Methods of synthesizing insulin polypeptide-oligomer conjugates, and proinsulin polypeptide-oligomer conjugates and methods of synthesizing same

Methods for the synthesis of products of the combination of insulin polypeptide with an oligomer, and products of the combination of proinsulin polypeptide with an oligomer

(and ways to create it)

Full description

Background of the invention:

This invention relates to products of a combination of insulin, methods for their synthesis, and methods for treating diseases thereof including diabetes.

Polypeptide insulin is the main hormone responsible for controlling the transport, use and storage of glucose in the body. The beta cells of the pancreatic islets secrete the first single-chain compound of insulin, known as proinsulin or proinsulin. Proteolysis of proinsulin results in the removal of some basic amino acids in the proinsulin chain and the attachment of the C-peptide to give the biologically active polypeptide insulin.

The insulin molecule has been largely conserved in development and generally consists of two chains of amino acids linked by double sulfur bonds. In humans, the two chains of the insulin molecule (molecular weight 5800 daltons) are: Chain (A) consists of 21 amino acids and has glycine at the amino terminus. The B chain consists of 30 amino acids and has phenylalanine at the amino terminus.

Insulin may exist in the form of a monomer, or it may be aggregated in the form of a dimer or hexamer consisting of three dimers. The biological activity, that is, the ability to bind to receptors and stimulate the biological action of insulin, lies in the monomer.

Diabetes is a disease that involves incorrect metabolism of carbohydrates. Diabetes results from a lack of insulin production or a lack of response to it. In people with diabetes, the normal ability to use glucose is lost, which increases blood sugar levels. As sugar accumulates in the blood, the excess levels are excreted in the urine. Other symptoms of diabetes include increased urine volume and frequency of urination, thirst, itching (scratching), hunger, weight loss, and weakness (general weakness).

There are two types of diabetes. The first type is insulin-dependent diabetes mellitus, or IDDM. IDDM was previously known as juvenile onset diabetes. In IDDM, insulin is not secreted by the pancreas and must be supplied from an external source. Type 2, or adult diabetes, can usually be controlled with food, although some advanced cases may require insulin.

Before insulin was isolated in the 1920s, most patients died within a short time of its introduction. Untreated cases of diabetes lead to ketosis, which is the accumulation of ketone bodies, which are the products of fat breakdown, in the blood. This is followed by acid buildup in the blood (aciduria) with nausea and vomiting. With the accumulation of toxic products of irregular carbohydrate and fat metabolism, the patient enters a diabetic coma, which leads to death.

The use of insulin as a treatment for diabetes dates back to 1922. When Banting and his colleagues (Can. Med. Assoc J., Diabetes, 12:141-146 (1922)) demonstrated that active extracts of the pancreas had therapeutic effects in diabetic dogs. In the same year, treating a diabetic patient with pancreatic extracts resulted in significant clinical improvement that saved the patient's life.

Until recently, only bovine and porcine insulin were used to treat diabetes in humans. However, there are many differences between the types of insulin depending on its class, which are currently known. Each type differs from human insulin by containing an amino acid substitution(s) at one or more sites in the A and/or B chain. Despite these differences, most mammalian insulin has the same biological activity. The advent of genetic engineering technology allowed the large-scale commercial manufacture of human insulin

eg, Humulin™ insulin, commercially available from Eli Lilly and Company,)

(Indianapolis, IN

Or genetically engineered insulin that has biological activity similar to natural human insulin.

Diabetes treatment requires regular frequent injections of insulin. Due to the uncomfortable nature of injections, extensive efforts have been made to improve insulin administration and its metabolism has been taken into account.

Attempts have been made to give insulin orally, and the problems of giving insulin orally to achieve a normal blood sugar level in diabetics are known and proven in pharmaceutical and medical references. Digestive enzymes quickly break down insulin, resulting in inactive metabolites. In the stomach, for example, insulin administered orally is subjected to enzymatic protein breakdown and acid breakdown. The same protein breakdown of insulin occurs in the intestine. In the intestinal lumen, insulin attacks several enzymes, including gastric and pancreatic enzymes, exogenous and endogenous peptidases, and peptidases of the ciliary wall of the intestine. Even if insulin bypasses this enzymatic attack, the biological barriers that insulin must cross before reaching its receptors within the body may limit its bioavailability after oral administration. For example, it might be insulin

Insulin has low membrane permeability, which limits its ability to cross from the intestinal lumen into the bloodstream.

Some efforts to provide an oral form of insulin have focused on providing insulin-oligomer combination products. Human insulin and many closely related insulins used therapeutically contain three amino acid moieties bearing free primary amino groups. All three primary amino groups, namely the amino terminus (alpha amino groups) of the A and B chains (GlyA1 and pheB1) and the epsilon-amino group of LysB29, can be modified by association with oligomers. Depending on the reaction conditions, the N-acyl of unprotected insulin results in a complex mixture of mono-, di-, and triple-conjugated products (for example: insulin mono-conjugated at GlyA1, insulin mono-conjugated at PheB1, insulin mono-conjugated at LysB29, insulin di-conjugated at GlyA1). And PheB1, insulin double-bonded at GlyA1 and PheB1, insulin double-bonded at GlyA1 and LysB29, insulin double-bonded at PheB1 and LysB29, and insulin triple-bonded at GlyA1, PheBI, and LysB29). When it is desired to obtain a specific conjugation product, for example an insulin monomerized at LysB29, it is difficult and/or expensive to separate (purify) such a complex mixture of conjugation products to obtain the desired conjugation product.

As a result, many efforts have been made to selectively synthesize the desired insulin conjugate. For example, Muranishi and Kiso, in Japanese patent application 254,699, point out a five-step synthesis for preparing insulin derivatives from a fatty acid. The Al and BI amino groups are protected (or locked) by para-methoxybenzoxycarbonyl azide (pMZ). After acylation of the fatty acid ester, the protecting groups are removed to yield insulin mono-acylated at LysB29 with a fatty acid. As another example, US Patent 5,750,497 in the name of Havelund and colleagues refers to the treatment of human insulin with Boc reagent (di-tert-butyl dicarbonate) to form (B1, Al) “second Boc human insulin, i.e. human insulin in which the amino terminus is protected.”

amino terminus of both chains A and B of the Boc group. After enantioselective purification, for example by high-pressure liquid chromatography, a lipophilic acyl group is introduced into the epsilon amino group of LysB29 by letting the product react with an N-hydroxysuccinimide ester with the formula X-OSu, where X is the lipophilic acyl group to be introduced. In the final step, trifluoroacetic acid is used to remove the Boc group and the resulting -X N-B29 human insulin is separated.

Many other efforts have been made to preferentially synthesize the desired insulin conjugate to provide a mixture of conjugate products in which the desired insulin conjugate is the preferred product. For example, Baker and co-workers in US Patent No. US 5,646,242 refer to a reaction performed without the use of amine protecting groups. Baker refers to the reaction of an activated acid ester with the epsilon-amino group in insulin under basic conditions in a polar solvent. The acylation of the amino-epsilon group depends on the basicity of the reaction. At a pH greater than 9.0, the reaction involves differential acylation of the amino-epsilon group in lysine B29-lysine on the amino-alpha groups. Examples 1 to 4 indicate the reaction products of monoconjugated insulin as a percentage of the initial amount of insulin between 67.1% and 75.5%. In Example 5, Baker also refers to the acylation of human proinsulin by N-succinimidyl palmitate. The exact ratios of the amino groups were not calculated. Epsilon acylation to amino-alpha acylation groups. The sum of all acylated amino-epsilon groups in the chromatogram equals 87-90% of the total area, while the sum of all related substances (which are assumed to include alpha-amino-acylated groups) which represents < 7% of the total area, at any time.

It is desirable to provide methods for the site-specific synthesis of desired insulin-oligomer adducts that may be less laborious and/or more effective than conventional methods described previously.

General description of the invention:

When compared with conventional schemes described previously, embodiments of this invention provide a less commercially expensive and/or more productive manufacturing scheme for preparing insulin-oligomer conjugates when a site-specific combination is desired (e.g., when it is preferable to provide a monomeric insulin-oligomer conjugate product combined with B-29Lys in the insulin molecule). Unlike previous conventional schemes, which suggest selective conjugation of insulin by closing the amino terminus of insulin with compounds such as PARA. Methoxybenzoxycarbonyl azide (Muranishi and Kiso) or by attempting to control the reaction conditions to reduce, but not prevent, the combination at the amino terminus of Baker insulin), embodiments of this invention combine the oligomer with B-29Lys in proinsulin or synthetic proinsulin (e.g., insulin). A primer fused at the amino terminus of its B chain to a leader peptide. The C-peptide (and the leader peptide, if present) is then cleaved from the oligomer–insulin combination to give insulin monoubiquitously bonded at B-29Lys to the oligomer. Embodiments of the invention may provide high site selectivity for B-29Lys modification. Methods of embodiments of this invention using the first insulin polypeptide, Proinsulin, can provide a high conversion of the modified yield at 29-B, for example, with yields of up to 0.8% or more, compared to that produced by conventional insulin pathways.

According to embodiments of this invention, the method for synthesizing the product of combining an insulin polypeptide with an oligomer includes connecting a proinsulin polypeptide to an insulin polypeptide conjugated to one or more peptides by a peptide bond(s) that can be broken to obtain a polypeptide. An insulin polypeptide with an oligomer under conditions sufficient for the oligomer to combine with the insulin polypeptide portion of the insulin polypeptide. The initial product gives the proinsulin polypeptide-oligomer, and one or more peptides are part of the polypeptide.

Insulin polypeptide-oligomer to give the product of insulin polypeptide-oligomer.

According to other embodiments of this invention, a method for synthesizing the insulin polypeptide-acyl oligomer conjugate includes enzymatically cleaving one or more peptides from the insulin polypeptide-acyl oligomer conjugate to obtain the insulin polypeptide conjugate. insulin polypeptide - acyl oligomer.

According to other embodiments of this invention, the method for synthesizing a proinsulin polypeptide with an oligomer includes connecting a proinsulin polypeptide ignited on an insulin polypeptide conjugated to one or more peptides by a peptide bond(s) that can be broken to obtain An insulin polypeptide with an oligomer under conditions sufficient for the oligomer to combine with the insulin polypeptide portion of the polypeptide. Proinsulin and the product of the combination gives proinsulin polypeptide-oligomer.

According to other embodiments of the invention, the product of a proinsulin polypeptide-oligomer is ignited on a proinsulin polypeptide and includes an insulin polypeptide, an oligomer combined with the insulin polypeptide portion of the insulin polypeptide.

Initial.

According to other embodiments of this invention, a method for synthesizing the product of a C-peptide conjugate to an oligomer includes a primary C-polypeptide conjugate comprising a C-peptide conjugated to one or more peptides by a peptide bond(s) which It can be cleaved to yield a C-peptide, with an oligomer under conditions sufficient for the oligomer to combine with the polypeptide moiety

C-peptide of a polypeptide, the first C-peptide and providing the product of the polypeptide conjugation, the C-peptide oligomer, and one or more peptides from the product of the tenacious conjugation of the peptide C-peptide, the first C-peptide - oligomer to obtain the product of the polypeptide conjugation. -C oligomer.

Brief explanation of the drawings:

Figure 1 shows an embodiment of a synthetic route for preparing insulin modified at B-29Lys using a proinsulin with a leader peptide;

Figure 2 shows the HPLC analysis of Proinsulin II conjugate.

Figure 3 shows the mass spectrum of the pure monomeric conjugate product of proinsulin II;

Figure 4 shows the mass spectrum of the pure dimeric product of proinsulin II;

Figure 5: shows the HPLC pattern for the production of the monomeric product of insulin-hexyl PEG7 - Insulin-hexyl.

Figure 6: Shows the mass spectrum of the trypsin moiety of proinsulin II;

Figure 7: HPLC pattern of the PEG7-hexyl moiety of insulin (Arg31) by carboxypeptidase B;

Figure 8: Shows the mass spectrum of the carboxypeptidase moiety product of insulin (Arg31), acylated at 29-B-hexyl (PEG7-hexyl).

Figure 9: HPLC pattern for the production of PEG7-hexyl insulin (poly-diffusion) from the monomeric product of primary insulin II activated by an enzyme cocktail of carboxypeptidase B and trypsin.

Figure 10: Shows the mass spectrum of the PEGn-insulin-hexyl product (polydiffusion) via proinsulin II;

Figure 11: HPLC pattern of production of acylated insulin at 29-B-hexyl PEG7-hexyl by proinsulin 1;

Figure 12: Mass spectrum of the resulting acylated insulin at 29-B-hexyl PEG7-hexyl via proinsulin I;

Figure 13: shows the mass spectrum of insulin (side product) derived from the insulin conjugate product mixture;

Figure 14: HPLC pattern of proinsulin I conjugate product; (A) Monomer and proinsulin I conjugate; (B) Monomer and proinsulin I dimer;

Figure 15: HPLC pattern for the production of PEG7-insulin-hexyl from the reaction of primary insulin I with an enzyme cocktail of carboxypeptidase B and trypsin.

Figure 16: HPLC pattern of insulin production (side product) from the reaction of primary insulin monomer A (I) with an enzyme cocktail of carboxypeptidase B and trypsin.

Detailed Description:-

This invention is now described in detail below with reference to the drawings, in which preferred embodiments of the invention are illustrated. However, this invention may be illustrated in many different ways and should not be understood to be limited to the embodiments described hereinafter; But these embodiments will clarify the invention so that the description is complete and fully representative of the field of the invention for those with experience in the field.

All amino acid abbreviations used in this description are those accepted by the US Patent and Trademark Office as described below in 37 CFR § 1.822(b).

The phrase &between& in this description describes various types of range which should be interpreted as including the final boundaries of the range described.

The phrase “essentially monodisperse” in this description is used to describe a mixture of compounds in which at least 95% of the compounds have the same molecular weight.

The phrase “monodisperse” describes a mixture of compounds in which approximately 100% of the compounds in the mixture have the same molecular weight.

The phrase “insulin polypeptide” in this description means a polypeptide that has at least some of the biological activity of insulin (eg, the ability to act on the body through the primary mechanism of insulin action). For example, an insulin polypeptide may be a polypeptide such as insulin with a polypeptide chain (A) and a polypeptide chain (B) joined to the chain (A) by double sulfur bonds. In various embodiments of this invention, the insulin polypeptide is preferably having most of the biological activity of insulin, preferably having substantially all of the biological activity of insulin and preferably all of the biological activity of insulin.

In this description, the phrase “proinsulin polypeptides” means an insulin polypeptide bound to one or more peptides (eg, leader peptides and/or contact peptides or C-peptides) by a laboratory-cleavable peptide bond(s). And inside the body. For example, the primary insulin polypeptide, an insulin polypeptide such as insulin, may include a polypeptide (A) chain joined to a polypeptide (B) chain, ligands such as double disulfide bonds, and a connecting peptide joined to the C-terminus ( Carboxy) of a polypeptide (B) chain and bound to the N-terminus (amine) of a polypeptide (A) chain by peptide bonds that can be cleaved in vitro or in vivo. As another example, the proinsulin polypeptide may be ignited on an insulin polypeptide, e.g., insulin. insulin, by A polypeptide chain (A) bonded to a polypeptide chain (B) by ml bonds, disulfide bonds, and a contact peptide bonded to the C-terminus (carboxy) of a polypeptide chain (B) and bonded to the N-terminus (amine) of a polypeptide chain (A). ) Peptide ligands that can be cleaved in vitro or in vivo, and a leader peptide fused to the N-terminus of the polypeptide (B) chain. Examples of proinsulin polypeptides include, without limitation, proinsulins, proinsulin analogues, fragments of proinsulin, fragments of a proinsulin homolog, or any

Proinsulin, proinsulin homologs, proinsulin fragments, fragments of a proinsulin homologue having a leader peptide; Protoinsulin, protoinsulin isomers, protoinsulin fragments, protoinsulin homologs, miniprotoinsulins, and fusion proteins.

The phrase “insulin” in this description means insulin of one of the following types: human, bovine, porcine, sheep, horses, dogs, chickens, ducks or whales, provided by natural, synthetic or genetically engineered sources. In various embodiments of this invention, human insulin is preferred.

The phrase “insulin-like” in this description means an insulin in which one or more amino acids have been replaced while maintaining some or all of the activity of insulin. The analogue is described by marking the replacement of amino acids with the location of the substitution as a superscript number followed by the description of insulin. For example, “insulin B29, human” means that the lysine at position B29 of the human insulin molecule has been replaced by a proline.

Insulin analogues can be obtained in different ways, as is clear to those with experience in the field. For example, some amino acids can be replaced by other amino acids in the insulin structure without an obvious loss in the ability to interact with structures such as, for example, antigen-binding regions. In antibodies or binding sites on the main substance molecules. Whereas the reactivity and nature of insulin determine its functional bioactivity, some amino acid sequence substitutions can be made in the amino acid sequence and still have the same polypeptide properties.

When making replacements, the water treatment factor can be taken into account. The importance of the amino acid parameter used in hydrotreatment, in the biological interactive function of the polypeptide, is generally understood in the field. It is accepted that the relative hydrophilicity of the amino acid contributes to the secondary structure of the resulting polypeptide, which in turn determines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA,

Antibodies, antigens, and the like. For each amino acid, a hydrophilicity index was determined based on its hydrophobicity and charge characteristics as follows: Isoleucine (+ 4.5); valine (+ 4.2); leucine (3.8+); phenylalanine (2.8+); cysteine/cystine (+ 2.5); methionine (+1.9); alanine (+ 1.8); glycine (- 0.4) threonine (- 0.7); serine (- 0.8); tryptophan (- 0.9); tyrosine (-1.3); proline protein (- 1.6); histidine (-3,2); glutamate (- 3.5); glutamine (- 3.5); aspartate (- 3.5); Asparagine (-3.5); lysine (-3.9); And arginine (-4.5). As will become clear to those with experience in the art, some amino acids can be replaced by amino acids with a similar hydrophilic index or measure and result in a polypeptide with similar biological activity, i.e., a polypeptide with equivalent biological functions is obtained. When making these changes, substituting amino acids whose hydrotreatment indices are approximately ±2 of each other is preferred, and those whose indices are approximately ±0.5 of each other are specifically preferred, and those whose indices are approximately ±0.5 are most preferred.

It is also understood in the field that such amino acids can be effectively replaced based on their affinity for water. US patent 1 0 4.554.1, which is included as a reference in this description, describes the large average local affinity for water of a protein, as we refer to its affinity. Hydrophilicity of neighboring amino acids, related to a biological property of a protein. As US Patent 4,554,101 describes the following hydrophilicity values for amino acid moieties:

arginine (+3.0); lysine (±3.0); aspartate (±3.0±1); glutamate (±3.0±1); serine (±0.3); asparagine (+2.0); glutamine (0.2 +) glycine (zero); threonine (- 0.4); Proline (- 0.5 ± 1); alanine (- 0.5); histidine (- 0.5);

cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); Isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3,4). As it is clear to those with experience in the field, it is possible to replace an amino acid with another with a similar hydrophilicity value and at the same time obtain a biologically equivalent, and specifically immunologically equivalent, polypeptide. When these changes are made, substituting amino acids whose hydroprocessing indices are approximately ±2 of each other are preferred, those whose indices are approximately ±2 of each other are particularly preferred, and those whose indices are approximately ±0.5 are most preferred.

As previously described, amino acid substitutions therefore depend on the relative similarity of the amino acid side substituents, for example, their hydrophilicity, hydrophobicity, charge, size and the like. Examples of substitutions (i.e. amino acids that are changed without significantly changing the biological activity of the polypeptide) that take different properties into account are well known to those with experience in the art. It includes, for example: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; And valine, leucine and isoleucine

As will be apparent to those with experience in the art, insulin analogues can be prepared by several known peptide synthesis methods including, but not limited to, classical (solution) methods, solid-phase methods, semi-synthetic methods, and cloned DNA methods.

Examples of human insulin homologues include, without limitation, human insulin GlyA21; GlnB3 GlyA21 insulin, human; AlaA21 insulin, human; AlaA21 insulin, human; GinB3 insulin, human; GlnB20 insulin, human; GluB30 GlyA21 insulin, human; GinB30 GluB3 GlyA21 insulin; Human; GluB30 GlnB3 insulin; Human; AspB28 insulin, human; LysB28 insulin, human; LeuB28 insulin, human; ValB28 insulin,insulin

Human; AlaB28 insulin, human; ProB29 AspB28 insulin, human; LysB2S ProB29 insulin, human; ProB29 LeuB29 insulin, human; ProB29 ValB28 insulin, human; ProB29 AlaB28 insulin, human.

In this description, the phrase “insulin fragment” means a piece of the amino acid sequence found in insulin that has some or all insulin activity. Insulin fragments are labeled with a location(s) in an amino acid sequence followed by a description of the amino acid. For example, the B30-B25 human insulin fragment is a sequence of six amino acids corresponding to positions B28, B27, B26, B25, B29, and B30 in the amino acid sequence of human insulin.

The phrase “insulin fragment” in this description means a piece of the amino acid sequence found in the insulin molecule in which one or more amino acids have been replaced while maintaining some or all of insulin activity.

The phrase “primary insulin” in this description means primary insulin from one of the following classes:

Human, bovine, porcine, sheep, horses, dogs, chickens, ducks or whales, which are provided by natural, artificial or genetically engineered sources. In general, proinsulin consists of an insulin with a C-peptide connecting the N-terminus of the A chain to the C-terminus of the B chain of insulin. In various embodiments of this invention described herein, the primary insulin is preferably human.

