Glucagon-like peptide-1 analogs

Description of the invention

The invention relates to a glucagon-like peptide 1 (GLP-1) compound.

Glucagon-like peptide 1 (GLP-1), a 37 amino acid peptide, is secreted by the L cells of the intestine in response to food intake. It has been found to stimulate insulin secretion (insulinotropic effect), whereby it causes glucose uptake by cells and a reduction in serum glucose levels [see, for example: S. Mojsov, Int. J. Peptide Protein Research, 40, 333-343 (1992)]. However, GLP-1 (1-37) exhibits only little activity, so attention has focused on its truncated analogs, known as GLP compounds, which are much more biologically active than GLP-1 itself. Examples of such compounds include GLP-1 (7-37), GLP-1 (7-36) NH2, Gly⁸-GLP-1 (7-37) OH and Ser³⁴-GLP-1 (7-37) OH. Due to their ability to stimulate insulin secretion, it is believed that GLP compounds can be used successfully as agents in the treatment of diabetes, obesity and related conditions.

GLP-1 compounds can exist in at least two different forms. The first form is physiologically active and dissolves easily in an aqueous solution with a physiological pH value (7.4). In contrast, the second form exhibits no insulinotropic activity, even if only slightly, and is essentially water-insoluble at pH 7.4. Unfortunately, formation of an inactive form readily occurs when aqueous GLP-1 solutions are agitated, exposed to hydrophobic surfaces, or have large air / water interface. This tendency to convert to an insoluble form greatly complicates the commercial production of active GLP-1 compounds. Mixing operations or continuous flow through pumps are common operations in large-scale manufacturing processes and these operations result in mixing, air / water interfaces and / or contact with hydrophobic surfaces, all of which result in an insoluble form. Conversion to the inactive form may also occur during storage or after administration to a subject, which further complicates the use of these compounds as medicaments. Thus, there is a great need for biologically active GLP-1 analogs that are more difficult to convert to the insoluble form than is the case with the GLP-1 compounds available to date.

It has now been found that a number of GLP-1 analogues with modifications to one or more of the following positions: 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36 or 37, exhibit significant lower aggregation tendency compared to GLP-1 (7-37) OH.

Many of these analogs maintain a GLP-1 receptor activation capacity comparable to, and in some cases even greater than, that of known GLP-1 compounds such as GLP-1 (7-37) OH and Val⁸-GLP-1 (7-37) OH. For example, the aggregation time of Val⁸-Glu²²-Glp (7-37) OH is more than 20 times longer than the aggregation time of GLP-1 (7-37) OH, and its ability to activate the GLP-1 receptor is about 25% greater than that of GLP-1 (7 -37) OH. Based on these findings, novel GLP-1 compounds and their use in treatment methods are disclosed herein.

The invention relates to a GLP-1 compound comprising the amino acid sequence of formula III (SEQ ID NO: 3):

His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Xaa22-Xaa23-Ala-Ala-Lys-Xaa27-Phe-Ile-Xaa30-Trp- Leu-Val-Lys-Gly-Arg-R

Formula III (SEQ ID NO: 3) in which:

Xaa8 is: Gly or Val;

Xaa22 is: Glu;

Xaa23 is: His, Asp, Lys, Glu or Gln;

Xaa27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg or Lys

Xaa30 is: Ala, Glu, As p, Ser or His;

R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro, Gly-Pro-NH2, or is removed.

Preferably, the GLP-1 compound is selected from:

Val⁸-Glu²²-GLP-1 (7-37) OH (SEQ ID NO: 5),

Gly⁸-Glu²²-GLP-1 (7-37) OH (SEQ ID NO: 9),

Val⁸-Glu²²-GLP-1 (7-36) NH2 (SEQ ID NO: 32),

Gly⁸-Glu²²-GLP-1 (7-36) NH2 (SEQ ID NO: 37),

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Val⁸-Glu²²-Lys²³-GLP-1 (7-37) OH (SEQ ID NO: 48),

Val⁸-Glu²²-Glu²³-GLP-1 (7-37) OH (SEQ ID NO: 49) or

Val⁸-Glu²²-Ala²⁷-GLP-1 (7-37) OH (SEQ ID NO: 50).

More preferably it has the sequence designated SEQ ID NO: 5.

Even more preferably it forms a complex with a divalent metal cation.

Most preferably it forms a complex with the divalent zinc cation.

According to the invention, a polypeptide having the amino acid sequence of formula I (SEQ ID NO: 1) is described:

His-Xaa8-Glu-Gly-Xaa11-Xaa12-Thr-Ser-Asp-Xaa16-Ser-Ser-Tyr-Leu-Glu-Xaa22-Xaa23-Xaa24-Ala-Xaa₂₆-Xaa₂₇-Phe-Ile-Ala-Xaa₃₁-Leu-Xaa₃₃ - Xaa₃₄ -Xaa₃₅-Xaa₃₆-R formula I (SEQ ID NO: 1) where:

Xaa8 is: Gly, Ala, Val, Leu, Ile, Ser or Thr;

Xaa11 is: Asp, Glu, Arg, Thr, Ala, Lys or His;

Xaa12 is: His, Trp, Phe or Tyr;

Xaa16 is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu or Ala;

Xaa22 is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys or cysteic acid;

Xaa23 is: His, Asp, Lys, Glu or Gln;

Xaa24 is: Glu, His, Ala or Lys;

Xaa26 is: Asp, Lys, Glu or His;

Xaa27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg or Lys;

Xaa30 is: Ala, Glu, Asp, Ser or His;

Xaa33 is: Asp, Arg, Val, Lys, Ala, Gly or Glu;

Xaa34 is: Glu, Lys or Asp;

Xaa35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His or Glu;

Xaa36 is: Arg, Glu or His;

R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro or Gly-Pro-NH2, or is absent and such polypeptide lacks the sequence GLP-1 (7-37) OH or GLP-1 (7-36) -NH2, and where such polypeptide is not

Gly⁸-GLP-1 (7-37) OH,

Leu⁸-GLP-1 (7-37) OH,

Gly⁸-GLP-1 (7-36) NH2,

Leu⁸-GLP-1 (7-36) NH2,

Val⁸-GLP-1 (7-37) OH,

How much⁸-GLP-1 (7-37) OH,

Thr⁸-GLP-1 (7-37) OH,

Cheese⁸-GLP-1 (7-37) OH, Ser⁸-GLP-1 (7-36) NH2,

Ala¹¹-GLP-1 (7-37) OH, Ala¹¹-GLP-1 (7-36) NH2

Val⁸-GLP-1 (7-36) NH2, Ile⁸-GLP-1 (7-37) NH2, Thr⁸-GLP-1 (7-36) NH2,

Glu²⁷-GLP-1 (7-37) OH, Glu²⁷-GLP- (7-36) NH

Ala³³-GLP-1 (7-37) OH or Ala³³-GLP-1 (7-36) NH2.

The invention also describes a polypeptide having the amino acid sequence of formula II (SEQ ID NO: 2): His-Xaa8-Glu-Gly-Thr-Xaa12-Thr-Ser-Asp-Xaa16-Ser-Ser-Tyr-Leu-Glu- Xaa22-Xaa23-Ala-AlaXaa26-Glu-Phe-Ile-Xaa30-Trp-Leu-Val -Lys-Xaa35-Arg-R formula II (SEQ ID NO: 2) where:

Xaa8 is: Gly, Ala, Val, Leu, Ile, Ser or Thr;

Xaa12 is: His, Trp, Phe or Tyr;

Xaa16 means: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu or Ala

Xaa22 is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys or cysteic acid;

Xaa23 is: His, Asp, Lys, Glu or Gln;

Xaa26 is: Asp, Lys, Glu or His;

Xaa30 is: Ala, Glu, Asp, Ser or His;

Xaa35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His or Glu;

R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro or Gly-Pro-NH2, or is absent and such polypeptide lacks the sequence GLP-1 (7-37) OH or GLP-1 (7-36) -NH2, and where such polypeptide is not

Gly⁸-GLP-1 (7-37) OH, Leu⁸-GLP-1 (7-37) OH, Ser⁸-GLP-1 (7-37) OH, Ala¹⁶-GLP-1 (7-37) OH or

Val⁸-GLP-1 (7-37) OH,

How much⁸-GLP-1 (7-37) OH,

Thr⁸-GLP-1 (7-37) OH,

Val⁸-GLP-1 (7-36) NH2,

How much⁸-GLP-1 (7-36) NH2,

Thr⁸-GLP-1 (7-36) NH2,

Gly⁸-GLP-1 (7-36) NH2,

Leu⁸-GLP-1 (7-37) NH2,

Cheese⁸-GLP-1 (7-36) NH2,

Ala¹⁶-GLP-1 (7-36) NH2.

The polypeptide of the invention has the amino acid sequence of formula III (SEQ ID NO: 3): His-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Xaa22-Xaa23 -Ala-Ala-Lys-Xaa27-Phe-Ile-Xaa30-Trp-Leu-Val-Lys-Gly-Arg-R

Formula III (SEQ ID NO: 3) in which:

Xaa8 is: Gly, Ala, Val, Leu, Ile, Ser or Thr;

Xaa22 is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, Cys or cysteic acid;

Xaa23 is: His, Asp, Lys, Glu or Gln;

Xaa27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg or Lys. Xaa30 is: Ala, Glu, Asp, Ser or His;

R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH2, Gly, Gly-Pro or Gly-Pro-NH2 or not. Such a polypeptide lacks the GLP-1 (7-37) OH or GLP-1 (7-36) NH2 sequence, and is not

Gly⁸-GLP-1 (7-37) OH, Gly⁸-GLP-1 (7-36) NH2, Leu⁸-GLP-1 (7-37) OH, Leu⁸-GLP-1 (7-36) NH2, Ser⁸-GLP-1 (7-37) OH, Ser⁸-GLP-1 (7-36) NH2, Ala¹⁶-GLP-1 (7-36) OH, Ala¹⁶-GLP-1 (7-36) NH2

Val⁸-GLP-1 (7-36) NH2,

How much⁸-GLP-1 (7-36) NH2,

Thr⁸-GLP-1 (7-36) NH2,

Glu²⁷-GLP-1 (7-36) NH2.

