A153-B103; A153-B104; A153-B105; A153-B106; A153-B107; A153-B108; A153- B109; A153-B110; A153-B111; A153-B112; A153-B113; A154-B1; A154-B2; A154-B3; A154-B4; A154-B5; A154-B6; A154-B7; A154-B8; A154-B9; A154-B10; A154-B11; A154-B12; A154- B13; A154-B14; A154-B15; A154-B16; A154-B17; A154-B18; A154-B19; A154-B20; A154-B21; A154-B22; A154-B23; A154-B24; A154-B25; A154-B26; A154-B27; A154-B28; A154-B29; A154-B30; A154-B31; A154-B32; A154-B33; A154-B34; A154-B35; A154-B36; A154-B37; A154- B38; A154-B39; A154-B40; A154-B41; A154-B42; A154-B43; A154-B44; A154-B45; A154-B46; A154-B47; A154-B48; A154-B49; A154-B50; A154-B51; A154-B52; A154-B53; A154-B54; A154-B55; A154-B56; A154-B57; A154-B58; A154-B59; A154-B60; A154-B61; A154-B62; A154- B63; A154-B64; A154-B65; A154-B66; A154-B67; A154-B68; A154-B69; A154-B70; A154-B71; A154-B72; A154-B73; A154-B74; A154-B75; A154-B76; A154-B77; A154-B78; A154-B79; A154-B80; A154-B81; A154-B82; A154-B83; A154-B84; A154-B85; A154-B86; A154-B87; A154- B88; A154-B89; A154-B90; A154-B91; A154-B92; A154-B93; A154-B94; A154-B95; A154-B96;A154-B97; A154-B98; A154-B99; A154-B100; A154-B101; A154-B102; A154-B103; A154-B104; A154-B105; A154-B106; A154-B107; A154-B108; A154- B109; A154-B110; A154-B111; A154-B112; A154-B113; Subcategory IA: Amino acid B of the dipeptide prodrug element is N-alkylated glycine In some embodiments, the amino acid B of the dipeptide prodrug element is N-alkylated glycine. A non-limiting example of a dipeptide prodrug element having N-alkylated glycine as the amino acid of B is shown in the table below.
<img id="000115" he="78" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />
Subcategory IB: Amino acid B of the dipeptide prodrug element is unsubstituted or mono-substituted at the β-position In some embodiments, the amino acid B of the dipeptide prodrug element is unsubstituted or mono-substituted at the β-position and has a relatively non-bulky side chain. A non-limiting example of a dipeptide prodrug element having an amino acid of B that is unsubstituted or monosubstituted at the β-position and a side chain that is not relatively bulky is shown in the table below.
<img id="000116" he="223" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000117" he="226" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000118" he="226" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000119" he="103" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />
In some embodiments, the amino acid B of the dipeptide prodrug element is mono-substituted at the β-position and has a relatively bulky side chain, as shown in the table below.
<img id="000120" he="200" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />
Subcategory IC: Amino acid B of dipeptide prodrug element disubstituted at β-position In some embodiments, the amino acid B of the dipeptide prodrug element is disubstituted at the β-position. A non-limiting example of a dipeptide prodrug element having an amino acid of B disubstituted at the β position is shown in the table below.
<img id="000121" he="200" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" />
In some exemplary embodiments, Aib-Gly (N-hexyl), dLys-Gly (N-hexyl), dCys-Gly (N-hexyl), dAla-Gly (N-hexyl), Aib-Gly ( N-methyl), dLys-Gly (N-methyl), dCys-Gly (N-methyl), dAla-Gly (N-hexyl), Aib-Phe (N-methyl), dLys-Phe (N-methyl), dCys-Phe (N-methyl) or dAla-Phe (N-methyl) binds to the N-terminal α-amino group of the peptide drug.
According to one embodiment, the dipeptide element has one of three amino acids in B of the AB dipeptide, Gly (N-hexyl), Gly (N-methyl) or Phe (N-methyl).
The dipeptide selected from one of these three dipeptide groups has a relative cleavage rate of Gly (N-hexyl)> Gly (N-methyl)> Phe (N-methyl) when all other factors are equal. )become. In one embodiment, Cys or Lys is provided at the first position (ie, the amino acid of A) as a site for acylation or PEGylation. Ala is used as the amino acid of A in one embodiment where acylation or PEGylation is not desired. In one embodiment, the Aib at position 1 (ie, the amino acid A) increases the cleavage rate compared to natural amino acids such as Ala, Cys, Lys.
Examples of dipeptides include the following.
dAla-Phe (N-methyl) dCys-Phe (N-methyl) dLys-Phe (N-methyl) Aib-Phe (N-methyl) dAla-Gly (N-methyl) dCys-Gly (N-methyl) dLys-Gly (N-methyl) Aib-Gly (N-methyl) dAla-Gly (N-hexyl) dCys-Gly (N-hexyl) dLys-Gly (N-hexyl) Aib-Gly (N-hexyl)
According to one embodiment, any of the novel insulin prodrug analogs disclosed herein are preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% purity levels. , 97%, 98% or 99%, and comprises a pharmaceutically acceptable diluent, carrier or excipient. Such compositions contain at least 0.5 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml of A19 insulin analogs as disclosed herein. , 7mg / ml, 8mg / ml, 9mg / ml, 10mg / ml, 11mg / ml, 12mg / ml, 13mg / ml, 14mg / ml, 15mg / ml, 16mg / ml, 17mg / ml, 18mg / ml, 19mg It may be contained in / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml or higher. In one embodiment, the pharmaceutical composition comprises an aqueous solution that is sterilized and optionally stored and housed in various packaging containers. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be packaged as part of a kit containing a disposable device for administering the composition to a patient. The container or kit may be labeled for storage at room temperature or refrigerator temperature.
In one embodiment, a composition comprising a mixture of first and second insulin prodrug analogs is provided, the first and second insulin prodrug analogs differing from each other in the structure of the prodrug elements. In particular, the first insulin prodrug analog may have a dipeptide prodrug element having a substantially different half-life than the dipeptide prodrug element of the second insulin prodrug analog. Therefore, by selecting different combinations of substituents on the dipeptide element, a composition containing a mixture of insulin prodrug analogs activated in a controlled manner at specified time intervals in a desired time frame is prepared. it can. For example, a composition of insulin that releases active insulin at mealtime and then releases and activates a suitable dose at night based on the time of activation can be prepared. In another embodiment, the pharmaceutical composition comprises a mixture of the insulin prodrug analogs disclosed herein with natural insulin or a known bioactive derivative of insulin.