In this description, we refer to the term “proinsulin analogue” to a proinsulin in which one or more amino acids have been replaced, as previously described for insulin analogues, while retaining some or all of the activity of the insulin portion of the proinsulin. The homolog is described by marking the amino acid replaced by the substitution site as a superscript followed by the description of the proinsulin. For example &ProB29 primary insulin, human& means lysine

Normally located at position B29 of the human proinsulin molecule, it has been replaced by a proline.

The phrase “preproinsulin” means a piece of amino acid sequence found in preproinsulin that retains some or all of the bioactivity of insulin, similar to the insulin or insulin portion of the preproinsulin. Primary insulin fragments are identified by identifying the location(s) in the amino acid sequence followed by the amino acid description. For example, the segment “human proinsulin B25 - B35” is a sequence of 1 amino acid corresponding to positions B29, B28, B27, B26, B25, B34, B33, B32, B31, and B35 in the insulin amino acid sequence. human insulin amino acid.

The phrase “insulin fragment analog” in this description means a piece of the amino acid sequence in the insulin fragment in which one or more amino acids are replaced as previously described for insulin analogues while maintaining some or all of the insulin activity. insulin, similar to insulin insulin, piece of insulin insulin or similar part piece of insulin in the piece of insulin initial insulin.

The phrase “proinsulin” in this description means proinsulin from one of these sources: humans, cows, pigs, sheep, horses, dogs, chickens, ducks or whales, and it is available from natural, industrial or genetically engineered sources. In general, proinsulin is a single-chain polypeptide (e.g., a polypeptide that has the leader peptide fused to the N-terminus of the B chain in insulin and has the C-terminus of the B chain fused to the N-terminus of the A chain by a contact peptide). Where chain (A) is united with chain (B), for example, with double sulfur bonds. In various embodiments of this invention in this description, human first insulin is preferred.

The phrase “pro-insulin analog” means a pro-insulin in which one or more amino acids have been replaced, as previously described for insulin analogues.

insulin while maintaining some or all insulin activity, or the insulin-like portion of the initial insulin homolog. The homologue is described by highlighting the amino acids replaced by the substitution site as a superscript followed by the description of insulin.

The term “primary insulin fragment” means a segment of the amino acid sequence found in the first insulin fragment that retains some or all of the bioactivity of insulin, or the insulin fragment, from the first insulin fragment. The initial proinsulin fragment is characterized by identifying the location(s) in the amino acid sequence followed by a description of the amino acid

amino acid

The phrase “initial insulin homolog” in this description means a segment of the amino acid sequence in the initial proinsulin fragment in which one or more amino acids are substituted as previously described for insulin analogues while maintaining some or all of the activity of insulin, analogous. insulin, insulin piece or similar part insulin piece in the initial initial piece of insulin.

The phrase "miniature insulin" refers to a single-chain proinsulin polypeptide with a polypeptide of the (A) chain and a polypeptide of the (B) chain, where the -N or -C end of the (A) chain combines with the -C or -N end of the chain. (B) A contact peptide containing amino acids between 1, 2, 3, 4, 5, 6, 7, 8 or 9 as a minimum and 3, 2, 4, 5, 9, 8, 7, 6 or 10 as a maximum, in which we combine A-chain polypeptide with B-chain polypeptide, linkages such as disulfide bonds. The mini-primary insulin may be diverse, as will be apparent to those with experience, and includes, without limitation, that described in US Patent 5,157,021.US in the name of Balschmidt and colleagues and US Patent 5,202,415.US in the name of Jonassen and colleagues, which are incorporated by reference in this description.

The phrase “C-peptide” in this description means a peptide that has the amino acid sequence of C-peptide in primary insulin from one of the following types: humans, monkeys, cows, pigs,

Sheep, horses, dogs, chickens, ducks or whales, provided by natural sources, synthetic or genetically engineered. In various embodiments of this invention, the preferred C-peptide is human.

The phrase “C-peptide analogue” in this description means a C-peptide in which one or more amino acids have been replaced, as previously described for insulin analogues, while maintaining some or all of the biological activity of the C-peptide. The C-peptide analog preferably includes a pentapeptide moiety at the C-terminus of the C-peptide and/or a non-peptide moiety located at positions 11-19 of the C-peptide. When the C-peptide analogue includes a pentapeptide fragment, it is preferable for the pentapeptide fragment to be at the C-terminus of the c-peptide analogue, and preferably for the C-peptide analogue to ignite on a tetrapeptide fragment at the C-terminus of the C-peptide. / Or the non-peptide fragment located at positions 11-19 of the c-peptide. When the C-peptide homologue includes a tetrapeptide segment, it is preferable for the tetrapeptide segment to be located at the C-terminus of the C-peptide analogue. The nonpeptide fragment located at positions 11-19 of the previously described C-peptide would preferably be the nonpeptide fragment located at positions 11-19 of the human C-peptide.

The term C-peptide fragment in this description means a segment of the amino acid sequence of the C-peptide that retains some, most or all of the biological activity of the C-peptide. The C-peptide fragment preferably includes a pentapeptide fragment at the C-terminus of the C-peptide and/or a non-peptide fragment located at positions 11-19 of the C-peptide. When the C-peptide fragment includes a pentapeptide fragment, it is preferable for the pentapeptide fragment to be at the C-terminus of the c-peptide fragment, and it is preferable for the C-peptide fragment to be ignited on a quaternary peptide fragment at the C-terminus of the C-peptide. / Or the non-peptide fragment located at positions 11-19 of the c-peptide. When the C-peptide segment includes a tetrapeptide segment, it is preferable for the tetrapeptide segment to be located at the C-terminus of the C-peptide segment.

peptide. Ideally, the C-peptide fragment should consist of a selected peptide from the group

It consists of the pentapeptide segment from the C-terminus of the C-peptide, the non-peptide segment located at positions 11-19 of the C-peptide, and the tetrapeptide pause from the C-terminus of the C-peptide. It is preferable that the non-peptide fragment at positions 11-19 of the previous C-peptide be the non-peptide fragment located at positions 11-19 of the human C-peptide.

The phrase “C-peptide homolog” means a segment of the C-peptide amino acid sequence in which one or more amino acids are substituted as previously described for insulin analogues while maintaining some, most or all of the biological activity of insulin. The analogue of the C-peptide fragment preferably includes a pentapeptide fragment at the C-terminus of the C-peptide and/or a non-peptide fragment located at positions 11-19 of the C-peptide. When the C-peptide homologue includes a pentapeptide fragment, it is preferable for the pentapeptide fragment to be at the C-terminus of the C-peptide homologue, and it is preferable for the C-peptide homologue to include a tetrapeptide fragment at the C-terminus of the C-terminal peptide. -peptide and/or the non-peptide fragment located at positions 11-19 of the C-peptide. When the C-peptide homologue includes a tetrapeptide fragment, the tetrapeptide fragment is preferably located at the C-terminus of the C-peptide homologue. Preferably, the non-peptide fragment at positions 11-19 of the previous C-peptide is the non-peptide fragment located at positions 11-19 of the human C-peptide.

The phrase “C-peptide polypeptide” in this description means a polypeptide with a therapeutic use and biological activity similar to that of the C-peptides and/or C-peptide fragments described in J. Wahren and colleagues, “The Role of C-peptides in Human Physiology.” Am. J. physiol. Endocrinol. Metab., 278: 759 AH - 768 AH (2000) and/or .T. Forst and colleagues "New Discoveries in the Bioactivity of C-Peptide in IDDM Patients", Exp. Clin. Endocrinol. Diabetes, 106: 270-276 (1998), where they are included as references in this description. For example, C-peptide polypeptides have therapeutic uses that include, but are not limited to, decreasing renal ultrafiltration, increasing whole body utilization.

and/or glucose skeletal muscle, improved autonomic nerve function, and/or redistribution of blood flow in the skin capillaries. C-peptide polypeptides have biological activity that includes, but is not limited to, the ability to stimulate Na+ -K+ -ATPase activity.

Endothelial cell nitric oxide synthase activity, and/or the ability to bind specifically to cell surfaces (eg at a G protein-coupled surface receptor) and subsequent activation of intracellular Ca2+-dependent signaling pathways. Preferably, C-peptide polypeptides have a dissolution constant for binding to endothelial cells, renal tubule cells, and primary fibroblasts equal to about 3 x 910 1-M. Preferably, the C-peptide polypeptides are C-peptides, C-peptide homologues, C-peptide fragments, or C-peptide fragment homologs.

The first term C-peptide in this description means a C-peptide polypeptide combined with one or more peptides that can be cleaved to give a C-peptide.

The phrase “A-chain polypeptide” means a polypeptide biologically equivalent to the A-chain molecule of insulin. For example, A-chain polypeptides may be A-chain enantiomers, which may be provided as previously described for insulin analogues, A-chain segments, or A-chain enantiomers.

The phrase “B-chain polypeptide” means a polypeptide biologically equivalent to the B-chain molecule of insulin. For example, our B-chain polypeptides may be B-chain homologues, which may be provided as previously described for insulin analogues, B-chain segments, or B-chain homologs.

The term “polypeptide” means a peptide that has two or more amino acid moieties.

The phrase &amphiphilically balanced& means capable of

Mainly soluble in water and able to penetrate biological membranes.

The phrase “polyalkylene glycol” refers to straight or branched polyalkylene glycol polymers such as polyethylene glycol, polypropylene glycol, and polybutylene glycol, and includes the monoalkylether of polyalkylene glycol col. The phrase “polyalkylene glycol subunit” refers to a single polyalkylene glycol unit. For example, a polyethylene glycol unit is --O-CH2-CH2 -O.

The phrase “lipophilic” in this description means the ability to dissolve in fats and/or the ability to penetrate, interact with and/or cross biological membranes, and the phrase “lipophilic moiety” means a moiety that is attracted to lipids and/or when linked to another chemical moiety, increases the affinity of this moiety. The other part is for fat. Examples of lipophilic moieties include, but are not limited to, alkyls, fatty acids, esters of fatty acids, cholesteryl, adamantyl, and the like.

The phrase “lower alkyl” in this description means alkyl radicals replaced or unsubstituted with 1-5 carbon atoms.

The phrase “higher alkyl” in this description means alkyl radicals, whether substituted or unsubstituted, with 6 or more carbon atoms.

According to embodiments of the invention, methods for synthesizing an insulin polypeptide-oligomer conjugate include an insulin polypeptide combined with one or more peptides by a peptide bond(s) that can be broken to yield an insulin polypeptide with an oligomer in Conditions are sufficient for the oligomer to combine with the insulin polypeptide portion of the proinsulin polypeptide and give the combination product proinsulin. polypeptide- an oligomer, one or more peptide moieties

peptides from the product of the initial insulin polypeptide-oligomer to give the product of the insulin polypeptide-oligomer.

For example, insulin-oligomer conjugate products can be synthesized as shown in the examples below. An embodiment of a synthetic path is shown in Figure 1.

The primary insulin polypeptide may be diverse and include an insulin polypeptide combined with one or more peptides by a bond (peptide bonds) that can be cleaved to obtain the insulin polypeptide, as will be clear to those with experience in the field. It includes, without limitation, a primary insulin, Proinsulin homologs, proinsulin fragments, proinsulin homologs, mini-proinsulins, or fusion proteins. In some embodiments, the proinsulin polypeptide is a proinsulin analogue with a leader peptide. A similar pro-insulin with a leader peptide is produced by Itoham Foods Inc. of Ibaraki Pref, Japan. The leader peptide and C-peptide of the proinsulin homologue are both devoid of lysine moieties. In other embodiments, proinsulin polypeptide is a proinsulin polypeptide produced by Biobras of Belo Horizonte, Brazil. The proinsulin polypeptide has a leader peptide fused to the N-terminus of the B chain of insulin on the proinsulin polypeptide. The leader peptide is free of lysine moieties and preferably contains insulin polypeptide chain (A) and polypeptide chain (B). It is preferable that the A-chain polypeptide be free of lysine moieties. Preferably, the B-chain polypeptide contains one lysine moiety. The A-chain polypeptide and the B-chain polypeptide are preferably cross-linked, preferably cross-linked using one or more disulfide bonds. Ideally, both the A-chain polypeptide and the B-chain polypeptide contain cysteine moieties, one or more of which combine using one or more disulfide bonds to link the two chains. Preferably insulin polypeptide

insulin polypeptide is insulin, an insulin analogue, an insulin piece, or an insulin-like piece.insulin

In some embodiments, one or more peptides fused to the insulin polypeptide include an Enval peptide fused at one end to the C-terminus of the B-series polypeptide and the other end to the N-terminus of the A-series polypeptide. In general, the amino acid sequence of the contact peptide is not important and may be any contact peptide, as will be apparent to those with experience in the art, and includes, without limitation, C-peptide polypeptides, C-peptides, and contact peptides in mini-primary insulins. In some embodiments, the contact peptide is devoid of lysine moieties. These embodiments may use less oligomeric reagents by reducing the number of possible union sites on the proinsulin polypeptide molecule.

In other embodiments, one or more peptides fused to the insulin polypeptide comprise a leader peptide fused to the N-terminus of the B-chain polypeptide. In general, the amino acid sequence of the leader peptide is not important. In some embodiments, the leader peptide is devoid of lysine moieties. These embodiments may reduce the amount of oligomer reagent used by limiting the number of union sites on the proinsulin polypeptide molecule.

In other embodiments, one or more peptides bound to insulin polypeptide are ignited on both a contact peptide as described above and a leader peptide as described above. One or more peptides may consist primarily of a contact peptide and a leader peptide, or may consist of a contact peptide and a leader peptide.

Peptide bonds are bonds that can be broken in various ways, as will become clear to those with experience in the field. Preferably, the peptide bonds are bonds that can be enzymatically cleaved by enzymes including, but not limited to, trypsin, carboxypeptidase B, thrombin, pepsin, and chymo.

Chymotrypsin. Peptide bonds that can be enzymatically cleaved will be understood by those with expertise in the field and include, without limitation, Arg, Ala-Arg, Thr-Arg, Arg-Arg-Arg-Gly, Thr-Lys, Thr-Arg, and Arg-Phe.

An oligomer may be one of a variety of oligomers, as will become clear to those with experience in the field. And in general pain. An oligomer may be any oligomer capable of combining with a polypeptide as is apparent to those skilled in the art. For example, an oligomer may be a polyubiquitinated oligomer as described in US Patent No. 4,179,337 by Davis and co-workers; US Patent No. 5,567,422.US in Greenwold's name; US Patent No. 5,359,030.US in the name of Ekwuribe; US Patent No. 5,438,040.US in the name of Ekwuribe; US Patent No. 5,681,811.US In the name of Ekwuribe; US Patent No. 6,309,633 on behalf of Ekwuribe and colleagues; All of which are included as references in this description. As another example, an oligomer may be a non-polydisperse oligomer, as described in the US patent application, serial number 09/873,731, filed on June 4, 2001, by Ekwuribe and colleagues, under the title “Methods for the synthesis of essentially monodisperse mixtures of polyethylene glycol alloy polymers.” Glycol&; US Patent Application No. 09/873,797.US filed on June 4, 2001 on behalf of Ekwuribe and co-authors under the title "Mixture of drug-oligomer conjugate products comprising polyalkylene glycol Polyalkylene Glycol, its uses, and methods for preparing it&; The US patent application, serial number 09/873,899.US, was filed on June 4, 2001, in the name of Ekwuribe and his colleagues, under the title “A mixture of products of the combination of an insulin drug with an oligomer, including polyalkylene glycol, and its uses and methods for preparing it,” all of which are included as references in this invention.

In some embodiments, the oligomer includes a hydrophilic moiety, as will be apparent to those skilled in the art, including, without limitation, polyalkylene glycols such as polyethylene glycol or polypropylene

Polypropylene glycol, polyols treated with polyoxyethylenated, conjugated polymers thereof and template conjugated polymers thereof, provided that the hydrophilicity of the template conjugated polymers is maintained. The hydrophilic moiety is preferably the polykylene glycol moiety. A polyalkylene glycol moiety having at least 1, 2, 3, 4, 5, 6 or 7 polyalkylene glycol subunits. It is preferable that the polykylene glycol radical contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a maximum. Lowest and 10, 11, 12, 13, 14

15، 16، 17، 18، 19، 20، 21، 22، 23، 24، 25، 26، 27، 28، 29، 30، 31، 32

33، 34، 35، 36، 37، 38، 39، 40، 41، 42، 43، 44، 45، 46، 47، 48، 49، 50

or more polyalkylene glycol subunits. It is preferable for the part to contain polyalkylene glycol

Between 2, 3, 4, 5, or 6 as a minimum and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 17, 9, 1, or 20 as a maximum. Polyalkylene glycol subunits. It is preferable that it contain something between 5, 4, 3, or 6 as a minimum and 5, 6, 7, 8, 9, 10, 1, 1 or 12 as a maximum. The best option is between a minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polyalkylene glycol subunits. It is preferable for the polyglycol radical to contain 7 polykylene glycol subunits. It is preferable that the polykylene glycol radical in the oligomer be a low alkyl polykylene glycol radical, such as the polyethylene glycol radical, the polypropylene glycol radical, or the polybutylene glycol radical. When the polyalkylene radical is a polypropylene glycol radical, it is preferable for the radical to have a homogeneous structure (i.e., non-random). An example of cleaving polypropylene glycol with a homogeneous structure is as follows:

<img file="SA1946B1_D0001.tif" />

The structure of this homogeneous polypropylene glycol can be described as containing only one carbon atom replaced by a methyl near each oxygen atom in the polypropylene glycol chain. Such homogeneous moieties may exhibit both lipophilic and hydrophilic properties.

The oligomer may include one or more other moieties, as will become clear to those with experience in the field. They include, but are not limited to, additional hydrophilic radicals, lipophilic radicals, space-adding radicals, bonding radicals, and terminal radicals. The various moieties in the oligomer are covalently bonded to each other with either hydrolyzable or nonhydrolyzable bonds.

The oligomer may further include one or more hydrophilic addition moieties (i.e., moieties in addition to the polyalkylene glycol moiety) including, without limitation, sugars, polyalkylene glycols, and conjugated PEG/polyamine polymers. Adjacent polyalkylene glycol moieties are considered the same moiety if they are combined with ether bonds. For example, notch

—C2H4—O—C2H4—O—C2H4—O—C2H4—O—C2H4—O—C2H4—

It is a single polyethylene glycol radical with six polyethylene glycol subunits. If this moiety is the only hydrophilic moiety in the oligomer, the oligomer will not contain an additional hydrophilic moiety. Adjacent polyethylene glycol radicals are considered different radicals if they are united by bonds other than an ether bond. For example, notch

0

II

—O—C2H4—o—C2H4—o—C2H4—o—C2H4— C— o—C2H4—o—C2H4—

It is a single polyethylene glycol radical with 4 polyethylene glycol subunits and an additional hydrophilic radical with two polyethylene glycol subunits. The oligomers according to embodiments of this invention preferably include a polyalkylene glycol moiety and do not include additional hydrophilic moieties.

Preferably, the oligomer also includes one or more lipophilic moieties, as will become clear to those with experience in the art. The lipophilic moiety contains at least 1, 2, 3, 4, 5 or 6 carbon atoms. It is preferable for the lipophilic moiety to contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 11, 10, 13, 16, 15, 14, 17, 18, 19 or 20 as a minimum. And 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 29 or 30 carbon atoms maximum. Preferably between 2, 3, 4, 5, 6, 7, 8, 9 and 10 as a minimum and 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 21, 20 carbon atoms maximum. The best is between 3, 4, 5, 6, 7, 8 or 9 as a minimum and 4, 5, 6, 7, 8, 9, 10, 1 1, 2 1, 13 or 4 1 carbon atom as a maximum. The best is between 3, 4, 5, 6 or 7 as a minimum and 6, 7, 8, 9 or 10 carbon atoms as a maximum. It is preferable for the lipophilic moiety to contain 6 carbon atoms. It is preferable to choose the lipophilic moiety from the group consisting of saturated or unsaturated, linear or branched carbon atoms, linear or branched saturated or unsaturated fatty acid moieties, cholesterol, and adamantane. Examples of alkyl radicals include, but are not limited to, linear saturated alkyl radicals such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tert. tridecyl lycyl, tetradecyl tetradecyl tetradecyl tetradecyl tetradecyl tetradecyl tetradecyl tetradecyl tetradecyl octadecyl octadecyl octadecyl octadecyl octadecyl octadecyl octadecyl octadecyl lycyl and eicosyl lycyl. Branched alkyl radicals such as isopropyl, sec-butyl, and tert-butyl. tert-butyl; 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl,

2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl; ethynyl, l-propynyl, and 2-propynyl. Examples of fatty acid moieties include, but are not limited to, unsaturated fatty acid moieties such as lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosadenoate docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, arachidate, Behenate, lignocerate and cerotate

The oligomer may also include one or more space-adding moieties, as will be apparent to those with experience in the art. For example, space-adding moieties may be used to separate a hydrophilic moiety from a lipophilic moiety, to separate a lipophilic moiety or a hydrophilic moiety from proinsulin polypeptide. proinsulin polypeptide, to separate a first hydrophilic or lipophilic moiety from a second hydrophilic or lipophilic moiety, or to separate a hydrophilic or lipophilic moiety from a binding moiety. It is preferable to choose fractions that add space from the group consisting of sugar fractions, cholesterol, and glycerine. Sugar radicals may be diverse, as will become clear to those with experience in the field, and include, without limitation, monosaccharide radicals and disaccharide radicals. The preferred monosaccharide moieties contain between 4-6

carbon atoms-

The oligomer may also include one or more bond moieties that are used to combine the oligomer with the proinsulin polypeptide, as will become clear to those with experience in the art. It is preferable to choose the bonding moieties from the group consisting of alkyl moieties and a fatty acid. The linking alkyl moiety may be a moiety

The alkyl is saturated or unsaturated, linear or branched, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, and dodecyl. dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, vinyl, allyl, 1-butenyl l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. The alkoxy radical may be one of a variety of alkoxy radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methyl

3-methylpentyloxy, 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy, ethynyloxy, 1-l-propynyloxy , and 2-propynyloxy. The bonding alkyl moiety can contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 12, 11, 14, 15, 16, 17, 18, 19 or 20 as a minimum. And 5, 6, 7, 8, 9, 1, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or a maximum of 30 carbon atoms, preferably containing between 1, 2, 3, 4 or 5, 8, 9, 0 1, 1 1 or 2 1 carbon atoms. The linking fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate. , eicosapentaentoate, and docosahexaenoate;

And saturated fatty acid radicals such as acetate, caproate, caprylate, caprate, laurate, arachidate, behenate, lignocerate, and cerotate. The linking fatty acid radical may contain between 1, 2, 3, 4, 5, and 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minimum and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 20, 26, 27, 28, 29 or 30 carbon atoms, preferably between 1, 2, 3, 4 or 5 and 8, 10,

12, 14 or 16 carbon atoms.