Val⁸-GLP-1 (7-37) OH,

How much⁸-GLP-1 (7-37) OH,

Thr⁸-GLP-1 (7-37) OH,

Glu²⁷-GLP-1 (7-37) OH,

The invention also describes polypeptides having the amino acid sequence of formula IV (SEQ ID NO: 4):

Xaa7-Xaa8-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Xaa22-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Lys-Gly-Arg-R

Formula IV (SEQ ID NO: 4) in which:

Xaa₇ is L-histidine, D-histidine, desaminohistidine, 2-aminohistidine, β-hydroxyhistidine, homohistidine, α-fluoromethylhistidine and α-methylhistidine;

Xaa8 is glycine, alanine, valine, leucine, isoleucine, serine or threonine.

Xaa8 as recommended herein is glycine, valine, leucine, isoleucine, serine or threonine; Xaa22 is aspartic acid, glutamic acid, glutamine, asparagine, lysine, arginine, cysteine, or cysteic acid, and R is NH2 or Gly (OH).

The invention also describes a glucagon-like peptide 1 (GLP-1) compound having an amino acid other than alanine at position 8 and an amino acid other than glycine at position 22.

The invention also describes a method of stimulating a GLP-1 receptor in a subject in need of GLP-1 receptor stimulation. Such a method comprises the step of administering to a subject an effective amount of the GLP-1 compounds described herein, or a polypeptide having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4.

The invention also describes GLP-1 compounds, including compounds with the amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, for stimulating a GLP-1 receptor in a subject in need of it. stimulation of the GLP-1 receptor.

The GLP-1 compounds of the present invention retain the ability of GLP-1 to activate the receptor and, in addition, show a reduced tendency to aggregate compared to other GLP-1 compounds. As a result, solutions of the compounds in question can be mixed with only minimal conversion to insoluble inactive forms. This advantage greatly simplifies the manufacturing process. In addition, it is expected that when administered to a subject, no aggregation will occur in vivo, if indeed only to a small extent. This means increasing the activity of the compounds in question and minimizing their ability to cause adverse reactions. In addition, these GLP-1 compounds are resistant to degradation by diaminopeptidase IV and bind zinc and are believed to provide extended duration of action in vivo.

Figure 1 shows the amino acid sequences

Val⁸-Glu²² -GLP-1 (7-37) OH (SEQ ID NO: 5); Val⁸-Asp²² -GLP-1 (7-37) OH (SEQ ID NO: 6); Val⁸-Arg²² -GLP-1 (7-37) OH (SEQ ID NO: 7); and Val⁸-Lys²² -GLP-1 (7-37) OH (SEQ ID NO: 8).

Figure 2 shows the amino acid sequences

Gly⁸-Glu²² -GLP-1 (7-37) OH (SEQ ID NO: 9); Gly⁸-Asp²² -GLP-1 (7-37) OH (SEQ ID NO: 10); Gly⁸-Arg²² -GLP-1 (7-37) OH (SEQ ID NO: 11); and Gly⁸-Lys²² -GLP-1 (7-37) OH (SEQ ID NO: 12).

Figure 3 shows the amino acid sequences:

Val⁸-Glu³⁰-GLP-1 (7-37) OH (SEQ ID NO: 13);

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Gly⁸-Glu³⁰-GLP-1 (7-37) OH (SEQ ID NO: 14);

Val⁸-His³⁷-GLP-1 (7-37) OH (SEQ ID NO: 15); and

Gly⁸-His³⁷-GLP-1 (7-37) OH (SEQ ID NO: 16).

Figure 4 shows the amino acid sequences:

Val⁸-Glu²²-Ala²⁷-GLP-1 (7-37) OH (SEQ ID NO: 17);

Val⁸-Lys²²-Glu²³-GLP-1 (7-37) OH (SEQ IDNO: 18);

A GLP-1 compound is a polypeptide of from about 25 to about 39 amino acids, both natural and non-natural, and sufficiently homologous with GLP-1 (7-37) such that it exhibits insulinotropic activity. Examples of non-naturally occurring amino acids include α-methylamino acids (e.g., α-methylalanine), D-amino acids, histidine-like amino acids (e.g., 2-aminohistidine, β-hydroxyhistidine, homohistidine, α-fluoromethylhistidine, and α-methylhistidine), additional amino acids. a methylene group in the side chain ("homo amino acids) and amino acids, where the carboxylic acid functional group on the side chain is replaced with a sulfonic acid moiety (e.g., cysteic acid). However, the GLP-1 compounds of the present invention contain only naturally occurring amino acids, unless otherwise specified herein.

The term "GLP-1 compound typically includes a polypeptide with the amino acid sequence GLP-1 (7-37) OH, a GLP-1 (7-37) OH analog, a GLP-1 (7-37) OH fragment, or a GLP-1 analog fragment ( 7-37) OH.

GLP-1 (7-37) OH has the amino acid sequence of SEQ ID NO: 19:

⁷His-Ala-Glu-¹⁰Gly-Thr-Phe-Thr-Ser-¹⁵Asp-Val-Ser-Ser-Tyr-²⁰Leu-Glu-Gly-Gln-Ala-²⁵Ala-Lys-Glu-Phe-Ile-³⁰Ala-Trp-Leu-Val-Lys-³⁵Gly-Arg-³⁷Gly (SEQ ID NO: 19)

As is customary in the art, the N-terminus of GLP-1 (7-37) OH is given residue number 7 and the C-terminus is given residue number 37. Other amino acids in the polypeptide are numbered sequentially as shown in SEQ ID NO: 19 . For example, position 12 is phenylalanine and position 22 is glycine. Unless otherwise specified, the C-terminus is in the conventional carboxylic form.

"A GLP-1 fragment is a truncated polypeptide, obtained after cleaving one or more amino acids from the N-terminus and / or C-terminus of GLP-1 (7-37) OH or a GLP-1 (7-37) OH analogue). The nomenclature used to describe GLP-1 (7-37) OH is transferred to the GLP-1 fragments. For example, GLP-1 (9-36) OH is a GLP-1 fragment obtained by cleaving two amino acids from the N-terminus and one amino acid from the C-terminus. Amino acids present in the fragment are assigned the same number as the corresponding amino acids in GLP-1 (7-37) OH. For example, the N-terminal glutamic acid in GLP-1 (9-36) is at position 9, position 12 is occupied by phenylalanine and position 22 by glycine, as in GLP-1 (7-37) OH .

The term "GLP-1 compound also includes polypeptides with one or more amino acids added to the N-terminus or C-terminus of GLP-1 (7-37) OH, or to fragments thereof. GLP-1 compounds of this type contain up to about 39 amino acids. The amino acids in the "GLP-1 extended compound" are numbered with the same numbers as the corresponding amino acids in GLP-1 (7-37) OH. For example, the N-terminal amino acid of a GLP-1 compound obtained by adding two amino acids to the N-terminus of GLP-1 (7-37) OH is at position 5, and the C-terminal amino acid of the GLP-1 compound obtained by adding one amino acid to the C-terminus of GLP-1 (7-37) OH is at position 38. Thus, in both of these "extended GLP-1 compounds, position 12 is taken by phenylalanine and position 22 is taken by glycine. as in GLP-1 (7-37). Conveniently, amino acids 1-6 of the extended GLP-1 compound are the same or a conservative amino acid substitution at the corresponding GLP-1 (1-37) OH position. Conveniently, amino acids 38-45 of the GLP-1 extended compound are the same or a conservative amino acid substitution at the corresponding glucagon or exendin-4 position.

The "GLP-1 analog" is sufficiently homologous to GLP-1 (7-37) OH or to a GLP-1 (7-37) OH fragment to exhibit insulinotropic activity. Suitably, the GLP-1 analog has the amino acid sequence of GLP-1 (7-37) OH, or a fragment thereof, modified such that one, two, three, four, or five amino acids differ from the amino acids at the corresponding GLP-1 (7-37) position. 37) OH or a GLP-1 (7-37) OH fragment. In the nomenclature used herein to denote GLP-1 compounds, the substitution amino acid and its position are indicated before the parent structure. For example, Glu²²-GLP-1 (7-37) OH is a GLP-1 compound in which the glycine normally found at position 22 of GLP-1 (7-37) OH has been replaced with glutamic acid; Val⁸-Glu²²-GLP-1 (7-37) OH is the compound GLP-1 in which the alanine normally found at position 8 and the glycine normally found at position 22 of GLP-1 (7-37) OH have been replaced with valine and acid, respectively glutamine.

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Generally, the N-terminus of a GLP-1 compound is unsubstituted but may be alkylated or acylated (especially with a C1-C20 group). The C-terminus may be unsubstituted, as in the case of GLP-1 (7-37) OH, but may be amidated with -NH2, -NHR or NRR ', or esterified with -OR'. R and R 'are, independently, alkyl or acyl groups (especially C1-C20). R "is an alkyl group (C1-C20). GLP-1 (7-36) NH2 is an exemplary "amidated GLP compound). Preferred GLP-1 compounds have the C-terminus unsubstituted or substituted with -NH2.

The GLP-1 compounds of the present invention include GLP-1 analogs or fragments of GLP-1 analogs whose backbone contains at position 8 an amino acid other than alanine ("analogs at position 8). The skeleton may also contain at position 7 L-histidine, D-histidine or modified forms of histidine such as desaminohistidine, 2-aminohistidine, β-hydroxyhistidine, homohistidine, α-fluoromethylhistidine or α-methylhistidine). Suitably such analogs at position 8 may contain one or more changes at positions 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36 and 37 as compared to the corresponding native amino acid GLP-1 (7-37) OH. More suitably, such position 8 analogs may contain one or more additional changes at positions 12, 16, 22, 23, 30, 35 and 37 compared to the corresponding amino acid of native GLP-1 (7-37) OH. Even more conveniently such analogs at position 8 may contain one or more additional changes at positions 22, 23, 27, 30 and 37 compared to the corresponding amino acid of native GLP-1 (7-37) OH.

Suitably these analogs may contain 6 or fewer changes from the corresponding amino acids in native GLP-1 (7-37) OH. More suitably, the analogs may contain 5 or fewer changes from the corresponding amino acids in native GLP-1 (7-37) OH or 4 or fewer changes from the corresponding amino acids in native GLP-1 (7-37) OH . Even more conveniently, these analogs may contain 3 or fewer changes from the corresponding amino acids in native GLP-1 (7-37) OH, in particular these analogs contain 2 or fewer changes from the corresponding amino acids in native GLP-1. (7-37) OH.