The insulin prodrug analogs disclosed herein appear to be suitable for all applications previously described for insulin peptides. Therefore, the insulin prodrug analogs described herein can be used to treat hyperglycemia or other metabolic disorders resulting from high blood glucose levels. Accordingly, the present invention includes pharmaceutical compositions comprising the insulin prodrug analogs of the present disclosure and pharmaceutically acceptable carriers used in the treatment of patients with high blood glucose levels. According to one embodiment, the patient treated with the insulin prodrug analogs disclosed herein is a domesticated animal, and in another embodiment, the patient being treated is a human. One method of treating hyperglycemia according to the present disclosure includes parenteral (intravenous, intraperitoneal, subcutaneous or intramuscular, etc.), submucosal, transdermal, rectal, oral, nasal or inhalation administration. Includes the step of administering to a patient the insulin prodrug analogs disclosed herein using a standard route of administration. In one embodiment, the composition is administered subcutaneously or intramuscularly. In one embodiment, the composition is administered parenterally and the insulin prodrug analog composition is pre-encapsulated in a syringe.
The insulin prodrug analog of the present invention may be administered alone or in combination with other antidiabetic agents. As anti-diabetic agents known in the art or anti-diabetic agents under study, natural insulin, natural glipizide, functional derivatives thereof; tolubutamide (Orinase), acethexamide (Dymelor), trazamide (Tolinase), chlorpropa Sulfonylureas such as Diabinese, Glucotrol, Glybride (Diabeta, Micronase, Glynase), Glymepyrid (Amaryl) or Glycladide (Diamicron); Or biguanides such as phenformin; thiazolidinedione or other PPARγ inhibitors such as rosiglitazone (Avandia), pioglycazone (Actos) or troglitazone (Rezulin); Α-Glucosidase inhibitors that inhibit the digestion of carbohydrates such as plumlintide; dipeptidyl peptidase-4 (DPP-4) inhibitors such as bildagliptin or sitagliptin; SGLT (sodium-dependent glucose transporter 1) inhibitors; or FBPase (fluctose 1) , 6-bisphosphatase) inhibitors.
Pharmaceutical compositions containing the insulin prodrug analogs disclosed herein can be prepared and administered to patients using standard pharmaceutically acceptable carriers and routes of administration known to those of skill in the art. It is possible. Accordingly, the present disclosure comprises one or more of the insulin prodrug analogs disclosed herein, or pharmaceutically acceptable salts thereof, in combination with a pharmaceutically acceptable carrier. Also includes compositions. In one embodiment, the pharmaceutical composition comprises an insulin prodrug analog at a pH of about 4.0 to about 7.0 and a concentration of 1 mg / ml in a phosphate buffer system. The pharmaceutical composition may include the insulin prodrug analog as a single pharmaceutically active ingredient, or it may contain one or more other insulin prodrug analogs. May be combined with. According to one embodiment, one of the insulin prodrug analogs disclosed herein is preferably sterile and preferably at least 90%, 91%, 92%, 93%, 94%, 95%. , 96%, 97%, 98% or 99% purity levels, and also include pharmaceutically acceptable diluents, carriers or excipients. Such compositions may contain insulin prodrug analogs, in which case the resulting active peptide will be at least 0. 5mg / ml, 1mg / ml, 2mg / ml, 3mg / ml, 4mg / ml, 5mg / ml, 6mg / ml, 7mg / ml, 8mg / ml, 9mg / ml, 10mg / ml, 11mg / ml, 12mg / ml, 13mg / ml, 14mg / ml, 15mg / ml, 16mg / ml, 17mg / ml, 18mg / ml, 19mg / ml, 20mg / ml, 21mg / ml, 22mg / ml, 23mg / ml, 24mg / ml, It is present in concentrations of 25 mg / ml or higher. In one embodiment, the pharmaceutical composition comprises an aqueous solution that has been sterilized and optionally stored in various containers. The compounds of the invention can be used according to one embodiment to prepare prescription solutions for injection. In other embodiments, the pharmaceutical composition comprises a lyophilized powder. The pharmaceutical composition may be further packaged as part of a kit containing a disposable device for administering the composition to a patient. Containers or kits may be labeled for storage at ambient or refrigerated temperatures.
All of the therapeutic methods, pharmaceutical compositions, kits, and other similar embodiments described herein are intended to include insulin prodrug analogs that include all pharmaceutically acceptable salts thereof. There is.
In one embodiment, a kit is provided that includes a device for administering an insulin prodrug analog composition to a patient. The kit may include a wide variety of containers such as vials, tubes, bottles and the like. Preferably, the kit also includes an instruction manual. According to one embodiment, the device of the kit is a device for an aerosol, in which case the composition is pre-encapsulated in the aerosol device. In another embodiment, the kit comprises a syringe and a needle, and in one embodiment the insulin analog composition is pre-encapsulated in the syringe.
The compounds of the present invention can be prepared by standard synthetic methods, recombinant DNA techniques, or any other method of preparing peptides and fusion proteins. Although some unnatural amino acids cannot be expressed by standard recombinant DNA techniques, techniques for preparing them are known in the art. Compounds of the invention, including non-peptide moieties, may be synthesized by standard organic chemical reactions in addition to standard peptide chemical reactions, where appropriate.
<p num="0313">Example 1 Insulin A and B chain synthesis The A and B chains of insulin were synthesized on 4-methylbenzylamine (MBHA) or 4-hydroxymethylphenylacetamidemethyl (PAM) resins using the Boc method. Peptides were cleaved from the resin over 1 hour at 0 ° C. using 95: 5 HF / p-cresol. After HF removal and ether precipitation, the peptide was dissolved in a 50% aqueous acetic acid solution and lyophilized. Alternatively, peptides were synthesized using the Fmoc method. These peptides are combined with trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / H.<sub>2</sub>It was cut from the resin using O (95: 2.5: 2.5) at room temperature for 2 hours. Excess amount of diethyl ether was added to precipitate the peptide and the pellet was dissolved in an acidic buffer. Peptide quality was monitored by RP-HPLC and confirmed by mass spectrometry (ESI or MALDI).</p><p num="0314"> For the synthesis of the insulin A chain, free cysteine was used at the 7th amino acid, and cysteine protected with acetamide methyl was used elsewhere (A- (SH).<sup>7</sup>(Acm)<sup>6,11,20</sup>). For the synthesis of the B chain of insulin, free cysteine was used at the 7th position, and acetamide methyl-protected cysteine was used elsewhere (B- (SH).<sup>7</sup>(Acm)<sup>19</sup>). The crudely purified peptide was purified by conventional RP-HPLC.