Method of peptide acidifying

FIELD: chemistry, pharmaceutics.

SUBSTANCE: claimed invention relates to method of acidifying of one or several amino groups of peptide which is selected from group including exendin-3, exendin-4, Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide, Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), des(B30)-human insulin and their analogues, in which reaction of acidifying is carried out in water mixture containing less than 10% wt/wt aprotonic polar solvent, and interaction of peptide with acidifying agent of general formula I is carried out, where n is 0-8; R1 represents COOR4; R2 represents lipophilic part of molecule; R3 together with carboxyl group, to which R3 is bound, represents reaction-able ester or reaction-able N-hydroxyimidoesther; and R4 is selected from group including hydrogen, C1-12-alkyl and benzyl; in base conditions in water solution, acidifying agent being added to reaction mixture in form of solution stabilised by adding acid.

EFFECT: obtaining efficient method of peptide acidifying.

17 cl, 2 tbl, 8 ex

 

The technical field to which the invention relates

The present invention relates to a method of acylation of peptides and proteins. In particular, this invention relates to a method of introducing one or more acyl groups in the peptide or protein.

The level of technology

Many peptides recognized as suitable for use in medical practice, and these peptides can be produced in an acceptable cell host by methods of recombinant DNA or synthesized by well known methods of peptide synthesis. However, the native peptides and their analogs are characterized by a high rate of clearance, unacceptable for many clinical indications for which it is necessary to preserve a high concentration of peptide in the plasma for a long period of time. Examples of peptides that are in native form have a high ground clearance, include ACTH, a factor in the release of corticotropin, angiotensin, calcitonin, insulin, glucagon, like peptide-1 (GLP-1), and the like peptide-2 (GLP-2), insulin-like growth factor 1, insulin-like growth factor 2, a peptide that suppresses the secretion of gastric juice, the factor in the release of growth hormone, a peptide that activates pituitary adenylate cyclase, secretin, enterogastrone, somatostatin, somatotropin, somatomedin, parathyroid hormone thrombopoietin, erythropoietin, growth hormone-releasing factors of the hypothalamus, the hormone that stimulates the thyroid hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and their analogues, peroxide dismutase, interferon, asparaginase, arginase, argininosuccinate, adenoidectomies and ribonuclease.

It is established that various derivatives of the peptides and their analogues have a positive effect on the rate of clearance of the peptides. One way to obtain such a derivative is the introduction of lipophilic acyl groups in a therapeutic peptide that induces required more prolonged profile of action compared to palleroni peptide. Thanks to the more rare the introduction of a therapeutic protein improves compliance sick mode and scheme prescribed treatment and decreases the amount of injected peptide. This method is described in the application WO 98/08871, which covers the acylation of GLP-1 and its analogues in the application WO 98/08872, which covers the acylation of GLP-2 and its analogs, and in the application WO 99/43708, which covers the acylation of essendine and its analogues. It is established that between the peptide and the acyl group is preferably to use mono - and dipeptide spacer, such as aspartic acid and glutamic acid. The spacers comprising a free carboxylic acid group, it is necessary to protect up to acylation and then delete C the rights group.

In European patent No. 1227107 described acylation ε-amino group of human insulin.

In the application WO 00/55119 described method of acylation of peptides (e.g., GLP-1) and new alleluya agents.

To therapeutic peptides were profitable, the importance of production costs upon receipt of the peptides, as well as therapeutic dosage of the peptide. The largest cost in the production of therapeutic peptides belong to the stages of purification, necessary to separate the target protein from contaminants, cognate target protein, such as isomers, dazamide forms, etc. Cleaning is usually carried out by means of chromatography using expensive chromatographic matrices and solvents, which reduces the overall yield of product.

The aim of the present invention is to provide an efficient and economical way of introduction of lipophilic groups in peptides using spacers, representing α-aminoα,ω-dicarboxylic acid. This method is more specific, allows to obtain higher yields of the product and reduce the formation of closely related impurities. When this is achieved a significant cost reduction in the production of acylated peptides. Cheaper acylated peptides are highly desirable for Maxi is the real increase in the number of patients, which will be available for the treatment of the above drugs, and to realize the benefits of alternative delivery methods, characterized by a lower bioavailability compared with subcutaneous injections, such as cutaneous and pulmonary delivery.

The invention

The present invention relates to a method for acylation of one or more amino groups of the peptide or protein, which involves the following stages:

a) implementation of the interaction of a peptide containing at least one free amino group, with allermuir agent of General formula I

where

n is 0-8;

R1means COOR4;

R2means lipophilic portion of the molecule;

R3together with the carboxyl group is attached to R3means of a reactive ester or reactive N-hydroxyamide; and

R4selected from the group including hydrogen, C1-12-alkyl and benzyl;

in basic conditions in aqueous mixtures;

(b) saponification of the ester group acylated peptide (COOR4in basic terms, if R4is not hydrogen;

(C) the excretion of N-acylated peptide;

characterized in that the aqueous mixture used in stage (a)contains less than 10% wt./wt. aprotic polar solvent.