Such substitutions have been found to reduce the tendency of GLP-1 compounds to aggregate and form insoluble forms. In general, when evaluating them (e.g. in the aggregation assay described in Example 3, below), GLP-1 compounds of the present invention aggregate at least 5 times more slowly than GLP-1 (7-37) OH, in particular at least 20 times slower, in particular at least 40 times slower, also in particular about 50 times slower, especially about 60 times slower and also in particular at least 65 times slower. Suitably the GLP-1 compounds described herein are analogs of GLP-1 (7-36) NH2 or GLP-1 (7-37) OH.

In a GLP-1 compound described herein, the amino acid present at position 22 may have a side chain of at least two carbon atoms and polar or charged functional groups. This also applies to aspartic acid, which contains methylene and carboxylic carbon. More suitably, the side chain of the amino acid at position 22 may have a straight or branched chain alkyl group of 2 to 6 carbon atoms with a charged functional group such as, for example, a carboxylic acid, amine, guanidine or sulfonic acid moiety. Thus, examples of amino acids at position 22 include, in addition to glutamic acid, aspartic acid, arginine and lysine. Where position 22 is aspartic acid, glutamic acid, arginine or lysine, then position 8 is suitably glycine, valine, leucine, isoleucine, serine, threonine or methionine, especially valine or glycine. An exemplary amino acid with a sulfonic acid side chain moiety is cysteic acid [(-NH-CH (CH2SO3) -CO, abbreviated "Cya]". When position 22 is occupied by a sulfonic acid such as cysteic acid then position 8 is suitably glycine, valine, leucine, isoleucine, serine, threonine or methionine, especially valine or glycine.

In the compound described herein, the amino acid at position 8 is glycine or valine, or it may optionally be leucine, isoleucine, serine, threonine, or methionine, more preferably valine or glycine, and at position 30, glutamic acid, aspartic acid, serine or histidine, especially acid glutamine.

In another compound described herein, the amino acid at position 8 is glycine or valine, or optionally it may be leucine, isoleucine, serine, threonine, or methionine, more preferably valine or glycine, and at position 37, histidine, lysine, arginine, threonine, serine, glutamic acid , aspartic acid, tryptophan, tyrosine, phenylalanine and especially histidine.

In another compound described herein, the amino acid at position 8 is glycine labo-valine, or it may optionally be leucine, isoleucine, serine, threonine, or methionine, and more preferably valine or glycine, and at position 22, glutamic acid, lysine, aspartic acid, or arginine, especially glutamic acid or lysine, and at position 23, lysine, arginine, glutamic acid, aspartic acid and histidine, in particular lysine or glutamic acid.

In another compound described herein, the amino acid at position 8 is glycine or valine, or it may optionally be leucine, isoleucine, serine, threonine or methionine, especially valine or glycine, at position 22 glutamic acid, lysine, aspartic acid or arginine, and especially glutamic acid or lysine, and at position 27, alanine, lysine, arginine, tryptophan, tyrosine, phenylalanine or histidine, especially alanine.

Other GLP-1 compounds described herein contain an amino acid at position 8, and one, two, or three amino acids selected from the group consisting of position 11, position 12, position 16, position 22, position 23, position 24, position 26, position 27, position 30 , position 33, position 34, position 35, position 36 and position 37 which differ from the amino acid at the corresponding position of native GLP-1 (7-37) OH.

Other GLP-1 compounds described herein contain, in addition to the amino acid at position 8, one or two amino acids selected from the group consisting of position 11, position 12, position 16, position 22, position 23, position 24, position 26, position 27, position 30, position 33, position 34, position 35, position 36 and position 37 which differ from the amino acid at the corresponding position of native GLP-1 (7-37) OH.

As described above, the GLP-1 compounds described herein may contain, in addition to the amino acids present at positions 8, 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36, and 37, amino acids that differ from those at the corresponding positions in GLP-1 (7-37) or in the GLP-1 (7-37) fragment. Amino acids other than those found at positions 8, 11, 12, 16, 22, 23, 24, 26, 27, 30, 33, 34, 35, 36, and 37 in GLP, differing from the amino acid at the corresponding GLP- positions 1 (7-37) OH are suitably conservative substitutions, especially highly conservative substitutions.

The GLP-1 compounds described herein may contain 0, 1, 2, or 3 amino acids, in addition to the amino acids at positions 8 and 22, which differ from the amino acid at the corresponding GLP-1 (7-37) OH position or GLP-1 fragment (7 -37) OH. According to one exemplary such embodiment, one or more of the amino acids at positions 7, 21, and 27 of a GLP-1 compound differ from the corresponding amino acid in GLP-1 (7-37) OH or the GLP-1 (7-37) fragment. ) OH, in addition to the amino acids at positions 8 and 22.

Suitably only positions 7, 8 and 22 differ from the amino acid at the corresponding position GLP-1 (7-37) OH (or a fragment thereof). It is expected that other improved GLP-1 compounds with reduced aggregation properties can be obtained from known biologically active GLP-1 compounds by replacing glycine at position 22 and, suitably, alanine at position 8 of these compounds with the appropriate amino acid as described in this description. Known, biologically active GLP-1 compounds are disclosed in the following publications: US Patent No. 5,977,071 (Hoffmann et al.); U.S. Patent No. 5,545,618 (Buckley et al.); J. Biol. Chem, 269, 6275 (1994).

The term "conservative substitution" means the replacement of an amino acid with another amino acid having the same net electric charge and approximately the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains are approximately the same size when the total number of carbon and heteroatoms in their side chains differ by no more than 4. They are approximately the same shape when the number of branches in their side chains varies by no more than 1. It is believed that amino acids with phenyl groups or substituted phenyl groups in their side chains have about the same size and shape. Five groups of amino acids are listed below. Replacing an amino acid in a GLP-1 compound with another amino acid from the same group results in a conservative substitution.

Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, and non-naturally occurring amino acids with C1 - C4 aliphatic or hydroxyl substituted C1 - C4 aliphatic side chains (straight or monobranched).

Group II: glutamic acid, aspartic acid and non-naturally occurring amino acids with substituted C1 - C4 carboxylic acid-aliphatic side chains (straight or branched at one site).

Group III: lysine, ornithine, arginine and non-naturally occurring amino acids with substituted amino or guanidino group C1 - C4 aliphatic side chains (straight or branched at one site).

Group IV: glutamine, asparagine and non-naturally occurring amino acids with amide substituted C1 - C4 aliphatic side chains (straight or branched at one site).

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Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

Unless otherwise specified herein, conservative substitutions are preferably made with natural amino acids.

The term "highly conservative substitution" means the replacement of an amino acid with another amino acid with the same side chain functionality and about the same size and shape. Amino acids with aliphatic or substituted aliphatic amino acid side chains are about the same size when the total number of carbon and heteroatoms in their side chains differs by no more than two. They have almost the same shape if they have the same number of branches in their side chains. Examples of highly conservative substitutions include valine for leucine, threonine for serine, aspartic acid for glutamic acid, and phenylglycine for phenylalanine. Examples of substitutions that are not highly conserved include alanine for valine, alanine for serine, and aspartic acid for serine.

An exemplary GLP-1 compound described herein is a polypeptide with the amino acid sequence of SEQ ID NO: 1. A preferred exemplary GLP-1 compound is GLP-1 (7-37) OH, wherein Xaa8 is Gly or Val, Xaa22 is Glu or Lys, and Xaa23 is Glu or Lys. Another exemplary GLP-1 compound is GLP-1 (7-37) OH, with the proviso that Xaa8 is Gly or Val and Xaa30 is Glu. Additionally, an exemplary GLP-1 compound is GLP-1 (7-37) OH, wherein Xaa8 is Gly or Val and Xaa37 is His.

Another exemplary GLP-1 compound described herein is a polypeptide with an amino acid sequence

SEQ ID NO: 4. In a preferred exemplary compound of SEQ ID NO: 4, Xaa7 is L-histidine,

D-histidine, deazminohistidine, 2-aminohistidine, β-hydroxyhistidine, homohistidine, α-fluoromethylhistidine and α-methylhistidine, Xaa₈ means glycine, alanine, valine, leucine, isoleucine, serine or threonine, especially glycine, valine, leucine, isoleucine, serine or threonine, R is -NH2 or Gly (OH), and Xaa22 is lysine, glutamic acid, aspartic acid or arginine. In a more preferred exemplary compound, Xaa7 is L-histidine, Xaa8 is glycine or valine, Xaa22 is lysine, glutamic acid, aspartic acid or arginine, and R is Gly (OH). Alternatively, in SEQ ID NO: 4, Xaa7, Xaa8 and R are as defined above and Xaa22 is an amino acid with a side chain containing a sulphonic acid moiety, for example cysteic acid.

In other exemplary GLP-1 compounds described herein, the amino acid at position 8 is other than the D-amino acid and lacks a glycine, serine, threonine, cysteine, or betaalanine side chain when the amino acid at position 22 has a C1 - C2 alkyl chain side chain, hydroxylated C1 - C2 alkyl side chain or a thiolated C1 - C2 alkyl side chain (such as, for example, cysteine). In the preferred exemplary GLP-1 compounds described herein, the amino acid at position 8 is other than the D-amino acid and lacks a glycine, serine, threonine, cysteine, or betaalanine side chain when the amino acid at position 22 has a C1 - C4 alkyl side chain, hydroxylated A C1 - C4 alkyl side chain or a thiolated C1 - C4 alkyl side chain.

In other exemplary GLP-1 compounds described herein, the amino acid at position 8 is glycine, valine, or optionally leucine, isoleucine, methionine, serine, threonine, cysteine, aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine, phenylalanine, tyrosine, histidine or tryptophan and the amino acid at position 22 is aspartic acid, glutamic acid, lysine, arginine, asparagine, glutamine, or histidine.