</p><p num="0315"> The synthesized A and B chains were attached to each other via their natural disulfide bonds according to the general procedure outlined in Figure 1. Cys by dissolving each B chain in DMF or DMSO<sup>7</sup>It was activated into a -Npys derivative and reacted with 2,2'-dithiobis (5-nitropyridine) (Npys) at a molar ratio of 1: 1 at room temperature. Activation was monitored by RP-HPLC and the product confirmed by ESI-MS.</p><p num="0316"> Each A- (SH)<sup>7</sup>(Acm)<sup>6,11,20</sup>And B- (Npys)<sup>7</sup>(Acm)<sup>19</sup>Was dissolved in a 1: 1 molar ratio to a total peptide concentration of 10 mg / ml to form the first B7-A7 disulfide bond. When the chain binding reaction was complete, the mixture was diluted with 50% aqueous acetic acid. Iodine was added to form the last two disulfide bonds simultaneously. A 40-fold molar excess of iodine was added to the solution and the mixture was stirred at room temperature for an additional hour. An aqueous ascorbic acid solution was added to terminate the reaction. The mixture was purified by RP-HPLC and the final compound was confirmed by MALDI-MS. As shown in the data in Figure 2 and Table 1, the synthetic insulin prepared in this procedure is well comparable to purified insulin in terms of binding to the insulin receptor.</p><p num="0317"> Insulin peptides containing modified amino acids (such as 4-aminophenylalanine at position A19) can also incorporate unencoded amino acids into proteins, including the systems taught in US Pat. Nos. 7,045,337 and 7,083,970. It can be synthesized in vivo using the system that makes it possible.</p><p num="0318"><img id="000122" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0319">Example 2 PEGylation of amine groups (N-terminus and lysine) by reductive alkylation a. Synthesis Insulin (or insulin analog), MPEG20 k-aldehyde, NaBH<sub>3</sub>CN was dissolved in acetic acid buffer at pH 4.1-4.4 at a molar ratio of 1: 2:30. The reaction solution was 0.1N NaCl, 0.2N acetic acid, 0.1N Na.<sub>2</sub>CO<sub>3</sub>Consists of. The insulin peptide concentration was about 0.5 mg / ml. The reaction takes place at room temperature for 6 hours. When the degree of reaction was monitored by RP-HPLC, the reaction yield was about 50%.</p><p num="0320">b. Purification The reaction mixture was diluted 2-5 fold with 0.1% TFA and applied to a preparative RP-HPLC column. HPLC conditions: C4 column; flow velocity 10 ml / min; aqueous solution of A buffer 10% ACN and 0.1% TFA; ACN solution of B buffer 0.1% TFA; 0-40% linear gradient B% (0-80 minutes); PEG- Insulin or relatives were eluted with approximately 35% buffer B. The desired compound was confirmed by MALDI-TOF and subsequently chemically modified by sulfite degradation or trypsin degradation.</p><p num="0321">PEGylation of amine groups (N-terminus and lysine) by acylation of N-hydroxysuccinimide a. Synthesis Insulin (or insulin analog) and mPEG20k-NHS were dissolved in 0.1 N Bicine buffer (pH 8.0) at a molar ratio of 1: 1. The insulin peptide concentration was about 0.5 mg / ml. The progress of the reaction was monitored by HPLC. The reaction yield is about 90% after 2 hours at room temperature.</p><p num="0322">b. Purification The reaction mixture was diluted 2-5 times and RP-HPLC was performed.</p><p num="0323">HPLC conditions: C4 column; flow velocity 10 ml / min; aqueous solution of A buffer 10% ACN and 0.1% TFA; ACN solution of B buffer 0.1% TFA; 0-40% linear gradient B% (0-80 minutes); PEG- Insulin or relatives were recovered at approximately 35% B. The desired compound was confirmed by MAIDI-TOF and subsequently chemically modified by sulfite degradation or trypsin degradation.</p><p num="0324">Reductive amination of acetyl group of aromatic ring of phenylalanine PEGylation a. Synthesis Insulin (or insulin analog), MPEG20k-hydrazide, NaBH<sub>3</sub>CN was dissolved in acetic acid buffer (pH 4.1-4.4) at a molar ratio of 1: 2: 20. The reaction solution was 0.1N NaCl, 0.2N acetic acid, 0.1N Na.<sub>2</sub>CO<sub>3</sub>Consists of. The concentration of insulin or insulin analog was about 0.5 mg / ml. 24 hours at room temperature. The reaction process was monitored by HPLC. The conversion of the reactants was about 50% (calculated by HPLC).</p><p num="0325">b. Purification The reaction mixture was diluted 2-5 times and run on RP-HPLC.</p><p num="0326">HPLC conditions: C4 column; flow velocity 10 ml / min; aqueous solution of A buffer 10% ACN and 0.1% TFA; ACN solution of B buffer 0.1% TFA; 0-40% linear gradient B% (0-80 minutes); PEG- Insulin or PEG-insulin analogs were recovered at approximately 35% B. The desired compound was confirmed by MAIDI-TOF and subsequently chemically modified by sulfite degradation or trypsin degradation.</p><p num="0327">Example 3 Insulin receptor binding assay The scintillation proximity technique was used to measure the affinity of each peptide for the insulin or IGF-1 receptor in a competitive binding assay. A 3-fold serial dilution of the peptide was produced in Tris-Cl buffer (0.05M Tris-HCl, pH7.5, 0.15M NaCl, 0.1% w / v bovine serum albumin) and 96-well plates (Corning Inc., Acton). , MA) was mixed with 0.05 nM (3- [125I] -iodotyrosyl) A TyrA14 insulin or (3- [125I] -iodotyrosyl) IGF-1 (Amersham Biosciences, Piscataway, NJ). 1-6 micrograms of plasma membrane fragment prepared from cells overexpressing human insulin receptor or IGF-1 receptor was dispensed into each well and 0.25 mg / well of polyethyleneimine-treated wheat germ aglutinin. Type A scintillation proximity assay beads (Amersham Biosciences, Piscataway, NJ) was added. After shaking at 800 rpm for 5 minutes, the plates were incubated at room temperature for 12 hours and radioactivity was measured on a MicroBeta 1450 liquid scintillation counter (Perkin-Elmer, Wellesley, MA). Non-specific binding (NSB) radioactivity was measured in the wells using a "non-radioactive" natural ligand that was four times higher than the highest concentration of the test sample. Fully bound radioactivity was detected in the wells without competing material. The specific binding rate was determined as follows. Specific binding (%) = (binding-NSB / total binding-NSB) x 100. IC50 values were determined using Origin software (Origin Lab, Northampton, MA).</p><p num="0328">Example 4 Insulin receptor phosphorylation assay: To measure insulin or insulin-related receptor phosphorylation, receptor-transfected HEK293 cells were seeded into 96-well tissue culture plates (Costar # 3596, Cambridge, MA) at 100 IU / ml. 37 ° C, 5% CO in Dalveco's Modified Eagle's Medium (DMEM) with penicillin, 100 μg / ml streptomycin, 10 mM HEPES and 0.25% bovine growth serum (HyClone SH30541, Logan, UT).<sub>2</sub>Incubated at 90% humidity for 16 to 20 hours. Serial dilutions of insulin or insulin analogs were prepared in DMEM supplemented with 0.5% bovine serum albumin (Roche Applied Science # 100350, Indianapolis, IN) and added to wells containing adherent cells. 