In one the m variant implementation of the method according to the present invention the interaction on stage and perform in an aqueous mixture, containing less than 8% wt./wt. aprotic polar solvent, preferably less than 5% wt./wt. aprotic polar solvent, and more preferably less than 3% wt./wt. aprotic polar solvent.

In another embodiment of the method according to the present invention allerease agent is added to the reaction medium in the form of solids.

In another embodiment of the method according to the present invention allerease agent is added to the reaction mixture in the form of a solution in an aprotic polar solvent, which stabilizat the addition of acid.

Detailed description of the invention

Peptides and proteins

The present invention relates to the introduction of lipophilic acyl groups in any of the peptide (or protein) to reduce the speed of clearance in vivo. Examples of such peptides and proteins include ACTH, a factor in the release of corticotropin, angiotensin, calcitonin, Asendin and its analogues, insulin and its analogues, glucagon and its analogues, like peptide-1 and its analogs, and the like peptide-2 and its analogs, insulin-like growth factor 1, insulin-like growth factor 2, a peptide that suppresses the secretion of gastric juice, the factor in the release of growth hormone, a peptide that activates pituitary adenylate cyclase, secretin, enterogastrone, somatostatin, somatotropin, Somatom the Dean, parathyroid hormone, thrombopoietin, erythropoietin, growth hormone-releasing factors of the hypothalamus, the hormone that stimulates the thyroid hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and their analogues, peroxide dismutase, interferon, asparaginase, arginase, argininosuccinate, adenoidectomies and ribonuclease.

It is obvious that the peptide (or protein) must have at least one free amino group, which is the N-terminal amino group or the amino group of the side chain. The peptide or protein may contain amino acids that are not encoded by the genetic code such as D-amino acid, 3-hydroxyproline, ornithine and pencillin. Especially interesting are the amino groups of amino acid residues lysine and ornithine. This method, in particular, relates to N-acylation ε-amino groups of lysine residues. In addition, it should be clear that the peptide or protein may contain two or more lateral amino groups, all of which can be N-etilirovany the method according to the present invention.

The present invention particularly relates to the acylation of GLP-1 and its analogues. Examples of GLP-1 and its analogues, which may be N-etilirovany the method according to the present invention include GLP-1 and its analogs with shortened sequence, such as Arg26-GLP-1(7-37); Arg34-GLP-1(7-37);

and their derivatives. All of these analogues of GLP-1 and analogues with shorter sequence relate to possible alternative implementation of the present invention.

The present invention relates to the acylation of GLP-2 and its analogs. Examples of GLP-2 and its analogs, which may be N-etilirovany the method according to the present invention include analogs of GLP-2 and analogs with shortened sequence, such as Lys20GLP-2(1-33); Lys20Arg30GLP-2(1-33); Arg30Lys34GLP-2(1-34); Arg30Lys35GLP-2(1-35); Arg30,35Lys20GLP-2(1-35) and Arg35GLP-2(1-35). All of these analogs of GLP-2 and analogs with shortened sequence relate to possible alternative implementation of the present invention.

The present invention relates to the acylation of essendine-3, essendine-4 and their analogues. Examples of analogues of essendine, which may be N-etilirovany the method according to the present invention, are described, for example, in the application WO 99/43708. All of the above analogues of essendine and analogues with shorter sequence relate to possible alternative implementation of the present invention.

The present invention relates to the acylation of insulin and its analogues. Examples of insulin and its analogues, which may be N-etilirovany the method according to present the invention, are human insulin and des(B30)-human insulin.

In another embodiment, the present invention N-acylation subject ε-amino group of lysine residues.

Allerease agent

In the method according to the present invention, the peptide (or protein)having at least one free amino group, is subjected to the interaction with allermuir agent of General formula I

The integer n in formula I is preferably 0-8, in particular 0-6, which corresponds, for example, aspartic acid, glutamic acid, etc. the Number n is preferably equal to 0-4, preferably 0-2, for example 0 (aspartic acid) or 1 (glutamic acid). All these integers and ranges of these numbers belong to the alternatives of implementation of the present invention.

R1in the formula I is a free acid group (COOH) or ester group (COOR4). In those cases where R1is an ester group, R4choose from the groups that can be removed (in the form of corresponding alcohols) by hydrolysis in basic conditions. Examples of such groups include1-12-alkyl, for example methyl, ethyl, prop-1-yl, prop-2-yl, buta-1-yl, but-2-yl, 2-methylprop-1-yl, 2-methylprop-2-yl (tert-butyl), Gex-1-yl, benzyl, etc. All these groups belong to the viola is rnational variants of implementation of the present invention.

R2in the formula I is lipophilic portion of the molecule introduced into a peptide or protein. Such lipophilic part is usually chosen from a group including3-39-alkyl, C3-39alkenyl,3-39-alkadienes and steroid residues. Typical examples3-39-alkyl are heptyl, nonyl, undecenyl, tridecanol, pentadecanol, heptadecanol and nonadecane. All of these lipophilic part of the molecule are related to possible alternative implementation of the present invention.