The oligomer may also ignite on one or more terminal moieties at one or more ends of the oligomer. Which is not combined with insulin polypeptide. It is preferable for the final part to be the alkyl or alkyl part, and it is preferable for this part to contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 14, 13, 12, 11, 10, 15, 16, 17. 18, 19 or 20 minimum and 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 A maximum of 26, 27, 28, 29 or 30 carbon atoms, preferably between 1, 2, 3, 4, 5, 6 or 7 as a minimum and 5, 9, 8, 7, 6, 10, 11, 12, 3 or 1. 4 1 carbon atom maximum. It preferably contains between 4,3,2,1 or 5 as a minimum and a maximum of 5, 6, 7, 8, 9, 10 carbon atoms. Preferably between 1, 2, 3 or 4 and 5, 6 or 7 carbon atoms. The alkyl radical may be linear or branched, saturated or unsaturated, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl,

Butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, hexadecyl, octadecyl, nonadecyl eicosyl; Branched saturated alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl. 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . The alkoxy radical may be one of a variety of alkoxy radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tertiary. tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, ecosyloxy eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, l-butenyloxy, 2-butenyloxy, ethynyloxy, l-propynyloxy, 2-propynyloxy 2-propynyloxy. Preferably, the final radical should be a low alkyl radical, such as: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,

pentyl, tert-pentyl, or lower alkoxy radical, for example: methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy, or tert-pentyloxy. tert-pentyloxy. It is preferable that the final moiety be methyl or methoxy. While it is preferable for the final radical to be the alkyl or alkoxy radical, it must be understood that the final radical is one of a variety of radicals, as will become clear to those with experience, and includes, without limitation, sugars, cholesterol, alcohols, and fatty acids.

According to other embodiments of this invention, the oligomer ignites in a structure having the formula I:

(I) A—Lj—Gk—R—Gm—R—Gn—T

where A is an activatable cleft:

L is the link moiety;

G', G and &amp;G are individually selected space-added notches;

R is the lipophilic radical and R is the polyalkylene glycol radical, or 'R' is the lipophilic radical and R is the polyalkylene glycol radical; T is the terminal cleft; And

m, k, j and n individually are 0 or 1.

According to such embodiments of this invention, the polyalkylene glycol moiety contains at least 1, 2, 3, 4, 5, 6 or 7 polyalkylene glycol subunits. Preferably between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 17, 19 or 20 as a minimum and 10, 11, 13. 12, 14, 15, 18, 17, 16, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more polyalkylene glycol subunits. It is preferable for the polykylene glycol moiety to contain between 2, 3, 4, 5, or 6.

A minimum and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or a maximum of 20 polyalkylene units. It is preferable for the polykylene glycol moiety to contain a minimum of 3, 4, 5, or 6 and a maximum of 5, 65, 7, 8, 9, 10, 11, or 12 polykylene glycol subunits. It is preferable for it to contain between 4, 5, or 6 and 6, 7, or 8. It is best for polyalkylene glycol to contain 7 polyalkylene glycol subunits. The polykylene glycol radical from the oligomer is preferably a low alkyl polyglycol radical, such as a polyethylene glycol radical, a polypropylene glycol radical, or a polybutylene glycol radical. When the polyalkylene glycol radical is a polypropylene glycol radical, it is preferable for the radical to have a homogeneous composition (i.e., non-random). An example of cleaving polypropylene glycol with a homogeneous structure is as follows:

<img file="SA1946B1_D0002.tif" />

The structure of this homogeneous polypropylene glycol can be described as containing only one carbon atom replaced by a methyl near each oxygen atom in the polypropylene glycol chain. Such homogeneous moieties may exhibit both lipophilic and hydrophilic properties. According to these embodiments of this invention, the lipophilic moiety is the lipophilic moiety that will be apparent to those with experience in the art.Preferably, the oligomer also ignites on one or more lipophilic moieties, as will be apparent to those experienced in the art. The lipophilic moiety contains at least 1, 2, 3, 4, 5 or 6 carbon atoms. It is preferable for the lipophilic moiety to contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum. And 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms maximum. Preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 0, 1, 1, 12, 13, 4, 1, 15, 16, 19, 18, 17, 20, 21 carbon atoms maximum. The best is between 3, 4, 5, 6, 7, 8 or 9 as a minimum and 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms as a maximum. The best is between 3, 4, 5, 6 or 7 as a minimum and 6, 7, 8, 9 or 10 carbon atoms as a maximum. It is preferable for the lipophilic moiety to contain 6 carbon atoms. It is preferable to choose the lipophilic radical from the group consisting of saturated or unsaturated, linear or branched alkyl radicals, linear or branched saturated or unsaturated fatty acid radicals, cholesterol and adamantane. Examples of alkyl radicals include, but are not limited to, linear saturated alkyl radicals such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and tridecyl. tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched saturated alkyl radicals such as isopropyl, sec-butyl, tert-butyl, and 2-methylbutyl. 2-methylbutyl, tert-pentyl, 2-methyl—pentyl

2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and ignite without specificity vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . Examples of fatty acid moieties include, but are not limited to, unsaturated fatty acid moieties such as lauroleate, myristoleate, palmitoleate, oleate, and ylides.

elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; and saturated fatty acid radicals such as acetate, caproate,

Caprylate, caprate, laurate, arachidate, behenate, lignocerate and cerotate.

According to embodiments of the invention, additional slits of area, G', G and &amp;G, are as will be apparent to those with experience in the art. It is preferable to choose space-added fractions from the group consisting of sugar, cholesterol, and glycerine fractions. The sugar radicals may be radicals, as will be clear to those with experience in the field, and include, without specifying, monosaccharide radicals and disaccharide radicals. It is preferable for the monosaccharide moieties to contain between 4-6 carbon atoms. Preferably, these oligomers do not include space-adding moieties (i.e., m, k, and n should preferably be zero).

According to these embodiments of the invention, the linking moiety, L, may be used to combine the oligomer with the drug as will be apparent to those with experience in the art. It is preferable to test the bonding radicals from the group consisting of alkyl radicals and a fatty acid. The bonding alkyl radical may be a saturated or unsaturated alkyl radical, linear or branched, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl 2-propylpentyl, phenyl

vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. The alkoxy radical may be one of a variety of alkoxy radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, or disyloxy.

decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, ter-butoxy t-butoxy, 2-methylbutoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, l-butenyloxy, 2-butenyloxy, ethynyloxy, l-propynyloxy, and 2-propynyloxy. The bonding alkyl moiety can contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 12, 11, 14, 15, 16, 17, 18, 19 or 20 as a minimum. And 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Or a maximum of 30 carbon atoms, preferably containing between 1, 2, 3, 4 or 5 and 8, 9, 10, 11 or 12 carbon atoms. The linking fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, elaidate,

erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, arachidate, behenate, lignocerate and cerotate. The linking fatty acid moiety may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 at a minimum. And 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms, preferably between 1, 2, 3, 4 or 5 and 8, 0, 1, 12, 4, 1 or 16 Carbon atom. According to these embodiments, the final radical, T, is preferably an alkyl or alkoxy radical, preferably containing between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms maximum, preferably between 1, 2, 3, 4, 5, 6 or 7 as a minimum and 5, 6, 7, 8 , 9, 10, 11, 12, 3 1 or 4 1 carbon atom maximum. It is preferable to contain between 3,2,1,

4 or 5 as a minimum and 5, 9, 8, 7, 6 or 10 carbon atoms as a maximum. Preferably between 1, 3, 2 or 4 and 5, 6 or 7 carbon atoms. The alkyl radical may be linear or branched, saturated or unsaturated, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, lysyl. decyl, undecyl, dodecyl, tridecyl, tetradecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Isopropyl, sec-butyl, tert-butyl, 2- 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl 2-propylpentyl; Unsaturated alkyl radicals are derived

The above saturated alkyl radicals include, without limitation, vinyl, allyl, 1-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. The alkoxy radical may be one of a variety of alkoxy radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy

tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy, 2-propylpentylxy phenyloxy

vinyloxy, allyloxy, l-butenyloxy, 2-butenyloxy, ethynyloxy, l-propynyloxy, and 2-propynyloxy. It is preferable that the final radical be a low alkyl radical, such as: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl, or a low alkoxy radical, for example: methoxy. methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy, or tert-pentyloxy. It is preferable for the final moiety to be methyl or methoxy. While it is preferable for the final radical to be the alkyl or alkoxy radical, it must be understood that the final radical is one of a variety of radicals, as will become clear to those with experience, and includes, without limitation, sugars, cholesterol, alcohols, and fatty acids.

According to these embodiments, the inhibitory cleft, A, is a cleft that allows the oligomer to combine with an activating factor to form an activating oligomer capable of combining with a proinsulin polypeptide. The labile crack may be one of a variety of cracks, as will become clear to those with experience in the field, including, without limitation, c(s)-, -C(O)-OH SH, -OH, -C(S)-SH, OH-, and NH2. .

In other embodiments, the oligomer comprises a structure of formula II:

(II) A—X(CH2)m Y(C2H4O)nR

wherein: A is SH, -OH, -C(S)-SH, C(S)-OH, -C(O)-OH-, or NH2;

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is the moiety of an ester, ether, carbamate, carbonate, or amide linkage, preferably the ether moiety;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16,

17, 18, 19 or 20 minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 as a maximum, and preferably 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5, 13, 12, 11, 10, 9, 8, 7, 6 or 14 as a maximum, and the best is between 4, 3, 6, or 7 as a minimum and 9, 8, 7, 6 or 10 as a maximum.

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 13, 12, 14, 15, 16, 17, 18, 19 or 20 as a maximum, and the best thing is between 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8, 9, 10 1,1 or a maximum of 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polyalkylene glycol subunits is best, and 7 is best;

R is an alkyl moiety, a sugar moiety, a cholesterol moiety, adamantane moiety, an alcohol moiety, or a fatty acid moiety. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl,

hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3- 3-methylpentyl, 2-ethylhexyl 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propenyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. It is preferable for the alkyl radical to be the C1-C3 alkyl. It is preferable that the alkyl radical be a methyl. The fatty acid moiety may be a fatty acid moiety, saturated or unsaturated, linear or branched, as will become clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate.

oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, arachidate, behenate, lignocerate and cerotate.

In other embodiments, the oligomer comprises a structure of formula III:

(A(CH2)m(OC2H4O)nR(III-

In which A is SH, -OH, -C(S)-SH, C(S)-OH, -S(O)-OH, or NH2;

wherein: A is SH, -OH, -C(S)-SH, C(S)-OH, -C(O)-OH-, or NH2;

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is an ester, ether, carbamate, carbonate, or amide bond, preferably an ether bond;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 12,11 10, 13, 15, 14, 18, 17, 16, 19, 20, 21, 22 as a maximum, and preferably 5, 4, 3 or 6 as a minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5. 13, 12, 11, 10, 9, 8, 7, 6 or 14 as a maximum, and the best is between 4, 3, 6, or 7 as a minimum and 9, 8, 7, 6 or 10 as a maximum.

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 0 2 as a maximum, and the best thing is between 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8, 9, A maximum of 10, 1 or 2 1 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is the alkyl radical, sugar radical, cholesterol radical, adamantine radical, alcohol radical, or fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and nonyl. nonyl, decyl lysyl, undecyl undecyl; dodecyl, tridecyl,

tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3- 3-methylpentyl,2-ethylhexyl,2-

2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. It is preferable for the alkyl radical to be the C1-C3 alkyl. It is preferable that the alkyl radical be a methyl. The fatty acid moiety may be a fatty acid moiety, saturated or unsaturated, linear or branched, as will become clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate.

erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, arachidate, behenate, lignocerate and cerotate.

In other embodiments, the oligomer includes a structure having the formula IV

<img file="SA1946B1_D0003.tif" />

And in it: where in it: A is SH, -OH, -C(S)-SH, C(S)-OH, -C(O)-OH-, or

NH2;

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is the radical of an ester, ether, carbamate, carbonate, or amide linkage, preferably the ether linker;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 minimum and 4, 5, 6, 8, 9, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2 and 3.

4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 15, 14, 13, 12, 11, 10,

16, 17, 18, 19, 20, 21, 22 as a maximum, and preferably 5, 4, 3 or 6 as a minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5, 6, 7, 8, 9, 0 1, 1 1, 2 1, 3 1 or 4 1 as a maximum, and the best is between 4,3, 6, or 7 as a minimum and 9,8,7,6 or 10 as a maximum,

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 0 2 as a maximum, and the best is between 3, 4, 5 or 6 as a minimum and 5, 6, 9, 8, 7, 10, 1 or a maximum of 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is the alkyl radical, sugar radical, cholesterol radical, adamantine radical, alcohol radical, or fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and nonyl.

nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl.

2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl; Propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. It is best for the alkyl radical to be the C1-C3 alkyl. It is better for the alkyl cleavage to be identical. The fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate,

linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, arachidate, behenate, lignocerate, and cerotate.

In other embodiments, the oligomer includes a structure having the formula (V):

<img file="SA1946B1_D0004.tif" />

In various embodiments described previously, the oligomer is covalently coupled to the insulin polypeptide. In some embodiments the oligomer is combined with the insulin polypeptide using a hydrolyzable bond (eg, an ester or carbonate bond). The hydrolyzable combination may give the product of combining an insulin polypeptide with an oligomer that acts as a prodrug. In some cases, such as when the insulin polypeptide–oligomer conjugate is biologically inactive (i.e., the conjugate lacks the ability to act on the body through the insulin polypeptide's mechanism of action), the hydrolyzable conjugate may provide a neutralizing effect Timed or controlled release, giving biologically active insulin polypeptide over a specified period of time when one or more oligomers of the insulin polypeptide conjugate cleavage. The bioactive inactive form is combined with an oligomer to provide the bioactive insulin polypeptide. And in other embodiments. The oligomer combines with insulin polypeptide using a nonhydrolyzable bond (eg, carbamate, amide, or ether bond). The use of a nonhydrolyzable conjugate may be preferred when it is required to leave the bioactive insulin polypeptide-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least two hours. When an oligomer combines with an insulin polypeptide using a linker moiety that includes a carbonyl moiety, such as an ester moiety, carbamate, carbonate, or amide linker, the resulting combination product is an insulin polypeptide - acyl oligomer. oligomer

The oligomers used in the various previous embodiments are commercially available or can be synthesized in various ways, as will become clear to those with experience in the field. For example, the oligomer may be a polydisperse oligomer as described in U.S. Pat. No. 4,179,337. US in the name of Davis and his colleagues; US Patent No. 5,567,422 in Greenwold's name; US Patent No. 5,359,030 in the name of Ekwuribe; US patent no

5,438,040 in the name of Ekwuribe; US Patent No. 5,681,811 in the name of Ekwuribe; US Patent No. 6,309,633 on behalf of Ekwuribe and colleagues; All of which are included as references in this description. As another example, an oligomer may be a non-polydisperse oligomer, as described in U.S. Patent Application No. 09/873,731 filed on June 4, 2001 by Ekwuribe and his co-authors, under the title “Methods for Synthesizing a Single-Diffusion Mixture Essentially of Polyethylene Glycol Mixed Polymers.” US Patent Application No. 09/873,797.US filed on June 4, 2001, on behalf of Ekwuribe and co-authors, under the title “Mixtures of oligomer-drug conjugate products comprising polyalkylene glycol, their uses, and methods.” To prepare it&amp;; The US patent application, serial number 09/873,899.US, was filed on June 4, 2001, in the name of Ekwuribe and his co-authors, under the title “A mixture of products of the combination of an insulin drug with an oligomer, including polyalkylene glycol, and its uses and methods for its preparation. The oligomers, according to the embodiments of this invention, should preferably be single.” Essentially diffusive and preferably monodiffusional and examples of methods for synthesizing preferably monodiffusional oligomers are provided in Examples 1-10 below.

The connection of the proinsulin polypeptide with the oligomer can be carried out under conditions sufficient to provide the proinsulin polypeptide-oligomer combination product, using a variety of conditions, as will become clear to those with experience in the art. The proinsulin polypeptide-oligomer conjugation preferably includes contact of the oligomer with an activating agent under conditions sufficient to provide an activating oligomer; The activated oligomer connects with the proinsulin polypeptide under conditions sufficient to provide the product of the proinsulin polypeptide. The activated oligomer can be formed ex situ or in situ.

The activating factor may be one of various activating agents capable of activating one or more of the oligomers described previously, such that the oligomer is able to react with the hydroxyl and/or amino nucleophilic functional groups in the proinsulin polypeptides, as will be apparent to those with experience. In scope, it includes, without limitation, N-hydroxysuccinimide, p-nitrophenyl chloroformate, 1,3-dihexylcyclocarbodiimide. 1,3-dicyclohexylcarbodiimide, and hydroxybenzotriazide

hydroxybenzotriazide

It will become clear to someone experienced in the field that the conditions are sufficient for the activating agent to combine with the oligomer to provide an activated oligomer. For example, an experienced person might refer to:

Comprehensive Organic Transformations...'' R. c. “Larock”

“A Guide To Functional Group Preparations” (2nd ed., New York, VCH Wiley, 1999), whose description is included by reference herein.

Sufficient conditions for the union of the activated oligomer with the proinsulin polypeptide will become clear to those with experience in the field. For example, proinsulin polypeptide can be dissolved in a bipolar aprotic solvent, such as dimethylsulfoxide, to give a proinsulin polypeptide solution. A buffer such as triethylamine may be added to the primary insulin polypeptide solution. The activated oligomer can then be added in an anhydrous solvent such as acetonitrile to the proinsulin polypeptide solution. And it is possible

Experience also refer to:

Comprehensive Organic Transformations.....'' R. c. “Larock”

“A Guide To Functional Group Preparations” (2nd ed., New York, VCH Wiley, 1999). It is preferable that the molar ratio of the activated oligomer to the polymer be

The proinsulin polypeptide is greater than about 1:1, and preferably greater than 2:1; The best is greater than 3:1, the best is greater than 4:1, and the best is greater than 5:1.

In the various foregoing embodiments, more than one oligomer may be combined (i.e.

A group of oligomers) with the insulin polypeptide portion of the proinsulin polypeptide. Preferably, the oligomers in the group should be identical. However, it must be understood that the oligomers in the group may be different from each other, or, alternatively, some oligomers in the group may be the same and some different. When the group of oligomers is bound to the insulin polypeptide portion of the proinsulin polypeptide, the combination of one or more oligomers with the insulin polypeptide portion of the proinsulin polypeptide may be preferred. Hydrolyzable ligands and a single conjugate or more oligomers with the insulin polypeptide portion of the primary insulin polypeptide linked to non-hydrolyzable ligands. Alternatively, all bonds connecting the oligomer group to the insulin polypeptide portion of the primary insulin polypeptide are hydrolyzable, but have different degrees of hydrolysis. For example, one or more oligomers have been removed from the insulin polypeptide or portion. Insulin polypeptide is hydrolyzed in the body and one or more oligomers are slowly removed from the insulin polypeptide or insulin polypeptide fragment. Polypeptide is hydrolyzed within the body.

In various previous embodiments, the oligomer may combine with the insulin polypeptide portion of the proinsulin polypeptide at different nucleophilic moieties of the insulin polypeptide portion including without limitation the nucleophilic hydroxide and/or amine functional groups. The nucleophilic hydroxide group may be present, for example, at the histidine moiety and/or

Lysine and/or at one or more of the N-termini of a polypeptide. When an oligomer combines with one or more of the N-termini of the proinsulin polypeptide, the combination forms a secondary amine. When the proinsulin polypeptide contains a leader peptide with the N-terminus of the B-chain polypeptide, the N-terminus of the insulin molecule can be protected from association (eg, acylation). When the proinsulin polypeptide is a human proinsulin with a leader peptide fused to the N-terminus of the B chain, for example, the oligomer can combine with the three functional amino groups of proinsulin: The N-terminus of the leader peptide, the amino group in the Lys moiety of the C-peptide, and the amino group in LysB29. Upon cleavage of the leader peptide and the C-peptide, the oligomer is shown to combine site-dependently with LysB29 in insulin to give a single insulin conjugate. Insulin is monoubiquitinated to an oligomer at LysB29.

