Similar-amylin agonist, its pharmaceutically acceptable salts, the composition exhibiting the properties of an amylin agonist

 

(57) Abstract:

Similar is the Amylin agonist has the amino acid sequence of General formula I:

A1-X - Asn - Thr -5Ala - Thr - Ala - Thr - 10Gln - Arg - Leu-B1- Asn -15Phe - Leu - C1- D1- E1-20F1- G1- Asn - H1- Gly - 25J1- J1-Leu - K1- L1-30Thr - M1- Val - Gly - Ser -35Asn - Thr - Tyr - Z

where A1- M1represent the amino acid residues specified in paragraph I of the claims.

Composition based on compounds of formula I exhibits the property of Amylin agonist. 2 C. and 8 C.p. f-crystals, 3 ill., table 1.

The invention relates to the field of medicine, and in particular, to the treatment and prevention of hypoglycemic events, as well as other States in which it is desirable increase in the activity of Amylin, including conditions caused by a lack of insulin, such as diabetes. More specifically, the present invention relates to the production and use of analogues of agonist peptide hormone Amylin.

Diabetes mellitus is a disease characterized by a gross violation of metabolism with chronically elevated levels of glucose in tste activity of the peptide hormone, of insulin. Insulin is produced and secreted by cells in the pancreas, it is Known that insulin stimulates the uptake of glucose by tissues, protein synthesis and the formation and preservation of neutral lipids. Glucose, which is the main source of carbohydrate energy contained in the body as glycogen, i.e., polymerized form of glucose, which can be converted back into glucose in accordance with the needs of the metabolism. Under normal conditions, insulin is secreted both at the basal level and at higher levels after stimulation sugar load, and all of these levels secretiruetsa to maintain metabolic homeostasis by transforming glucose into glycogen.

The term "diabetes" encompasses several different hyperglycemic States. Such States are Type 1 diabetes (insulin-dependent diabetes mellitus or IDDM) and Type 2 diabetes (insulin-dependent diabetes mellitus or NIDDM). The presence of hyperglycemia in people with Type 1 diabetes due to lack of insulin, either low-level insufficient to maintain the levels of blood glucose in the desired physiological limits. Treatment of diabetes Type 1 is the introduction of the complementary doesa with normal or elevated insulin level; however, these people have noted the inability to maintain metabolic homeostasis caused by a state of insulin resistance in peripheral tissues and liver, as well as the development of the disease, this inability to maintain homeostasis caused by progressive destruction of pancreatic cells, which are responsible for insulin secretion. Thus, pre-treatment of patients with Type 2 diabetes can be based on the diet regime, supported by oral administration of hypoglycemic agents such as sulfonylureas. Diseases of this type often require insulin therapy, however, usually, it is prescribed only in the later stages of the disease in order to eliminate hyperglycemia and minimization of complications. Thus, in the end, many patients with type 2 diabetes need medication insulin.

Amyloid is called extracellular deposition of fibers folds of proteins. These amyloid accumulation of material were detected in the pancreas of patients with diabetes mellitus Type 2. Other studies have shown that the degree of amyloid deposits increases with the degree of hyperglycemia in man is pignolo hormone, Amylin. Clark A. and others , Lancet 11: 231 - 234 (1987). It was found that this peptide contains 37 amino acids, none of which is an acid residue, and has a disulfide bond between cysteine residues at positions 2 and 7, and aminirovanie C-end. Amylin is a major protein component of amyloid, which, as shown, is located in the islets of Langerhans of the pancreas in patients with diabetes mellitus Type 2.

It was found that the presence of intramolecular cysteine bridge and carboxy-terminal amide group in the peptide structure of synthetic molecules contributes the greatest biological activity in ignorirovanie glycogen synthesis in skeletal muscle. See, for example. Cooper, G. J. S. , and others, Proc. Natl. Acad. Sci, (USA) 84: 8628-8632 (1987); Cooper, G. J. S., and others, Diabetes 1988, ed. Larkins R., Zimmet, P. and Chisholm, D. (Elsevier, Amsterdam, p, 493-496 (1989)). Amino acid sequence of Amylin (see Fig. 1) has 46% homology with the sequence of peptide 2, associated with the gene calcitonin (CGPP-2).