5% CO<sub>2</sub>After incubation at 37 ° C for 15 minutes under humidified conditions, cells were fixed in 5% paraformaldehyde at room temperature for 20 minutes, washed twice with phosphate buffered saline (pH 7.4), and 2% in PBS. Blocked with bovine serum albumin for 1 hour. The plate was then washed 3 times and filled with Horseradish peroxidase-binding antibody (Upstate biotechnology # 16-105, Temecula, CA) against phosphotyrosine reconstituted with PBS containing 2% bovine serum albumin as recommended by the manufacturer. .. After incubation at room temperature for 3 hours, the plates were washed 4 times and 0.1 ml TMB single solution substrate (Invitrogen, # 00-2023, Carlbad, CA) was added to each well. After 5 minutes, 0.05 ml of 1N HCl was added to stop color development. Absorbance at 450 nm was measured with Titertek Multiscan MCC340 (Thermo Fisher, Pittsburgh, PA). Plot the peptide concentration-dose-response curve for absorbance and origin software (Origin Lab, Northampton, EC using MA)<sub>50</sub>The value was calculated.</p><p num="0329">Example 5 Judgment of cleavage rate of model dipeptide (in PBS) As a model peptide, a specific hexapeptide (HSRGTF-NH)<sub>2</sub>; SEQ ID NO: 59) was used. With this peptide, it was possible to study the cleavage rate of the extension of the N-terminal of the dipeptide. This dipeptide extension model peptide was prepared and Boc-protected sarcosin and lysine were continuously added to the model peptide binding resin to peptide A (Lys-Sar-HSRGTF-NH).<sub>2</sub>; SEQ ID NO: 60) was synthesized. Peptide A was cleaved by HF and purified by preparative HPLC.</p><p num="0330"> The cleavage rate was determined for each propeptide. The concentration of the propeptide and the concentration of the parent peptide of the model were determined from the respective peak areas. The logarithm of the concentration of the prodrug was plotted at various time intervals to determine the first-order dissociation rate constant of the prodrug. The rate constant "k" is obtained from the gradient of this plot. Expression t<sub>1/2</sub>The cleavage half-lives of various prodrugs were calculated using = 0.693 / k. This model peptide HSRGTF-NH<sub>2</sub>The half-life of the Lys-Sar extension with respect to (SEQ ID NO: 59) was determined to be 14.0 hours.</p><p num="0331">Example 6 Rate of dipeptide cleavage half-life in plasma, as determined by all d-isoform model peptides Another model of hexapeptide (dHdTdRGdTdF-NH) to determine the rate of dipeptide cleavage in plasma<sub>2</sub>, SEQ ID NO: 63) was used. Except for the prodrug extension, the d-isomer of each amino acid was used to prevent enzymatic cleavage of the model peptide. The d-isomer hexapeptide of this model was synthesized in the same procedure as the l-isomer. In the case of peptide A, sarcosine and lysine are continuously added to the N-terminus as described above, and peptide B (dLys-dSar-dHdTdRGdTdF-NH) is added.<sub>2</sub>, SEQ ID NO: 64).</p><p num="0332"> The cleavage rate was determined for each propeptide. The concentration of the propeptide and the concentration of the parent peptide of the model were determined from the respective peak areas. The logarithm of the concentration of the prodrug was plotted at various time intervals to determine the first-order dissociation rate constant of the prodrug. The rate constant "k" is obtained from the gradient of this plot. This model peptide dHdTdRGdTdF-NH<sub>2</sub>The half-life of the Lys-Sar extension with respect to (SEQ ID NO: 63) was determined to be 18.6 hours.</p><p num="0333">Example 7 Using the procedure described in Example 5, the model hexapeptide (HSRGTF-NH)<sub>2</sub>The cleavage rate of another dipeptide bound to SEQ ID NO: 59) was determined. The results obtained in these experiments are listed in Tables 2 and 3.</p><p num="0334">Table 2. Model peptide (HSRGTF-NH)<sub>2</sub>Cleavage of the dipeptide OU bound to the side chain of N-terminal paraaminophenylalanine from SEQ ID NO: 59) (in PBS)<img id="000123" he="50" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000124" he="136" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0335"><img id="000125" he="212" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0336"> In addition, the dipeptide element prepared various prodrug derivatives of the IGF1YL insulin analog, which are bound by the 4-aminophenylalanine residue present at A19 of IGF1YL via an amide bond. Analysis of these compounds in vitro using the procedure of Example 5 reveals that the activity of these compounds increases over time when incubated with either PBS buffer or 20% plasma. See Figures 8-11. Also, regarding the binding of insulin receptors to natural insulin over time (1 hour, 3 hours, 6 hours, 9 hours, 10.5 hours) when incubated with 20% plasma, the IGF analog prodrug MIU30: A1 (aF19). The in vitro activity of -dLys (Ac), Sar) (a dipeptide bound to A19 4-aminophenylalanine via an amide bond) was measured. Table 3A compares the relative binding strength to insulin receptors over time when incubated in 20% plasma / PBS at 37 ° C. Data from in vitro binding assay (see Table 3A) and in As shown in the in vitro phosphorylation assay data (see Table 3B), when the prodrug form is converted to the active IGF1YL peptide, it becomes more active from the A19 IGF prodrug derivative sample over time. Recover to the state.</p><p num="0337"><img id="000126" he="91" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0338"> C57 administered with the insulin analog MIU-30a: B1 (Y16, L17, Y25) 29a: A1 (dLys (Ac), Sar-aF19) (dipeptide bound to A19 4-aminophenylalanine via amide bond) An in vivo glucose tolerance test was performed using / Blk mice. MIU 30 was dissolved in PBS (pH 7.4) containing 20% plasma and incubated at 37 ° C for 48 hours (producing "MIU-30c"). To measure glucose reduction, samples incubated for 0 hours (MIU 30a) and samples incubated for 48 hours (MIU 30c) were removed and injected into C57 / Blk mice at 90 nmol / kg and 270 nmol / kg (insulin resistance test). ). FIG. 20A shows the glucose-lowering profiles of MIU 30a and MIU 30c at various time points up to 8 hours. The parent compound has low activity, but increases activity after 48 hours of incubation in 20% plasma (forming "MIU-30c") (see Figure 20A). Figure 20B shows MIU 30a and MIU. 30c of total blood glucose is shown as a difference in area under the curve (AUC) in comparison with the solvent. At 90 nmol / kg, MIU 30a shows almost no change in glucose, but MIU 30c causes a considerable decrease. At 270 nmol / kg, both MIU 30a and MIU 30c show a decrease in glucose, but the latter sample has significantly higher hypoglycemic activity. Thus, the prodrug form of the insulin analog MIU30 is clearly less glucose-lowering when injected under physiological conditions prior to ex vivo conversion to the parent insulin analog. These in vivo results are consistent with in vitro analysis. The half-life of this prodrug is estimated to be about 20 hours.