Cleavage of one or more peptides from the proinsulin polypeptide-oligomer can be performed to provide the insulin polypeptide-oligomer through several processes, as will become clear to those with experience in the field. Preferably, the cleavage of one or more peptides from the proinsulin polypeptide-oligomer is ignited in contact with one or more enzymes capable of cleaving the bond(s) between One or more peptides and an insulin polypeptide under sufficient conditions to moiety one or more peptides from the product of the primary insulin polypeptide conjugate proinsulin polypeptide - oligomer. As described in various references, for example, Kemmler and colleagues, "Studies on the Conversion of Protoinsulin to Insulin", J. Biol. Chem, 246: 6786-6791 (971 1), the description of which is included in this invention as a self-referential reference, and it will become clear to those experienced in the field how to select suitable enzymes in light of the links

The specific peptide to be cleaved and how to provide sufficient conditions for the cleavage of one or more peptides from the product of the proinsulin polypeptide with the oligomer. Preferably, one or more of the enzymes may include different enzymes including, but not limited to, trypsin, chymotrypsin, carboxypeptidase B; And a mixture of them. Ideally, one or more of the enzymes selected are trypsin, carboxypeptidase B, and a mixture thereof.

In some embodiments, such as those previously described, that have a contact peptide, the contact peptide contains a terminal amino acid moiety at the first end. In some of these embodiments, the cleavage of the contact peptide from the proinsulin polypeptide - oligomer is ignited in contact with the proinsulin polypeptide - oligomer under sufficient conditions to provide the product of the combination of a terminal amino acid moiety - insulin polypeptide - oligomer. This product is connected to an enzyme in sufficient conditions to provide the product of the insulin polypeptide - oligomer and the connection of the product of the proinsulin polypeptide. The polypeptide - oligomer with the first enzyme and the connection of the product of the terminal amino acid moiety - insulin polypeptide - oligomer with the second enzyme may occur simultaneously, for example when the first enzyme and the second enzyme are provided as a mixture or blend. Preferably, the first enzyme is trypsin and the second enzyme is carboxypeptidase B. The terminal amino acid moiety may be one of a variety of moieties, such as the arginine moiety. For example, the terminal amino acid moiety is an arginine moiety when the insulin polypeptide is insulin and the contact peptide is a human C-peptide.

Preferably, the cleavage of one or more peptides from the proinsulin polypeptide - oligomer provides a product of the insulin polypeptide - oligomer that consists of a single insulin polypeptide - oligomer (i.e., free). Mainly from the products

Insulin polypeptide (oligomer) conjugate. Preferably, the product of the insulin polypeptide - oligomer combination consists of the product of a monomeric union of insulin polypeptide with an oligomer. For example, in the previously described embodiments in which the proinsulin polypeptide comprises an insulin polypeptide having an A-chain polypeptide devoid of a lysine moiety and a B-chain polypeptide having a single lysine moiety, preferably composed of Insulin polypeptide - oligomer The product of a monomeric insulin polypeptide with a single oligomer, where the oligomer combines with a lysine moiety Lysine is a B-series polypeptide. As another example, when the proinsulin polypeptide is a proinsulin with a leader peptide, the cleavage of the C-peptide and the leader peptide from the product of the proinsulin-oligomer combination provides the product of a monomeric combination of insulin and the oligomer, where the insulin is monomeric at LysB29.

The embodiments and methods of synthesis of insulin polypeptide-oligomer conjugate products described previously preferably result in a yield of insulin polypeptide-oligomer conjugate products that is greater than 75, 76, 77, 78 or 79%. The best result is greater than 86, 87, 88, 89 or 0.9%. Ideally, the result is greater than 91, 92, 93, 94, or 95%. When the proinsulin polypeptide-oligomer is provided in contact with an activating oligomer with the proinsulin polypeptide-oligomer, it may be preferable to use an excess amount of activating oligomers to obtain higher products. For example, the previously described products are preferably obtained using a molar ratio of activator oligomer to proinsulin polypeptide greater than 2:1, preferably greater than 3:1, preferably greater than 4:1, and preferably greater than 5:1. It is best to obtain products greater than 91, 92, 93, 94, and 95% using a molar ratio of the activated oligomer to

proinsulin polypeptide. Greater than 4:1, and better than 5:1.

According to embodiments of the invention, methods for synthesizing an insulin polypeptide-oligomer conjugate include an insulin polypeptide combined with one or more peptides by a peptide bond(s) that can be broken to yield an insulin polypeptide with an oligomer in Conditions are sufficient for the oligomer to combine with the insulin polypeptide portion of the proinsulin polypeptide and give the resulting combination proinsulin polypeptide. proinsulin polypeptide - oligomer, and cleavage one or more peptides from the proinsulin polypeptide - oligomer to give the product of insulin polypeptide - oligomer.

For example, insulin-oligomer conjugate products can be synthesized as shown in the examples below. An embodiment of a synthetic path is shown in Figure 1.

The proinsulin polypeptide may be diverse and include an insulin polypeptide combined with one or more peptides by a bond (peptide bonds) that can be cleaved to obtain an insulin polypeptide, as will become clear to those with experience in the field, and includes, without limitation, proinsulin. , proinsulin homologues, proinsulin fragments, proinsulin homologs, miniature proinsulins, or fusion proteins. In some embodiments, the proinsulin polypeptide is a proinsulin analogue with a leader peptide. A similar proinsulin with a leader peptide is produced by Itoham Foods Inc. of Ibaraki Pref, Japan. The leader peptide and C-peptide of the proinsulin homologue are both devoid of lysine moieties. In other embodiments, proinsulin polypeptide is a proinsulin polypeptide produced by Biobras of Belo Horizonte; Brazil. And polypeptide

A proinsulin polypeptide has a leader peptide fused to the N-terminus of the B chain of insulin on the proinsulin polypeptide. The leader peptide is free of lysine moieties and preferably contains insulin polypeptide chain (A) and polypeptide chain (B). It is preferable that the A-chain polypeptide be free of lysine moieties. Preferably, the B-chain polypeptide contains one lysine moiety. The A-chain polypeptide and the B-chain polypeptide are preferably cross-linked, preferably cross-linked using one or more disulfide bonds. Ideally, both the A-chain polypeptide and the B-chain polypeptide contain cysteine moieties, one or more of which combine using one or more disulfide bonds to link the two chains. Preferably, the insulin polypeptide is insulin, similar to Insulin, piece of insulin, or piece of similar insulin.

In some embodiments, one or more peptides fused to the insulin polypeptide include a contact peptide fused at one end to the C-terminus of the B-series polypeptide and the other end to the N-terminus of the A-series polypeptide. In general, the amino acid sequence of the contact peptide is not important and may be any contact peptide, as will be apparent to those with experience in the art and includes, without limitation, C-peptide polypeptides, C-peptides, and contact peptides in mini-primary insulins. In some embodiments, the contact peptide is devoid of lysine moieties. These embodiments may use less oligomeric reagents by reducing the number of possible union sites on the proinsulin polypeptide molecule.

In other embodiments, one or more peptides fused to the insulin polypeptide comprise a leader peptide fused to the N-terminus of the B-chain polypeptide. In general, the amino acid sequence of the leader peptide is not important. In some embodiments, the leader peptide is devoid of lysine moieties. These embodiments may reduce the amount of oligomeric reagent used by limiting the number of union sites on the proinsulin polypeptide molecule.

In other embodiments, one or more peptides bound to the insulin polypeptide comprise both a contact peptide as described above and a leader peptide as described above. One or more peptides may consist primarily of a contact peptide and a leader peptide, or may consist of a contact peptide and a leader peptide.

Peptide bonds are bonds that can be broken in various ways, as will become clear to those with experience in the field. Preferably, the peptide bonds are bonds that can be enzymatically cleaved by enzymes including, but not limited to, trypsin, carboxypeptidase B, thrombin, pepsin, and chymotrypsin. Peptide bonds that can be enzymatically cleaved will be understood by those with expertise in the field and include, without limitation, Thr-Arg, Arg-Arg, Arg-Gly, Thr-Lys, Thr-Arg- Arg, Ala-Arg, and Arg-Phe.

The oligomer may be one of a variety of oligomers, as will become clear to those with experience in the field. And in general pain. An oligomer may be any oligomer capable of combining with a polypeptide as is apparent to those skilled in the art. For example, an oligomer may be a polyubiquitinated oligomer as described in US Patent No. 4,179,337 by Davis and co-workers; US Patent No. 5,567,422 in Greenwold's name; US Patent No. 5,359,030 in the name of Ekwuribe; US Patent No. 5,438,040 in the name of Ekwuribe; US Patent No. 1 5,681.81 in the name of Ekwuribe; US Patent No. 6,309,633 on behalf of Ekwuribe and colleagues; All of which are included as references In this description. As another example, the oligomer may be a non-polydisperse oligomer, as described in the US patent application, serial number 873.731/9 0, filed on June 4, 10 02, by Ekwuribe and colleagues, under the title "Methods for the synthesis of essentially monodisperse mixtures of polyethylene blend polymers. Polyethylene Glycol &amp; US Patent Application No. 873,797 / 9 0 filed on June 4, 2001 on behalf of Ekwuribe and co-authors under the title "Mixtures of drug-oligomer conjugate products comprising polyalkylene glycol, its uses, and methods for preparing it" And he asked US patent serial number 873.899/9 0 filed on June 4 1 0 0 2 in the name of Ekwuribe

And his colleagues under the title “A mixture of products of the insulin drug and an oligomer that includes polykylene glycol, its uses and methods for its preparation.”

According to other embodiments of this invention, the oligomer comprises a composition having formula VI:

VI) A—Lj—Gk —R—G'm- R—G&amp;n—T) where A is an activatable moiety:

L is the link moiety;

G' and G' are individually selected notches to add space;

R is the lipophilic moiety and R is the polyalkylene glycol moiety, or 'R is the lipophilic moiety and R is the polykylene glycol moiety; T is the final cleft; And

m, k, j and n individually are 0 or 1.

According to such embodiments of this invention, the polykylene glycol moiety contains at least 1, 2, 4, 3, 5, 6, or 7 polykylene glycol subunits. Preferably between 1, 5, 4, 3, 2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum and 10, 11.

١٢، ١٣، ١٤، ١٥، ١٦، ١٧، ١٨، ١٩، .٢، ٢١، ٢٢، ٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩،

30، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، 40، ٤١، ٤٢، ٤٣، ٤٤، ٤5،٤٦، ٤٧،

48, 49, 0 5 or more polyalkylene glycol subunits. It is better for it to contain several cracks

Alkylene glycol at a minimum of 5, 4, 3, 2 or 6 and a maximum of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Alkylin. It is preferable for the polykylene glycol moiety to contain a minimum of 5, 4, 3, or 6 and a maximum of 5, 65, 9, 8, 7, 10, 1, or 12 polykylene glycol subunits. It is preferable that it contain between 4, 5, or 6 and 6, 7, or 8. It is best for polykylene glycol to contain 7 polykylene glycol subunits. Preferably, the polyalkylene glycol radical from the oligomer is a low-alkylene glycol radical, such as a polyethylene glycol radical.

Polyethylene Glycol, polypropylene glycol radical or polybutylene glycol radical. When the polyalkylene glycol radical is a polypropylene glycol radical, it is preferable for the radical to have a homogeneous composition (i.e., non-random). An example of a stubborn polypropylene glycol crack with a homogeneous composition is as follows:

<img file="SA1946B1_D0005.tif" />

The structure of this homogeneous polypropylene glycol can be described as containing only one carbon atom replaced by a methyl near each oxygen atom in the polypropylene glycol chain. Such homogeneous moieties may exhibit both lipophilic and hydrophilic properties.

According to these embodiments of this invention, the lipophilic moiety is the lipophilic moiety that will be apparent to those with experience in the art.

Preferably, the oligomer also includes one or more lipophilic moieties, as will become clear to those with experience in the field. The lipophilic moiety contains at least 1, 4, 3, 2, 5 or 6 carbon atoms. It is preferable that the lipophilic fraction contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum. And 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 29 or 30 carbon atoms maximum. Preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19, 20, 21 carbon atoms maximum. The best is between 3, 4, 5, 6, 7, 8 or 9 as a minimum and 4, 9, 8, 7, 6, 5, 10, 11, 12, 13 or 14 carbon atoms as a maximum. The best is between 4,3, 6,5 or 7 as a minimum and 9,8,7,6 or 10 carbon atoms as a maximum. It is preferable for the lipophilic moiety to contain 6 carbon atoms. It is preferable to choose the lipophilic part

The group consisting of saturated or unsaturated, linear or branched alkyl radicals, linear or branched saturated or unsaturated fatty acid radicals, cholesterol and adamantane. Examples of alkyl radicals include, without limitation, linear saturated alkyl radicals such as methyl, ethyl; propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl; dodecyl, tridecyl; tetradecyl, pentadecyl pentadecyl, hexadecyl hexadecyl, octadecyl eighth, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl; 2- 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl; 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, ethynyl, l-propynyl, and 2-propynyl. . Examples of fatty acid moieties include, but are not limited to, unsaturated fatty acid moieties such as lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosades Ynwat docosahexaenoate; and saturated fatty acid moieties such as acetate, caproate; Caprylate, caprate, laurate, arachides arachidate, behenate, lignocerate and cerotate.

According to embodiments of the invention, additional slits of area, G', G and &amp;G; This is as it will become clear to those with experience in the field. It is preferable to choose space-added fractions from the group consisting of sugar, cholesterol, and glycerine fractions. Sugar cracks may be cracks, as will become clear to those with experience in the field, and include, without limitation, monosaccharide cracks and cracks.

Disaccharide. It is preferable for the monosaccharide moieties to contain between 4-6 carbon atoms. Preferably, these oligomers do not include space-adding moieties (i.e., m, k, and n should preferably be zero).

According to these embodiments of the invention, the linking moiety, L, may be used to combine the oligomer with the drug as will be apparent to those with experience in the art. It is preferable to test the bonding radicals from the group consisting of alkyl radicals and a fatty acid. The bonding alkyl radical may be a saturated or unsaturated alkyl radical, linear or branched, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl; 2-propylpentyl, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. The alkoxy radical may be one of a variety of alkyl radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, disyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy. tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, ecosyloxy eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy; tert pentyloxy, 2-methyl-pentyloxy, 3-methyl-pentyloxy

methylpentyloxy, 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy, ethynyloxy, l-propynyloxy, and 2 -2-propynyloxy. The linking alkyl moiety can contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 2 1, 13, 4 1,

15, 16, 17, 18, 19 or 20 minimum and 5, 6, 7, 8, 9, 10, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms maximum, preferably containing between 1, 2, 4,3 or 5 and 9, 8, 10, 1 or 12 carbon atoms. The linking fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience, and it includes, without limitation, lauroleate, myrticoleate, palmitoleate, oleate, ilides, eucatenate, linoleate, linolenate, arachidonate, eicosa hexaenoate, and docoza hexaenoate. ; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, arachidate, behenate, lignocerate and cerotate. The linking fatty acid moiety may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 at a minimum. And 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms, preferably between 1,

4, 3, 2 or 5 and 10, 8, 14, 12 or 16 carbon atoms.

According to these embodiments, the final radical, T, is preferably an alkyl or alkoxy radical, and this radical preferably contains between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19 or 20 minimum and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms as a maximum, preferably between 2,1,3,5,4,6 or7 as a minimum and 5,6,7,8,9, 12,11,10, 3 1 or 4 1 carbon atom maximum. It is preferable that it contains between 4, 3, 2, 1 or 5 as a minimum and 5, 9, 8, 7, 6 or 10 carbon atoms as a maximum. Preferably between 1, 2, 3 or 4 and 6.5

Or 7 carbon atoms. The alkyl radical may be linear or branched, saturated or unsaturated, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl. decyl, undecyl, dodecyl, tridecyl, tetradecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, butyl Secondary sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl -ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . The alkoxy radical may be one of a variety of alkyl radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy. tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, ecosyloxy eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, tert pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy, ethynyloxy; 1-l-propynyloxy, 2-propynyloxy. Preferably a slit

The final is the low alkyl radical, such as: methyl, ethyl, propyl, isopropyl, butyl, di-butyl, tert-butyl, pentyl, or tert-pentyl, or the low alkyl radical, such as: methoxy, ethoxy, propoxy, isopropoxy, butoxy. , sec-butoxy, tert-butoxy, pentyloxy; Or tert-pentyloxy. It is preferable for the final moiety to be methyl or methoxy. While it is preferable for the final radical to be the alkyl or alkoxy radical, it must be understood that the final radical is one of a variety of radicals, as will become clear to those with experience, and includes, without limitation, sugars, cholesterol, alcohols, and fatty acids.

According to these embodiments, the labile moiety, A, is a moiety that allows the oligomer to combine with an activating factor to form an activating oligomer capable of combining with a proinsulin polypeptide. The labile radical may be one of a variety of radicals, as will become clear to those with experience in the field, including, without limitation, C(S)-OH, -C(O)-OH, -SH'-OH, C(S)-SH-, and NH2. .

In other embodiments, the oligomer comprises a structure having the formula: VII VII) A— X(CH2)m Y(C2H40)nR)

Where: A is SH, -OH, -C(S)-SH, C(S)-OH, -C(O)-OH-, or NH2;

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is an ester, ether, carbamate, carbonate, or amide bond, preferably an ether bond;

m is between 5,4,3,2,1, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 12,11 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 as a maximum, and preferably 4,3, 5 or 6 as a minimum and 5, 6,

7, 8 or 9 as a minimum and 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13 or 4 1 as a maximum, and the best is between 4,3, 6, or 7 as a minimum and 6, 7, 8, 9 or 10 maximum,

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، 30، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a maximum, and the best thing is between 3, 4, 5 or 6 as a minimum and 5, 8, 7, 6, 9, 10. 1,1 or a maximum of 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is an alkyl radical, a sugar radical, a cholesterol radical, adamantine radical, an alcohol radical, or a fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl. decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl; 2-methyl pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and ignite without specification vinyl, allyl, l-butenyl; 2-Butenyl; ethynyl, l-propynyl, and 2-propynyl. It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl,

Pentyl, or tert-pentyl. It is best for the alkyl radical to be the C1-C3 alkyl. It is better for the alkyl cleavage to be identical. The fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate. arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, and caprate. caprate, laurate, arachidate; Behenate, lignocerate and cerotate

In other embodiments, the oligomer comprises a structure having the formula: VIII VIII) A—(CH2)m (OC2H4)nOR)

In which: A is SH, -OH, C(S)-SH, C(S)-OH, -C(O)-OH-, or NH2;

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is an ester, ether, carbamate, carbonate, or amide bond, preferably an ether bond;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 11, 10, 13, 14, 15, 16,

17, 18, 19 or 20 minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2 and 3.

4, 5, 6, 7, 8, 9 or 10 minimum and 4, 5, 6, 7, 8, 9, 12, 11, 10, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22 as a maximum, and preferably 4,3, 5 or 6 as a minimum and 5, 6,

7, 8 or 9 as a minimum and 4, 5, 13, 12, 11, 10, 9, 8, 7, 6 or 14 as a maximum, and the best option is between 4, 3, 6, or 7 as a minimum and 9, 8, 7, 6 or 10. Maximum,

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a maximum, and the best thing is between 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8, 9, 10 1,1 or a maximum of 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is an alkyl radical, a sugar radical, a cholesterol radical, adamantine radical, an alcohol radical, or a fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl. decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl pentyl, 3-methylpentyl, 2-ethylhexyl 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl; Propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. It is best for the alkyl radical to be the C1-C3 alkyl. It is better for the alkyl cleavage to be identical. The fatty acid moiety may be a fatty acid moiety

Saturated or unsaturated, linear or branched, as will be apparent to those with experience in the art, and including, without limitation, lauroleate, myristoleate, palmitoleate, and oleate; elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; and saturated fatty acid moieties such as acetate, caproate; Caprylate, caprate, laurate, arachides arachidate, behenate, lignocerate and cerotate.

In other embodiments, the oligomer comprises a structure of formula IX

<img file="SA1946B1_D0006.tif" />

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is an ester, ether, carbamate, carbonate, or amide bond, preferably an ether bond;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 19, 18, 17 or 20 as a minimum and 4, 5, 9 8,6, 10, 14,13,12,11, 15, 17,16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 01 as a minimum and 4, 5, 6, 7, 8, 9, 15, 14, 13, 12, 11, 10, 16, 17, 18 19, 20, 21, 22 as a maximum, and preferably 4,3, 5 or 6 as a minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5, 9, 8, 7, 6, 10, 13, 12.11 or 14 as a maximum, and the best is between 4.3, 6, or 7 as a minimum and 9.8.7.6 or 10 as a maximum.

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2, 21,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، 40،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 0 1, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 maximum, and best between 3,

A minimum of 4, 5 or 6 and a maximum of 5, 6, 7, 8, 9, 10, 1 or 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is an alkyl radical, a sugar radical, a cholesterol radical, adamantine radical, an alcohol radical, or a fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and nonyl. decyl, undecyl, dodecyl, tridecyl, tehadecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl; 2-methylpentyl, 3-methylpentyl; 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. It is best for the alkyl radical to be the C1-C3 alkyl. It is better for the alkyl cleavage to be identical. The fatty acid radical may be a fatty acid radical

Saturated or unsaturated, linear or branched, as will be apparent to those with experience in the field, it includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, and elaidate; erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate, and arachides. arachidate, behenate, lignocerate and cerotate.