Lipophilic Deputy or part of a molecule is characterized by a solubility in water at 20°in the range from about 0.1 mg/100 ml water to about 250 mg/100 ml of water, preferably in the range from about 0.3 mg/100 ml water to about 75 mg/100 ml of water. For example, octanoic acid (C8) is characterized by solubility in water at 20°equal to 68 mg/100 ml, cekanova acid (C10) is characterized by solubility in water at 20°equal to 15 mg/100 ml, and octadecanoic acid (C18) is characterized by solubility in water at 20°equal to 0.3 mg/100 ml All limits of solubility of these lipophilic substituents are alternatives to the implementation of the present invention.

The terms "C3-39-alkyl", "C3-39alkenyl" and "C3-39-alkadienes" means saturated, monounsaturated and Dimensione hydrocarbon radicals with a straight and osvetleni chain preferably a straight chain containing 3-39 carbon atoms. Typical examples3-39-alkyl are heptyl, nonyl, undecenyl, tridecanol, pentadecanol, heptadecanol and nonadecane.

Used here is the term "steroid residue" means a lipophilic group, which together with the carbonyl group to which is attached R2represents a carboxylic acid derivative of the steroid, i.e. tri-, Tetra - and Pyh, fully saturated or partially unsaturated With16 to 36the hydrocarbon. Examples of such groups R2-C(=O)- are lithology, desoxycholic and Halol.

Among the above-mentioned lipophilic groups7-25-alkyl, C7-25alkenyl,7-25-alkadienes and steroid residues are particularly suitable for use in the present invention. Particularly interesting examples are the heptyl, nonyl, undecenyl, tridecanol, pentadecanol, heptadecanol, nonadecane, lithology, desoxycholic and Halol. All of these lipophilic group are alternatives to the implementation of the present invention.

R3in the formula I together with a carboxyl group is attached to R3means of a reactive ester or reactive N-hydroxyamides. All of these esters include alternatives to the wasp is estline of the present invention. Reactive esters and reactive N-hydroxymaleimide well known in the field of organic chemistry (especially in the chemistry of peptides as functional groups used in the acylation of amino-, thio -, and hydroxyl groups. In the context of the present invention, the term "reactive ester or reactive N-hydroxyamides" means functionally active ester form of the group of carboxylic acids that are suitable for the acylation of an amine, preferably a primary amine. Thus, it should be clear that the selective acylation of primary amines is preferred over the acylation of hydroxyl and tigroup. Especially preferred are reactive N-hydroxymaleimide.

Examples of reactive esters are esters of 1-hydroxybenzotriazole and their derivatives. There are a number of highly effective reagents, such as tetrafluoroborate 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium used to obtain the activated esters of carboxylic acids. Such reactive esters usually formed in situ in the presence of a base, for example, organic bases such as trialkylamine.

Examples kidney part of the reactive N-hydroxyaminoimino given in EV is Orascom patent No. 0511600 A2, on page 3-7. Particularly interesting examples imenik parts are succinimide, phthalimide etc. All these kidnie parts relate to possible alternative implementation of the present invention.

Reactive N-hydroxymaleimide formula I can be obtained in accordance with the description given in the applications WO 00/55119 and WO 98/02460.

When using Alliluyeva reagent of the formula I in the form of the free α-carboxylic acid (R4= hydrogen) compound of formula I, where R4means a group that can be selectively removed, converted into the corresponding compound where R4means hydrogen. A protective group of carboxylic acid may be a benzyl group which can be removed by catalytic hydrogenation, or allyl group, which can be selectively removed. Benzyl protective group can be removed by catalytic hydrogenation in an aprotic polar solvent, such as acetone, at room temperature using palladium-on-coal and hydrogen. This reaction can be performed in a closed vessel in an atmosphere of hydrogen (typically under a pressure of 0.1-10 ATM) under vigorous stirring. This reaction is usually continued for 0.5-12 hours depending on the quality palladium catalyst. This produces the usual processing.

The condition is I reactions

The interaction between allermuir agent of formula I and the peptide or protein is carried out in basic conditions in an aqueous solution containing less than 10% wt./wt. aprotic polar solvent.

In one embodiment of the invention the reaction in stage a) is performed in an aqueous solution containing from 0% wt./wt. up to 10% wt./wt. aprotic polar solvent.

In another embodiment of the invention the reaction in stage a) is performed in an aqueous solution containing from 1% wt./wt. up to 10% wt./wt. aprotic polar solvent.

In another embodiment of the invention the reaction in stage a) is performed in an aqueous solution containing from 1% wt./wt. up to 8% wt./wt. aprotic polar solvent.

Allerease agent of formula I is usually used in a small excess in relation to the number alleluiah amino group of the peptide. The ratio of excess Alliluyeva agent is usually from 1:1 to 1:20, preferably from 1:1.2 to 1:5 depending on the number of amino groups in the peptide. Allerease agent can be added to the reaction mixture in the form of solid or solution. Adding Alliluyeva agent in the form of a solution the specified agent is dissolved in an aprotic polar solvent and preferably stabilizat by adding acid. To stabilize typically use mineral acid, e.g. the sulphuric acid.