In one study indicates that a limited segment of the Amylin molecule, namely, residues 20-29, as a possible participant in the formation of fibrils of amyloid in the islets of Langerhans in capacilities sequence of milinov, derived from mammalian species differ from each other, and further research was directed at identifying residues that are associated with the formation of amyloid. See, Westermark and others, Proc. Natl. Acad. Soc. (USA) 87: 5036-5040 (1990). In their work, Westermark and others report their attempts to synthesize various 20-29-amino acid segments milinovich sequences from different species, with subsequent comparison of their ability to form amyloid fibrils. It has been suggested that the most amyloidogenesis are the remains of 25-29 Amylin person, and that the substitution of serine for Proline at position 28, as is the case in some species of rodents, contributes to a significant inhibition of education fibrillin in the investigated Decapeptide.

Amylin is a complex of the peptide and the synthesis of bioactive drugs Amylin is a time-consuming operation. It was also found that Amylin has limited solubility and limited stability in solution. The authors of this application it was found that Amylin in rats has higher solubility and stability in solution than Amylin person. To some extent, this may be due, although not yet proven, according to which the ATA besides man, and cats are capable of aggregation with the formation of amyloid islets in vivo. In the present work was selected sequence of Amylin from different species, which is shown in Fig. 2.

In patients with Type 1 diabetes Amylin levels are significantly reduced or absent compared to normal control levels. In patients with diabetes mellitus Type 1 cells, which produce insulin and Amylin are destroyed by an autoimmune process. In line with this, it was suggested that Amylin may be used for the treatment of diabetes and hypoglycemia, including insulin-induced hypoglycemia. It was also suggested that the joint administration of insulin with Amelina is predominant in comparison with the commonly practiced by introducing one only insulin, and that co-administration with Amylin glucagon for the treatment of hypoglycemia is predominant in comparison with the commonly practiced by the introduction of only one of glucagon. Hence, for these and other purposes it is preferable to use a less complex compounds that have the activity of native human Amylin, as well as connections that would have enhanced solubility and/or ptx2">

The present invention relates to novel analogs of the peptide hormone Amylin. These compounds have activity Amylin and can be qualified as Amylin agonists or analogs, Amylin agonists.

The present invention relates to pharmaceutical compositions containing agonists analogs of the present invention, and to methods of treatment and prevention of hypoglycemic events and other disorders in which the desired increased activity of Amylin, including conditions caused by a lack of insulin, such as diabetes; moreover, these methods provide for the introduction of the animal analogue of Amylin agonist alone or in combination with insulin or glucagonoma).

Definition

If it is not specifically mentioned, it is used in the present description, the terms have the following meanings:

The term "alkyl" refers to straight or branched alkyl groups. The term "lower alkyl" refers to straight or branched groups having generally from 1 to 6 carbon atoms, and represents a primary, secondary, and tertiary alkyl groups. Typical examples of such groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t is 6-14 carbon atoms, such as phenyl and naphthyl, and heterocyclic aromatic groups containing 1-3 heteroatoms (nitrogen, oxygen, sulfur, etc.,), such as pyridyl, triazolopyridine, pyrimidine, etc.

The term "aralkyl" refers to an "aryl" group with 6-10 carbon atoms, directly linked to the "alkyl" group with 1-4 carbon atoms and represents, for example, benzyl, p-Chlorobenzyl, p-methylbenzyl, and 2-phenylethyl.

The term "cycloalkyl" refers to cyclic alkyl groups with 5 to 8 carbon atoms.

In Fig.1 depicts the amino acid sequence of human Amylin.

In Fig. 2 shows a comparison of amino acid sequences of amelino isolated from several mammals.

In Fig. 3 shows the amino acid sequence of new peptide Amylin agonists.

In accordance with the present invention were obtained new analogs, Amylin agonists. These analogs can be used as Amylin agonists, such as hyperglycemic funds, and their structure is shown in Fig. 3.