In other embodiments, the oligomer comprises a structure of formula X:

<img file="SA1946B1_D0007.tif" />

In various embodiments described previously, the oligomer is covalently coupled to the insulin polypeptide. In some embodiments the oligomer is combined with the insulin polypeptide using a hydrolyzable bond (eg, an ester or carbonate bond). The hydrolyzable combination may give the product of combining an insulin polypeptide with an oligomer that acts as a prodrug. In some cases, such as when the insulin polypeptide–oligomer conjugate is biologically inactive (i.e., the conjugate lacks the ability to act on the body through the insulin polypeptide's mechanism of action), the hydrolyzable conjugate may provide a neutralizing effect Timed or controlled release, giving biologically active insulin polypeptide over a specified period of time when one or more oligomers of the inactive insulin polypeptide conjugate cleavage. Biotinylated with oligomer to provide polypeptide

Bioactive insulin polypeptide. And in other embodiments. The oligomer combines with insulin polypeptide using a nonhydrolyzable bond (eg, carbamate, amide, or ether bond). The use of a nonhydrolyzable conjugate may be preferred when it is required to leave the bioactive insulin polypeptide-oligomer conjugate to move in the blood stream for an extended period of time, preferably at least two hours. When an oligomer combines with an insulin polypeptide using a linker moiety that includes a carbonyl moiety, such as an ester moiety, ether moiety, carbamate, carbonate, or amide binder, the resulting conjugation product is an insulin polypeptide - oligomer. Ask.

The oligomers used in the various previous embodiments are commercially available or can be synthesized in various ways, as will become clear to those with experience in the field. For example, the oligomer may be a polyubiquitinated oligomer as described in US Pat. No. 4,179,337 by Davis and co-workers; US Patent No. 5,567,422 in Greenwold's name; US Patent No. 5,359,030 in the name of Ekwuribe; US Patent No. 5,438,040 in the name of Ekwuribe; US Patent No. 1 5,681.81 in the name of Ekwuribe; US Patent No. 90,633,6030 on behalf of Ekwuribe and colleagues; All of which are included as references in this description. As another example, an oligomer may be a non-polydisperse oligomer, as described in US Patent Application No. 873.731/09 filed on June 4, 2001 by Ekwuribe et al., under the title “Methods for the synthesis of essentially monodisperse mixtures of polyethylene glycol mixtures.” US Patent Application No. 8730797/9 0 filed on June 4, 2001 on behalf of Ekwuribe and co-authors under the title "Mixtures of drug-oligomer conjugate products comprising polyalkylene glycol, uses thereof, and methods for preparing the same" And they asked US patent, serial number 8730899/9 0, filed on June 4, 2001, in the name of Ekwuribe and his co-authors, under the title “A mixture of products of the combination of an insulin drug with an oligomer, including polykylene glycol, and its uses and methods for preparing it, and oligomers according to embodiments of this invention.”

It is preferable to be essentially monodisperse and preferably monodisperse. Examples of methods for synthesizing preferred monodisperse oligomers are provided in the following Examples 1-10.

The connection of the proinsulin polypeptide with the oligomer can be carried out under conditions sufficient to provide the product of the proinsulin polypeptide-oligomer combination, using a variety of conditions, as will become clear to those with experience in the art. The proinsulin polypeptide-oligomer connection preferably includes contact of the oligomer with an activating agent under conditions sufficient to provide an activating oligomer; The activated oligomer connects with proinsulin polypeptide under conditions sufficient to provide the proinsulin polypeptide. The activated oligomer can be formed ex situ or in situ.

The activating factor may be one of various activating agents capable of activating one or more of the previously described oligomers such that the oligomer is capable of reacting with the hydroxyl and/or amine nucleophilic functional groups in the proinsulin polypeptides, as will be apparent to those with experience in the field, including without limitation. , N-hydroxysuccinimide, p-nitrophenyl cholroformate, 1,3-dihexylcyclocarbodiimide, and hydroxybenzotriazide.

It will become clear to anyone who has experience in the field that the conditions are sufficient for the activation moiety to combine with the oligomer to provide an activating oligomer. For example, an experienced person might refer to:

Comprehensive Organic Transformations.....&amp; R.c. “Larock”

“A Guide To Functional Group Preparations” (2nd ed., New York, VCH Wiley, 1999), whose description is included by reference herein.

Sufficient conditions for the union of the activated oligomer with the proinsulin polypeptide will become clear to those with experience in the field. For example, proinsulin polypeptide can be dissolved in a bipolar aprotic solvent, such as dimethylsulfoxide, to give a proinsulin polypeptide solution.

A buffering agent such as triethylamine may be added to the initial insulin polypeptide solution. The activated oligomer can then be added in an anhydrous solvent such as acetonitrile to the initial insulin polypeptide solution. Those with experience can also refer to:

“Comprehensive Organic Transformations...”R. c. Larock “A Guide To Functional Group Preparations” (2nd ed., New York, VCH Wiley, 1999). Preferably, the molar ratio of the activated oligomer to the proinsulin polypeptide should be greater than about 1:1, and preferably greater than 2:1. The best is greater than 3:1, the best is greater than 4:1, and the best is greater than 5:1.

In various embodiments of the synthesis of proinsulin polypeptide-oligomer combination products described previously, more than one oligomer (i.e., group of oligomers) may be combined with the insulin polypeptide portion of the proinsulin polypeptide. Preferably, the oligomers in the group should be identical. However, it must be understood that the oligomers in the group may be different from each other, or, alternatively, some oligomers in the group may be the same and some different. When the oligomer group is combined with the insulin polypeptide portion of the proinsulin polypeptide, it may be preferable to combine one or more oligomers with the insulin polypeptide portion of the proinsulin polypeptide. Hydrolyzable ligands and the combination of one or more oligomers may be preferred. With the insulin polypeptide portion of the proinsulin polypeptide with non-hydrolyzable ligands. Alternatively, all bonds connecting the oligomer group to the insulin polypeptide portion of the primary insulin polypeptide are hydrolyzable bonds, but have different degrees of hydrolysis. For example, one or more oligomers are removed from the insulin polypeptide or insulin polypeptide portion. The polypeptide is hydrolyzed in the body and one or more are removed

Slowly oligomers of insulin polypeptide or insulin polypeptide fraction are hydrolyzed within the body.

In various embodiments of the synthesis of proinsulin polypeptide-oligomer combination products described previously,

The oligomer may combine with the insulin polypeptide portion of the proinsulin polypeptide at various nucleophilic moieties of the insulin polypeptide portion including, without limitation, the nucleophilic hydroxide and/or amino functional groups. The nucleophilic hydroxide group may be located, for example, at the histidine and/or lysine moieties, and/or at one or more of the N-termini of the polypeptide. When the oligomer combines with one or more of the N-termini of the proinsulin polypeptide; It is preferable for the union to form a secondary secretary. When the proinsulin polypeptide contains a leader peptide with the N-terminus of the B-chain polypeptide, the N-terminus of the insulin molecule can be protected from association (eg, acylation). When the proinsulin polypeptide is a human proinsulin with a leader peptide fused to the N-terminus of the B chain, for example, the oligomer can combine with the three functional amino groups of proinsulin Proinsulin: the N-terminus of the peptide Leader, the amino group in the Lys moiety of the C-peptide, and the amino group in LysB29. When cleaving the leader peptide and the C-peptide; It is shown that the oligomer combines site-dependently with LysB29 in insulin to give a single insulin conjugate product, insulin monoubiquitinated to the oligomer at LysB29.

A proinsulin polypeptide comprising an insulin polypeptide combined with one or more peptides by peptide bond(s) that can be cleaved to yield an insulin polypeptide, which may be one of various proinsulin polypeptides and includes, without limitation, several

Proinsulin peptides described previously in relation to methods for the synthesis of proinsulin polypeptide and oligomer products. The oligomer may be one of the oligomers including, but not limited to, the oligomers described previously regarding methods for synthesizing the products of combining the proinsulin polypeptide with the oligomer. The oligomer preferably includes a hydrophilic moiety and a lipophilic moiety. The products of the conjugation of proinsulin polypeptide with the oligomer can be synthesized in accordance with the invention, by various methods known to those with expertise in the field, including, without limitation, the methods of synthesizing the conjugation products of proinsulin polypeptide described previously.

According to embodiments of this invention, the method for synthesizing the product of combining an insulin polypeptide with an oligomer includes connecting a proinsulin polypeptide comprising an insulin polypeptide conjugated to one or more peptides by a peptide bond(s) that can be broken to yield an insulin polypeptide. insulin polypeptide with an oligomer under conditions sufficient for the oligomer to combine with the insulin polypeptide portion of the primary insulin polypeptide and give the resulting polypeptide proinsulin polypeptide - oligomer, and cleavage one or more peptides from the proinsulin polypeptide - oligomer to give the product insulin polypeptide - oligomer.

According to other embodiments of this invention, the proinsulin polypeptide - oligomer comprises an ignited proinsulin polypeptide, an insulin polypeptide combined with one or more peptides by a peptide bond(s) that can be cleaved to yield an insulin polypeptide. , an oligomer fused to the insulin polypeptide portion of the proinsulin polypeptide.

The primary C-peptide polypeptide may be one of the various primary C-peptides known to those with expertise in the field. Preferably, the primary C-peptide is a proinsulin polypeptide, and preferably, the primary C-peptide is proinsulin.

C-peptide polypeptide may be one of the C-peptide polypeptides known to those with experience in the field. Preferably, it is C-peptide.

One or more of the peptides combined with a C-peptide polypeptide may be one of several different peptides as known by those with expertise in the art. It is preferable that one or more of the peptides include an insulin polypeptide. It is preferable that it is an insulin polypeptide. The insulin polypeptide may be devoid of lysine moieties, which may reduce the amount of oligomeric reagents used to combine the initial C-peptide. Ideally, one or more of the peptides is insulin or insulin fused at the N-terminus of the B chain to a leader peptide.

Peptide bonds are bonds that can be broken in various ways known to those with experience in the field. Preferably, the peptide bonds are bonds that can be enzymatically cleaved by enzymes including, but not limited to: trypsin, carboxypeptide B, thrombin, pepsin, and chymotrypsin. Peptide bonds that can be enzymatically cleaved and known to those with experience include, but are not limited to: Thr-, Thr-Arg-Arg, Ala-Arg, Thr-Arg, Arg-Arg, Arg-Gly, Lys, and Arg-Phe.

It is preferable that this part contain between 1, 2, 4, 3, 5, 9, 8, 7, 6, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum and 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms maximum, preferably between 1, 2, 3, 4, 5, 6 or 7 as a minimum and 5, 6, 7, 8 9, 10, 11, 12, 13 or 14 carbon atoms maximum. It is preferable to contain between 3,2,1,

A minimum of 4 or 5 and a maximum of 5, 6, 7, 8, 9 or 10 carbon atoms. Preferably between 1, 3, 2 or 4 and 5, 6 or 7 carbon atoms. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl. decyl, undecyl, dodecyl, tridecyl, tetradecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, butyl Secondary sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl pentyl, 3-methylpentyl, 2-ethylhexyl -ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . The alkoxy radical may be one of a variety of alkoxy radicals, including but not limited to methoxy; Ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy xadecyloxy, eighth Octadecyloxy, nonadecyloxy, ecosyloxy eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy, 2-propyl

pentyloxy, phenyloxy, allyloxy, 1-butenyloxy, and 2-butenyloxy, ethyloxy, 1-

Propainyloxy, 2-Propainyloxy. It is preferable that the final incision be a low incision, such as; methyl, ethyl, propyl, isopropyl, butyl, butyl

di-sec-butyl; tert-butyl, pentyl, or tert-pentyl or lower alkoxy radical, for example: methoxy, ethoxy, propoxy, isopropoxy, butoxy, sec-butoxy, tert-butoxy, pentyloxy, or tert-pentyloxy. It is preferable that the final moiety be a methyl or methoxy. While it is preferable for the final radical to be the alkyl or alkyl radical, it must be understood that the final radical is one of a variety of radicals, as will become clear to those with experience, and it ignites without specifying sugars, cholesterol, alcohols, and fatty acids.

According to other embodiments of this invention, the oligomer comprises a composition having the formula XI:

XI) A— L— Gk —R— G’m— R— G&amp;n— T)

where A is an activatable moiety: L is a binding moiety;

G', G' and 'G' are individually selected space-added notches;

R is a lipophilic moiety and 'R' is a polyalkylene glycol moiety, or 'R' is a lipophilic moiety and 'R' is a polykylene glycol moiety; T is the final cleft; And

m, k, j and n separately are 0 or 1.

According to such embodiments of this invention, the polykylene glycol moiety contains at least 1, 2, 4, 3, 5, 6, or 7 polykylene glycol subunits. Preferably between 1, 3, 2, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum and 10, 11,

١٢، ١٣، ١٤، ١٥، ١٦، ١٧، ١٨، ١٩، ٢٠، ٢١، ٢٢، ٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩،

٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠، ٤١، ٤٢، ٤٣، ٤٤، ٤٥، ٤٦، ٤٧،

48, 9 4, 0 5 or more polyalkylene glycol subunits. It is preferable that the polykylene glycol radical contain between 2, 3, 4, 5, or 6 at a minimum, and 5, 6, 7, 8, 9, 10, 11, 12,

13, 14, 15, 16, 18, 17, 9 1, or a maximum of 20 polyalkylene units. It is preferable for the polykylene glycol moiety to contain between 4,3,5, or 6 as a minimum and a maximum of 5,65,7,8,9,01,11, or 21 of polykylene glycol subunits. It is preferable that it contains between 4, 5, or 6 and 6, 7, or 8. It is best for polykylene glycol to contain 7 polykylene glycol subunits. The polykylene glycol radical from the oligomer is preferably a low alkyl polyglycol radical, such as a polyethylene glycol radical, a polypropylene glycol radical, or a polybutylene glycol radical. When the polyalkylene glycol radical is a polypropylene glycol radical, it is preferable for the radical to have a homogeneous composition (i.e., non-random). An example of cleaving polypropylene glycol with a homogeneous structure is as follows:

<img file="SA1946B1_D0008.tif" />

The structure of this homogeneous polypropylene glycol can be described as containing only one carbon atom replaced by a methyl near each oxygen atom in the polypropylene glycol chain. Such homogeneous moieties may exhibit both lipophilic and hydrophilic properties.

According to these embodiments of this invention, the lipophilic moiety is the lipophilic moiety that will be apparent to those with experience in the art.

Preferably, the oligomer also includes one or more lipophilic moieties, as will become clear to those with experience in the art. The lipophilic moiety contains at least 1, 2, 3, 4, 5 or 6 carbon atoms. It is preferable that the lipophilic fraction contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum. And 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms maximum. Preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 10, 12, 11, 13, 4, 1, 15, 16, 18,17, 19, 20, 1 2 carbon atoms maximum. The best is between 3, 4, 5, 6, 7, 8 or 9 as a minimum and 4, 5, 6, 7, 8, 9, 10, 1 1, 2 1, 13 or 4 1 carbon atom as a maximum. The best is between 4, 3, 5, 6 or 7 as a minimum and 9, 8, 7, 6 or 10 carbon atoms as a maximum. It is preferable for the lipophilic part to contain 6 carbon atoms. It is preferable to choose the lipophilic moiety from the group consisting of saturated or unsaturated, linear or branched alkyl moieties, linear or branched saturated or unsaturated fatty acid moieties, cholesterol and adamantane. Examples of alkyl radicals include, but are not limited to, linear saturated alkyl radicals such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, undecyl, dodecyl, and tridecyl. tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2- 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl 2-ethylhexyl-2

ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . Examples of fatty acid moieties include, but are not limited to, unsaturated fatty acid moieties such as lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosadenoate docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, caprate, laurate.

laurate, arachidate, behenate, lignocerate and cerotate.

According to embodiments of the invention, additional slits of area, G', G and &amp;G, are as will be apparent to those with experience in the art. It is preferable to choose space-added fractions from the group consisting of sugar, cholesterol, and glycerine fractions. The sugar radicals may be radicals, as will become clear to those with experience in the field, and include, without limitation, monosaccharide radicals and disaccharide radicals. It is preferable for the monosaccharide moieties to contain between 4-6 carbon atoms. It is preferable that the oligomers of these embodiments do not include space-adding moieties (i.e., m, k and n should preferably be zero).

According to these embodiments of the invention, the linking moiety, L, may be used to combine the oligomer with the drug as will be apparent to those with experience in the art. It is preferable to test the bonding radicals from the group consisting of alkyl radicals and a fatty acid. The bonding alkyl radical may be a saturated or unsaturated alkyl radical, linear or branched, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl, phenyl vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. The alkoxy radical may be one of a variety of alkoxy radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy; pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy,

undecyloxy; Dodecyloxy, tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy -methylbutoxy, tert pentyloxy, 2-methyl-pentyloxy, 3-methylpentyloxy, 2-ethylhexyloxy 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-butenyloxy, 2-butenyloxy, ethynyloxy, l-propynyloxy, and 2-propynyloxy. The linking alkyl moiety can contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 11, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum. And 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Or a maximum of 30 carbon atoms, preferably containing between 1, 2, 4, 3, 5 and 9, 8, 10, 1, 1 or 2 1 carbon atoms. The linking fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience, and it includes, without limitation, lauroleate, myrticoleate, palmitoleate, oleate, ilides, eucatenate, linoleate, linolenate, arachidonate, ecosa hexaenoate, and docosa hexaenoate; and saturated fatty acid moieties such as acetate, caproate; caprylate, caprate; Laurate, arachidate, behenate, lignocerate, and cerotate. The linking fatty acid moiety may contain between 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16, 17, 18, 19 or 20 as a minimum and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms, preferably between 1, 2, 3, 4 or 5 and 8, 10, 12 14 or 16 carbon atoms.

According to these embodiments, the final moiety, T, is preferably an alkyl or alkoxy moiety and this moiety preferably contains between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19 or a minimum of 02 and 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 carbon atoms maximum, preferably between 4, 3, 2, 1, 5, 6 or 7 as a minimum and 5, 9, 8, 7, 6, 10, 11, 12, 13 or 4 1 carbon atom maximum. It is preferable that it contains between 4, 3, 2, 1 or 5 as a minimum and 5, 9, 8, 7, 6 or 10 carbon atoms as a maximum. Preferably between 1, 2, 3 or 4 and 5, 6 or 7 carbon atoms. The alkyl radical may be linear or branched, saturated or unsaturated, as will become clear to those with experience, and includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl. decyl, undecyl, dodecyl, tridecyl, tetradecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, butyl Secondary sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl pentyl, 3-methylpentyl, 2-ethylhexyl -ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl; 1-l-butenyl, 2-butenyl, 2-butenyl ethynyl, 1-l-propynyl, and 2-propynyl. The alkoxy radical may be one of a variety of alkyl radicals, including, without limitation, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy. tridecyloxy, tetradecyloxy, pentadecyloxy, hexadecyloxy, octadecyloxy, ninth

nonadecyloxy, eicosyloxy, isopropoxy, sec-butoxy, tert-butoxy, 2-methylbutoxy, tert-pentyloxy, 2-methyl pentyloxy, 3-methyl pentyloxy 3-methylpentyloxy, 2-ethylhexyloxy, 2-propylpentyloxy, vinyloxy, allyloxy, 1-l-butenyloxy, 2-butenyloxy, ethynyloxy, 1-propenyloxy l-propynyloxy, 2-propynyloxy. It is preferable that the final radical be a low alkyl radical, such as: methyl, ethyl, propyl, isopropyl, butyl, di-butyl, tert-butyl, pentyl, or tert-pentyl, or a low alkyl radical, such as: methoxy, ethoxy, propoxy, isopropoxy. isopropoxy, butoxy, sec-butoxy, tert-butoxy; Pentyloxy, or tert-pentyloxy. It is preferable for the final moiety to be methyl or methoxy. While it is preferable for the final radical to be the alkyl or alkoxy radical, it must be understood that the final radical is one of a variety of radicals, as will become clear to those with experience, and includes, without limitation, sugars, cholesterol, alcohols, and fatty acids.

According to these embodiments, the activatable moiety, A, is a moiety that allows the oligomer to combine with an activating metabolite to form an activating oligomer capable of combining with a proinsulin polypeptide. The labile radical may be one of a variety of radicals, as will become clear to those with experience in the field, including, without limitation, C(S)-OH, -C(O)-OH, -SH, -OH, C(S)-SH-, and NH2. .

In other embodiments, the oligomer includes a structure of the formula XII: XII) A—X(CH2)mY(C2H4O)nR)

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is the moiety of an ester, ether, carbamate, carbonate, or amide linkage, preferably the ether moiety;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 as a minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 17, 16, 19, 20, 22, 21 as a maximum, and preferably 4, 3, 5 or 6 as a minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5, 9, 8, 7, 6, 10, 13, 12, 11 or 14 as a maximum, and the best is between 4, 3, 6, or 7 as a minimum and 9, 8, 7, 6 or 10 as a maximum.