It should be clear that the peptide can be fully N-allerban or only partially N-allerban depending on the number Alliluyeva agent and the reaction conditions. It is desirable that N-acylation was essentially stoichiometric.

Aprotic polar solvents are solvents with a moderately high dielectric constants, which do not contain acidic hydrogen (see, for example, Morrison and Boyd, Organic Chemistry, 5thed. p. 229). Aprotic polar solvent is typically selected from the group comprising anhydrous tetrahydrofuran (THF), anhydrous dimethylformamide (DMF), acetone, dichloromethane, dimethyl sulfoxide (DMSO), dioxane, dimethylacetamide, N-methyl-2-pyrrolidone and mixtures thereof, of which preferred are dimethylformamide, dimethylsulfoxide, dimethylacetamide and N-methyl-2-pyrrolidone, and especially preferred is N-methyl-2-pyrrolidone.

The temperature usually ranges from -10°C to 50°C.

It is important that the pH value of the mixture of solvents was within 7-14, for example 9-13, preferably within 10-12, for a calmer reaction. The yield and purity of product are usually best when the pH value of the mixture of solvents within 10-12. The desired pH value receive, adding hydroxides of alkali metals such as sodium hydroxide and guide the oxide of potassium, and/or organic bases, such as trialkylamine (for example, triethylamine, N,N-diisopropylethylamine etc). In addition, it may be desirable to add a buffer that allows you to keep the pH is almost the original value before the start of the reaction. Examples of buffers that can be used for this purpose include phosphate buffer, borate buffer, and the like.

As a typical example can be noted that the reaction in stage (a) is carried out using protein and Alliluyeva agent of formula I in a molar ratio of from 1:1 to 1:5. The peptide is typically dissolved in water at a temperature of from -10°With 30°With, for example, when 0-25°and the pH value was adjusted to the desired level by adding an alkali metal hydroxide (e.g. sodium hydroxide or potassium hydroxide). The pH may be further adjusted using acids, for example acetic acid, and bases, such as trialkylamine, but the temperature is preferably in the above range. Alternative peptide is dissolved directly in an aqueous solution of an appropriate number of the above acids or bases. Then add allerease agent in the form of solids or in solution in an aprotic polar solvent. The reaction is usually performed to complete (the course of the reaction can control the encoded HPLC), usually achieved through 0.2 to 4 hours, in particular through the 0.2-1 hour, then add water and acid, for example acetic acid, to a pH of 6.5 to 9.0. The product is typically isolated and purified using HPLC, precipitated at the isoelectric pH or hydrolyzing (stage (b)) before cleaning.

When using Alliluyeva agent of the formula I, in which R4means hydrogen, immediately get N-acylated peptide or protein containing a lipophilic part and a free carboxyl group. Thus, a variation in which R4means hydrogen is the preferred embodiment of the method according to the present invention.

In the alternative case, i.e. when R4means1-12-alkyl or benzyl, an ester of N-acylated peptide (or an ester of protein) is subjected to saponification in basic conditions, resulting in a gain of N-acylated peptide or N-acylated protein. Saponification is usually performed in 0.01-4.0 M solution of alkali metal hydroxide, for example sodium hydroxide or potassium. The pH of the solution is usually equal to 10-14. The reaction is usually performed for 0.1 to 12 hours, preferably for 0.5-4 hours, at 0-40°With, for example at room temperature. After the reaction product is purified, for example by precipitation at the isoelectric pH value and/or using the product the main HPLC. Thus, a variation in which R4means1-12-alkyl or benzyl, is another preferred embodiment of the method according to the present invention.

In one embodiment of the method according to the present invention allerease agent is added to the reaction mixture in the form of solids.

In another embodiment of the method according to the present invention the reaction in stage a) is performed in water mixtures containing from 0% wt./wt. up to 8% wt./wt. aprotic polar solvent, preferably from 0 wt.%/wt. up to 5% wt./wt. aprotic polar solvent, and more preferably from 0 wt.%/wt. up to 3% wt./wt. aprotic polar solvent.

In another embodiment of the method according to the present invention the reaction in stage a) is performed in the presence of aprotic polar solvent chosen from the group comprising N-methyl-2-pyrrolidone, tetrahydrofuran, and dimethyl sulfoxide.

In another embodiment of the method according to the present invention, the entire aprotic organic solvent is added to the reaction mixture as a solvent for Alliluyeva agent.

In another embodiment of the method according to the present invention allerease agent is added to the reaction mixture in the form of a solution, stabilized by adding acid

In another embodiment of the method according to the present invention the acid is added to the aprotic polar solvent at a concentration of from 0.01% wt./wt. up to 1% wt./wt., preferably in a concentration of from 0.05% wt./wt. up to 0.5% wt./wt.