</p><p num="0339">Example 8 Identification of insulin analogs with structures suitable for prodrug construction The 19th position of the A chain is known to be an important site for insulin activity. Therefore, it is desirable to modify this site to allow binding of prodrug elements. Specific analogs of insulin in A19 have been synthesized and their activity on the insulin receptor has also been characterized. In A19, two highly active structural analogs have been identified. Similar structural changes in the aromatic residue (B24) of the second active site failed to identify insulin analogs with similarly complete activity.</p><p num="0340"> Tables 4 and 5 show a high degree of structural conservation at position A19 to obtain full activity on the insulin receptor (binding to the receptor was determined by the assay described in Example 3). Table 4 shows that only two A19-modified insulin analogs have receptor-binding activity similar to that of native insulin. For 4-aminoinsulin analogs, here are the data obtained from three separate experiments. The column labeled "Activity (Test)" compares the binding rates of insulin analogs to natural insulin for two separate experiments performed at the same time. The column labeled "Activity (0.60 nM)" is the relative binding rate of the insulin analog to the previous average value obtained for insulin binding in this assay. In both analyzes, the two A19 insulin analogs (4-aminophenylalanine and 4-methoxyphenylalanine) show almost equivalent receptor binding to native insulin. FIG. 3 is a graph showing the specific binding of natural insulin and A19 insulin analog to the insulin receptor. Table 5 shows data that two A19 insulin analogs (4-amino and 4-methoxy), which exhibit equivalent binding activity to natural insulin, also exhibit equivalent activity to insulin receptors (receptor activity). Was determined by the assay described in Example 4).</p><p num="0341"><img id="000127" he="104" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0342"><img id="000128" he="58" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0343">Example 9 Insulin-like growth factor (IGF) analog A common purification scheme for isolating the A chain of insulin is NH<sub>4</sub>HCO<sub>3</sub>Use buffer (pH = 7.8). Under these conditions, the dipeptide prodrug element is cleaved from the A chain in a short time. To simplify the purification of prodrugs used to study the activity of prodrugs on the insulin receptor, Applicants have identified IGF analogs that exhibit similar activity to insulin receptors as natural insulin. A study was conducted using. Specifically, this IGF analog (IGF1 (Y)<sup>B16</sup>L<sup>B17</sup>) Have the A and B chains of the native IGF (SEQ ID NOS: 61 and 62, respectively). In this case, the 15th and 16th natural glutamine and phenylalanine in the B chain of natural IGF (corresponding to the 16th and 17th in the B chain of natural insulin) are replaced with tyrosine and leucine residues, respectively. IGF1 (Y), as shown in Figure 4 and Table 6 below.<sup>B16</sup>L<sup>B17</sup>) Binding activity indicates that this compound is a very strong insulin analog.</p><p num="0344"><img id="000129" he="41" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0345">Example 10 IGF prodrug derivative Based on the activity of the A19 insulin analog (see Example 5), IGF1 A: B (Y)<sup>B16</sup>L<sup>B17</sup>) The analogs were similarly modified to examine their ability to bind to the insulin receptor and stimulate activity. Figure 5 shows the natural tyrosine with 4-aminophenylalanine [IGF1 A: B (Y).<sup>B16</sup>L<sup>B17</sup>) (P-NH<sub>2</sub>-F)<sup>A19</sup>Replace with [amide], IGF1 A: B (Y)<sup>B16</sup>L<sup>B17</sup>), And its dipeptide extension derivative [IGF1 A: B (Y)<sup>B16</sup>L<sup>B17</sup>)<sup>A19</sup>-Aib Ala amide] shows a general synthetic scheme for preparing. Here, the dipeptide having Aib and Ala binds to the peptide via an amide bond with A19 4-aminophenylalanine. As shown in Figure 6 and Table 7, IGF1 (Y) is an IGF analog.<sup>B16</sup>L<sup>B17</sup>) A (p-NH<sub>2</sub>-F)<sup>19</sup>Binds specifically to the insulin receptor. In this case, the dipeptide extension derivative of this analog cannot specifically bind to the insulin receptor. Note that this dipeptide extension lacks the proper structure to allow autonomous cleavage of the dipeptide (there is no N-alkylated amino acid second in the dipeptide) and therefore insulin receptor binding is not restored. I want to be.</p><p num="0346"><img id="000130" he="54" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0347"> A dipeptide prodrug element (alanine-proline) was attached to the amino terminus of the A chain via an amide bond (IGF1 (Y).<sup>B16</sup>L<sup>B17</sup>) (AlaPro)<sup>A-1,0</sup>), IGF<sup>B16B17</sup>Another prodrug derivative of the derivative peptide was prepared. IGF1 (Y) as shown in Table 8<sup>B16</sup>L<sup>B17</sup>) (AlaPro)<sup>A-1,0</sup>In, the affinity for the insulin receptor was substantially reduced. Based on the data in Table 3, this dipeptide prodrug element does not have a suitable structure that allows autonomous cleavage of the dipeptide prodrug element, and therefore the insulin receptor binding detected is a prodrug. Note that it is not the result of element disconnection.</p><p num="0348"><img id="000131" he="40" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0349">Example 11 Another IGF insulin analog IGF1 (Y<sup>B16</sup>L<sup>B17</sup>) (YL) B<sup>16</sup>B<sup>17</sup>Further modification of the peptide sequence reveals another IGF insulin analog with different activity on the insulin receptor and the IGF-1 receptor. Binding data for each of these analogs is shown in Table 9 (using the assay in Example 3). The position of modification is displayed based on the corresponding position in the native insulin peptide (DPI = des B26-B30). For example, without further detail, the term "position of B28" herein means the position of B27 in the B chain of an insulin analog lacking the first amino acid of SEQ ID NO: 2. Therefore, the term "B (Y16)" generally refers to the substitution of the tyrosine residue at position 15 of the B chain of the natural IGF-1 sequence. Table 9 shows data on the relative binding of insulin to the IGF analog to the receptor, and Table 10 shows data on IGF analog-stimulated phosphorylation (using the assay in Example 4).</p><p num="0350"><img id="000132" he="201" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000133" he="201" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000134" he="196" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /><img id="000135" he="196" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0351">Example 12 Cleavage of the dipeptide from the prodrug form of the IGFB16B17 derivative peptide Cleavage of the (pNH2-Phe) amide-binding dipeptide AibPro from various IGF-1 peptides was measured to determine the effect of the peptide sequence or heteroduplex on dipeptide cleavage. The results for the test peptides are shown in Table 11. This data shows IGF1 B: A (YL) for the IGF1-A chain alone.<sup>B16,17</sup>It has been shown to be an excellent model for studying the prodrug half-life of peptides.