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 13, 12, 14, 15, 16, 18, 17, 19 or 20 as a maximum, and the best thing is between 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8, 9, 10, 11 or a maximum of 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is an alkyl radical, a sugar radical, a cholesterol radical, adamantine radical, an alcohol radical, or a fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl. , decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl; octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-

2-methylbutyl, tert-pentyl, 2-methyl pentyl; 3-methylpentyl, 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl, propyl; isopropyl; Butyl, sec-butyl, tert-butyl, pentyl, or tert-pentyl. It is preferable that the coronal incision be C1-C3. It is better for the alkyl cleavage to be identical. The fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will be clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate, and palmitoleate; oleate; elaidate, erucate, linoleate, linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, and caprate. caprate, laurate, arachidate, behenate, lignocerate and cerotate.

In other embodiments, the oligomer comprises a structure of the formula XIII: XIII) A—(CH2)m (OC2H4)n OR)

In which: A is SH, -OH, C(S)-SH, C(S)-OH, -C(O)-OH-, or NH2;

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH—;

Y is an ester, ether, carbamate, carbonate, or amide bond, preferably an ether bond;

m is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 14, 13,12,11,10, 15, 16, 17, 18, 19, 20, 21, 22 as a maximum, and preferably 4,3, 5 or 6 as a minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5, 9, 8, 7, 6, 10, 13, 12, 11 or 14 as a maximum, and the best is between 3, 4, 6, or 7 as a minimum and 6, 7, 8, 9 or 10 as a maximum.

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 10, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 13, 12, 14, 15, 16, 18, 17, 19 or 20 as a maximum, and the best thing is between 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8, 9, 10 11 or a maximum of 12 polyalkylene glycol subunits. A minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polykylene glycol subunits is best, and 7 is best;

R is an alkyl radical, a sugar radical, a cholesterol radical, adamantine radical, an alcohol radical, or a fatty acid radical. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, and pentyl; hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl pentyl, 3-methylpentyl, 2-ethylhexyl 2-ethylhexyl, 2-propylpentyl

2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, 2-butenyl, ethynyl, l-propynyl, and 2-propynyl. . It is preferable that the alkyl radical be a low alkyl radical, for example: methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl; Or tert-pentyl. It is best for the alkyl radical to be the C1-C3 alkyl. It is better for the alkyl cleavage to be identical. The fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate; palmitoleate, oleate, elaidate, erucate, linoleate; linolenate, arachidonate, eicosapentaentoate, and docosahexaenoate; and saturated fatty acid moieties such as acetate, caproate; Caprylate, caprate caprate, laurate, arachidate, behenate, lignocerate and cerotate.

In other embodiments, the oligomer comprises a structure having the formula XIV:

<img file="SA1946B1_D0009.tif" />

X is an oxygen atom or a covalent bond, provided that X is not an oxygen atom when A is OH-;

Y is the moiety of an ester, ether, carbamate, carbonate, or amide linkage, preferably the ether moiety;

m is between 4,3,2,1, 5, 6, 7, 9,8, 10, 14,13,12,11, 15, 16,

17, 18, 19 or 2 as a minimum and 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, preferably between 2, 3, 4, 5, 6, 7, 8, 9 or 10 as a minimum and 4, 5, 6, 7, 8, 9, 14, 13, 12, 11, 10, 15, 6 1, 17, 18, 9 1, 0 2, 21, 22 maximum, and preferably 5, 4, 3 or 6 maximum. minimum and 5, 6, 7, 8 or 9 as a minimum and 4, 5, 13, 12, 11, 10, 9, 8, 7, 6 or 14 as a maximum, and the best is between 4, 3, 6, or 7 as a minimum and 9,8,7,6 or 10 maximum,

n is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19 or 20 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,

٢٣، ٢٤، ٢٥، ٢٦، ٢٧، ٢٨، ٢٩، ٣٠، ٣١، ٣٢، ٣٣، ٣٤، ٣٥، ٣٦، ٣٧، ٣٨، ٣٩، ٤٠،

41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 and the best is between 2, 3, 4, 5, 6, 7, 8,

9,10,11, 13,12, 14, 15, 18,17,16, 19 or 20 as a maximum, and the best is between 3, 4, 5 or 6 as a minimum and 5, 6, 7, 8, 9, 10 , a maximum of 1 1 or 2 1 polyalkylene glycol subunits. The best is between a minimum of 4, 5, or 6 and a maximum of 6, 7, or 8 polyalkylene glycol subunits, and the best is 7;

R is an alkyl moiety, sugar moiety, cholesterol moiety, adamantane moiety, alcohol moiety, or fatty acid moiety. The alkyl radical may be a linear or branched alkyl radical, saturated or unsaturated, as will become clear to those with experience in the field, and it includes, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl. , nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl and eicosyl; Saturated branched alkyl radicals such as isopropyl, sec-butyl, tert-butyl, 2-methylbutyl, tert-pentyl, 2-methyl-pentyl, 3-methylpentyl, 2-ethylhexyl 2-ethylhexyl, 2-propylpentyl; The unsaturated alkyl radicals are derived from the previous saturated alkyl radicals and include, without limitation, vinyl, allyl, l-butenyl, and 2-butenyl.

butenyl, ethynyl; 1- l-propynyl, and 2-propynyl. It is preferable that the alkyl radical be a lower alkyl radical, for example: methyl, ethyl, propyl, isopropyl, butyl, di-butyl, tert-butyl, pentyl, or tert-pentyl. It is best for the alkyl radical to be the C1-C3 alkyl. It is better for the alkyl cleavage to be identical. The fatty acid moiety may be a saturated or unsaturated fatty acid moiety, linear or branched, as will become clear to those with experience in the field, and includes, without limitation, lauroleate, myristoleate, palmitoleate, oleate, elaidate, erucate, linoleate, linolenate. ; arachidonate, eicosapentaentoate, and docosahexaenoate; And saturated fatty acid moieties such as acetate, caproate, caprylate, and caprate. caprate, laurate, arachidate, behenate, lignocerate and cerotate.

In various embodiments described previously, the oligomer is covalently coupled to the insulin polypeptide. In some embodiments, the oligomer is combined with a C-peptide using a hydrolyzable bond (eg, an ester or carbonate bond). The hydrolyzable combination may give the product of a C-peptide combined with an oligomer that acts as a prodrug. In some cases, such as when the C-peptide-oligomer conjugate product is not biologically active (i.e., the product lacks the ability to act on the body through the C-peptide mechanism of action), a hydrolyzable conjugate may provide Timed release or controlled release effect, giving a biologically active C-peptide over a specified period of time when one or more oligomers cleavage from the products of the non-biologically active C-peptide combining with the oligomer to provide Bioactive C-peptide. And in other embodiments. The oligomer combines with a C-peptide using a nonhydrolyzable bond (eg, a carbamate, amide, or ether bond). It may be preferred

Use a non-hydrolyzable bond when it is desired to leave the bioactive C-peptide-oligomer conjugate to circulate in the bloodstream for an extended period of time, preferably at least two hours. When an oligomer combines with a C-peptide using a linker moiety that includes a carbonyl moiety, such as an ester moiety, ether moiety, carbamate, carbonate, or amide binder, the resulting conjugation product is an insulin polypeptide conjugate. - Acyl oligomer.

The oligomers used in the various previous embodiments are commercially available or can be synthesized in various ways, as will become clear to those with experience in the field. For example, the oligomer may be a polyubiquitinated oligomer as described in US Patent No. 4,179,337 by Davis and co-workers; US Patent No. 5,567,422.US in Greenwold's name; US Patent No. 5,359,030.US in the name of Ekwuribe; US Patent No. 5,438,040.US in the name of Ekwuribe; US Patent No. 5,681,811.US in the name of Ekwuribe; US Patent No. 6,309,633 on behalf of Ekwuribe and colleagues; All of which are included as references in this description. As another example, the oligomer may be a non-polydisperse oligomer, as described in US patent application No. 09/873,731 filed on June 4, 1 0 0 2 by Ekwuribe and his colleagues under the title “Methods for the synthesis of essentially monodisperse mixtures of polyethylene glycol mixtures.” ; US Patent Application No. 873,797 / 9 0 filed on June 4, 2001 on behalf of Ekwuribe and co-authors under the title "Mixtures of drug-oligomer conjugate products comprising polyalkylene glycol, uses thereof, and methods for preparing the same" And he asked US patent, serial number 873,899/09, filed on June 4, 2001, in the name of Ekwuribe and his colleagues, under the title “A mixture of products of the combination of the drug insulin with an oligomer, including polykylene glycol, and its uses and methods for its preparation.” The oligomers, according to embodiments of this invention, should preferably be basically monodisperse. Mono-diffusion and examples of preferred mono-diffusion oligomer synthesis methods are provided in Examples 1-10 that follow.

The connection of the primary C-peptide with the oligomer can be carried out under conditions sufficient to provide the product of the primary C-peptide-oligomer combination, using a variety of conditions, as will be apparent to those with experience in the art. Preferably, the C-peptide conjugation to a primary-oligomer involves contacting the oligomer with an activating agent under conditions sufficient to provide an activating oligomer; The activated oligomer connects with proinsulin polypeptide under conditions sufficient to provide the product of the primary C-peptide union. The activated oligomer can be formed ex situ or in situ.

The activating agent may be one of various activating agents capable of activating one or more of the previously described oligomers such that the oligomer is capable of reacting with the hydroxyl and/or amine nucleophilic functional groups in the primary C-peptide polypeptide, as will be apparent to those with experience in the art, including without Determination, N-hydroxysuccinimide, para-nitrophenyl chloroformate, 1,3-dihexylcyclocarbodiimide, and hydroxybenzotriazide.

It will become clear to those with experience in the field that the conditions are sufficient for the activating agent to combine with the oligomer to provide an activating oligomer. For example, an experienced person might refer to:

Comprehensive Organic Transformations...” R.c. “Larock”

“A Guide To Functional Group Preparations” (2nd ed., New York, VCH Wiley, 1999), whose description is included by reference in this description.

Sufficient conditions for the combination of the activated oligomer with the primary C-peptide will become clear to those with experience in the art. For example, the primary C-peptide polypeptide can be dissolved in a bipolar aprotic solvent, such as dimethylsulfoxide, to give a primary C-peptide solution. A buffer such as triethylamine can be added to the initial C-peptide solution. then

The oligomer activated in an anhydrous solvent such as acetonitrile can be added to a primary C-peptide solution. Those with experience can also refer to:

Comprehensive Organic Transformations.......” R. c. “Larock”

“A Guide To Functional Group Preparations” (2nd ed., New York, VCH Wiley, 1999). Preferably, the molar ratio of the activated oligomer to the initial C-peptide should be greater than about 1:1, and preferably greater than 2:1; The best is greater than 3:1, the best is greater than 4:1, and the best is greater than 5:1.

More than one oligomer (i.e., a group of oligomers) may be combined with the insulin polypeptide portion of the primary C-peptide. Preferably, the oligomers in the group should be identical. However, it must be understood that the oligomers in the group may be different from each other, or, alternatively, some oligomers in the group may be the same and some different. When the group of oligomers is combined with the C-peptide portion of the primary C-peptide, it may be preferable to combine one or more of the oligomers with the C-peptide portion of the primary C-peptide with hydrolyzable ligands. The combination of one or more oligomers with the C-peptide portion of the primary C-peptide by non-hydrolyzable linkages. Alternatively, all bonds linking the oligomer group to the C-peptide portion of the primary C-peptide are hydrolyzable, but have different degrees of hydrolysis. For example, one or more oligomers are removed from the C-polypeptide. A peptide or C-peptide fraction is hydrolyzed in the body. One or more oligomers are slowly removed from the C-peptide or C-peptide by hydrolysis in the body.

In various embodiments of the methods for synthesizing C-peptide-oligomer combination products described above, the oligomer may combine with the C-peptide portion of the primary C-peptide at various different moieties of the C-peptide portion.

A C-peptide polypeptide including, without limitation, nucleophilic hydroxyl and/or amine functional groups. The nucleophilic hydroxide group may be located, for example, at the serine and/or tyrosine moiety, and the nucleophilic amine functional group may be located, for example, at the histidine and/or lysine moiety, and/or at one or more of the amino ends of the polypeptide. When the oligomer combines with one or more of the N-termini of the proinsulin polypeptide, the combination preferably forms a secondary amine.

It is possible to perform the cleavage of one or more peptides from the product of the primary C-peptide-oligomer combination to provide the C-peptide-oligomer combination product through several processes, as will become clear to those with experience in the field. Preferably, the cleavage of one or more peptides from the C-peptide-oligomer conjugate involves contacting the C-peptide-oligomer conjugate with one or more enzymes capable of cleaving the bond(s). Between one or more peptides and a C-peptide polypeptide under sufficient conditions to split one or more peptides from the initial C-peptide polypeptide. Oligomer. As described in various references, for example, Kemmler and colleagues, “Studies on the Conversion of Primary C-peptide to Insulin,” J. Biol. Chem, 246: 6786-1 679 (1 971), the description of which is included in this invention as a self-reference, and it will become clear to those experienced in the field how to select suitable enzymes in light of the specific peptide bonds to be cleaved and how to provide adequate conditions for cleaving one or more peptides from The product of the primary C-peptide combining with an oligomer. Preferably, one or more of the enzymes may include different enzymes including, but not limited to, trypsin, chymotrypsin, carboxypeptidase B, and a mixture thereof. Ideally, one or more of the enzymes selected are trypsin, carboxypeptidase B, and a mixture thereof.

This invention will now be described with reference to the following examples. It must be understood that these examples are for the purpose of illustrating this invention, and are not intended to define the scope of the invention as defined by the elements of protection.

Examples Example - 1

Synthesis of 2,5-dioxo-pyrrolidine-1-yl acid ester 6_(2- (2- [2- (2-{2-[2~ (2-methoxyethoxy)ethoxy] - ethoxy} - ethoxy) - ethoxy]-ethoxy}- ethoxy)-hexanoic (8)

6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoic acid 2,5-dioxo -pyrrolidin-l-ester (8)

Monobenzyl ether hexaethylene glycol (1):

(1) Hexaethylene glycol monobenzyl ether

An aqueous sodium hydroxide solution prepared by slowly dissolving 3.99 (0.01 mmol) NaOH in 4 ml of water was added to monodispersed hexaethylene glycol (28.175 g, 100 mmol). Benzyl chloride (3.9 g, 30.8 mmol, 3.54 ml) was added and the reaction mixture was heated with stirring to 100°C for 18 hours. The reaction mixture is then cooled, diluted with salt solution (250 L) and extracted with methylene chloride (200 ml twice). The combined organic layers were washed with salt solution once, dried over Na2SO4, filtered and vacuum concentrated to obtain dark brown oil. The resulting crude mixture was purified by flash chromatography (silicagel, gradient perfusion: ethyl acetate to 1/9 ethyl acetate/methanol) to yield 80.099 g (70%) of compound (1) monodispersed in the form of a smaller oil.

6-Methyl sulfonyloxy hexanoate ethyl (2):

(2) Ethyl 6-methylsulfonyloxyhexanoate

A solution of monodispersed ethyl hydroxyhexanoate (50.76 mL, 50.41 g, 227 mmol) in dry dichloromethane (75 mL) was cooled in an ice bath and placed in a nitrogen atmosphere. Triethylamine (34.43 ml, 24.99 g, 247 mmol) was added. A solution of methanesulfonyl chloride (19.15 ml, 28.3 g, 247 mmol) in dry dichloromethane (75 ml) was added in the form of drops from an adding funnel. The mixture was stirred for 3 and a half hours, and left to reach room temperature slowly while the ice bath melted. The mixture was filtered through silica gel, and the resulting product was washed successively with water, saturated NaHCO3, water and salt solution. The organic extracts were dried over Na2SO4, filtered and vacuum concentrated to obtain a fainter oil. A final purification of the final product was carried out by flash chromatography (silicagel, 1/1 hexane/ethyl acetate) to obtain compound (2), monodisperse (3 46.1 g, 85%) in the form of a clear, colorless oil.

FAB MS: mass/charge 239 (M+H), 93 1 (M-C2H5O)

Acid ethyl ester 6-(2-[2-(2- {2- [2- (2-benzyloxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoic (3):

Sodium hydride (3.225 g or 60% diffusion) was suspended in oil,

80.6 mmol) in 80 ml of anhydrous toluene, placed in a nitrogen atmosphere and cooled in an ice bath. A solution of monodisperse alcohol (9) (27.3 g, 73.3 mmol) in 0.8 ml of dry toluene was added to the NaH suspension. The mixture was stirred at 0°C for 30 minutes, left to reach room temperature and stirred for another 5 hours, during which time the mixture turned into a clear brown solution. Monodisperse mesylate (10) (19.21 g, 80.6 mmol) in 80 ml of dry toluene was added to

The mixture was NaH/alcohol, and the combined solution was stirred at room temperature for 3 days. The reaction mixture was quenched with 0.5 ml of methanol and filtered through basic alumina. The filtrate was concentrated by vacuum and purified by flash chromatography (silica gel, gradient perfusion: 1/3 ethyl acetate/hexanes to ethyl acetate) to obtain compound (3) monodisperse in the form of a faint smaller oil (16.52 g, 44 g). %). FAB MS: mass/charge 515 (M+H).

Acid ethyl ester 6-(2-[2-(2- (2- [2- (2-hydroxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy]-hexanoic acid (4):

6-{2-[2-(2{-2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}_

(4) hexanoic acid ethyl ester

Essentially monodisperse benzyl ether (3) (1.03 g, 2.0 mmol)

In 25 ml of ethanol. To this solution, 270 mg 0 1% Pd/c was added, and the mixture was placed in a hydrogen atmosphere and stirred for 4 hours, then TLC showed that the starting material had completely disappeared. The reaction mixture was filtered through 545 Celite to remove the catalyst, and the filtrate was concentrated by vacuum to obtain compound (4) monodisperse in the form of pure oil (0.67 g, 79%).

FABMS: mass/charge 425 (M+H), 447 (M+Na)

Acid ethyl ester 6-{2-[2-(2-{2- [2-(2-methylsulfonylethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-hexanoic (5):

6-{2-[2-(2-{2-[2-(2-methylsulfonylethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]_. ethoxy}-hexanoic acid ethyl ester (5).

Dissolve monodisperse alcohol (4) (0.835 g, 1.97 mmol) in 3.5 ml of dry dichloromethane and place in a nitrogen atmosphere. Triethylamine (0.301 ml, 0.219 g, 2.16 mmol) was added and the mixture was cooled in an ice bath. After 2 minutes, methanesulfonyl chloride (0.165 ml,

0.248 g, 2.16 mmol). The mixture was stirred for 15 minutes at 0°C, then at room temperature for two hours. The reaction mixture was filtered through silica gel to remove triethylammonium chloride, and the filtrate was washed successively with water, saturated NaHCO3, water, and a salt solution. The organic extracts were dried on Na2SO4, filtered and concentrated by vacuum. The remaining fraction was purified by column chromatography (silica gel; 1/9 ethyl acetate/methanol) to obtain compound (5) monodisperse in the form of pure oil (9 1 8, * g, 83%) FAB MS: mass/ Charge 3 0 5 (M+H).

Acid ethyl ester 6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-

Ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoic (6):

6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxyl-ethoxy}-ethoxy)-ethoxy]-

(6) ethoxy}-ethoxy)-hexanoic acid ethyl ester

NaH (88 mg of 0.6% diffusion in oil, 2.2 mmol) was suspended in anhydrous toluene (3 ml) in a N2 atmosphere and cooled to 0 C. Diethylene glycol monomethyl ether (0.26 ml, 0.26 g, 2.2 mmol) was added, which was dried by azeotropic distillation with toluene. The reaction mixture was left to warm to room temperature and stirred for 4 hours, during which the hazy gray suspension became clear and yellow in color and then turned brown. Mesylate (5) (0.50 g, 1.0 mmol) was added in 2.5 ml of dry toluene. After stirring at room temperature overnight, the reaction was quenched by adding 2 ml of methanol and the resulting solution was filtered through silica gel. The filtrate was concentrated by vacuum and BAB MS: mass/charge 499 (M+H), 521 (M+Na).

By further purification by preparatory chromatography (silica gel; 19/

3. Chloroform/methanol) Compound (6) was supplied monodispersely in the form of a smaller pure oil (0.302 g, 57%).

FAB MS: mass/charge 527 (M+H), 549 (M+Na).

6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-hexanoic acid (7):

6-{2-[2-(2{-2-[2-(2-hydroxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy[_

(7) ethoxy}-ethoxy)-hexanoic acid

The monodispersed ester (6) (0.25 g, 0.46 mmol) was stirred for 18 h in 0.17 mL of 1 N NaOH. After 8 1 h, the mixture was vacuum concentrated to remove the alcohol and the remaining moiety was concentrated in another 10 ml of water. The aqueous solution was made acidic to pH 2 with standard HCl 2, and the product was extracted in dichloromethane (30 ml twice). Then the combined organic extracts were washed with a salt solution (25 ml twice), dried with Na2SO4, filtered and concentrated by vacuum to obtain compound (15) monodisperse in the form of a smaller oil (0.147 g, 62%).

FAB MS: mass/charge 499(M+H), 521(M+Na).

2,5-Dioxo-pyrrolidine-1-yl acid ester 6-(2-{2-[2-(2-{2-[2-(2-methoxyethoxy)-ethoxy]-ethoxy}-ethoxy)- Ethoxy]-ethoxy}-ethoxy)-hexanoic (8):

6-(2-{2-[2-(2{-2-[2-(2-methoxyethoxy)ethoxy]ethoxy}-ethoxy)-ethoxy]-ethoxy}_

(8) ethoxy)-hexanoic acid 2,5-dioxo-pyrrolidin-l-yl ester.