In one of its variants the present invention relates to analogs-agonists is shown in Fig. 3, where A1Val, Leu or lle; D1is His or Arg; E1is Ser or Thr; F1is Ser, Thr, Gln or Asn; G1is Asn, Gln or His; H1is Phe, Leu or Ter; I1is Ala or Pro; J1is lle, Val, Ala or Leu; K1is Leu, Pro, Leu, lle, or Thr; L1is Ser, Pro or Thr; M1is Asn, Asp or Gln; X and Y are independently selected residues having side chains which are chemically bonded to each other to form an intramolecular linkage; and Z is hydroxy, amino, alkylamino, dialkylamino, cyclooctylamine, arylamino, aralkylamines, alkyloxy, aryloxy or aralkylated; provided that (a) if A1is Lys, B1is Ala, C1is Val, D1is His, E1is Ser, F1is Ser, G1is Asn, H1is Phe, I1is Ala, J1is lle, K1is Ser, L1is Leu and M1is Asn; (b) if A1is Lys; B1is Ala, C1is lle, D1is Arg, E1is Ser, F1is Ser, G1is Asn, H1is Leu, I1is Ala, J1is lle, K1is Ser, L1is Pro, and M1is Asn; (c) if A1is Lys, B1Java Asn, H1is Leu, I1is Ala, J1is lle, K1is Ser, L1is Pro, and M1is Asn; (d) if A1is Lys, B1is Ala, C1is Val, L1is Arg, E1is Ser, F1is Ser, G1is Asn, H1is Leu, I1is Pro, J1is Val, K1is Pro, L1is Pro, and M1is Asn; (e) if A1is Lys, B1is Ala, C1is Val, D1is His, E1is Ser, F1is Asn, G1is Asn, H1is Leu, I1is Pro, J1is Val, K1is Ser, L1is Pro, and M1is Asn; or (f) if A1is Les, B1is Thr, C1is Val, D1is Arg, E1is Ser, F1is Ser, G1is His, H1is Leu, I1is Ala, J1is Ala, K1is Leu, L1is Pro, and M1is Asp; that one or more of any A1-M1not an L-amino acid, a Z is not amino.

Suitable side chains for X and Y are groups originating from alkylsulfides that can form disulfi kilgallen and bonds alkylamines, which may be condensed with education alkylamino bridge; or side chains which may be associated with the formation of alkilani, alkenilovyh, alkinilovymi, broadcasting or thioester linkages. Preferred alkylamine chains are lower alkyl groups having from about 1 to about 6 carbon atoms.

In yet another embodiment, the present invention relates to analogs-agonists is shown in Fig. 3, which does not have a bridge connection, and in which X and Y independently are selected from Ala, Ser, Cys, Val, Leu and lle, or from alilovic, arolovich or aralkylated esters and ethers Ser or Cys.

The present invention also includes biologically active derivatives, analogs-agonists, shown in Fig. 3, in which the stereochemistry of the individual amino acids can be inverted from one or more specific sites.

In the scope of the present invention also includes analogs, agonists, modified by glycosylation residues Asn, Ser and/or Thr.

In the scope of the present invention also includes biologically active analogs, Amylin agonists, which have less of peptide nature. Such pseudopeptide may have, for example, one or Kenny (-CH=CH-), - enaminoketone (-C(=CH-CN)-NH-), thioamides (-CS-NH-), dimethylene (-S-CH2- or-CH2-S-), methylene (-CH2-CH2- ), retro amide (-NH-CO-).

Compounds of the present invention form salts with various inorganic and organic acids and bases, These salts may be derived from such organic and inorganic acids, such as HCl, HBr, H2SO4H3PO4, triperoxonane acid, acetic acid, formic acid, methanesulfonate acid, toluensulfonate acid, Malinova acid, fumaric acid and camphorsulfonic acid. Examples of salts that can be obtained using bases are ammonium salts, alkali metal salts (such as sodium and potassium salts), and salts of alkaline earth metals (such as calcium and magnesium salts). The preferred salts are the hydrochloride and triptorelin.

These salts can be obtained by traditional methods, for example, through reaction of the free acid or base product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble or in a solvent such as water, kotohime ions on the appropriate ion-exchange resin.

Compounds of the present invention may exist in the form of various stereoisomers. In the preferred compounds of the present invention chiral centers on the peptide backbone are all s

Compounds of the present invention can be obtained by using some standard reactions for the synthesis of well known professionals peptides. Analogs of the present invention can be obtained by successively attaching the necessary amino acids to a growing peptide chain. Usually, N-carbarnoyl-protected amino acid and the amino acid that is associated with the growing peptide chain on the polymer carrier, and subjected to reaction at room temperature in an inert solvent, such as N-organic, dimethylformamide or methylene chloride in the presence of a crosslinking agent, such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, in the presence of a base such as diisopropylethylamine. - N-carbamoyl protective group is removed from the resulting peptide using a reagent such as triperoxonane acid or piperidine, and the specified reaction accession repeat with the next N-protected amino acid. Suitable N-protective groups are well known experts who haunted the methods of synthesis described in the assigned situated on the simultaneous consideration and assigned patent application reg. N 667040 ("Syntetic Preparation of Amylin and Amylin Analogs" filed on March 8, 1991). These methods belong to the solid phase synthesis of a peptide containing Amylin or an Amylin analogue, which has a high biological activity and generally does not contain deletions and other impurity peptides; and the specified peptide synthesized using successive cycles of synthesis, where in each cycle the desired amino acid attached to the growing peptide chain associated with an insoluble polymer carrier by formation of a peptide bond between the amino group of the growing peptide chain and carboxyla desired amino acid; and where each cycle of synthesis includes; (a) processing the growing peptide chain in the conditions - amino-unlock with removal of the amino group; (b) activating - carboxyl group of the amino - protected right amino acids; (c) the interaction of the growing peptide chain and the necessary amino acids under the reaction conditions of accession with the formation of the peptide bond between the free amino group of the peptide chain and activated - carboxyl group of the desired amino acids; and (d) repeating steps (b) and (c), if the efficiency of the synthesis stage (C) is less than about 97%. Stage (b) and (c) is preferably in the I present invention stage (b) and (c) are repeated at each cycle of the synthesis. The efficiency of synthesis is usually measured, although it is not necessary, after each stage of the merger.