</p><p num="0352"><img id="000136" he="42" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0353"> Disulfide bond A chain and B chain constructs (IGF1 A: B (Y)<sup>B16</sup>L<sup>B17</sup>Comparing the prodrug derivatives of the IGF A chain with respect to)), it was revealed that these two compounds have similar half-lives in the prodrug form. Therefore, IGF1A alone, IGF1 B: A (Y)<sup>B16</sup>L<sup>B17</sup>) Was determined to be an excellent model for studying the prodrug half-life. The AibAla derivative is not cleaved and is not a prodrug, but the modification is the insulin analog IGF1 A: B (Y)<sup>B16</sup>L<sup>B17</sup>) (P-NH<sub>2</sub>-F)<sup>A19</sup>Note that it plays a role in indicating that the amide can be inactivated. For convenience, the half-life of the prodrug was determined using only the A chain of IGF1 in the absence of the B chain. The half-life of each propeptide was determined as described in Example 5. The data are shown in Table 12.</p><p num="0354"><img id="000137" he="77" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0355"> The data show that the half-life of a prodrug can be varied from 2 hours to> 100 hours by altering the substituents on the dipeptide prodrug element.</p><p num="0356"> IGF1-A (pNH)<sub>2</sub>-F)<sup>19</sup>Using a systemic peptide, another prodrug derivative peptide was prepared by altering the amino acid composition of the dipeptide prodrug element bound via 4-aminophenylalanine at position A19. Dipeptide half-lives were measured in PBS and in 20% plasma / PBS for different constructs (ie, in the presence of serum enzymes. Results are shown in Table 13. These results are of the four peptides tested. Three species show that they were not affected by serum enzymes.</p><p num="0357"><img id="000138" he="102" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0358">Example 13 IGF over time<sup>B16B17</sup>Binding of derivative peptides to the receptor IGF<sup>B16B17</sup>A prodrug preparation of the derivative peptide was prepared and its degradation over time was measured using the insulin receptor binding assay of Example 3. The peptides used in this assay were prepared as follows.</p><p num="0359">Dipeptide-IGF1A analog Unless otherwise specified, the Boc method was used for the synthesis of design peptide analogs. Selected dipeptide H<sub>2</sub>N-AA1-AA2-COOH, IGF1A (Ala)<sup>6,7,11,20</sup>Above (pNH<sub>2</sub>-Phe)<sup>19</sup>In addition to. The IGF-1 A chain C-terminal tripeptide Boc (Fmoc-pNH-Phe) -Ala-Ala was synthesized on MBHA resin. After removing Fmoc by treatment with 20% piperidine / DMF at room temperature for 30 minutes, Fmoc-AA2 is p-aminobenzyl side with A19 using a 3-fold excess of amino acid, PyBop, DIEA, and catalytic amount of pyridine. Bonded to the chain. Remaining IGF-1 A chain (Ala)<sup>6,7,11,20</sup>Boc synthesis of the sequence was completed using Applied Biosystems 430A Peptide Synthesizer and IGF-1 A chain (Boc).<sup>0</sup>(Ala)<sup>6,7,11,20</sup>(Fmoc-AA2-pNH-Phe)<sup>19</sup>-I got MBHA. After removing the Fmoc group from the N-terminus of AA2, Boc-AA1 was attached to the amine using a 3-fold excess of amino acids, DEPBT and DIEA. After removing the two Boc groups remaining in the A chain with TFA and then HF cleavage, IGF-1 A chain (Ala)<sup>6,7,11,20</sup>(H<sub>2</sub>N-AA1-AA2-pNH-Phe)<sup>19</sup>An amide was obtained. If AA1 was d-lysine, acetylation to ε-amine was performed prior to Boc removal. Dipeptide IGF-1 A chain analogs were purified by half-taken RP-HPLC and characterized by analytical RP-HPLC and MALDI mass spectrometry.</p><p num="0360">Dipeptide-IGF-1 (YL) analog The dipeptide H selected by the method described above, except that a PAM resin was used to synthesize the IGF-1 A chain so that a C-terminal acid was obtained upon HF cleavage.<sub>2</sub>N-AA1-AA2-COOH IGF-1 A chain (Acm)<sup>6,11,20</sup>(PNH<sub>2</sub>-Phe)<sup>19</sup>In addition to. IGF-1 B chain (YL)<sup>16,17</sup>(Acm)<sup>19</sup>Was synthesized on MBHA resin to obtain a C-terminal amide. Cys<sup>B7</sup>Free thiols in 100% DMSO were modified with Npys by reaction with DTNP in a molar ratio of 1: 1. Purified Dipeptides-IGF-1 A Chain and IGF-1 B Chain (YL)<sup>16,17</sup>Derivatives were bound using the "1 + 2" two-step chain combination strategy shown in Scheme 1. Intermediate and final purifications were performed on half-take RP-HPLC and characterized by analytical RP-HPLC and MALDI mass spectrometry.</p><p num="0361"> IGF<sup>B16B17</sup>Derivative peptide prodrugs are incubated in PBS at pH 7.4, 37 ° C, small fractions are taken at predetermined time intervals, further degradation is stopped at 0.1% TFA, and the liquids are analyzed. Analyzed by HPLC for use. IGF<sup>B16B17</sup>Peaks a and b representing the prodrug and active form of the derivative peptide were identified by LC-MS and quantified by integrating the peak area of HPLC. Figures 98 to 8C show IGF<sup>B16B17</sup>Derivative peptide prodrug: IGF1A (Ala)<sup>6,7,11,20</sup>(Aib-Pro-pNH-F)<sup>19</sup>The output of the HPLC analysis for the decomposition of is shown. Small doses were taken at 20 minutes (Fig. 8A), 81 minutes (Fig. 8B) and 120 minutes (Fig. 8C) after the start of incubation of the prodrug in PBS. These data are based on IGF1A (Ala)<sup>6,7,11,20</sup>(Aib-Pro-pNH-F)<sup>19</sup>From amide to IGF1A (Ala)<sup>6,7,11,20</sup>(pNH<sub>2</sub>-F)<sup>1</sup>It shows autonomous and enzyme-independent conversion over time to amides.</p><p num="0362"> IGF<sup>B16B17</sup>Degradation of the derivative peptide from prodrug form to active form was also measured based on the ability of the compound to bind to the insulin receptor, as measured in the in vitro assay of Example 3. 9A and 9B are graphs showing the in vitro activity of the prodrugs Aib, dPro-IGF1YL (A19 4-aminophenylalanine-bound dipeptide). Figure 9A shows the temporal (0 hours, 2.5 hours, 10.6) of natural insulin (measured at 4 ° C at 1 hour) and A19 IGF prodrug analog (Aib, dPro-IGF1YL) incubated in PBS. Time) It is a graph comparing the relative binding force to an insulin receptor. Figure 9B shows natural insulin (measured at 4 ° C for 1.5 hours) and A19 incubated in 20% plasma / PBS. It is a graph comparing the relative binding force to the insulin receptor over time (0 hour, 1.5 hours, 24.8 hours) of the IGF prodrug analog (Aib, dPro-IGF1YL). As is clear from the data shown in the graph, when the prodrug form is converted to the active IGF1YL peptide, the A19IGF prodrug analog sample recovers to a more active state. IGF for binding to insulin receptor<sup>B16B17</sup>The activity of the derivative peptide was measured. Original IGF<sup>B16B17</sup>Derivative peptides are more active than natural insulin and can be more than 100% active against insulin.</p><p num="0363"> 10A and 10B are graphs showing the in vitro activity of the prodrug dK, (N-isobutyl G) -IGF1YL (A19 4-aminophenylalanine-bound dipeptide). Figure 10A shows natural insulin (measured at 4 ° C at 1 hour) and A19 IGF prodrug analog (IGF1YL: dK, (N-isobutyl G)) incubated in PBS over time (0 hours,). 