Specific exemplary GLP-1 compounds described herein include:

Glu²²-GLP-1 (7-37) OH (SEQ ID NO: 20),

Asp²²-GLP-1 (7-37) OH (SEQ ID NO: 21),

Arg²²-GLP-1 (7-37) OH (SEQ ID NO: 22),

Lys²²-GLP-1 (7-37) OH (SEQ ID NO: 23),

Cya²²-GLP-1 (7-37) OH (SEQ ID NO: 24),

Val⁸-Asp²²-GLP-1 (7-37) OH (SEQ ID NO: 6),

Val⁸-Arg²²-GLP-1 (7-37) OH (SEQ ID NO: 7),

Val⁸-Lys²²-GLP-1 (7-37) OH (SEQ ID NO: 8),

Val⁸-Cya²²-GLP-1 (7-37) OH (SEQ ID NO: 25),

Gly⁸-Asp²²-GLP-1 (7-37) OH (SEQ ID NO: 10),

Gly⁸-Arg²²-GLP-1 (7-37) OH (SEQ ID NO: 11),

Gly⁸-Lys²²-GLP-1 (7-37) OH (SEQ ID NO: 12),

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Gly⁸-Cya²²-GLP-1 (7-37) OH (SEQ ID NO: 26),

Glu²²-GLP-1 (7-36) NH2 (SEQ ID NO: 27),

Asp²²-GLP-1 (7-36) NH2 (SEQ ID NO: 28),

Arg²²-GLP-1 (7-36) NH2 (SEQ ID NO: 29),

Lys²²-GLP-1 (7-36) NH2 (SEQ ID NO: 30),

Cya²²-GLP-1 (7-36) NH2 (SEQ ID NO: 31),

Val⁸-Asp²²-GLP-1 (7-36) NH2 (SEQ ID NO: 33),

Val⁸-Arg²²-GLP-1 (7-36) NH2 (SEQ ID NO: 34),

Val⁸-Lys²²-GLP-1 (7-36) NH2 (SEQ ID NO: 35),

Val⁸-Cya²²-GLP-1 (7-36) NH2 (SEQ ID NO: 36),

Gly⁸-Asp²²-GLP-1 (7-36) NH2 (SEQ ID NO: 38),

Gly⁸-Arg²²-GLP-1 (7-36) NH2 (SEQ ID NO: 39),

Gly⁸-Lys²²-GLP-1 (7-36) NH2 (SEQ ID NO: 40), and Gly⁸-Cya²²-GLP-1 (7-36) NH2 (SEQ ID NO: 41),

Val⁸-Lys²³-GLP-1 (7-3 (7) OH (SEQ ID NO: 42),

Val⁸-Ala²⁷-GLP-1 (7-3 (7) OH (SEQ ID NO: 43),

Val⁸-Glu³⁰-GLP-1 (7-3 (7) OH (SEQ ID NO: 44),

Gly⁸-Glu³⁰-GLP-1 (7-37) OH (SEQ ID NO: 45),

Val⁸-His³⁵-GLP-1 (7-3 (7) OH (SEQ ID NO: 46),

Val⁸-His³⁷) -GLP-1 (7-3 (7) OH (SEQ ID NO: 47),

Val⁸-Gly³⁴-Lys³⁵-GLP-1 (7-37) OH (SEQ ID NO: 51),

Val⁸-His³⁷) -GLP-1 (7-3 (7) OH (SEQ ID NO: 52),

Gly⁸-His³⁷) -GLP-1 (7-3 (7) OH (SEQ ID NO: 53).

The term "GLP-1 compound as used herein also includes the pharmaceutically acceptable salts of the compounds described herein. A GLP-1 compound described herein may contain a sufficiently strong acid functional group, a sufficiently strong basic functional group, and both, whereby it may accordingly react with a variety of inorganic bases as well as inorganic and organic acids to form a salt. Acids commonly used to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc., as well as organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc. Examples of this type of salt include sulfate, metabisulfite, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, octanoate, acrylate, formate, hexanoate, isobutyrate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-danoate, hexine-1,6-danoate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate etc.

Basic addition salts include those derived from inorganic bases such as ammonium, alkali or alkaline earth metal hydroxides, carbonates and bicarbonates and the like. Thus, the bases useful for the formation of the salts described herein include sodium hydroxide, potassium hydroxide, ammonium hydroxide , potassium carbonate etc.

GLP-1 compounds can be used to treat subjects suffering from a wide variety of diseases and conditions. GLP-1 compounds, including the compounds of the present invention, are believed to exert their biological effects by acting on a receptor referred to as the "GLP-1 receptor [see US Patent No. 5,670,360 (Thorrens)]). Thus, subjects suffering from diseases and / or conditions with a beneficial response to GLP-1 receptor stimulation or administration of GLP-1 compounds may be treated with the GLP-1 compounds of the present invention. Such subjects are referred to as "in need of treatment with GLP-1 compounds or" in need of GLP-1 receptor stimulation. " These terms also include subjects with non-insulin dependent diabetes, insulin dependent diabetes mellitus, stroke [see WO 00/16797 (Efendic)], myocardial infarction [see WO 98/08531 (Efendic)], obesity [see: patent document WO 98/19698 (Efendic)], catabolic changes after surgery [see US Patent10

In U.S. Patent No. 6,006,753 (Efendic)], functional dyspepsia and irritable bowel syndrome [see WO 99/64060 (Efendic)]. Also encompassed by this term are subjects in need of prophylactic treatment with a GLP-1 compound, for example subjects at risk of developing insulin-dependent diabetes (see WO 00/07617). Subjects with impaired glucose tolerance or impaired fasting glycaemia, subjects weighing 25% over normal body weight for the subject's height and body build, hereinafter subjects with partial pancreatic resection, having one or both parents with non-insulin dependent diabetes mellitus , women who have had gestational diabetes and subjects who have had acute or chronic pancreatitis they are all at risk of developing non-insulin dependent diabetes mellitus.

The term "effective amount of a GLP-1 compound" as used herein is that amount which, when administered to a subject in need of GLP-1 receptor stimulation, results in the desired therapeutic and / or prophylactic effect without causing undesirable side effects. The term "desired therapeutic effect" encompasses one or more of the following events: 1) alleviation of symptom (s) associated with a given disease or condition; 2) delay in the onset of symptoms related to a given disease or condition; 3) increase in life expectancy compared to life expectancy in the absence of treatment; and 4) better quality of life compared to quality of life in the absence of treatment. For example, "an effective amount of a GLP-1 compound in the treatment of diabetes is that amount which will provide better blood glucose control than it would be without treatment, thereby delaying the onset of diabetic complications such as such as retinopathy, neuropathy, or kidney disease. "An effective amount of a GLP-1 compound in the prevention of diabetes is that amount that will delay, compared to no treatment, the onset of elevated blood glucose levels requiring treatment with antihypoglycemic drugs such as sulfonylureas, thiazolidinediones, insulin and / or biguanides.

The "effective amount of GLP-1 compound" administered to a subject will also depend on the nature and severity of the disease, and on specific characteristics such as general health, age, sex, body weight, and drug tolerance. One skilled in the art can determine appropriate dosages depending on these and other factors. Typically, a therapeutically effective amount of a GLP-1 compound administered to an adult may range from about 0.01 mg / day to about 1000 mg / day. A recommended dosage is in the range of about 0.1 mg / day to about 100 mg / day, especially in the range of about 1.0 mg / day to about 10 mg / day.

The GLP-1 compounds of the present invention can, for example, be administered orally, nasally, by inhalation, or parenterally. Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, or intraperitoneal injection. GLP-1 compounds can be administered to a subject in conjunction with a pharmaceutically acceptable carrier, diluent or excipient as part of a pharmaceutical composition for the treatment of the above-discussed diseases. The pharmaceutical composition may be a solution or, when administered parenterally, a suspension of a GLP-1 compound or a suspension of a GLP-1 compound complexed with a divalent metal cation, as described below. Suitable pharmaceutical carriers can contain inert ingredients which do not interact with the peptide or peptide derivative. Conventional pharmaceutical drug formulation methods can be used herein, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Suitable pharmaceutical carriers include, for example, sterile water, physiological sodium chloride solution, bacteriostatic sodium chloride solution (containing about 0.9% mg / ml benzyl alcohol), phosphate buffered sodium chloride solution, Hank's solution, Ringer's lactate, etc. Some examples of suitable excipients include lactose, glucose, sucrose, trehalose, sorbitol, and mannitol.

As used herein, the term "subject" refers to a mammal, especially a human, as well as domestic animals (e.g., dogs, cats, etc.), farm animals (such as cows, sheep, pigs, horses, etc.), and laboratory animals ( such as rats, mice, guinea pigs, etc.).

Preferably, GLP-1 compounds of the present invention can be complexed with a suitable divalent metal cation. The complexes formed by GLP-1 compounds with divalent metal cations are generally insoluble in aqueous solutions at approximately physiological pH values. Thus, these complexes can be administered subcutaneously in the form of suspensions and exhibit a reduced release rate in vivo thereby prolonging the duration of action of the compound. Examples of suitable divalent metal cations include: Zn⁺⁺, Me⁺⁺, Fe⁺⁺, Ca⁺⁺, What⁺⁺, Cd⁺⁺, Ni⁺⁺ etc. Zn cations are preferred⁺⁺.

PL 206 302 B1

To obtain complexes of GLP-1 compounds of the present invention with divalent metal cations, GLP-1 is dissolved in an appropriate buffer in the presence of a metal salt. The mixture is allowed to stand at ambient temperature to allow the reaction to take place and to precipitate the resulting complex. Suitable buffers are those buffers that maintain the pH of the mixture in the range of about 3.0 to about 9.0 and that do not interfere with the complexation reaction. Exemplary buffers include phosphate buffers, acetate buffers, citrate buffers, and Goode buffers, such as, for example, HEPES, Tris, and Tris acetate. Suitable metal salts are those for which the metal is available for the complexation reaction. Examples of suitable zinc salts include zinc chloride, zinc acetate, zinc oxide, and zinc sulfate. Conveniently, a divalent metal salt such as zinc chloride is introduced in excess to give a molar ratio of up to about 50 divalent metal cation molecules for each GLP-1 molecule.

The term "insulinotropic activity" refers to the stimulation of insulin secretion in response to increased glucose levels, thereby causing cellular glucose uptake and a reduction in serum glucose levels. Insulinotropic activity can be assessed by methods known in the art, including carrying out in vivo and in vitro experiments that measure GLP-1 receptor binding activity or receptor activation, such as assays using islet cells or insulinoma cells as described, respectively. , in EP 619322 (Gelfand et al.) and in US Patent No. 5,120,712.

The GLP-1 compounds of the present invention can be prepared using conventional solid phase peptide synthesis methods. Peptide synthesizers are commercially available, for example from Applied Biosystems in Foster City CA. Reagents for solid phase peptide synthesis are commercially available, for example, from Midwest Biotach (Fishers, IN). Solid-phase peptide synthesizers can be used, according to the manufacturer's instructions, to block interfering groups, protect the amino acids to be reacted, conjugate, detach, and "capping unreacted amino acids."