Appropriate conditions of synthesis include the use of solvents, which maximizes the swelling of the solid carrier, minimizes the elements of the secondary structure of the peptide chain during cycles of synthesis and minimizes vnutriplevralnoe and interpeptide hydrogen binding. Preferably, if the synthesis loop includes a phase lock after stage(stages) of the accession where the unreacted-amino group of the peptide chain become directionspanel. Synthesis cycle is then repeated using the appropriate protected amino acids, resulting in a gain Amylin or an Amylin analogue sequence. After successive cycles of synthesis specified Amylin or an Amylin analogue otscheplaut from solid media. Preferably, the cysteine residues of the peptide chains were selectively unlocked and intramolecular disulfide bonds were formed before it can be derived peptide bond from solid media.

Suitable - amino protective groups are t-butoxylate - amino-protective group using t-butoxycarbonyl - carboxyl group activated using of DICYCLOHEXYL carbodiimide and 1-hydroxybenzotriazole with the formation of esters of 1-hydroxybenzotriazole. A particularly preferred solvent system is a system containing N-organic.

In Examples 1-17 of this application describes some analogues (agonists. In addition, other analogs, agonists, which can be obtained in accordance with the procedures outlined above (see table 2). Compounds of the present invention can also be obtained using the techniques of recombinant DNA, are well known in the art. See, for example, Sambrook and other , Molecular Cloning: A Laboratory Manual, 2nd ed., Gold Spring Harbor (1989).

The nomenclature of the compounds of the present invention can be used to refer to a peptide from the sequence of which was derived compound, and modifications introduced in the peptide sequence of Amylin, such as Amylin person. The amino acid with the previous Superscript index indicates that this amino acid substitutes are usually present in the primary amino acid sequence of the amino acid in position, Lucen of the sequence including Amylin" (Amylin person), with the following substitution: His was replaced in Arg at position 18, Ala was replaced by Pro in position 25 and Ser was replaced by Pro in position 28. The term "des-1Lys-including Amylin" refers to a peptide derived from the sequence of human Amylin with deletional first (or N-terminal) amino acid,

Analogs, Amylin agonists of the present invention have valuable pharmacological properties. In particular, the compounds of the present invention are active Amylin agonists, as evidenced by the analysis of binding with the receptor and analysis using the soleus muscle, as described below in Examples 18 and 19, respectively. Amylin-agonistic activity of the compounds can also be estimated by identifying the ability to induce hyperlactemia and/or hyperglycemia in a mammal, In addition to the description of the compounds depicted in Fig. 3, Table I presents some of the preferred compounds. All of these preferred compounds, namely: des-1lys-including Amylin,28Pro-am Amylin,25,28,29Pro-am Amylin,18Arg25,28Pro-am Amylin, des-1Lys18Arg25,28Pro-am Amylin was found amelineau activity in vivo in animals that have ecternal for Amylin, compounds of the present invention also have better solubility and stability compared with amilina person. Such preferred compounds are25Pro26Val28,29Pro-am Amylin,25,28,29Pro-am Amylin and 18Arg25,28Pro-am Amylin.

Particularly preferred compounds of the present invention are18Arg25,28Pro-am Amylin, des-1Lys18Arg25,28Pro-am Amylin, 18Arg25,28,29Pro-am Amylin, des-1Lys18Arg25,28,29Pro-am Amylin, 25,28,29Pro-am Amylin, des-1Lys25,28,29Pro-am Amylin and25Pro26Val25,28Pro-am Amylin. Additional peptide compounds-Amylin agonists are presented in Table 2. These compounds are:

23Leu25Pro26Val28,29Pro-am Amylin;

23Leu25Pro26Val28Pro-am Amylin;

des1Leu23Leu25Pro26Val28Pro - am Amylin;

18Arg23Leu25Pro26Val28Pro - am Amylin;