In another embodiment of the method according to the present invention, the above acid is chosen from the group comprising sulfuric acid, methanesulfonate acid and triperoxonane acid.

In another embodiment of the method according to the present invention the reaction at the stage and perform without aprotic polar solvent.

In another embodiment of the method according to the present invention R4in the formula I is hydrogen.

In another embodiment of the method according to the present invention R4choose from C1-8-alkyl and benzyl.

In another embodiment of the method according to the present invention R3together with the carboxyl group is attached to R3means of a reactive N-hydroxymethyl.

In another embodiment of the method according to the present invention is an ester of acylated peptide is subjected to saponification when the pH value is within 10 to 14, preferably at a pH in the range 9-13.

In another embodiment of the method according to the present invention is an ester of acylated peptide to podvergautsya when the pH value in the range 9-14, in particular at pH 10-13.

In another embodiment of the method according to the present invention the pH value of the reaction mixture in stage (a) is from pH 9 to pH 13, preferably from pH 10 to pH 12 or more preferably from pH to a pH of 11.0 to 11.5.

In another embodiment of the method according to the present invention, the reaction mixture used in stage (a), contains a buffer suitable for maintaining essentially constant pH during the reaction. In one embodiment of the method according to the present invention the specified buffer is a phosphate buffer, borate buffer or mixtures thereof.

In another embodiment of the method according to the present invention, the temperature of the reaction mixture in stage (a) is in the range of 0-50°C, preferably in the range of 5-40°s and more preferably in the range of 10-30°C.

In another embodiment of the method according to the present invention R2selected from the group including3-39-alkyl, C3-39alkenyl,3-39-alkadienes and steroid residue.

In another embodiment of the method according to the present invention R2-C(=O)- chosen from the group including lithology and hexadecanol.

In another embodiment of the method according to the present invention, the peptide used as the starting material in stage (a), meetcustomer, equal to at least 80%, at least 90%, at least 93%, at least 95% or at least 97%, at determination by HPLC with reversed phase.

In another embodiment of the method according to the present invention the specified peptide is chosen from the group comprising GLP-1, Asendin-4, GLP-2, glucagon, insulin, analogs and derivatives of any of the foregoing substances.

In another embodiment of the method according to the present invention the specified peptide is an agonist of GLP-1.

In another embodiment of the method according to the present invention the specified peptide is chosen from the group comprising Asendin-3, Asendin-4, Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide, Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His22-GLP-1(7-37), (B30)-human insulin and derivatives thereof.

In another embodiment of the method according to the present invention the specified peptide selected from the group Val8Trp19Glu22-GLP-1(7-37), Val8Glu22Val25-GLP-1(7-37), Val8Tyr16Glu22-GLP-1(7-37), Val8Trp16Glu22-GLP-1(7-37), Val8 16Glu22-GLP-1(7-37), Val8Tyr18Glu22-GLP-1(7-37), Val8Glu22His37-GLP-1(7-37), Val8Glu22Ile33-GLP-1(7-37), Val8Trp16Glu22Val25Ile33-GLP-1(7-37), Val8Trp16Glu22Ile33-GLP-1(7-37), Val8Glu22Val25Ile33-GLP-1(7-37), Val8Trp16Glu22Val25-GLP-1(7-37) and their analogues.

In another embodiment of the method according to the present invention the specified peptide selected from HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH2 (ZP-10) and its analogs.

In another embodiment of the method according to the present invention the specified peptide is insulin peptide, i.e. it is not insulin or its analogue. In another embodiment of the method according to the present invention the specified peptide consists of only one polypeptide chain. In another embodiment of the method according to the present invention the specified peptide contains two polypeptide chains that are covalently linked by at least one disulfide bond.

Another object of the present invention is the application of the described method of acylation to obtain a derivative of a peptide selected from the group comprising Arg34, Lys26(Nε-(γ-Glu(Nα-hexadecanoyl)))-GLP-1(7-37) and LysW(Nεthe deletion) (B30)-human insulin and LysW(NB5; [Nα-lithocholyl-Glu-OH])des(B30)-human insulin.

EXAMPLES

Alleluya reagents obtained in accordance with the description given in the application WO 00/55119.

In the examples, the final purification of the product was made using chromatography on columns.

Example 1

Arg34GLP-1(7-37)expressed in yeast (S. cerevisiae) with conventional methods of recombinant DNA, for example, as described in the application WO 98/08871. Arg34GLP-1(7-37)cultivated in a fermentation broth was purified by chromatography with reversed phase and then besieged at the isoelectric pH of the peptide, i.e. at pH of 5.4. The precipitate was separated by centrifugation and frozen.

Arg34GLP-17-37(1.47 g frozen isoeugenol peptide, approximately 0.10 mmol) was dissolved in 0.1 mol/kg of triethylamine (23 ml) at 10-15°C. the pH Value of the solution was equal to 11.6. Added γ-N-hydroxysuccinimide N-hexadecanesulfonate acid (63.7 mg, 0.13 mmol). The reaction mixture was stirred for 20 minutes at room temperature and added water (42 ml), the pH was brought to 8.0 by adding 1.0 M solution of acetic acid.