Dissolve monodisperse acid (7) (9 0.2 g, 0.42 mmol) in 4 ml of dry dichloromethane and add to a dry vial containing N-hydroxysuccinimide (57.8 mg). , 0.502 mmol) and EDC

(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (98.0 mg, 0.502 mmol) in an N2 atmosphere. The solution was stirred at room temperature overnight and filtered through silica gel to remove excess reagents and urea formed from the EDC. The filtrate was concentrated by vacuum to provide the activated monodisperse oligomer (8).

Dark yellow oil form (0.235 g, 94%). FAB MS: mass/charge 596 (M+H), 8 1 6

(M+Na).

Example 2:

MPEG7-C8 Synthesis Activator (4 1)

Mesylate of monomethyl ether triethylene glycol (9):

To a solution of CH2CI2 (100 ml) cooled to 0°C in an ice bath, monomethyl ether (25 g, 0.15 mol) was added. Then triethylamine (29.5 ml, 0.22 mol) was added and the solution was stirred for 15 minutes at 0 C, then drops of methanesulfonyl chloride (1 3.8 ml, 0.18 mol, dissolved in 20 ml of CH2CI2). The reaction mixture was stirred for 30 minutes at 0°C, left to warm to room temperature, and then stirred for two hours. The crude reaction mixture was filtered through Celite (washed with approximately 0.2 ml of CH2Cl2), then washed with water (300 ml), 5% NaHCO3 (300 ml), water (300 ml), saturated NaCl (300 ml), dry MgSO4. Steam it until dry. Then the oil was placed on a vacuum line for approximately two hours to ensure dryness and the mono-diffuse compound (9) was obtained in the form of yellow oil (29.15 g, yield 80%). Ethylene glycol monomethyl ether (10):

(10) Heptaethylene glycol monomethyl ether

to a solution of monodispersed tetraethylene glycol (51.5 g,

0.27 mol) in THF (1 L) potassium t-butoxide (4.8 1 g, 0.13 mol, small portions over about 30 minutes) was added. Then the reaction mixture was stirred for an hour, then (9) was dissolved in THF (0.9 ml) and added in drops, and the reaction mixture was stirred over night. The crude reaction mixture was filtered through washed celite (CH2Cl2), about 0.2 L) and evaporated to dryness. Then I dissolve the oil in HCl (250 L, 1 M) and wash it

Ethyl acetate (250 ml) to remove excess (9). An additional rinse of ethyl acetate (1 25 ml) was used to remove the remaining (9). The aqueous phase was washed successively with CH2CI2 (in volumes of 1.25 ml). Until most of the compound (18) is removed from the aqueous phase. The first extraction will contain (9), (10) and a dimerization side product and must be extracted again with HCl (125 ml, 1 M). The organic layers were mixed and evaporated until dry. The resulting oil was then dissolved in CH2CI2 (100 ml) and washed repeatedly with water (in volumes

0 5 ml) until it is removed (10). The aqueous fractions were mixed with a total volume of 0 0 5 ml, NaCl was added until the solution became cloudy, then washed with CH2CI2 (twice of 500 ml each). The organic layers were mixed, dried on MgSO4, and evaporated until dry to obtain the monoclonal compound (10) in the form of oil (6.9 1 g, yield 1 4%). It may be necessary to repeat one or more steps of the purification process to ensure high purity.

8-Bromooctoanate (11):

To a solution of monodispersed 8-bromooctanoic acid (5.0 g, 22 mmol) in ethanol (100 ml), H2SO4 (0.36 ml, 7.5 mmol) was added and the reaction was reflux heated with stirring for 3 hours. The crude reaction mixture was cooled to room temperature, washed with water (100 ml), saturated NaHCO3 (100 ml twice), water (100 ml), dried with MgSO4, and evaporated to dryness to obtain pure oil (11) (5.5 g, yield 98%). . MPEG7-C8 ester(12):

To a solution of monodispersed compound (10) (3.0 g, 8.8 mmol) in ether (90 ml), potassium t-butoxide (2.1 g, 9.6 mmol) was added and the mixture was stirred. Interaction for an hour. Then the monodisperse compound (11) (2.4 g, 9.6 mmol) dissolved in ether (10 ml) was added in drops, and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through washed celite (CH2CH2), approx

0 0 2 ml) and evaporate it until dry. The resulting oil was dissolved in ethyl acetate and washed

With water (twice, 200 ml each), dried on MgSO4, and evaporated until dry. Through column chromatography (silica, ethyl acetate to ethyl acetate/methanol, 10:1), then the compound (12) was obtained monodispersely in the form of pure oil (0.843 g, yield 19%). MPEG7-C8 Acid (13)

To oil of compound (12) monodiffused (0.70 g, 1.4 mmol), 1 M NaOH (2.0 ml) was added and the reaction mixture was stirred for 4 hours. The crude reaction mixture was concentrated, acidified (pH about 2), saturated with NaCl, and washed with CH2Cl2 (twice, each time 0.5 mL). The organic layers were mixed, washed with saturated NaCl, dried with MgSO4, and evaporated until dry to obtain compound (13) monodisperse in the form of pure oil (0.35 g, yield 53%). Activation of MPEG7-C8 acid

Monodispersed MPEG7-C8 (13) (0.31 g, 0.64 mmol) was dissolved in 3 mL of anhydrous methylene chloride and then a solution of N-hydroxysuccinimide (0.079 g, 0.69 mmol) and EDCl was added. HCl (135.6 mg, 0.71 mmol) in anhydrous methylene chloride. The reaction was stirred for several hours, then washed with 1 m HCl and water, dried with MgSO4, filtered and concentrated. The raw material was purified by column chromatography, concentrated to obtain the monodiffusion activated MPEG7-C8 (14) in the form of pure oil and dried by vacuum. Example 3

Synthesis of MPEG7-C10 activator (9 1) 0 1-hydroxydecanoate (15):

To a solution of monodisperse 10-hydroxydecanoic acid (5.0 g, 26.5 mmol) in ethanol (1 0 ml) add H2SO4 (0.43

ml, 8.8 mmol) and the reaction was reflux heated with stirring for 3 hours. The raw reaction mixture was cooled to room temperature, washed with water (100 ml), saturated NaHCO3 (twice, 100 ml), H2O, dried (MgSO4) and evaporated to dryness to obtain the compound (5 1) monodisperse (15) in the form of pure oil. (6.9 g, 98%). Mesylate of 10-hydroxydecanoate (16):

To a solution of CH2CI2 (27 ml) add 10-hydroxydecanoate monoclonal (5 1) (5.6 g, 26 mmol) and cool to 0 C in an ice bath. Then triethylamine (5 ml, 37 mmol) was added and the reaction mixture was stirred for 15 minutes at 0°C. Then methanesulfonyl chloride (2.7 ml, 2 mmol) dissolved in CH2CI2 (3 ml) was added and

The reaction mixture was stirred at 0°C for 30 minutes, the ice bath was removed, and the reaction was stirred for an additional two hours at room temperature. The crude reaction mixture was filtered through Celite (washed with CH2CI2, 80 ml) and the filtrate was washed with water (100 ml), NaHCO3 5% (twice each 0.1 ml), water (100 ml), saturated NaCl (100 ml). ) and dried it on MgSO4, and evaporated it until dry to obtain the compound (16) monodisperse in the form of a yellowish oil (7.42 g, 97%). MPEC7 C10 Ester (17):

To a solution of heptaethylene glycol (10) (2.5 g, 7.3 mmol) in THF (100 ml), sodium hydride (0.194 g, 8.1 mmol) was added and the mixture was stirred. Interaction for several hours. Then mesylate of monodispersed 10-hydroxydecanoate (6 1) (2.4 g, 8.1 mmol) dissolved in THF (10 ml) was added dropwise and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through Celite (washed with CH2CI2, approximately 0.2 ml) and evaporated to dryness. The resulting oil was dissolved in ethyl acetate, washed with water (2 times, 200 ml each), and dried on MgSO4.

It was evaporated until dry, and separated chromatographically (silica, ethyl acetate/methanol, 10:1) and separated chromatographically (silica, ethyl acetate) to obtain compound (17) monodisperse in the form of pure oil (0.570 g, with a yield of 5 1 %). MPEG7-C10 ACID (18):

To the oil of the MPEG7-C10 ester (17) (0.570 g, 1.1 mmol) 1 M NaOH (1.6 ml) was added and the reaction mixture was stirred overnight. The crude reaction mixture was concentrated, made acidic (pH about 2), saturated with NaCl, and washed with CH2CI2 (two times each 0 5 mL). The organic layers were mixed, washed with saturated NaCl (twice, each time 0.5 ml), dried over MgSO4, and evaporated until dry to obtain compound (18) in the form of pure oil (0.340 g, yield 2.6%). MPEG7-C10 acid activation:

Mono-diffusion acid (18) was activated using methods similar to those previously described in Example 10 to provide an activated MPEG7-C10 oligomer (19). Example 4:

Synthesis of C18(PEG6) activator oligomer (22) Synthesis of C18(PEG6) oligomer (20)

Monodispersed stearoyl chloride (0.7 g, 2.31 mmol) was added slowly to a mixture of monodispersed PEG6 (5 g, 17.7 mmol) and pyridine (0.97 g, 12.4 mmol) in benzene. The reaction mixture was stirred for several hours (about 5). The reaction was followed by TLC using ethyl acetate/methanol as the rising solvent. The reaction mixture was then washed with water, dried over MgSO4, concentrated and vacuum dried. The pure monodiffusion compound (02) was analyzed by FAB MS: mass/charge 9 4 5/M+H.

Activation of C18(PEG6) oligomer:

The monoubiquitinated C18(PEG6) oligomer was activated in two steps:

(1) Mono-diffuse PEG6 stearoyl (0.2) (0.8 g, 1.46 mmol)

in toluene and added to a phosgene solution (0.1 ml, 2.0% in toluene), which was cooled with an ice bath. The reaction mixture was stirred for several hours at 0°C and then for 3 hours at room temperature. Then the phosgene and toluene were eliminated by distillation, and the monodisperse PEG6 stearoyl chloroformate (1 2) remaining on P2O5 was dried over night.

(2) To a solution of monodispersed PEG6 stearoyl chloroformate (21) (0.78 g, 1.27 mmol) and TEA (128 mg, 1.27 mmol) in anhydrous methylene chloride, a solution was added. NHS in methylene chloride. The reaction mixture was stirred for 16 hours, then washed with water, dried with MgSO4, filtered, concentrated, and dried by vacuum to obtain (22). Example 5:

Synthesis of activated C18(PEG8) oligomer (28)

Tetraethylene glycol monobenzylether (23):

to oil of monodispersed tetraethylene glycol (19.4 g,

0.10 mol) A solution of NaOH (4.0 g in 4.0 ml) was added and the reaction was stirred for 15 minutes, then benzyl chloride (3.54 ml, 30.8 mmol) was added and the reaction mixture was heated at 0 1 C and stirred overnight. The reaction mixture was cooled at room temperature, diluted with saturated NaCl (250 ml), and washed with CH2CI2 (twice, each time 200 ml). The organic layers were mixed, washed with saturated NaCl, dried with MgSO4 and separated chromatographically (silica; ethyl acetate) to obtain the monodisperse compound 23 in the form of a smaller oil (6.21 g, yield 71%).

Mesylate of tetraethylene glycol monobenzylether (24):

To a solution of CH2CI2 (20 mL) (23) (6.21 g, 22 mmol) was added and cooled to 0 C in an ice bath. Then triethylamine (3.2 ml, 2 mmol) was added and the reaction mixture was stirred for 15 minutes at 0 C. Then methanesulfonyl chloride (7.1 ml, 4.2 mmol) dissolved in CH2CI2 (2 ml) was added, the reaction mixture was stirred at 0 C for 30 minutes, the ice bath was removed and the reaction was stirred for an additional two hours at room temperature. The crude reaction mixture was filtered through Celite (washed with CH2CI2, 0.8 L) and washed with water (100 ml), NaHCO3 5% (twice of 1 0 ml), water (100 ml), saturated NaCl (100 ml), And dried MgSO4. The resulting yellow oil was separated chromatographically on a layer of silica containing activated carbon (0.1 g) to obtain compound (24) monodisperse in the form of pure oil (7.10 g, 89%). Octaethylene glycol monobenzylether (5 2): To a solution of THF (tetrahydrofuran) (140 ml) containing sodium hydride (0.43 g, 1.8 mmol), drops of a solution of tetrahydrofuran were added. Monodisperse tetraethylene glycol (3.5 g, 18 mmol) in THF (10 ml) and the reaction mixture was stirred for 1 h. Then mesylate of monodispersed tetraethylene glycol monobenzyl ether (24) (6.0 g, 16.5 mmol) dissolved in THF (10 ml) was added in droplets, and the reaction mixture was stirred overnight. The crude reaction mixture was filtered through Celite (washed, CH2CI2, 250 ml) and the filtrate was washed with water, dried over MgSO4 and evaporated to dryness. The resulting oil was separated chromatographically (silica, ethyl acetate/methanol, 1:1) and separated chromatographically (silica, chlorofom/methanol, 25:1) to obtain compound (25) monodisperse in the form of pure oil. (2.62 g, yield 34%). Synthesis of stearates from benzyl PEGs (26):

To a cold stirred solution of (25) (0.998 g, 2.07 mmol) and pyridine, monodisperse stearoyl chloride (627.7 mg, 2.07 mmol) was added.

mmol) in benzene. The reaction mixture was stirred over night (18 hours). The next day, the reaction mixture was washed with water, dried with MgSO4, concentrated, and dried by vacuum. The crude product was then separated chromatographically on a silica gel column and chromatographically, using 10% methanol/90% chloroform. The parts containing the product were mixed, concentrated and dried by vacuum to obtain (26). Hydrogen analysis of the compound PEG8-benzyl stearate:

To a methanol solution of (26) (0.854 g, 1.138 mmol) add Pd/c (10%) (palladium, 1% by weight on activated carbon). The reaction mixture was stirred over night (18 hours) in a hydrogen atmosphere. The solution was then filtered, concentrated, and purified by flash column chromatography using 10% methanol/90% chloroform, and fractions with Rt = 0.6 were collected, concentrated, and dried to obtain the monodisperse acid (27). C18CPEG oligomer activation:

Two activation steps (27) were performed as described for PEG6-stearate in Example 4 above to obtain a monodisperse activated C18(PEG8) oligomer (28). Example 6:

Synthesis of triethylene glycol monomethyl activated oligomers:

A solution of toluene containing 0.2% phosgene (100 ml, about 1.8 g, 189 mmol of phosgene) was cooled to 0 C in an atmosphere of nitrogen N2. Dissolve mTEG (triethylene glycol, monomethyl ether, 7.8 g, 47.5 mmol) in 25 mL of anhydrous ethyl acetate and add to the cold phosgene solution. The mixture was stirred for an hour at 0°C, then left to warm to room temperature and stirred for another two and a half hours. The remaining phosgene, ethyl acetate, and toluene were removed by distillation.

Vacuuming leaves mTEG chloroformate monodisperse in the form of a clear oily crack.

The monodisperse mTEG chloroformate was dissolved in 50 ml of dry dichloromethane to which TEA (triethylamine, 6.62 ml, 47.5 mmol) and N)H-hydroxysuccinimide, 5.8 g, 50.4 mmol were added. mmol). The mixture was stirred at room temperature in a dry atmosphere for 20 hours, during which a large amount of white precipitate appeared. The mixture was filtered to remove this precipitate and concentrated by vacuum. The resulting oil was treated in dichloromethane and washed twice with cold deionized water, twice with 1 m HCl and once with a salt solution. The organic extracts were dried on MgSO4, filtered and concentrated to obtain the monodisperse title compound in the form of a clear, light, finer oil. If necessary, the NHS ester can be purified by curing and flash chromatography on silica gel using EtOAc for quenching. Example 7

Synthesis of palmitate oligomers TEG - Palmitate activator

Solvent monodisperse palmitic anhydride (5 g, 1 mmol)

in dry THF (20 ml) and stirred at room temperature. To the stirred solution, 3 moles of pyridine were added diversified with monodisperse triethylene glycol (1.4 ml). The reaction mixture was stirred for 1 hour (the reaction was monitored by TLC; ethyl acetate-chloroform; 3:7). At the end of the reaction, the THF was removed and the product was mixed with 1% H2SO4 and extracted with ethyl acetate (3 times, 30 ml each). The combined extract was successively washed with water, salt solution, dried on MgSO4, and otherwise to obtain monodisperse palmitate-TEG oligomers.

Add a solution of N',N-disuccinimidyl carbonate (3 ml) in DMF (about 10 ml) to a solution of palmitate oligomers.

Mono-diffuse TEG (1 mmol) in 0.1 mL anhydrous DMF with stirring. Sodium hydride (3 mmol) is added slowly to the reaction mixture. The reaction mixture is stirred for several hours (eg, 5 hours). Diethyl ether is added to precipitate the single-diffusion activated title oligomer. This process is repeated 3 times, finally drying the result. Example 8

Synthesis of monomethyl hexaethylene glycol oligomers

Monomethyl Oligomers activator

The monomethyl ether hexaethylene glycol monodisperse activator is prepared similarly to the monodisperse triethylene glycol in Example 14. A solution of 20% phosgene in toluene solution (35 ml, 6.66 g, 67.4 mmol of phosgene) was cooled under an N2 atmosphere in an ice/salt water bath. I dissolve monodispersed hexaethylene glycol (1.85 ml, 2.0 g, 6.74 mmol) in 5 ml of anhydrous EtOAc and add it to the phosgene solution via a syringe. The reaction mixture was kept agitated in an ice bath for one hour, removed and stirred for another 2.5 hours at room temperature. EtOAc, phosgene, and toluene were removed by vacuum distillation, leaving chloroformate, methyl hexaethylene glycol, monodisperse as a clear oily fraction.

The chloroformate tube was monodispersed in 20 ml of dry dichloromethane and placed in a dry inert atmosphere, then Triethylamine (0.94 ml, 0.68 g, 6.7 mmol) was added, then NHS (0.82 g, 7.1 mmol) was added. The reaction mixture was stirred at room temperature for 18 hours. The mixture was filtered through silica gel to remove the white precipitate and concentrated by vacuum. The remaining incision was treated in dichloromethane and washed twice with cold water and twice with 1 N HCl.

And once with a salt solution. The organic extracts were dried over Na2SO4, filtered and concentrated. Final purification was carried out by flash chromatography (EtOAc, silica gel) to obtain the activated monodiffusion hexaethylene monomethyl ether. Example 9

Synthesis of Heptaethylene Glycol Monomethyl Ether Activated 8-Methoxy-1-(methylsulfonyl)oxy-3,6-Dioxaoctane (29):

(29) 8-Methoxy-l-(methylsulfonyl)oxy-3,6-dioxaoctane.

A solution of monomethyl ether molecules monodisperse triethylene glycol (4.00 ml, 9 4.1 g, 25.5 mmol) and triethylamine (4.26 ml, 9 3.0 g, 30.6 mmol) was cooled ) in dry dichloromethane (0.5 ml) in an ice bath and placed in a nitrogen atmosphere. A solution of methanesulfonyl chloride (2.37 ml, 3.51 g, 30.6 mmol) in dry dichloromethane (0.2 ml) was added in drops from an adding funnel. 10 minutes after the chloride addition was completed, the reaction mixture was removed from the ice bath and allowed to come to room temperature. The mixture was stirred for another hour, during which TLC (CHCl3 with 15% MeOH for quenching) showed that there was no remaining triethylene glycol monomethyl ether.

The reaction mixture was diluted with another 75 ml of dichloromethane and washed successively with saturated NaHCO3, water and a salt solution. The organic extracts were dried over Na2SO4, filtered, and vacuum concentrated to obtain a monodisperse mixture of compounds (29) in the form of pure oil (5.31 g, 86%). Heptaethylene glycol mono methyl ether (0 3): To a solidified solution of monodiffused tetraethylene glycol (35.7 mmol) in dry DMF (25.7 ml) in a nitrogen atmosphere added in portions hanging

60 % of NaH in mineral oil, and the mixture was stirred at room temperature for one hour. To the resulting sodium salt of tetraethylene glycol, a solution of monoclonal mesylate (29) (23.36) in dry DMF (4 ml) was added in one part, and the mixture was stirred at room temperature for 3.5 hours. The reaction progress was followed by TLC (2 1% CHCI3-CH3OH). The reaction mixture was diluted with an equal amount of 1 m HCl, extracted with ethyl acetate (twice, 20 ml each time) and discarded. Extraction and treatment of the aqueous solution resulted in compound (30) (82-84%). oil; Rf 0.46 (methanol:chloroform = 3:22); MS The calculated mass/charge of the formula C15H32O8 is 340.21 (+M+1), and is found to be 341.2.