18Arg23Leu25,28,29Pro-am Amylin;

18Arg23Leu25,28Pro-am Amylin;

17lle23Leu25,28,29Pro-am Amylin;

17lle25,28,29Pro-am Amylin;

des-1Lys17lle23Leu25,28,29Pro - am is Amylin;

17lle18Arg23Leu25Pro26Val28,29Pro-am Amylin;

13Thr21His23Leu26Ala28Leu29Pro31Asp-including Amylin;

13Thr21His23Leu26Ala29Pro31Asp-including Amylin;

des-1Lys13Thr21His23Leu26Ala28Pro31Asp-including Amylin;

13Thr18Arg21His23Leu26Ala29Pro31Asp-including Amylin;

13Thr18Arg21His23Leu25Pro26Ala28,29Pro31Asp-including Amylin;

13Thr18Arg21His23Leu25Pro26Ala28,29Pro31Asp-including Amylin.

Compounds of the present invention can be mixed with pharmaceutical carriers in order to obtain the pharmaceutical dosage forms suitable for parenteral administration. The results of experiments conducted with the use of these compounds, suggest the possibility of clinical use of such pharmaceutical compositions in the treatment of diabetes and other conditions caused by a lack of insulin, as well as for the prevention and treatment of attacks of hypoglycemia. Compounds of the present invention can also be used in combination with insulin is h" means a polypeptide or its equivalent, necessary to regulate glucose levels in the blood. General description of insulin is held in Goodman and Gilman The Pharmacological Basis of Therapeutic, 8th ed., Pergamon Press (1990). Such insulin can be a hormone quick action, interim action or prolonged action. In the present invention may be used various derivatives of insulin. Cm. for example, the U.S. patents NN 5 049 547, 5 028 587 and 5 016 643. Can also be used peptides of insulin (see, for example, U.S. patent No. 5 008 241) as analogues (see, for example, U.S. patent No. 4 992 417 and 4 992 418). These compositions can be introduced by any conventional method, including the introduction through the nose (see, for example, U.S. patent No. 4 988 512 and 4 985 242, and 2 Bio World Today, 125 N (1991)). Compounds of the present invention can also be used in combination with glucagon for the prevention and treatment of hypoglycemia. [See, Young and others, the application of U.S. reg. N 07/640478, filed January 10, 1991, entitled "Hyperglycemic Compositions" ("hyperglycemic composition"), which is introduced in the present description by reference].

Compositions or products of the present invention can be obtained as solutions suitable for parenteral (including intravenous, intramuscular and subcutaneous administration), naati together with insulin or glucagonoma in a single composition or solution. In other cases, it may be preferable to introduce insulin or glucagon separately from the specified analog-agonist. A suitable mode of administration of the medicinal product could be best determined by the attending physician for each patient individually. Suitable pharmaceutically acceptable carriers and their composition are described in the monographs on the technology of drug production, see , for example, Remington's Pharmaceutica Science. Martin E. W., Cm., also Wang Y. J. and Hanson, M. A. "Parenteral Formulation of Proteins and Peptides: Stability and Stabilizers", Journal of Parenteral Science and Technologi Technical Report No. 10, Supp. 42:2S (1988). Suitable preparations containing insulin or glucagon, well known to experts.

Drugs are agonists of the present invention can be stable at neutral pH. Because the products of the present invention are amphoteric, they can be used as free bases, in the form of acid additive salts or as salts of the metals. Needless to say, these salts must be pharmaceutically acceptable, and, as the metal salts may be used salts of alkaline and alkaline-earth metals, for example, salts of sodium or potassium. As mentioned above, for the purposes of the present invention MoE is Holocene with organic and inorganic acids, preferably mineral acids. Typical examples of acids that can be used for the purposes of the present invention are citric, succinic, lactic, hydrochloric and Hydrobromic acid. These products can be easily obtained using standard procedures well known to specialists.

The products of the present invention are primarily designed for administration in the form of parenteral compositions by injection or infusion. They can be, for example, suspended in an inert oil, preferably a vegetable oil such as sesame oil, peanut oil or olive oil. Alternatively, these products may be suspended in the aqueous isotonic buffer solution at a pH of from about 5.6 to 7.4. Suitable buffers include sodium citrate - citric acid and sodium phosphate - phosphoric acid. Can also be used long-acting drugs or so-called "deposited" drugs slow release, such that a therapeutically effective amount of drug contained in the drug, enter the bloodstream over many hours or days after subcutaneous injection.

Not the money, such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols such as mannitol and sorbitol, or other inorganic or organic substances. From buffers containing sodium ions, especially preferred is sodium chloride.