Typically, an α-N-carbamoyl protected amino acid and an N-terminal amino acid are coupled on the nascent peptide chain on a resin at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling reagents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, and bases such as diisopropylethylamine. The α-N-carbamoyl protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid or piperidine and the coupling reaction is repeated to add the next desired N-protected amino acid to the peptide chain. Suitable amino protecting groups are well known in the art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis, John Wiley and Sons (1991). The guidance contained therein is incorporated herein by reference in its entirety. Exemplary groups here are: tert-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).

Peptides are also synthesized by conventional automated solid phase synthesis protocols using tert-butoxycarbonyl- or fluorenylmethoxycarbonyl-α-amino acids with appropriate side chain protection. After the synthesis is complete, the peptides are cleaved from the solid phase support while unblocking the side chain, using conventional hydrofluoride procedures. The obtained crude peptides are further purified by reverse phase chromatography on Vydac C18 columns using acetonitrile gradients in 0.1% trifluoroacetic acid (TFA). To remove acetonitrile, peptides are lyophilized from a solution containing 0.1% TFA, acetonitrile and water. Purity is checked by an analytical reverse-phase chromatography method. The identity of the peptides can be checked by mass spectrometry. Peptides can be dissolved in aqueous buffers at neutral pH.

The invention is further illustrated by the following Examples which should not be construed as limiting the scope of the invention in any way.

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Example 1. Preparation of GLP-1 compounds of the present invention using t-Boc solid phase chemistry

Approximately 0.5-0.6 g (0.38-0.45 mmol) of Boc-Gly-PAM resin was placed in a standard 60 ml reactor and the double coupling was initiated on Applied Biosystems ABI430A peptide synthesizer. The following side chain protected amino acids (2 mM charges of Boc-amino acids) obtained from Midwest Biotach (Fishers, IN) were used for the synthesis:

Arg-tosyl (TOS), Asp1-cyclohexyl ester (CHXL), Glu6-cyclohexyl ester (CHXL), Hisbenzyloxymethyl (BOM), Lys-2-chlorobenzyloxycarbonyl (2Cl-2), Methsulfoxide (O), Ser ether -O-benzyl (OBz1), Thr-O-benzyl ether (OBz1), Trp-formyl (CHO) and Tyr-2-bromobenzyloxycarbonyl (2Br-Z) and Boc-Gly-PAM resin. Trifluoroacetic acid (TFA), diisopropylethylamine (DIEA), 0.5M hydroxybenzotriazole (HOBt) in DMF, and 0.5M dicyclohexylcarbodiimide (DCC) in dichloromethane were supplied by PE-Applied Biosystems (Foster City, CA). Dimethylformamide (DMF-Burdick and Jackson) and dichloromethane (DCM-Mallinkrodt) were supplied by Mays Chemical Co. (Indianapolis, IN).

Typical double couplings were performed using either symmetric anhydride or HOBt esters, both formed using DCC. A second series of double couplings (not deprotected with TFA) were performed with Trp31, Thr13 and Thr11. After the synthesis was complete, the N-terminal Boc group was removed and the peptidyl-resin was treated with a 20% solution of piperidine in DMF to deformyl the Trp side chain. After washing with DCM, the resins were transferred to a Teflon reactor and dried in vacuo.

For the Met-containing analogs, reductions on the resin were performed using TFA / 10% dimethylsulfoxide (DMS) / 2% concentrated HCl. Cleavage was accomplished by connecting the reactor to an HF (hydrofluoric acid) apparatus (Penninsula Laboratories). 1 ml m-cresol / gram resin was added and 10 ml HF (supplied by AGA, Indianapolis, IN) was condensed into a pre-chilled reactor. In the case of presence of methionine, 1 ml of DMS / g resin was added. The reaction mixtures thus formed were stirred for 1 hour in an ice bath, then the HF was removed in vacuo. The residues were dissolved in diethyl ether and the solids were filtered off and washed with ether. Each peptide was extracted into aqueous acetic acid and either lyophilized or loaded directly onto a reverse phase chromatography column.

Purification was performed on a 2.2 x 25 cm VYDAC C18 column in buffer A (0.1% trifluoroacetic acid in water, B: 0.1 TFA in acetonitrile). A gradient of 20% to 90% B (Waters) was applied to HPLC over 120 minutes at 10 ml / min, with UV monitoring at 280 nm (4.0 A) and collection of one minute fractions. The appropriate fractions were combined together, frozen and lyophilized. Dry products were analyzed by HPLC (0.46 x 15 cm, METASIL AQ C18) and MALDI mass spectrometry.

Example 2. Preparation of GLP-1 compounds of the present invention using F-Moc solid phase chemistry

Approximately 114 mg (50 mmol) of FMOC-Gly-Wang resin (supplied from NovaBiochem, LaJolla, CA) was placed in the programmed wells of a 96-well reaction block and double couplings were performed on an Advanced ChemTech 396 peptide synthesizer. C-terminal amide analogs were obtained using (75 mg (50 µmol) of Rink Amide AM resin (NovaBiochem; LaJolla, CA).

The following Fmoc-amino acids were supplied by Advanced ChemTech (Louisville, KY), NovaBiochem (La Jolla, CA), and Midwest Biotach (Fishers, IN):

Arg-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Asn-trityl (Trt), Asp-e-tertbutyl ester (tBu), Glu-e-tert-butyl ester (tBu), Gln-trityl (Trt), His-trityl (Trt), Lys-tert-butyloxycarbonyl (Boc), Ser-tert-butyl ether (OtBu), Thr-tert-butyl ether (OtBu), Trp-tert-butyloxycarbonyl (Boc ), Tyr-tert-butyl ether (OtBu).

Solvents: dimethylformamide (DMF - Burdick and Jackson), N-methylpyrrolidone (NMP - Burdick and Jackson) and dichloromethane (DCM - Mallinkrodt) were supplied by Mays Chemical Co. (Indianapolis).

Hydroxybenzotriazole (HOBt), diisopropylcarbodiimide (DIC), diisopropylethylamine (DIEA) and piperidine (Pip) were supplied by Aldrich Chemical Co. (Milwaukee, WI).

All amino acids were dissolved in 0.45 M HOBt in NMP and after 20 min deblocking with 20% Pip / DMF 50 min activation couplings (DIC / HOBt) were performed. Each resin was washed with DMF after deblocking and coupling.

After the final coupling and deprotection, the peptidyl resins were washed with DCM and dried in vacuo in the reaction block.

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In the appropriate reaction / cleavage block kit, 2 ml of K reagent was introduced into each well and the cleaved reaction mixture was stirred for 2 hours [K reagent = 0.75 g phenol, 0.5 ml thioanisole, 0.25 ml ethanedithiol, 0 , 5 mL of water per 10 mL of trifluoroacetic acid (TFA); all components were supplied by Aldrich Chemical Co., Milwaukee, WI]. The TFA filtrates were introduced into 40 ml of diethyl ether and the precipitates were centrifuged for 2 minutes at 2000 rpm. Subsequently, the supernatants were decanted, the pellets were suspended in 40 ml of ether, re-centrifuged and, after decantation, dried under nitrogen and then under vacuum.

0.3-0.6 mg portions of each of the above-mentioned products were dissolved in 1 ml aliquots of 0.1% TFA / acetonitrile (ACN) and 20 μl of the sample was analyzed by HPLC [0.46 x 15 cm METASIL AQ C18, 1 mL / min, 45 ° C, 214 nM (0.2 A), A = 0.1% TFA, B = 0.1% TFA / 50% ACN. Gradient = 50% B to 90% B for 30 minutes].

Purification operations were performed on a 2.2 x 25 cm VYDAC C18 column in buffer A (0.1% trifluoroacetic acid in water, B: 0.1% TFA in acetonitrile). A gradient of 20% to 90% B (Waters) was applied after HPLC over 120 minutes at 10 ml / min, with UV monitoring at 280 nm (4.0 A) and collection of one minute fractions. The appropriate fractions were combined together, frozen and lyophilized. Dry products were analyzed by HPLC (0.46 x 15 cm, METASIL AQ C18) and MALDI mass spectrometry.

Example 3. GLP Aggregation Test

The GLP peptides of the present invention were analyzed for their ability to aggregate in solution. Generally speaking, the peptides in solution were mixed at an elevated temperature in an appropriate buffer, recording turbidity at 350 nm as a function of time. The start time of aggregation was measured to quantify the ability of a given GLP molecule to aggregate under these constrained conditions.

Protocol:

First, GLP-1 Compound was dissolved under alkaline conditions (pH 10.5) for 30 minutes to dissolve any aggregated material. Thereafter, the pH of the solution thus obtained was adjusted to 7.4 and filtered. More specifically, 4 mg of the lyophilized GLP-1 compound was dissolved in 3 ml of a 10 mM phosphate / 10 mM citrate system and the pH was adjusted to a value between 10.0 and 10.5 and held at this level for 30 minutes. The solution was then adjusted with HCl to pH (7.4 and filtered through a suitable filter such as, for example, Millex GV syringe filter (Millipore Corporation, Bedford, MA). The solution was then diluted to give a final sample of 0.3 mg / ml protein in the system 10 mM citrate, 10 mM phosphate, 150 mM NaCl and the solution was adjusted to pH 7.4-7.5 The sample was incubated at 37 ° C in a quartz cuvette. The turbidity of the solution was measured every 5 minutes at 350 nm using an AVIV Model 14DS spectrophotometer. UV-VIS (Lakewood, NJ). 30 seconds before and during the measurement, the solution was mixed using a magnetic stirrer from Starna Cells, Inc. (Atascadero, CA). An increase in the OD value at 350 nm indicates aggregation of the GLP-1 peptide. The time to aggregation is approximately determined by the intersection of the lines from the linear fits to the pre-growth phase and the growth phase according to the Drake method [T. Arvinte, A. Cudd, and AF Drake, J. Bio. Chem., 268,6415-6422 (1993)].

Between two experiments, the litter box was cleaned with a caustic soap solution (for example, Contrad-70).

The results obtained for a number of GLP-1 compounds of the present invention are shown in Table 1 below as the time (h) required for the compound to aggregate. As can be seen from the data in this table, the compounds of the present invention show an aggregation time much longer than that exhibited by the prior art GLP-1 compounds.