Output: the results of the analysis by HPLC with reversed phase showed that the reaction mixture contained 84% (by area) Arg34Lys26-[N-ε(γ-Glu(N-hexadecanoyl))]-GLP-17-37and 0.5% (by area) Arg34Lys -[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37.

Example 2

Arg34GLP-1(7-37)expressed in yeast (S. cerevisiae) with conventional methods of recombinant DNA, for example, as described in the application WO 98/08871. Arg34GLP-1(7-37)cultivated in a fermentation broth was purified by chromatography with reversed phase and then besieged at the isoelectric pH of the peptide, i.e. at pH of 5.4. The precipitate was separated by centrifugation and frozen.

Arg34GLP-17-37(1,57 g frozen isoeugenol peptide, approximately 0.14 mmol) was dissolved in 0.1 mol/kg of triethylamine (23 ml) at 10-15°C. the pH Value of the solution was brought to 11.5 by adding triethylamine. Then was added 1.7 ml γ-N hydroxysuccinimide N-hexadecanesulfonate acid (to 92.1 mg, 0,19 mmol), dissolved in N-methyl-2-pyrrolidone containing 0,105% wt./wt. 1 M solution of N2SO4. The reaction mixture was stirred for 20 minutes at room temperature and added water (42 ml), the pH was brought to 8.0 by adding 1.0 M solution of acetic acid.

Output: the results of the analysis by HPLC with reversed phase showed that the reaction mixture contained 83% (by area) Arg34Lys26-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-37and 0.4% (by area) Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37.

Example 3

Arg34GP-1 (7-37)expressed in yeast (S. cerevisiae) with conventional methods of recombinant DNA, for example, as described in the application WO 98/08871. Arg34GLP-1(7-37)cultivated in a fermentation broth was purified by chromatography with reversed phase and then besieged at the isoelectric pH of the peptide, i.e. at pH of 5.4. The precipitate was separated by centrifugation and frozen.

Arg34GLP-17-37(1,53 g frozen isoeugenol peptide, approximately 0.13 mmol) was dissolved in 0.05 mol/kg of triethylamine (25 ml) at room temperature. The pH value of the solution was equal to 10.9. Then added 1.8 ml γ-N hydroxysuccinimide N-hexadecanesulfonate acid cases (94.2 mg, 0,19 mmol), dissolved in N-methyl-2-pyrrolidone without the addition of N2SO4. The reaction mixture was stirred for 30 minutes at room temperature and added to water (48 ml), the pH was brought to 8.0 by adding 1.0 M solution of acetic acid.

Output: the results of the analysis by HPLC with reversed phase showed that the reaction mixture contained 75% (by area) Arg34Lys26-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-37and 4.0% (by area) Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37.

Example 4. A comparative example using an aprotic polar solvent at a concentration of >10% wt./wt.

Arg34GLP-1(7-37) expressed in yeast (S. cerevisiae) with conventional methods of recombinant DNA, for example, as described in the application WO 98/08871. Arg34GLP-1(7-37)cultivated in a fermentation broth was purified by chromatography with reversed phase and then besieged at the isoelectric pH of the peptide, i.e. at pH of 5.4. The precipitate was separated by centrifugation and frozen.

Arg34GLP-17-37(1,57 g frozen isoeugenol peptide, approximately 0.14 mmol) was dissolved in 0.1 mol/kg of triethylamine (23 ml) at 10-15°C. was Added N-methyl-2-pyrrolidone (6.8 ml) and the pH value of the solution was brought to 11.5 by adding triethylamine. Then added γ-N-hydroxysuccinimide N-hexadecanesulfonate acid (to 92.1 mg, 0,19 mmol), dissolved in 1.7 ml of N-methyl-2-pyrrolidone containing 0,105% wt./wt. 1 M solution of N2SO4. The reaction mixture was stirred for 20 minutes at room temperature and added water (54 ml), the pH was brought to 8.0 by adding 1.0 M solution of acetic acid.

Output: the results of the analysis by HPLC with reversed phase showed that the reaction mixture contained 87% (by area) Arg34Lys26-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-37and 0.5% (by area) Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37.

Example 5. Comparative example using aprotic polar solution is a dye in a concentration of > 10% wt./wt.

Arg34GLP-1(7-37)expressed in yeast (S. cerevisiae) with conventional methods of recombinant DNA, for example, as described in the application WO 98/08871. Arg34GLP-1(7-37)cultivated in a fermentation broth was purified by chromatography with reversed phase and then besieged at the isoelectric pH of the peptide, i.e. at pH of 5.4. The precipitate was separated by centrifugation and frozen.