Activation of monomethyl ether hexaethylene glycol: (30) is activated in a manner similar to that used in the previous example 6 to activate monomethyl ether triethylene glycol to obtain activated monomethyl ether hexaethylene glycol. Example 10

Synthesis of Ethylene Glycol Monomethyl Ether -

Tonic (33)

20 - Methoxy-1-(methylsulfonyl)oxy-3,6,9,12,15,18-six-oxa

Ecozan (31):

20-methoxy-l-(methylsulfonyl)oxy-3,6,9,12,15,18-hexaoxaeicosane(31)

The monodisperse compound (31) was obtained with a quantitative product of compound (30) and methanesulfonyl chloride as described for compound (29) in Example 9 above; Rf 0.4 (ethyl acetate: acetonitrile = 1:5); MS The calculated mass/charge of the formula C17H37C10 is 433,021 (+1+M), and in practice it is found to be 433,469. Decaethylene glycol monomethyl ether (32):

The monodispersed compound (32) was prepared from compound (31) and monodispersed triethylene glycol using the method of Example 7 1 described previously. oil; Rf 0.41 (methanol:chloroform = 6:01); MS The calculated mass/charge of the formula C21H44O11 is 472.29 (+1+M), and in practice it is found to be 472.29. Activation of monomethyl ether ethylene glycol:

The monodiffusion monomethyl ether (32) is activated in a manner similar to that used in Example 6 to activate the triethylene glycol monomethyl ether to provide the activated monomethyl ether (33). Example 1 1

Preparation of LysB29-oligomer-insulin conjugate (A) and cloned insulin conjugate I (I):

Proinsulin clone I (molecular weight 10,642 Da) was obtained from Biobras of Belo Horizonte, Brazil. Equip 2.32 x 0 1 -3 mmol of Insulin I Proinsulin from 10 ml of DMSO. To the solution, 324 microliters of triethylamine were added. The resulting solution was stirred for 5 minutes, then a solution of activated methylheptaethylene glycol (PEG7-hexyl oligomer) (9.30 The course of the acylation reaction was followed by HPLC. When the reaction is complete, it is quenched by adding 3.54 ml of a 5% aqueous trifluoroacetic acid solution. The reaction mixture was then treated and replaced in 100 mmol of Tris-HCl buffer, pH 7.6. The HPLC pattern of the resulting mixture, the product of the oligomer and Proinsulin clone I, is shown in Figure 11.

(b) Enzymatic cleavage of the cloned Proinsulin I oligomer:

A standard amount of Tris-HCl solution from the resulting mixture from Example 11a was analyzed by HPLC to determine the polypeptide concentration. A solution of treated trypsin (TPCK) was prepared; from bovine pancreas) in 0.1 mmol of Tris-HCl buffer, pH 7.6. A solution of carboxypeptidase B (from pig pancreas) was prepared in 0.1 mmol of Tris-HCl buffer, pH 7.6. The resulting mixture of Example 11-a (0.424 µmol/ml) was then left to react with trypsin (5.97 x 10-10 µmol/ml) and carboxypeptidase B (1.93 x 0-1-4).

µmol/ml). After 30 minutes, the reaction was quenched by adding 1.58 ml of 1% trifluoroacetic acid in acetonitrile. The major products were determined by HPLC retention time (relative to the retention times of standard reference methods) and mass spectrometry analysis, thus obtaining insulin (10%) and the LysB29-hexyl-PEG7-oligomer-insulin (84 5) product (Figures 1 1 - 13). Example 12

Separation of oligomer products with cloned Proinsulin I:

Reverse-phase HPLC was used to separate the main products from the mixture resulting from the combination reaction in Example 1.1a. The HPLC column (1.0 cm inside diameter In HPLC system. The balance of the system was adjusted with a quenching regulator, which is a mixture that burns in a mobile phase (A) 72% (water with trifluoroacetic acid 0.1%) and a mobile phase (B) 28% (acetonitrile with trifluoroacetic acid 1, 0%) which was delivered at a flow rate of 5 ml/min. A solution of the resulting mixture was placed in 100 mmol Tris-HCl buffer; With a pH of 7.6, on the reverse phase column, the products were separated and quenched using a gradient in which the acetonitrile component in the quenching buffer (mobile phase (b)) was increased as follows:

Mobile phase (B) 28%-0.3% over 60 minutes, then

Mobile phase (B) 30%-32% over 30 minutes, then mobile phase (B) 32%-36% over 40 minutes.

The fractions were collected and individually analyzed by HPLC to determine the identity and purity of the product contained within. Then, common fractions were collected containing one of the four products (single-combination product A (­product of insulin I&amp;), stylar product-B (­product of insulin I­), dual product (­product of insulin I­ The dimer of insulin I&amp;) and the tertiary product (&amp;product of insulin I), and the solvent was removed by rotary evaporation. HPLC analysis (Figure 14) and mass spectra were used to determine the identity and purity of each separation product. Example 13

Enzymatic cleavage of the separated cloned Proinsulin I products:

Each conjugate (mono-A, mono-B, dimer or triple) that was separated using the method Example—12, was dissolved in 100 mmol of Tris-HCl buffer, pH 7.6, and analytical HPLC was used to determine the polypeptide concentration of the solution The resulting. A solution of treated trypsin (TPCK) was prepared; from bovine pancreas) in 100 mmol of Tris-HCl buffer; pH 7.6. A solution of carboxypeptidase B (from porcine pancreas) was prepared in 1 mmol of Tris-HCl buffer, pH 7.6. The crude mixture (1 mmol) was then left to react with trypsin (39.1 x 0 1 -3 mmol) and carboxypeptidase B (4.56 x 10 -4 mmol). After 30 minutes, the reaction was quenched by adding 1% trifluoroacetic acid in acetonitrile. The resulting mixture of each reaction was treated and analyzed by HPLC. The HPLC retention time for these standard methods and mass spectra analysis were used to determine the identity and purity of each product (Table-1)

Table-1

Proinsulin I oligomer-binding products, and the cleavage products (or expected products) with the enzymatic mixture for each

<img file="SA1946B1_D0010.tif" />

Example 14

Cleavage of proinsulin I separated by trypsin

Each conjugation product (Insulin I Proinsulin mono-A, mono-B,

binary, ternary) using the method described in Example 21, each is dissolved in 100 mmol of Tris-HCl buffer, pH 7.6, and the resulting solution is analyzed by HPLC to determine the polypeptide concentration. A solution of treated trypsin (TPCK) is prepared; from bovine pancreas) in 0.1 mmol of Tris-HCl buffer, pH 7.6. Then each combination product (300 mmol) is left to react with trypsin (1 mmol). After 20 minutes, the reaction is quenched by adding 1% trifluoroacetic acid in acetonitrile. The reaction products are separated and analyzed by HPLC and mass spectrometry analysis for identification. The expected products are insulin (Arg) or the LysB29-hexyl-PEG7-oligomer-insulin (Arg31) shown in Table 2.

<img file="SA1946B1_D0011.tif" />

Example 15

Cleavage of the trypsin cleavage mixture by carboxypeptidase B

A standard amount of the reaction mixture containing the LysB29-hexyl-PEG7-oligomer-insulin (Arg31) conjugate (300 mmol) (from Example 14) was eluted in 100 mmol Tris-HCl buffer, pH 7.6. A solution of carboxypeptidase B (from porcine pancreas) is prepared in 100 mmol of Tris-HCl buffer; pH 7.6. Carboxypeptidase B (1 mmol) is added to the reaction mixture. The reaction was left to continue for 15 hours, then it was quenched by adding 1% trifluoroacetic acid of acetonitrile. The expected products for each reaction are shown in Table 3. Table-3

<img file="SA1946B1_D0012.tif" />

Example 16

Preparation of the LysB29-oligomer-insulin conjugate (a) Proinsulin II clone:

Proinsulin clone II (molecular weight 11,133 da) was obtained from Itoham Foods Corporation of Ibaraki Pref, Japan. The first insulin, Proinsulin II, contained the leader peptide, and the C-peptide was devoid of lysine moieties. Pour a portion of 2.55 x 10-3 mmol Proinsulin II into 10 ml of DMSO. To the solution, 355 microliters of triethylamine were added. The resulting solution was stirred for 5 minutes, then a solution of activated polyethylene glycol (PEGn-hexyl) (n = 7 ± 3) and n = 7 (5.10 × 10-3 mmol) in acetonitrile was added. acetonitrile. The reaction course was followed by HPLC. After the reaction was completed, it was quenched by adding 3.7 ml of a 5% aqueous trifluoroacetic acid solution. The mixture was then treated and replaced in 1 mmol of Tris-HCl buffer, pH 7.6. The HPLC pattern of the oligomer-proinsulin II mixture is shown in Figure 2.

(b) Cleavage by an enzymatic mixture of the oligomer-proinsulin II product:

The Tris-HCl solution of the mixture obtained in Example 16-(a) was analyzed by HPLC to determine its peptide number concentration. A solution of processed trypsin (TPCK) from bovine pancreas was prepared in 0.1 mmol of Tris-HCl buffer; pH 7.6. A solution of carboxypeptidase B (from porcine pancreas) in 0.1 mmol of Tris-HCl buffer was prepared; pH 7.6. The resulting mixture (0.399 µmol/ml) was left to react with trypsin (5.57 × 10-4 µmol/ml) and carboxypeptidase B (1.82 After 30 minutes, the reaction was quenched by adding 0.55 μl of 1% trifluoroacetic acid in acetonitrile. The major products were identified by HPLC retention time (relative to that of known reference standard methods) and mass spectrometry analysis. Thus, insulin (23%) and the combination product LysB29-hexyl-PEGn-oligomer-insulin (0.6%) and others (17%) were obtained (Figures 9-10) Example 17

Separation of the combination products between the oligomer and Proinsulin II:

Reverse-phase HPLC was used to separate the main products from the mixture resulting from the combination reaction in Example 6-1a. The HPLC column (1.0 cm inside diameter In HPLC system. The balance of the system was adjusted with a perfusion regulator, which is a mixture of mobile phase A 75% (water with 0.1% trifluoroacetic acid) and mobile phase B 25% (acetonitrile with 0.1% trifluoroacetic acid). Which was connected at a flow rate of 5 ml/min and the Tris-HCl solution from the mixture obtained from Example 16-A was placed on the column and the main products were separated and eluted using.

Gradual percolation in which the composition of the percolation regulator changed from a 25% mobile phase to a 35% mobile phase over a period of 120 minutes. Each of the collected fractions was analyzed by HPLC to determine the purity of the product contained in it. Then the common fractions of each product were collected: Proinsulin II monomer and Proinsulin II monomer, and the solvent was removed by rotary evaporation. The identity and purity of each product was determined by HPLC and mass spectrometry analysis (Figures 2-4). Example 18

Cleavage of the products of the first insulin conjugate, Proinsulin II, separated by an enzyme mixture

All proinsulin II conjugates (mono-, di- or tri-proinsulin) separated using Example 17 were dissolved in 100 mmol of Tris-HCl buffer, pH 7.6, and a standard amount of the solution was analyzed by HPLC to determine Polypeptide concentration. A solution of trypsin (TPCK) processed from bovine pancreas was prepared in 100 mmol of Tris-HCl buffer; pH 7.6. A solution of carboxypeptidase B (from porcine pancreas) was prepared in 1.0 mmol of Tris-HCl buffer, pH 7.6. The combination product (0.127 µmol/ml) was left to react with trypsin (1.77 x 10-4 µmol/ml) and carboxypeptidase B (5.77 x 10-5 µmol/ml). After 30 minutes, the reaction was quenched by adding 0.25 μl of 1% trifluoroacetic acid in acetonitrile. Separation of the main products followed by identification by HPLC retention time against reference standard methods and mass spectrometry analysis showed the production of insulin or 29-B-acylated PEG-7-hexyl insulin in the reaction. The products in each reaction are shown in Table 4.

<img file="SA1946B1_D0013.tif" />

Example 19

Cleavage of the separated proinsulin II products, by trypsin

Each conjugate product (mono-, di-, or tri-proinsulin II) of example 17 was dissolved in * 10 mmol of Tris-HCl buffer, pH 7.6, and the resulting solution was analyzed by HPLC to determine its polypeptide concentration. A solution of trypsin (processed TPCK, from bovine pancreas) was prepared in 100 mmol of Tris-HCl buffer, pH 7.6. Then each combination product (0.127 µmol/ml) was left to react with trypsin (4.23 After 20 minutes, the reaction was quenched by adding 250 μl of 1% trifluoroacetic acid in acetonitrile. Separation of the main products followed by their identification by HPLC and mass spectrometry analysis showed that insulin (Arg31) or LysB29-hexyl PEGn-hexyl oligomer-insulin (Arg31) was produced in the reaction. The products for each reaction are shown in Table 5.

<img file="SA1946B1_D0014.tif" />

Example 20

Cleavage of the trypsin cleavage mixture by carboxypeptidase B

A standard amount of LysB29-hexyl PEG7-hexyl oligomer-insulin (Arg31) (3.10 × 10-5 mmol) was removed from Example 19. A solution of carboxypeptidase B (from porcine pancreas) was prepared in 1.0 mmol of Tris-HCl buffer, pH 7.6. Carboxypeptidase B (1.03×7-10) was added to the reaction mixture. The reaction was left to continue for 15 hours, then it was quenched by adding 1% trifluoroacetic acid in acetonitrile. After processing, the reaction products were analyzed by HPLC. Retention time and mass spectrum analysis were used for identification. Insulin (23%) and LysB29-hexyl PEGn-hexyl oligomer insulin (60%) (Figures 5, 7, 8) were produced from the reaction of monoconjugated Proinsulin II. The expected products of the proinsulin II reaction are shown in Table 6.

<img file="SA1946B1_D0015.tif" />

Example 21

Preparation of the LysB29 conjugate - oligomer - insulin (A) Natural human proinsulin conjugate

Natural human Proinsulin (Sigma Chemical Co.)

(0 3.2 x 0 1 -4 mH) in 5 ml of DMSO. To the solution, 45 microliters of triethylamine were added. The solution was stirred for 5 minutes before adding a solution of activated PEG7 oligomer-hexyl oligomer (6.4 x 1 -4 mmol) in acetonitrile. After the reaction progresses such that HPLC analysis indicates that the proinsulin has been consumed (or the concentration of the proinsulin no longer decreases further), the reaction is quenched by adding 0.5 ml of a 5% aqueous trifluoroacetic acid solution. The reaction mixture was then treated and replaced in 1 0 mmol of Tris-HCl buffer, pH

٧,٦.

(B) Cleavage of the natural oligomer-insulin product

With enzyme mixture:

A standard amount of Tris-HCl solution from the mixture obtained in Example 12a is analyzed by HPLC to determine its polypeptide concentration. A solution of trypsin (TPCK) is prepared

Processor; From bovine pancreas) in 0 0 1 mol of Tris-HCl buffer, pH 7.6. A solution of carboxypeptidase B (from porcine pancreas) is prepared in 1.0 mmol of Tris-HCl buffer, pH 7.6. The crude mixture (1 mol eq) was then left to react with trypsin (1.39 x 10-3 mol eq) and carboxypeptidase B (4.56 x 10-4 mol eq). After 30 minutes, the reaction is quenched by adding 1% trifluoroacetic acid in acetonitrile. The resulting reaction mixture is treated and analyzed by HPLC. Retention time (versus that of reference standard methods) and mass spectrum analysis are used for identification. The expected products of the reaction are insulin and the product LysB29-hexyl PEG7-hexyl-oligomer-insulin.

Example 22

Separation of natural human Proinsulin products

Each major product of the combination reaction described in Example 11a is separated using reverse-phase HPLC, and a column (1.0 cm diameter x 2.5 cm length) is filled with a commercially available C18 stationary phase known to be useful for the purification of polypeptides and proteins. Enter it into the HPLC system. The system is balanced with a quenching regulator containing a mixture of mobile phase A-75% (water with 0.1% trifluoroacetic acid) and mobile phase B-25% (acetonitrile with 0.1% trifluoroacetic acid). . A Tris-HCl solution from the mixture resulting from Example 21-A is placed on the column, and the main products are separated and quenched using a gradual quenching in which the percentage of the acetonitrile component increases from 25% - 35% over a period of 120 minutes. The fractions are collected and analyzed by HPLC to determine the identity and purity of the product. The common fractions of each product are combined, and the solvent is removed by rotary evaporation. The identity and purity of each resulting peak are determined by HPLC and mass spectrometry. The expected outcomes consist of 2 unary union outcomes

For human insulin, one dimer of human proinsulin and one dimer of human proinsulin. Example 23

Cleavage of natural human proinsulin, separated by an enzymatic mixture:

The entire product of the combination obtained using the method of Example 2 2 is dissolved in 0 0 1 mmol of Tris-HCl buffer; pH 7.6, and the resulting solution is analyzed by HPLC to determine the polypeptide concentration. A solution is prepared from processed trypsin (TPCK). from bovine pancreas) in 0–1 mmol of Tris-HCl buffer; pH 7.6. A solution of carboxypeptidase B (from porcine pancreas) is prepared in 0.1 mmol of Tris-HCl buffer; pH 7.6. The crude mixture (1 mol eq) is then left to react with trypsin (1.39 x 3-10 mol eq) and carboxypeptidase B (4.56 x 0-1 mol eq). After 30 minutes, the reaction is quenched by adding 1% trifluoroacetic acid in acetonitrile. The products are processed and analyzed by HPLC. Retention time (compared to that of standard methods ) and mass spectrum analysis are used for identification. The expected products are insulin or the LysB29-hexyl PEG7-hexyl-oligomer-insulin. Example 24

Cleavage of natural human proinsulin products separated by trypsin

Each product obtained by the method of Example 2 is dissolved in 1 mmol of Tris HCl buffer, pH 7.6, and the resulting solution is analyzed by HPC to determine the polypeptide concentration in it. A solution of processed trypsin (TPCK) from bovine pancreas is prepared in 0.1 mmol of Tris-HCl buffer; pH 7.6. Then leave the result of the union

(300 mol equivalents) to react with trypsin (1 mol equivalent). After 20 minutes, the reaction is quenched by adding 1% trifluoroacetic acid in acetonitrile. The products were treated and analyzed by HPLC. Detention time and mass spectrometry are used to determine the identity of the compound. The expected products of the reaction are insulin (Arg31) or LysB29-hexyl PEG7-hexyl oligomer-insulin (Arg31). Example 25

Cleavage of the trypsin cleavage mixture by carboxypeptidase B

A standard amount of the reaction mixture of LysB29-hexyl PEG7-hexyl oligomer-insulin (Arg31) (300 mmol) is removed from Example 24. A solution of carboxypeptidase B (from porcine pancreas) is prepared in 1-0 mmol of Tris-HCl buffer, pH 7.6. Carboxypeptidase B (1 mmol) is added to the reaction mixture. The reaction is left to continue for 15 hours. Then it is quenched by adding 1% of trifluoroacetic acid in acetonitrile, and the products are treated and analyzed by HPLC. Detention time and mass spectrometry analysis are used to determine the identity and the expected products are insulin or LysB29-hexyl PEG7-hexyl oligomer-insulin. Example 26

Ideal preparation of the LysB29 conjugate - insulin oligomer

Analysis of the experimental data from Example 11 indicates that the LysB29-hexyl PEG7-hexyl-oligomer insulin and the Lys-hexyl PEG7-hexyl oligomer-peptide-C can be obtained in high yield and high purity by: (a) Acylation of the epsilon amino group in all lysine moieties present in the crude proinsulin, and (b) cleavage of the resulting proinsulin completely bound to the oligomer by an enzyme mixture consisting of trypsin and carboxypeptidase B. Practical confirmation of this theory was obtained as follows:

(A) Proinsulin clone I:

Proinsulin clone I (molecular weight 10,642 Da) was obtained from Biobras of Belo Horizonte, Brazil. Equip 2.32 x 0.1 -3 mmol of Proinsulin I from 1.0 ml of DMSO. To the solution, 4.32 microliters of triethylamine were added. The resulting solution was stirred for several 5 minutes, then a solution of activated methylheptaethylene glycol (PEG7-hexyl oligomer) was added (4-6 mol equivalents; sufficient to convert all of the proinsulin I to the triadduct) in aceto. acetonitrile nitrile. The acylation reaction was followed by HPLC. When the reaction is complete, it is quenched by adding 3.54 ml of a 5% aqueous trifluoroacetic acid solution. The reaction mixture was then treated and replaced in 1 0 mmol of Tris-HCl buffer; pH 7.6. The HPLC pattern of the resulting mixture, the product of the clone Proinsulin I with the oligomer, is expected to show peaks corresponding to the triple union product (all Lys and N-terminus combined) and the double union product only.

(b) Cleavage of the oligomer product with Proinsulin I, by an enzyme mixture:

A standard amount of Tris-HCl solution from the resulting mixture of Example 16-a was analyzed by HPLC to determine the polypeptide concentration. A solution of treated trypsin (TPCK) was prepared; from bovine pancreas) in 0.1 mmol of Tris-HCl buffer, pH 7.6. A solution of carboxypeptidase B (from pig pancreas) was prepared in 1.0 mmol of Tris-HCl buffer, pH 7.6. The resulting mixture of Example 6-1a (0.424 µmol/ml) was then left to react with trypsin (5.97 x 1-4 µmol/ml) and carboxypeptidase B (1.93 x 1-4 µmol/ml) . After 30 minutes, the reaction was quenched by adding 1.58 ml of 1% trifluoroacetic acid in acetonitrile. The major outcomes were identified

By HPLC retention time (relative to the retention times of standard reference methods) and mass spectrometry analysis. It is expected to obtain the product of the combination of LysB29 - PEG7-hexyl oligomer - insulin; The only insulin conjugate in existence, with a yield of approximately 95%. Lys1-hexyl PEG7-hexyl-oligomer peptide-C is also obtained as an adduct.

The invention has been described above with reference to preferred embodiments. These embodiments are not intended to identify the invention, but are for the purpose of illustration. The scope of the invention is determined by the following elements of protection.