Example 4. GLP-1 receptor activation with GLP-1 compounds of the present invention

The ability of GLP-1 compounds of the present invention to activate the GLP-1 receptor has been assessed in in vitro assays such as those described in EP 619322 (Gelfand et al.) And in US Patent No. 5,120,712, respectively. Compounds in relation to GLP-1 (7-37) OH activity are shown in Table 1 below. As can be seen from the data in this table, the activity of the GLP-1 compounds of the present invention is generally as good as or even better than GLP-1 (7-37) OH.

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Table 1

<td>GLP-1 compound</td><td>Aggregation time (h)</td><td>GLP-1 receptor activation</td>

<td>GLP-1 (7-37) OH</td><td> 1</td><td> 1.0</td>

<td>Val⁸-GLP-1 (7-37) OH</td><td>0.9 ± 0.2 (n = 6)</td><td> 0.47</td>

<td>Gly⁸-His^11-GLP-1 (7-37) OH</td><td> 9*</td><td> 0.282</td>

<td>Val⁸-Ala¹¹-GLP-1 (7-37) OH</td><td> 10</td><td> 0.021</td>

<td>Val⁸-Lys¹³-GLP-1 (7-37) OH</td><td> 13</td><td> 0.001</td>

<td>Val⁸-Tyr¹²-GLP-1 (7-37) OH</td><td> 6</td><td> 0.81</td>

<td>Val⁸-Glu¹⁶-GLP-1 (7-37) OH</td><td> 12</td><td> 0.047</td>

<td>Val⁸-Ala¹⁶-GLP-1 (7-37) OH</td><td> 16</td><td> 0.112</td>

<td>Val⁸-Tyr¹²-GLP-1 (7-37) OH</td><td> 5</td><td> 1.175</td>

<td>Val⁸-Lys²⁰-GLP-1 (7-37) OH</td><td> 5</td><td> 0.33</td>

<td>Main²²-GLP-1 (7-37) OH</td><td> 7</td><td> 0.42</td>

<td>Val⁸-Ala²²-GLP-1 (7-37) OH</td><td> 19</td><td> 0.56</td>

<td>Val⁸-Cheese²²-GLP-1 (7-37) OH</td><td> 22</td><td> 0.50</td>

<td>Val⁸-Asp²²-GLP-1 (7-37) OH</td><td> &gt;90</td><td> 0.40</td>

<td>Val⁸-Glu²²-GLP-1 (7-37) OH</td><td> 72</td><td> 1.29</td>

<td>Val⁸-Lys²²-GLP-1 (7-37) OH</td><td> 100, 54</td><td> 0.58</td>

<td>Val⁸-Pro²²-GLP-1 (7-37) OH</td><td> &gt;75</td><td> 0.01</td>

<td>Val⁸-His²²-GLP-1 (7-37) OH</td><td> &gt;75</td><td> 0.14</td>

<td>Val⁸-Lys²²-GLP-1 (7-36) NH2</td><td> 24</td><td> 0.53</td>

<td>Val⁸-Glu²²-GLP-1 (7-36) NH2</td><td> &gt;65</td><td> 1.0</td>

<td>Gly⁸-Glu²²-GLP-1 (7-37) OH</td><td> 19</td><td> 1.07</td>

<td>Val⁸-Glu²³-GLP-1 (7-36) OH</td><td> 65</td><td>0.2B</td>

<td>Val⁸-Lys²³-GLP-1 (7-37) OH</td><td> &gt;45</td><td> 0.18</td>

<td>Val⁸-His²⁴-GLP-1 (7-37) OH</td><td> 3</td><td> 0.007</td>

<td>Val⁸-Lys²⁴-GLP-1 (7-37) OH</td><td> 22</td><td> 0.02</td>

<td>Ala⁸-His²⁶-GLP-1 (7-37) OH</td><td> &gt;24</td><td> 0.8</td>

<td>Ala⁸-Glu²⁶-GLP-1 (7-37) OH</td><td> &gt;24</td><td> 0.7</td>

<td>Val⁸-His²⁷-GLP-1 (7-37) OH</td><td> 10</td><td> 0.37)</td>

<td>Val⁸-Ala²⁷-GLP-1 (7-37) OH</td><td> 2</td><td> 0.47</td>

<td>Gly⁹-Glu³⁰-GLP-1 (7-37) OH</td><td> &gt;40</td><td> 0.29</td>

<td>Val⁸-Glu³⁰-GLP-1 (7-37) OH</td><td> 30</td><td> 0.29</td>

<td>Val⁸-Asp³⁰-GLP-1 (7-37) OH</td><td> &gt;45</td><td> 0.15</td>

<td>Val⁸-Cheese³⁰-GLP-1 (7-37) OH</td><td> 8</td><td> 0.19</td>

<td>Val⁸-His³⁰-GLP-1 (7-37) OH</td><td> 13</td><td> 0.19</td>

<td>Val⁸-Glu³³-GLP-1 (7-37) OH</td><td> &gt;70</td><td> 0.039</td>

<td>Val⁸-Ala³³-GLP-1 (7-37) OH</td><td> 20</td><td> 0.1</td>

<td>Val⁸-Gly³³-GLP-1 (7-37) OH</td><td> 9</td><td> 0.01</td>

<td>Val⁸-Glu³⁴-GLP-1 (7-37) OH</td><td> &gt;40*</td><td> 0.17</td>

<td>Val⁸-Pro³⁵-GLP-1 (7-37) OH</td><td> 14</td><td> 0.094</td>

<td>Val⁸-His³⁵-GLP-1 (7-37) OH</td><td> &gt;45, 30</td><td> 0.41</td>

<td>Val⁸-Glu³⁵-GLP-1 (7-37) OH</td><td> 63</td><td> 0.15</td>

<td>Val⁸-Glu³⁶-GLP-1 (7-37) OH</td><td> &gt;45</td><td> 0.11</td>

<td>Val⁸-His³⁶-GLP-1 (7-37) OH</td><td> 8</td><td> 0.22</td>

<td>Val⁸-His³⁷-GLP-1 (7-37) OH</td><td> &gt;40</td><td> 0.33</td>

<td>Val⁸-Leu¹⁶-Glu²⁶-GLP-1 (7-37) OH</td><td> &gt;20</td><td> 0.23</td>

<td>Val⁸-Lys²²-Glu³⁰-GLP-1 (7-37) OH</td><td> 4</td><td> 0.37)</td>

<td>Val⁸-Lys²²-Glu²³-GLP-1 (7-37) OH</td><td> &gt;30</td><td> 0.35</td>

<td>Val⁸-Glu²²-Gln²³-GLP-1 (7-37) OH</td><td> &gt;20</td><td> 0.47</td>

<td>Val⁸-Glu²²-Ala²¹-GLP-1 (7-37) OH</td><td> &gt;45</td><td> 1.02</td>

<td>Val⁸-Glu²²-Lys²³-GLP-1 (7-37) OH</td><td> &gt;65</td><td> 1.43</td>

<td>Val⁸-Lys³³-Val³⁴-GLP-1 (7-37) OH</td><td> 22</td><td> 0.08</td>

<td>Val⁸-Lys³³-Asn³⁴-GLP-1 (7-37) OH</td><td> &gt;48</td><td> 0.09</td>

<td>Val⁸-Gly³⁴-Lys³⁵-GLP-1 (7-37) OH</td><td> 27</td><td> 0.34</td>

<td>Val⁸-Gly³⁶-Pro³⁷) -GLP-1 (7-37) NH</td><td> 2 2</td><td> 0.53</td>

* Aggregation time was determined at 30 ° C.

Example 5. Precipitation of GLP-1 compounds with zinc

Individual GLP-1 compounds were prepared as described in Examples 1 or 2. In 3 ml of 0.1 M HEPES buffer pH 10.5, 3 mg of individual freeze-dried GLP particles were dissolved. Thereafter, the pH of the resulting solution was adjusted to a value in the range of 10.0 to 10.5 using 0.2 N NaOH. The resulting solution was stirred at room temperature for 30 minutes and then its pH was adjusted to 7.4 with 0.2 N HCl. The solution was filtered through a suitable syringe filter, for example a Millex GV syringe filter (Millipore Corporation, Bedford, MA) and the concentration of the GLP-1 compound was determined by measuring the absorption at 280 nm using a spectrophotometer such as a Beckman DU640. Then, the protein concentration was adjusted to

200 μΜ in HEPES pH 7.4.

Then, 100 μL of filtered GLP-1 solutions on an ELISA plate (for example Falcon Microtest ™ 96) was diluted with 100 μL of 0.1 M HEPES pH 7.4, containing various concentrations of zinc chloride, to obtain 200 μL of a solution containing various zinc chloride concentration and 100 μM of GLP-1 compounds. These solutions were kept at ambient temperature (22 ° C) for 18 hours and then centrifuged, for example, in a Jouan CR412 centrifuge with microplate adapters. After centrifugation, 150 µl of the supernatant was transferred to a UV-readable ELISA microtiter plate (e.g. Costar UV plate) and determined the OD at 280 nm using a microplate reader (e.g. Molecular Devices SPECTRAmax PLUS, SOFTmax PRO). The obtained results of the experiment are presented in Table 2 below. The A250 values ​​are the result of two independent determinations.