Arg34GLP-17-37(1.51 g frozen isoeugenol peptide, approximately 0.13 mmol) was dissolved in 0.1 mol/kg of triethylamine (20 ml) and N-methyl-2-pyrrolidone (100 ml) at 10-15°C. Then added γ-N-hydroxysuccinimide N-hexadecanesulfonate acid (74 mg, 0.15 mmol), dissolved in 1.4 ml of N-methyl-2-pyrrolidone containing 0,105% wt./wt. 1 M solution of N2SO4. The reaction mixture was stirred for 45 minutes at room temperature and added water (206 ml)while the pH was brought to 8 by adding 1.0 M solution of acetic acid.

Output: the results of the analysis by HPLC with reversed phase showed that the reaction mixture contained 57% (by area) Arg34Lys26-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-37and 14% (by area) Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37.

Example 6

Table 1 summarizes the experimental conditions used for the acylation Arg34-GP-1 7-37in the experiments of examples 1-5, and the content of impurities Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37(abbreviation α-glu). The table shows that without the addition of H2SO4as the stabilizer, the amount of impurities α-glu can be further reduced by using lower concentrations of the aprotic polar solvent. Adding stabilizer formation of significant quantities of impurities α-glu begins at much higher concentrations aprotic polar solvent is at a concentration of greater than 28% wt./wt.

Table 1

The content of the aprotic polar solvent in the reaction mixture when the acylation of peptides GLP-1 and the fraction of contaminants Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37(α-glu). (Data from examples 1-5)
# exampleAn aqueous solution of the peptide (ml)The added amount of NMP (ml)The NMP content (% wt./wt.)Add the amount H2SO4α-glu
12300%-0,5%
2231,77%+0,4%
3251,87%-4%
4236,8+1,728%+0,5%
520100+1,484%+14%

Example 7

Crystallized sodium(B30)-human insulin (1,74 g frozen peptide selected isoelectric precipitation, approximately 0,088 mmol) was dissolved in 0.1 mol/kg of triethylamine (10,85 ml) at room temperature. The pH value of the solution was equal to 10.9. Then was added methyl-(2S)-5-[(2,5-dioxo-1-pyrrolidinyl)oxy]-2-{[(3a,5b)-3-hydroxy-9-methyl-24-Oklahoman-24-yl]amino}-5-oxopentanoate (55,1 mg, 0,089 mmol), dissolved in N-methyl-2-pyrrolidone (1.25 ml). The reaction mixture was stirred for 30 minutes at room temperature and was added water (5°C, 85 ml).

Output: the results of analysis by HPLC with reversed-phase reaction mixture contained 34% (by area) N-ε-(lithocholyl-γ-glutamyl)-LysW-des(B30)-human insulin.

Example 8

Arg34GLP-1(7-37)expressed in yeast (S. cerevisiae) with conventional methods of recombinant DNA, for example, as described in the application WO 98/08871. Arg34GP-1 (7-37)cultivated in a fermentation broth was purified by chromatography with reversed phase and then besieged at the isoelectric pH of the peptide, i.e. at pH of 5.4. The precipitate was separated by centrifugation and frozen.

The precipitate obtained by isoelectric precipitation and containing Arg34GLP-1(7-37)(5,70 g frozen precipitated peptide, approximately 0.38 mmol), was dissolved in 0.1 M solution of the dihydrate of dinitrigenoxide (170 ml, the pH was brought to 11.35, adding 1 M NaOH solution) at 15°C. the pH Value of the solution is again brought up to 11.4 by adding 1 M NaOH solution. Then within 8 minutes was added dropwise 3 ml γ-N hydroxysuccinimide N-hexadecanesulfonate acid (248,1 mg, 0.51 mmol), dissolved in N-organic containing 0,105% wt./wt. 1 M solution of H2SO4.

The samples were taken for 21.5 hours performing the reaction at 15°and added a 0.1 M solution of the dihydrate of dinitrigenoxide containing 1 mg/ml glycine, pH 7.5. The final reaction mixture was diluted with water (300 ml) and the pH was brought to 8.0 by adding glacial acetic acid.

Output: the results of analysis methods HPLC with reversed-phase reaction mixture contained 78-80% (by area) Arg34Lys26-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-370.24-1,67% (by area) Arg34Lys26-[N-ε-(α-Glu(N-hexade anoil))]-GLP-1 7-37.

Table 2

The content of Arg34Lys26-[N-ε-(γ-Glu(N-hexadecanoyl))]-GLP-17-37(γ-Glu and Arg34Lys26-[N-ε-(α-Glu(N-hexadecanoyl))]-GLP-17-37(α-Glu) depending on the reaction time. (Data from example 8)
Time (hours)γ-Glu (% by area)α-Glu (% by area)
0,1778,010,24
179,350,31
279,450,36
480,490,45
a 21.580,271,67