If necessary, the solutions of the above compositions can be thickened with the use of such thickeners, as methylcellulose. They can be obtained in emulsifiable form or in the form of an emulsion water in oil, or in the form of an emulsion oil-in-water". Can be used by any agent from a wide range of existing pharmaceutically acceptable emulsifiers, including, for example, powdered Arabian gum, nonionic surfactant (such as tween) or ionic surfactant (such as basic salts of sulfates or sulfonates polyepitopic, for example, Triton).

Therapeutically valuable compositions of the present invention is prepared by mixing ingredients according to traditional procedures. For example, the selected components may be simply mixed in the mixer, or other standard device to obtain a concentrated mixture, which can be then goodiegoodie pH, or more soluble substances for regulation of toychest.

For use by the attending physician compositions of the present invention can be manufactured in the form of a unified standard dosage forms, containing a number of connection-agonist, taken separately or in combination with insulin or glucagon, which at its introduction in single or multiple dose is effective to regulate or restore blood sugar at the right level. A therapeutically effective amount of an analogue of Amylin agonist of the present invention for the treatment of hypoglycemia correspond to those quantities that contribute to the increase of sugar level in the blood, and preferably up to about 80 mg/DL. Therapeutically effective amounts of such analogs-agonists for the treatment of diabetes mellitus and other disorders associated with insulin deficiency, are the amounts sufficient to reduce cases of pleaserobme or unwanted hypoglycemia. As known in the art, the effective amount of therapeutic agent can vary depending on many factors, such as age and weight of the patient, physical the AI contain about 0.5-1.0 mg of the compound of the Amylin agonist and if necessary, commonly used, or fewer glucagon.

As mentioned above, the composition used for the present invention can be manufactured using standard techniques. These compositions can also be introduced by conventional methods. The desired dose can be easily determined by the expert, on the basis of the above examples.

For a clearer understanding of the present invention the following examples, which describe the results of a series of experiments. However, the following examples should not be construed as a limitation of the invention, and it can be made various changes that previously described or will be described below, in accordance with the essence and scope of the invention defined in the following claims.

Examples

Example 1

Getting28Pro-people Amylin

Solid-phase synthesis of the specified analog of the human person Amylin about using methylbenzhydrylamine-linked prepolymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis,2,7[disulfide]Amylin-MBHA-resin has been obtained by processing the Acm is emer and the protective groups of the side chain was tsalala with liquid hydrofluoric acid (HF) in the presence of dimethyl sulfide and anisole28Pro including Amylin was purified using preparative HPLC. The homogeneity of the peptide was determined by analytical HPLC and capillary electrophoresis, and the structure was confirmed using amino acid analysis and sequence analysis. The resulting product had the desired mass of the ion. FAB-mass-SPECT.: (M + 1)/e=3914.

Example 2

Getting25Pro26Val28,29-people Amylin

Solid-phase synthesis of the specified analog of Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide] Amylin-MBHA-resin has been obtained by processing trifurcation waist(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole.25Pro26Val28,29Pro-people Amylin was purified using preparative HPLC. Using analytical HPLC and capillary electrophoresis was installed, the homogeneity of the peptide, and its structure was confirmed using amino acid analysis and analysis of its sequence. The resulting product had the desired mass of the ion. FAB-mass-SPECT.: M+1/antes specified analog of Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2Asp and7Lys were introduced with Boc-2Asp (Fmoc)-OH and Boc-7Lys (Fmoc)-OH. After the election unlock the side chain using piperidine was carried out by the cyclization of the side chain - side chain"2Asp7Lys using hexafluorophosphate benzotriazol-Lil-oxy-Tris dimethylamino of phosphonium (BOP-reagent). Description cyclization has to Do Maio, J., and others, J. Med.chem. 33: 661-667 1990; Felix, A. M., and others , Int J. Pent. Prot. Res. 32: 441 (1988).2,7cyclo-[2Asp7Lys] Amylin-MBHA-polymer obtained after cyclization, were digested liquid HF in the presence of dimethyl sulfide and anisole.2,7cyclo-[2Asp7Lys]-people Amylin was purified using preparative HPLC. Using analytical HPLC and capillary electrophoresis was installed, the homogeneity of the peptide, and its structure was confirmed using amino acid analysis and analysis of its sequence. The resulting product had the desired mass of the ion. FAB-mass SPECT.: (M + 1)/e=3925.