5 hours, 52 hours) Graph comparing relative binding to insulin receptor. Figure 10B shows natural insulin (measured at 4 ° C at 1.5 hours) incubated in 20% plasma / PBS and A19. It is a graph comparing the relative binding force of the IGF prodrug analog (IGF1YL: dK, (N-isobutyl G) to the insulin receptor over time (0 hour, 3.6 hours, 24.8 hours). Data shown in the graph. As is clear from the above, when the prodrug form is converted to the active IGF1YL peptide, the A19 IGF prodrug analog sample recovers to a highly active state.</p><p num="0364"> 11A and 11B are graphs showing the in vitro activity of the prodrug dK (e-acetyl), Sar) -IGF1YL (A19 4-aminophenylalanine-bound dipeptide). Figure 11A shows the temporal (0 hours) of natural insulin (measured at 4 ° C at 1 hour) and A19 IGF prodrug analog (IGF1YL: dK (e-acetyl), Sar) incubated in PBS. , 7.2 hours, 91.6 hours) It is a graph comparing the relative binding force to the insulin receptor. Figure 11B shows the time course of natural insulin (measured at 4 ° C for 1.5 hours) and A19 IGF prodrug analog (IGF1YL: dK (e-acetyl), Sar) incubated in 20% plasma / PBS. It is a graph comparing the relative binding force to the insulin receptor (0 hour, 9 hours, 95 hours). As evidenced by the data shown in the graph, conversion of the prodrug form to the active IGF1YL peptide restores the active state from the A19 IGF prodrug analog sample.</p><p num="0365">Example 14 Comparison of insulin tolerance of insulin prodrug analogs Insulin heterodimer analogs in healthy mice [B<sup>1</sup>(Y16, L17, Y25) 29a: A<sup>1</sup>(aF19-NH2)], or a prodrug derivative thereof, was administered. Prodrug derivative MIU-29: [B<sup>1</sup>(Y16, L17, Y25) 29a: A<sup>1</sup>(aF19-dLys (Ac), NLeu)] has a 4-aminophenylalanine substitution at the A19 position, and the dipeptide dLys (Ac), NLeu is covalently linked at the 4-amino position of the A19 residue. This dipeptide cleaves autonomously under physiological conditions and has a half-life of approximately 4.4 hours (see Figure 19A). Prodrug derivative [B<sup>1</sup>(Y16, L17, Y25) 29a: A<sup>1</sup>(aF19-dLys (Ac), NLeu)] was incubated ex vivo for 24 hours, and the obtained compound was administered to mice, and the function of this compound to lower blood glucose was compared with that of the parent compound. As shown in FIG. 19B, the two compounds functioned in much the same way.</p><p num="0366"> Acylation of insulin prodrug analogs was investigated to determine if the in vivo residence time could be increased. MIU42 [B1 (Y16, L17, Y25) 29a: A1 (dLys (rE-C14), Sar-aF19)] with acylated dipeptide prodrug elements compared to unacylated prodrugs in In vitro activity increases over time (provides time for prodrug conversion) when incubated ex vivo at pH 7.4, 37 ° C in 30% ACN / PBS (see Figure 21). Insulin activity comparison studies performed with MIU42 prodrugs administered without a pre-incubation step show that this prodrug is not as potent as the non-prodrug parent compound (MIU-27). I understand. This also applies to MIU-46 [B1 (H5,10 Y16, L17, Y25, K29-C14) 28a: A1 (N18,21, aF19NH2)], which is an insulin analog in which the position of B29 is acylated. It was. This compound is desired in vivo when tested in vivo in mice. It showed no vivo activity or basic properties (Fig. 23C). Therefore, at least in mice, acylation does not produce the desired profile.</p><p num="0367">Example 15 Biosynthesis and purification of PEGylated insulin prodrug analogs The solid phase Boc method was used to synthesize the B chain (2-25) H5,10 Y16 L17 SH7 Acm19 amide of IGF1 on MBHA resin. After removing the peptide from the resin and simultaneously deprotecting the side chains of the amino acids, the crude B chain is mixed with a DMSO solution of 2,2'-dithiobis (5-nitropyridine) and Cys7 is a cysteine-NpyS derivative. Got Boc-aminooxyacetyl (Aoa) was added to the N-terminus of the B chain by reacting the B chain with Boc-Aoa-OSu. Purified IGF1 B chain (2-25) (BocAoa) 0 H5,10 Y16 L17 SH7 Acm19 amide to IGF1 A chain Acm6,7, using the "1 + 2" method described herein (see Figure 1). 11 11 Combined with N18,21 (aa1aa2) -pNH-F19 acid, an insulin analog with Boc-Aoa at the N-terminus of the B chain was generated. In the presence of O- (carboxymethyl) hydroxylamine hemichloride as a scavenger, the peptide was lightly treated with 6N HCl to remove Boc. After removal and purification of Boc, the peptide was dissolved in 1% aniline / 30% ACN / 0.2M NaOAc (pH 4.6) at a concentration of 3 mg / ml. A 2-fold excess amount of 20KD PEG-propionaldehyde was added to this solution, and the mixture was reacted at room temperature for 1 hour with stirring, and then final purification was carried out to obtain a PEGylated insulin analog.</p><p num="0368"> As shown in FIGS. 26 and 27, addition of 20 kDa PEG to the amino terminus of a double-stranded insulin analog (Fig. 24) reduces the activity of the insulin analog (marked with , non-PEGylated parent). Comparison with compounds; PEGylated compounds are indicated by ). Addition of a self-cleavable dipeptide prodrug element (dLys (rE-C14), Nleu) at position A19 further reduces the activity of the compound by about one-hundredth (see in Figures 26 and 27). .. However, preincubation of the prodrug at 37 ° C in PBS for 78 hours (dipeptide half-life is about 4.4 hours) restores activity to values close to those of the PEGylated parent compound. See Table 14, which lists the EC50 of analogs to the A and B subtypes of the insulin receptor as measured by in vitro phosphorylation assays.</p><p num="0369"><img id="000139" he="48" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0370">Example 16 Biosynthesis and purification of single-chain insulin analogs Expression vector for the (B0-C1-A0) insulin IGF-I minigene, which contains the B and A chains of native insulin via the IGF-C chain for constitutive expression and purification of recombinant proteins in the yeast Pichia pastoris. It was cloned under the GAP promoter of pGAPZαA (purchased from Invitrogen) (promoter of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)). Minigene was bound to an N-terminal peptide encoding the Saccharomyces cerevisiae α conjugation factor leader signal for secreting recombinant proteins into the medium. The Kex2 cleavage site between the minigene and the α-conjugate leader sequence was used to cleave the leader sequence for the secretion of a minigene with a native amino terminus. B0-C1-A0 minigene 1 (G1A), 2 (Y2A), 3 (G3A), 4 (S4A), 5 (S5A), 6 (S6A), 7 ( It was introduced at the positions of R7A), 8 (R8A), 10 (P10A), 11 (Q11A), and 12 (T12A).</p><p num="0371"> Electroporation was used to transform yeast Pichia pastoris with minigenes containing B0-C1-A0, 11 alanine variants, and other selective derivatives. Positive transformants were selected in minimal methanol medium and genomes were isolated from clones of each yeast. It was confirmed by PCR that the construct was introduced into the yeast genome. The 833 base pair PCR product was visualized on an agarose DNA gel. Insulin analogs were produced by fermentation of the corresponding yeast line. Yeast cells were centrifuged in a 500 mL Beckman centrifuge tube at 5000 rpm for 20 minutes to pelletize and the medium was used for further protein purification.