Table 2

<td>Ratio molar Zn / GLP-1</td><td>GLP-1 (7-37) OH A280</td><td>Gly⁸- GLP-1 (7-37) OH A280</td><td>Val⁸- GLP-1 (7-37) OH A280</td><td>Main²²- GLP-1 (7-37) OH A280</td><td>Val⁸-Glu²²GLP-1 (7-37) OH A280</td><td>Val⁸-Ala²²GLP-1 (7-37) OH A280</td>

<td> 0</td><td> 0.337</td><td> 0.32</td><td> 0.3</td><td> 0.290</td><td> 0.295</td><td> 0.289</td>

<td> 0.3</td><td> 0.318</td><td> 0.166</td><td> 0.27</td><td> 0.390</td><td> 0.291</td><td> 0.202</td>

<td> 0.5</td><td> 0.329</td><td> 0.151</td><td> 0.26</td><td> 0.123</td><td> 0.292</td><td> 0.107</td>

<td> 0.7</td><td> 0.253</td><td> 0.156</td><td> 0.124</td><td> 0.076</td><td> 0.293</td><td> 0.104</td>

<td> 1</td><td> 0.148</td><td> 0.119</td><td> 0.06</td><td> 0.074</td><td> 0.26</td><td> 0.110</td>

<td> 2</td><td> 0.092</td><td> 0.089</td><td> 0.025</td><td> 0.095</td><td> 0.078</td><td> 0.110.</td>

<td> 3</td><td> 0.081</td><td> 0.085</td><td> 0.021</td><td> 0.095</td><td> 0.052</td><td> 0.104</td>

<td> 5</td><td> 0.074</td><td> 0.078</td><td> 0.019</td><td> 0.097</td><td> 0.035</td><td> 0.119</td>

Table 2 cont

<td>Ratio molar Zn / GLP-1</td><td>Val⁸-Cheese²²- GLP-1 (7-37) OH A280</td><td>Val⁸- Phe²²-GLP-1 (7-37) OH A280</td><td>Val⁸- Pro²²-GLP-1 (7-37) OH A280</td><td>Val⁸-Lys²²GLP-1 (7-37) OH A280</td><td>Val⁸-Asp²²GLP-1 (7-37) OH A280</td>

<td> 0</td><td> 0.2855</td><td> 0.31</td><td> 0.2595</td><td> 0.299</td><td> 0.288</td>

<td> 0.3</td><td> 0.2805</td><td> 0.1485</td><td> 0.2455</td><td> 0.0825</td><td> 0.2785</td>

<td> 0.5</td><td> 0.2665</td><td> 0.1165</td><td> 0.2325</td><td> 0.0905</td><td> 0.2845</td>

<td> 0.7</td><td> 0.1825</td><td> 0.1015</td><td> 0.219</td><td> 0.1195</td><td> 0.287</td>

<td> 1</td><td> 0.149</td><td> 0.1265</td><td> 0.1905</td><td> 0.1225</td><td> 0.291</td>

<td> 2</td><td> 0.0935</td><td> 0.092</td><td> 0.1695</td><td> 0.1675</td><td> 0.184</td>

<td> 3</td><td> 0.101</td><td> 0.061</td><td> 0.1615</td><td> 0.1475</td><td> 0.1485</td>

<td> 5</td><td> 0.0615</td><td> 0.00795</td><td> 0.171</td><td> 0.142</td><td> 0.1675</td>

PL 206 302 B1

These results show that only a small amount of zinc is needed to complex and substantially precipitate the various GLP-1 compounds from these dilute solutions.

Equivalent Modes for Carrying Out the Invention

While the present invention has been detailed and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various form and detail changes may be made thereto without departing from the spirit and scope of the invention as defined in the attached claims. Those skilled in the art will recognize, or will be able, by mere routine experimentation, to ascertain that multiple modes of carrying out the invention equivalent to those detailed herein will be possible.

PL 206 302 B1

Sequence Listing

<td> &lt;110&gt;</td><td>Eli Lilly and Company</td>

<td> &lt;120&gt;</td><td>GLUCAGON-LIKE PEPTIDE-1 ANALOGS</td>

<td> &lt;130)</td><td>Χ-13989</td>

<td> &lt;160&gt;</td><td> 53</td>

<td> &lt;170&gt;</td><td>Patentln version 3.0</td>

<td> &lt;210&gt;</td><td> 1</td>

<td> &lt;211&gt;</td><td> 31</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial</td>

&lt;220&gt;

<td> &lt;223&gt;</td><td>synthetic construction</td>

&lt;220&gt;

<td> &lt;221&gt;</td><td>VARIANT</td>

<td> &lt;222&gt;</td><td> (2) .. (2)</td>

<td> &lt;223&gt;</td><td>Xaa at position 2 is Gly, Ala, Val, Leu, Ile, Ser or</td>

Thr;

<td> &lt;220&gt;</td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td>

<td> &lt;222&gt;</td><td> (5) .. (5)</td>

<td> &lt;223&gt;</td><td>Xaa at position 5 is Asp, Glu, Arg, Thr, Ala, Lys or His;</td>

&lt;220&gt;

<td> &lt;221&gt;</td><td>VARIANT</td>

PL 206 302 B1

<td rowspan="2"> &lt;222&gt; &lt;223&gt;</td><td colspan="2"> (6) . . (6)</td><td rowspan="2">means His,</td><td rowspan="2">Trp,</td><td rowspan="2">Phe or Tyr;</td>

<td>Xaa in position</td><td> 6 &lt;</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (10)..(10)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 10</td><td>means Leu,</td><td>Cheese,</td><td>Thr, Trp, His, Phe,</td>

<td></td><td>Asp, Val, Glu</td><td colspan="2">or Ala;</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (16)..(16)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 16</td><td>means Gly,</td><td>Asp,</td><td>Glu, Gin, Asn, Lys,</td>

<td></td><td>Arg or Cys;</td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (17) . . (17)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 17</td><td>means His,</td><td>Asp,</td><td>Lys, Glu or Gin;</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (18)..(18)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 18</td><td>stands for Glu,</td><td>His,</td><td>Ala or Lys;</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (20) .. (20)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 20</td><td>means Asp,</td><td>Lys,</td><td>Glu or His;</td>

PL 206 302 B1 &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (21). . (21) &lt;223&gt; Xaa at position 21 is Ala, Glu, His, Phe, Tyr, Trp, Arg or Lys;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (25) .. (25) &lt;223&gt; Xaa at position 25 is Ala, Glu, Asp, Ser or His;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (27) .. (27) &lt;223&gt; Xaa at position 27 is Asp, Arg, Val, Lys, Ala, 'Gly or Glu;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (28) .. (28) &lt;223&gt; Xaa at position 28 is Glu, Lys or Asp;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (29) .. (29) &lt;223&gt; Xaa at position 29 is Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly, Pro, His or Glu;

&lt;220&gt;

&lt;221&gt; VARIANT

PL 206 302 B1

<td rowspan="2"> &lt;222&gt; &lt;223&gt;</td><td colspan="3"> (30)..(30)</td><td rowspan="2">Arg,</td><td rowspan="2">Glu</td><td rowspan="2">or</td><td rowspan="2">His;</td>

<td>Xaa</td><td>in position</td><td>30 oz nacz</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td colspan="2">VARIANT</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (31)</td><td> ..(31)</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa</td><td>in position</td><td>31 means</td><td>Lys,</td><td>Arg,</td><td>Thr</td><td>, Cheese, Glu, Asp,</td>

<td></td><td>Trp,</td><td>Tyr, Phe,</td><td>His, -NH₂,</td><td>Gly,</td><td>Gly-</td><td>-Pro</td><td>or Gly-Pro-</td>

NH₂or it isn't there.

&lt;400&gt; 1

His Xaa Glu Gly Xaa Xaa Thr Ser Asp Xaa Cheese Ser Tyr Leu Glu Xaa 15 10 15

Xaa Xaa Ala Xaa Xaa Phe Ile Ala Xaa Leu Xaa Xaa Xaa Xaa Xaa

25 30 &lt;210&gt; 2 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (2) .. (2) &lt;223&gt; Xaa in position 2 means Gly, Ala, Val, Leu, Ile, Ser or

Thr;

PL 206 302 B1 &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (6) .. (6) &lt;223&gt; Xaa at the β position is His, Trp, Phe or Tyr;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (10) .. (10)

<td> &lt;223&gt;</td><td>Xaa in position Val, Glu or j</td><td>10 ?\la;</td><td>means</td><td>Leu,</td><td>Cheese,</td><td>Thr,</td><td>Trp, His, Phe, Asp</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (16)..(16)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 16</td><td>means</td><td>Gly,</td><td>Asp,</td><td>Glu,</td><td>Gin, Asn, Lys, Arg</td>

<td></td><td>or Cys;</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (17) .. (17)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 17</td><td>means</td><td>His,</td><td>Asp,</td><td>Lys,</td><td>Glu or Gin;</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (20) .. (20)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 20</td><td>means</td><td>Asp,</td><td>Lys,</td><td>Glu</td><td>or His;</td>

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (24) .. (24)

PL 206 302 B1

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 24</td><td>means Ala, Glu, Asp,</td><td>Ser or His;</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (29)..(29)</td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 29</td><td>means Thr, Ser, Lys,</td><td>Arg, Trp, Tyr, Phe</td>

Asp, Gly, Pro, His or Glu;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (31) .. (31) &lt;223&gt; Xaa at position 31 is Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, -NH₂, Gly, Gly-Pro or Gly-Pro-NH_:or it isn't there.

&lt;400&gt; 2 .

His Xaa Glu Gly Thr Xaa Thr Ser Asp Xaa Ser Ser Tyr Leu Glu Xaa ¹⁵ 10 15

Xaa Ala Ala Xaa elu Phe Ile Xaa Trp Leu Val Lys Xaa Arg Xaa ²⁰ 25 jo &lt;210&gt; 3 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (2) .. (2) &lt;223&gt; Xaa at position 2 is Gly, Ala, Val, Leu, Ile, Ser or Thr;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (16) .. (16)

<td> &lt;223&gt;</td><td>Xaa in position or Cys;</td><td>16 means</td><td>Gly,</td><td>Asp, Glu, Gin, Asn, Lys,</td><td>Arg,</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (17)..(17)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td>17 means</td><td>His,</td><td>Asp, Lys, Glu or Glu;</td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (21) . . (21)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position or Lys;</td><td>21 means</td><td>Ala,</td><td>Glu, His, Phe, Tyr, Trp,</td><td>Arg</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (24) . . (24)</td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td>24 means</td><td>Ala,</td><td>Glu, Asp, Ser or His;</td><td></td>

&lt;220&gt;

PL 206 302 B1

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td>

<td> &lt;222&gt;</td><td> (31) .. (31)</td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa at position 31 is Lys, Arg, Thr, Ser, Glu,</td><td>Asp,</td><td>Trp</td>

<td></td><td>Tyr, Phe, His, -NH₂, Gly, Gly-Pro or Gly-Pro-NH₂,</td><td>or</td><td>him</td>

<td></td><td>there is no.</td><td></td><td></td>

&lt;400&gt; 1

His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 15 10 15

Xaa Ala Ala Lys Xaa Phe Ile Xaa Trp Leu Val Lys Gly Arg Xaa 20 25 30 &lt;210&gt; 4 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (1). (1) &lt;223&gt; Xaa at position 1 is L-histidine, D-histidine, deaminohistidine, 2-aminohistidine, betahydroxyhistidine, homohistidine, alphafluorethylhistidine, or alpha-methylhistidine;

&lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (2) .. (2)