Example 4

Receiving des-1Lys-people Amylin

Solid-phase synthesis of des-1Lys-people Amylin (also represented as2-37people Amylin using methylbenzhydrylamine-Sugo synthesis2,7[disulfide] Amylin-MBHA-resin has been obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole. Des-1Lys-people Amylin was purified using preparative reversed-phase HPLC. Using analytical HPLC and capillary electrophoresis was installed, the homogeneity of the peptide, and its structure was confirmed using amino acid analysis and analysis of its sequence. The product had the desired mass of the ion. FAB-mass spectrum.: (M + N)+= 3775.

Example 5

Getting1Ala-people Amylin

Tverdovsky synthesis1Ala-people Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide]Amylin-MBHA - polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole.1Ala-h the power of analytical HPLC and capillary electrophoresis, and its structure was confirmed using amino acid analysis and analysis of its sequence. This product had the desired mass of the ion. FAB-mass spectrum.: (M + N)+= 3847.

Example 6

Getting 1Ser-people Amylin

Solid phase synthesis1Ser-people Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis. 2,7[disulfide] Amylin - MBHA-polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole1Ser-people Amylin was purified using preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and its structure was confirmed using amino acid analysis and analysis of its sequence. This product had the desired mass of the ion. FAB-mass spectrum.: (M + N)+= 3863.

Example 7

Getting29Pro-people Amylin

Solid phase synthesis of this analogue of human amyl shall Westlake standard methods of peptide synthesis. 2,7[disulfide] Amylin-MBHA-polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole.29Pro-people Amylin was purified using preparative HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and the structure was confirmed using amino acid analysis and analysis of its sequence. The product had the desired mass of the ion. FAB-mass spectrum.: (M + N)+= 3916.

Example 8

Getting25,28-people Amylin

Solid phase synthesis25,28-people Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide Amylin-MBHA - polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole.25,28Pro-people Amylin was purified using preislerova electrophoresis, and the structure was confirmed using amino acid analysis and analysis of its sequence. The product had the desired mass of the ion. FAB-mass spec.: (M + N)+=3939.

Example 9

Receiving des-1Lys25,28-people Amylin

Solid-phase synthesis of des-1Lys25,28Pro-people.Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide]Amylin-MBHA - polymer was obtained by processing the Acm-protected cysteine triftoratsetata thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala using liquid HF in the presence of dimethyl sulfide and anisole. Des-1Lys25,28Pro-people Amylin was purified using preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and the structure was confirmed using amino acid analysis and analysis of its sequence. The resulting product had the desired ion mass. FAB-mass-SPECT.: (M + N)+= 3811.

Example 10

Getting18-Arg25,28Pro-people Amylin

Tverdofaznyi Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide]Amylin-MBHA - polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole.18-Arg25,28Pro-people Amylin was purified using preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and its structure was confirmed using amino acid analysis and analysis of its sequence. The resulting product had the desired ion mass. FAB-mass spectrum.: (M + N)+= 3959.

Example 11

Receiving des-1Lys18Arg25,28Pro - people Amylin

Solid-phase synthesis of des-1Lys18Arg25,28Pro - people Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis. 2,7[disulfide] - Amylin-MBHA-polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in tryforos liquid HF presence of dimethyl sulfide and anisole. Des-1Lys18Arg25,28-people Amylin was purified using preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and its structure was confirmed using amino acid analysis and analysis of its sequence. The resulting product had the desired ion mass. FAB-mass spectrum: (M + N)+= 3832.

Example 12

Getting18Arg25,28,29Pro-people Amylin

Solid phase synthesis 18Arg25,28,29Pro-people Amylin using methylbenzhydrylamine-linked polymer and protection of Na-Boc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide] Amylin-MBHA - polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala using liquid HF in the presence of dimethyl sulfide and anisole.18Arg25,28,29Pro-people Amylin was purified using preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary of electrophorese, and its structure was confirmed by the ACC-spectrum: (M + N)+= 3971.

Example 13

Receiving des-1Lys18Arg25,28,29Pro - people Amylin

Solid-phase synthesis of des-1Lys18Arg25,28,29Pro - people Amylin using methylbenzhydrylamine-linked polymer and protection of NaBoc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide] Amylin-MBHA-polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala using liquid HF in the presence of dimethyl sulfide and anisole. Des-1Lys18Arg25,28,29Pro-people Amylin was purified using preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and its structure was confirmed using amino acid analysis and analysis of its sequence. The resulting product had the desired ion mass. FAB-mass spectrum: (M + N)+= 3843.