</p><p num="0372"> The supernatant of the growth medium was filtered through a 0.2 μm millipore filter. Acetonitrile (ACN) was added to the supernatant (final concentration 20%). The supernatant was purified with Amberlite XAD7HP resin (Sigma) previously equilibrated with 20% ACN aqueous solution. The resin was washed twice with 30 ml of 20% ACN aqueous solution and contaminants were removed with 30% ACN aqueous solution containing 0.1% TFA. Partially purified insulin analogs were eluted from the column with 54% ACN aqueous solution containing 0.1% TFA and lyophilized. 0.025M lyophilized sample It was resuspended in NH3HCO3 (pH 8) and purified on a LunaC18 column (10 μm particle size, 300 angstrom pore size). Proteins were eluted from the column with a linear gradient of 20-60% ACN aqueous solution. The MALDI-MS positive fraction was pooled and transferred to a disposable scintillation vial for the next lyophilization. The lyophilized sample was then resuspended in 20% ACN aqueous solution containing 0.1% TFA and purified on a LunaC18 column (10 μm particle size, 300 angstrom pore size). Proteins were eluted from the column with a linear gradient of ACN aqueous solution containing 18-54% 0.1% TFA. The eluate was monitored at a wavelength of 280 mm. To confirm purity, MALDI-TOF MS positive fractions were analyzed on a C8 analytical column.</p><p num="0373"> FIG. 14 shows the activity of single chain insulin analogs. The B0-C1-A0 analog showed similarly effective activity on both the insulin receptor isoform and the IGF-1 receptor. Shortening of the C-terminus to 8 amino acids by a second tyrosine to alanine mutation or deletion of C9-12 selectively promotes insulin activity specificity by significantly reducing IGF-1 receptor activity. Indicated. See also the data presented in Tables 14A and 14B.</p><p num="0374"><img id="000140" he="140" wi="158" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0375"><img id="000141" he="139" wi="159" file="JP5912112B2_D0001.tif" img-format="tif" img-content="drawing" /></p><p num="0376"> FIG. 15 shows that the second and third C-peptides are most sensitive to modification to the IGF-I receptor, as well as the insulin receptor known to be relatively resistant to modification. .. Finally, FIGS. 16 and 17 show in vitro analysis of single-chain insulin variants as binding affinity (IC50) and biochemical signaling (EC50) through tyrosine phosphorylation. The homogeneity of the two independent measurements confirmed the in vitro approach to structural-functional analysis. All analogs maintained single-digit nM activity, along with specific analogs (nM below the single digit) that were found to be slightly enhanced in activity. The most insulin-selective analogs were those with the second alanine mutation in C-peptide or a combination of the two changes, with the last 4 residues of C-peptide deleted.</p><p num="0377"> The stability of the insulin / IGF chimera was investigated by exposing insulin analogs to insulin-specific degrading enzymes (IDEs) and assaying their activity.</p><p num="0378"> Insulin Degradation Assay: RDNA rat IDE was obtained from EMD Chemicals Inc. Peptides were prepared in 15 mM separate solutions in ammonium bicarbonate buffer. Initial concentrations were estimated based on UV absorbance at 276 nm and further confirmed by HPLC analysis using internal standards. The pH of the solution was maintained at 7.8-8.4. IDE was added and digested over time (12-48 hours) at 37 ° C. According to the experiment, the enzyme: substrate ratio was set to 1: 350 to 450. For HPLC analysis, small doses were taken at regular intervals with internal standard peptides and placed in TFA buffer. Also, in order to evaluate the activity, a small amount was taken separately and placed in DMEM assay medium. All small fractions were quickly frozen on dry ice and stored at -55 ° C until analysis.</p><p num="0379"> HPLC Assay: Degradation profiles of the peptides investigated were evaluated by HPLC assay. Each fraction was passed twice through an Agilant Zorbax C8 column attached to the Beckman-Coulter system using TFA buffer (gradient 20-60% B in 10 minutes, B = 90% AcN in this case).</p><p num="0380"> Biological activity: Insulin receptor phosphorylation ELISA assay determined the residual activity of analogs after incubation in IDE as half-effect concentration (EC50). When a bioactivity assay is performed on peptides prepared during enzymatic degradation by insulin receptor phosphorylation, virtually all A-chain analogs of insulin and IGF-2 lose virtually all activity, but IGF- It was observed that all A-chain analogs of 1 retained this. In particular, as shown in FIG. 17, in an insulin analog whose A chain is derived from a natural insulin sequence, the cleavage of the insulin analog by an insulin-specific degrading enzyme (IDE) is extremely firm and easily detected. In contrast, analogs whose A chain is derived from the IGF-1 sequence appear to be highly resistant to proteolysis.</p><p num="0381">421 Verify the expectation that increased stability will also increase the ability to promote cell division, and the results are shown in Figure 18. No correlation was found between the highly active insulin analogs and increased proliferation. Moreover, especially important for protein resolution, IDE-tolerant analogs did not appear to be mitogen-activated.</p><p num="0382"> A set of insulin analogs was tested in vivo using healthy mice (Melior Research Labs). Glucose-lowering activity was comparable or increased for all peptides of interest when compared to treatment with insulin as a reference material. However, when the analogs having the same B chain and having the A chain of insulin and the analogs having the A chain of IGF1 were compared, no significant difference in activity was observed. This supports the conclusion that IDE degradation and insulin excretion are not major or physiologically related mechanisms.</p>
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Every citation, both waysCites: the store holds 2 of 3
| Document | Relation | Office |
|---|---|---|
| JP2005524677A | Cites | Japan |
| WO2009099763A1 | Cites | World Intellectual Property Organization (WIPO) |
| Bioconjugate Chem., 2005, Vol.16, p.913-920 | Non-patent | – |
| FEMS Microbiol.Ecol.,2007 Jul,61(1),p.110-20 | Non-patent | – |
| Int. Pept. Res. Ther., 2008.08, Vol.14, p.255-262 | Non-patent | – |
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Numbers
- Publication
- 5912112
- Publication, DOCDB
- 5912112
- Publication, EPODOC
- JP5912112B
- Application
- 2013516766
- Application, DOCDB
- 2013516766
- Application, EPODOC
- JP20130516766
Titles2
- Japanese
- アミド系インスリンプロドラッグ
- English
- Amide insulin prodrug
Classification
- CPC, 3
- C07K14/62
- A61K38/00
- A61P3/10
- IPC, 3
- A61K38 28
- A61P3 10
- C07K14 62