PL 206 302 B1

<td>&lt;223&gt; Thr;</td><td>Xaa in position</td><td> 2</td><td>means</td><td>Gly, Ala,</td><td>Val, Leu, Ile,</td><td>Cheese</td><td>or</td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (16)..(16)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 16</td><td>means</td><td>Asp, Glu</td><td>, Gin, Asp, Lys,</td><td>Arg</td><td>or</td>

<td></td><td>Cys;</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;222&gt;</td><td> (31) .. (31)</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa in position</td><td> 31</td><td>means</td><td>-NH₂ or</td><td>Gly.</td><td></td><td></td>

&lt;4Q0&gt; 4

Xaa Zaa Glu Gly Thr Phe Thr Ser Aap Val Ser Ser Tyr Leu Glu Zaa

5 10 15

Glu Ala Ala Lya Glu Phe Ile Ala Trp Leu Val Lya Gly Ara Zaa

25 30 &lt;210&gt; 5 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223) synthetic construction

PL 206 302 B1 &lt;400&gt; 5

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu

5 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 6 &lt;211&gt; 31 &lt;222&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;40Q&gt; 6

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp 15 lo 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lya Gly Arg Gly 20 25 30 &lt;210&gt; 7 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;230&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 7

Hia Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Len Glu Arg ¹ 5 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 8 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;^ 30&gt; synthetic structure &lt;400&gt; 8

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 9 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220 &lt;223&gt; Synthetic &lt;400&gt; 5w

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu ^L. 5 io ₁₅

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30

PL 206 302 B1

<img file="PL206302B1_D0001.tif" />

PL 206 302 B1 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 12

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 &lt;210&gt; 13 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 13

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

5 10 ₁₅^J.

Gin Ala Ala Lys Glu Phe Ile Glu Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 14 &lt;211&gt; 31

PL 206 302 B1

<img file="PL206302B1_D0002.tif" />

<td> &lt;210&gt;</td><td> 16</td>

<td> &lt;211&gt;</td><td> 31</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial</td>

PL 206 302 B1 &lt;220&gt;

&lt;223&gt; synthetic structure &lt;40θ&gt; 16

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly ¹⁵ 10 15

Gin Ala Ala Lys Glu Phe ile Ala Trp Leu Val Lys Gly Arg His 20 - 25 30 &lt;210&gt; 17 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 17

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu ¹ 5 10 15

Gin Ala Ala Lys Ala Phe Ile Ala Trp Leu Val Lys Gly Aro His

25 30 &lt;210&gt; 18 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

PL 206 302 B1 &lt;223&gt; synthetic construction &lt;400-18

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys ¹⁵ 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Ara Hia 20. 25 30

<td> &lt;210&gt;</td><td> 19</td>

<td> &lt;211&gt;</td><td> 31</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Homo sapiens</td>

&lt;400&gt; 19

His Ala Cłu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly ^{1 5} 10 15

Gin Ala Ale Lys Glu Phe Ile Ale Trp Leu Val Lys Gly Aro Gly

25 lo &lt;210&gt; 20 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;40Q&gt; 20

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu ¹⁵ 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 &lt;210&gt; 21 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 21

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp ¹ 5 10 is

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu val Lys Gly Arg Gly

25 30 &lt;210&gt; 22 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 22

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Arg

S 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 23 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 23

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lya

5 10 1S

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 24 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;220&gt;

&lt;221&gt; VARIANT

PL 206 302 B1 &lt;222&gt; (16). (16) &lt;223&gt; Xaa at position 16 is cysteic acid.

&lt;400&gt; 24

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 &lt;210&gt; 25 &lt;211&gt; 31 &lt;212&gt; PRT.

&lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (16). (16) &lt;223&gt; Xaa at position 16 is cysteic acid.

&lt;400&gt; 25

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly &lt;210&gt;

&lt;210&gt;

&lt;211&gt;

&lt;212&gt;

PRT

PL 206 302 B1 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (16). (16) &lt;223&gt; Xaa at position 16 is cisteric acid.

&lt;400&gt; 26

Ή ** The Thr S &lt;r Asp Val Sar Sar Tyr Leu Glu Xaa

1Q ₁₅

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 &lt;210&gt; 27 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 27

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu ¹⁵ 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30

PL 206 302 B1 &lt;210&gt; 28 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 28

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3Q &lt;210&gt; 29 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 29

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Arg 15 10 15

Gin Ala Ala Lys Glu Phe ile Ala Trp Leu Val Lys Gly Arg 20 '25 30 &lt;210&gt; 30 &lt;211&gt; 30

PL 206 302 B1 &lt;2i2&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure ^ 400&gt; 30

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Lys 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 31 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (16). (16) &lt;223&gt; Xaa at position 16 is cisteric acid.

PL 206 302 B1 &lt;400&gt; 31

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 S 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu val Lys Gly Arg 20 25 30 &lt;210&gt; 32 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;4D0&gt; 32

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr.Leu Glu Glu 15 10 15

Gin Ala Ala Lya Glu Phe Ile Ale Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 33 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 33

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 34 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 34

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Arg 15 10 15 w

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 35 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 35

<td rowspan="2">His 1</td><td colspan="3">Val Glu Gly Thr Phe Thr Ser Asp Val Sar Ser tyr Leu Glu Lys</td>

<td> 5</td><td> 10</td><td> 15</td>

<td>Gin</td><td>Ala Ala Lya Glu Phe Ile Ala</td><td>Trp Leu Val Lys</td><td>Gly Arg</td>

<td></td><td> 20</td><td> 25</td><td> 30</td>

<td> &lt;210&gt;</td><td> 36</td><td></td><td></td>

<td> &lt;211&gt;</td><td> 30</td><td></td><td></td>

<td> &lt;212&gt;</td><td>PRT</td><td></td><td></td>

<td> &lt;213&gt;</td><td>Artificial</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td>

<td> &lt;223&gt;</td><td>synthetic construction</td><td></td><td></td>

<td> &lt;220&gt;</td><td></td><td></td><td></td>

<td> &lt;221&gt;</td><td>VARIANT</td><td></td><td></td>

<td> &lt;222&gt;</td><td> (16). .(16)</td><td></td><td></td>

<td> &lt;223&gt;</td><td>Xaa at position 16 is acid</td><td>cysteine.</td><td></td>

<td colspan="2"> &lt;400&gt; 3«</td><td></td><td></td>

<td>KiB 1</td><td>Val Glu Gly Thr phe Thr Ser Asp S</td><td>Val Cheese Cheese 10</td><td>Tyr Leu Glu Zaa 15</td>

Gin Ala Ala Lys Glu Phe He Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 37 &lt;211&gt; 30 &lt;212&gt; PRT

PL 206 302 B1 &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 37

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu ¹ ' ⁵ 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 38 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 38

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Asp ¹ 5 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Γτρ Leu Val Lys Gly Arg 20 25 30

<td> &lt;210&gt;</td><td> 39</td>

<td> &lt;211&gt;</td><td> 30</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial</td>

PL 206 302 B1 &lt;220&gt;

&lt;223&gt; synthetic structure &lt;40Q&gt; 19

His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Arg 1 5. 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 40 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;40O&gt; 40

His Gly Glu Gly Thr Phe Thr Sar Asp Val Ser Ser Tyr Leu Glu Lys 15 10 15

GIC Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 &lt;210&gt; 41 &lt;211&gt; 30 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;220&gt;

&lt;221&gt; VARIANT &lt;222&gt; (16). (16) &lt;223&gt; Xaa at position 16 is cisteric acid

<td colspan="2"> &lt;400&gt; 41</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>His</td><td>Gly Glu</td><td>Gly</td><td>Thr</td><td>Phe</td><td>Thr</td><td>Cheese</td><td>Asp</td><td>Val</td><td>Cheese</td><td>Cheese</td><td>Tyr</td><td>Leu</td><td>Glu Xaa</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td>

<td>Gin</td><td>Ala Ala</td><td>Lys</td><td>Glu</td><td>Phe</td><td>How much</td><td>Ala</td><td>Trp</td><td>Leu</td><td>Val</td><td>Lys</td><td>Gly</td><td>Arg</td><td></td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td>

&lt;210&gt; 42 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 42

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

5 10 15

Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

25 30 &lt;210&gt; 43 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial

PL 206 302 B1 &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 43

His Val Glu Gly Thr Phe Thr Ser 1 5

Gin Ala Ala Lys Ala Pfaa Ile Ala 20

Asp Val Cheese Cheese Tyr Leu Glu Gly 10 15

Tip Leu Val Lys Gly Arg Gly 25 30 &lt;210&gt; 44 &lt;211&gt; 31 &lt;212&gt; PRT &lt;2 ± 3&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 44

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu 1 5 and 10

Gin Ala Ala Lys Glu Phe Ile Glu Trp Leu Val Lya Gly Arg 20 25 30

Glu Gly 15

Gly

<td> &lt;210&gt;</td><td> 45</td>

<td> &lt;211&gt;</td><td> 31</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial</td>

&lt;220&gt;

PL 206 302 B1

<img file="PL206302B1_D0003.tif" />

&lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 47

Kia Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 15 10 15

Gin Ala Ala Lys Glu Phe Ile Ala Trp Lwu Val Lya Gly Arg Hie 20 25 30 &lt;210&gt; 48 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 48

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 15 10 15

Lys Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 &lt;210&gt; 49 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 49

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 15 10 15

Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lya Gly Arg Gly 20 25 30 &lt;210&gt; 50 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 50

His V * 1 Glu 1

Gin Ala Ala

Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 5 10 15

Lys Ala Phe Ile Ala Trp Leu Val Lye Gly Arg Gly 20 25 30

<td> &lt;210&gt;</td><td> 51</td>

<td> &lt;211&gt;</td><td> 31</td>

<td> &lt;212&gt;</td><td>PRT</td>

<td> &lt;213&gt;</td><td>Artificial</td>

&lt;220&gt;

&lt;223&gt; synthetic construction

PL 206 302 B1 &lt;400&gt; 51

His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser'Tyr Leu Glu Gly 15 io i®

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Vał Gly Lys Arg Gly ²⁰ 25 30 &lt;210&gt; 52 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic structure &lt;400&gt; 52

His val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly ¹ 5 10 ₁₅

Gin Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg His

25 30 &lt;210&gt; 53 &lt;211&gt; 31 &lt;212&gt; PRT &lt;213&gt; Artificial &lt;220&gt;

&lt;223&gt; synthetic construction

<img file="PL206302B1_D0004.tif" />