1. The method of obtaining the N-acylated peptide, which involves the following stages:

a) implementation of the interaction of the peptide that is chosen from the group comprising Asendin-3, Asendin-4, Arg34-GLP-1(7-37), Gly8-GLP-1(7-36)-amide, Gly8-GLP-1(7-37), Val8-GLP-1(7-36)-amide, Val8-GLP-1(7-37), Val8Asp22-GLP-1(7-36)-amide, Val8Asp22-GLP-1(7-37), Val8Glu22-GLP-1(7-36)-amide, Val8Glu22-GLP-1(7-37), Val8Lys22-GLP-1(7-36)-amide, Val8Lys22-GLP-1(7-37), Val8Arg22-GLP-1(7-36)-amide, Val8Arg22-GLP-1(7-37), Val8His22-GLP-1(7-36)-amide, Val8His2 -GLP-1(7-37), (B30)-human insulin and analogues, with allermuir agent of General formula I

where n is 0-8;

R1means COOR4;

R2means lipophilic portion of the molecule;

R3together with the carboxyl group is attached to R3means of a reactive ester or reactive N-hydroxyamide; and

R4selected from the group including hydrogen, C1-12alkyl and benzyl;

in basic conditions in aqueous mixtures;

(b) saponification of the ester group acylated peptide (COOR4in basic terms, if R is not hydrogen;

c) isolation of the N-acylated peptide;

characterized in that the aqueous mixture used in stage (a)contains less than 10% wt./wt. aprotic polar solvent, in which allerease agent is added to the reaction mixture in the form of a solution, stabilized by addition of an acid.

2. The method according to claim 1, wherein the reaction in stage a) is performed in aqueous mixtures containing less than 8% wt./wt. aprotic polar solvent, preferably less than 5% wt./wt. aprotic polar solvent, and more preferably less than 3% wt./wt. aprotic polar solvent.

3. The method according to claim 1, in which the reaction with the adiya's and perform in the presence of an aprotic polar solvent, moreover, the specified aprotic polar solvent is chosen from the group comprising N-methyl-2-pyrrolidone, tetrahydrofuran, and dimethyl sulfoxide.

4. The method according to claim 1, in which the aprotic organic solvent is added to the reaction mixture as a solvent for Alliluyeva agent.

5. The method according to claim 1, in which the specified acid is added to the aprotic polar solvent at a concentration of from 0.01 to 1% wt./wt., preferably in a concentration of from 0.05 to 0.5% wt./Mas..

6. The method according to claim 1, in which the specified acid selected from the group comprising sulfuric acid, methanesulfonate acid and triperoxonane acid.

7. The method according to claim 1, wherein the reaction in stage a) is performed without aprotic polar solvent.

8. The method according to claim 1, in which R4means hydrogen.

9. The method according to claim 1, in which R4selected from the group including C1-8-alkyl and benzyl.

10. The method according to claim 1, in which R3together with the carboxyl group is attached to R3means of a reactive N-hydroxyamides.

11. The method according to claim 1, in which the ester acylated peptide is subjected to saponification when the pH value is within 10 to 14, preferably at a pH in the range 9-13.

12. The method according to claim 1, wherein the pH of the reaction mixture in stage (a) is in the range from pH 9 to pH 13, preferably within the t pH 10 to pH 12 or more preferably in the range from pH to a pH of 11.0 to 11.5.

13. The method according to claim 1, in which the temperature of the reaction mixture in stage (a) is in the range of 0-50°C, preferably in the range of 5-40°s and more preferably in the range of 10-30°C.

14. The method according to claim 1, in which R2selected from the group including3-39-alkyl, C3-39alkenyl,3-39-alkadienes and steroid residue.

15. The method according to claim 1, in which R2-C(=O)- chosen from the group including lithology and hexadecanol.

16. The method according to claim 1, wherein the peptide used as the starting material in stage (a)has a purity equal to at least 80%, at least 90%, at least 93%, at least 95% or at least 97%, at determination by HPLC with reversed phase.

17. The method according to claim 1, wherein the reaction mixture used in stage (a), contains a buffer suitable to maintain essentially constant pH throughout the reaction.



 

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SUBSTANCE: invention relates to new polypeptides namely precursors of insulin or analogs thereof, whish are characterized by increased structure stability and high expression levels during production thereof in recombinant forms in yeast cells. Polypeptides of present invention unlike natural form of insulin precursors contain shortened C-peptide including aromatic amino acid group and have general formula B (1-27)-X2-X3-X1-Y-A (1-21), wherein X1 is peptide sequence of 1-3 amino acid rests, wherein aromatic acid rest is in close vicinity with Y; X2 represents Pro or Asp; X1 represents Lys, Y-Lys, or Arg; A (1-21) is A-chain of human insulin; B(1-27) is the first 27 amino acid rests of human insulin D-chain rests.

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(R2R3)-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-AND23-AND24-AND25-AND26-AND27-AND28-AND29-AND30-AND31-AND32-AND33-AND34-AND35-AND36-AND37-AND38-AND39-R1the values of the radicals indicated in the claims

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The invention relates to compounds of the formula (R2R3) AND7-AND8-AND9-AND10-AND11-AND12-AND13-AND14-AND15-AND16-AND17-AND18-AND19-AND20-AND21-AND22-AND23-AND24-AND25-AND26-AND27-AND28-AND29-AND30-AND31-AND32-AND33-AND34-AND35-AND36-AND37-R1,

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