Example 14

Getting25,28,29Pro-people Amylin

Solid phase synthesis25,28,29Pro-people Amylin using methylbenzhydrylamine-linked polymer and protection of NaBoc/the HA-polymer was obtained by processing the Acm-protected cysteine trifurcation thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala using liquid HF in the presence of dimethyl sulfide and anisole.25,28,29Pro-people Amylin was purified by means of preparative reversed-phase HPLC. The homogeneity of the peptide was established by analytical HPLC and capillary electrophoresis, and its structure was confirmed using amino acid analysis and analysis of its sequence, the resulting product had the desired ion mass. FAB - mass spectrum: (M + N)+= 3949.

Example 15

Receiving des-1Lys25,28,29Pro-people Amylin

Solid-phase synthesis Deux1Lys25,28,29Pro-people Amylin using methylbenzhydrylamine-linked polymer and protection of NaBoc/benzyl-side chain was carried out by standard methods of peptide synthesis.2,7[disulfide] Amylin-MBHA-polymer was obtained by processing the Acm-protected cysteine triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala using liquid HF in the presence of dimethyl sulfide and anisole. Des-1Lys25,28,29Pro-people Amylin was purified using preparative reversed-phase HPLC. Homage is, had been confirmed by using amino acid analysis and analysis of its sequence. The resulting product had the desired ion mass. FAB-mass spectrum: (M + N)+= 3823.

Example 16

Receiving des-1Lys25Pro26Val28,29Pro-people Amylin

Solid phase synthesis of this analogue people Amylin using methylbenzhydrylamine-linked polymer and protection of NaBoc/benzyl-side chain was carried out by standard methods of peptide synthesis. 2,7[disulfide]Amylin-MBHA - polymer was obtained by processing triptoreline thallium(111) in triperoxonane acid. After completion of the cyclization of the polymer and the protective groups of the side chain was tsalala with liquid HF in the presence of dimethyl sulfide and anisole. After that des-1Lys25Pro26Val28,29Pro - people Amylin was purified using preparative HPLC.

Example 17

Getting [(D)-11Arg]-Amylin

Solid phase synthesis of this analogue of human Amylin using methylbenzhydrylamine-linked polymer and protection of NaBoc/benzyl-side chain was carried out by standard methods of peptide synthesis.11Arg was introduced with Boc11Arg(Mtr)-OH. 2,7[disulfide]Amylin-MBHA-polymer obtained by processing triptoreline thallium(111) in triperoxonane acid, cyclized and anisole. Amylin was then purified using preparative HPLC.

Example 18

Analysis of binding with the receptor

Evaluation of the compounds of the present invention on the binding of Amylin receptors was carried out as follows.1251-Amylin in rats (Bolton-Hunter labeled at the N-terminal lysine) was obtained from Amersham Corporation (Arlington Heights, 1L). The specific activity in the period was in the range from 1950 to 2000 Ku/mm. Its peptides were obtained from BACHEM lnc. (Torrance, CA) and Peninsula Laboratories (Belmont, CA).

Male rats Sprague-Dawley (200-250 g) were killed by decapitate. The brain was removed and placed in cold phosphate buffer solution (PBS). From the ventral surface incisions were made Rostral to the hypothalamus, which on the one hand were limited to the olfactory tracts, and which stretched from these paths medial angle of 45o. This basal forebrain containing the basal nucleus and the surrounding area, weighed and homogenized in ice-HEPES buffer (20 mm HEPES-acid, the pH was brought to 7.4 with NaOH at 23oC). Membranes were washed three times in fresh buffer by centrifugation at 48000 x g for 15 minutes. The final precipitate resuspendable in 20 mm HEPES - buffer, containing 0.2 mm of the original wet weight of tissue, incubated with1251-Amelina at 12-16 PM in 20 mm HEPE - buffer containing 0.5 mg/ml bacitracin, 0.5 mg/ml bovine serum albumin, and 0.2 mm PMSF. The solutions were incubated for 60 minutes at 23oC. the Incubation was completed by filtration through filters made of glass fiber GF/B (Whatman Inc. , Clifton, N. J.), which previously was immersed for 4 hours in 0.3% polyethylenimine in order to reduce nonspecific binding of radioactively labeled peptides. Directly before filtration the filters were washed in 5 ml cold PBS and immediately after the filter 15 ml of cold PBS. Then the filters were removed and radioactivity was assessed using a gamma-counter efficiency accounts for 77%. Competitive binding curves constructed by measuring binding in the presence of 10-12-10-6its the test compounds and analyzed by the method of nonlinear regression, using the logical equation with 4 parameters (Inplot program, Graph-PAD Software, San Diego).

In this assay, purified human Amylin was associated with its receptor with IC50which measurement was about 50 PM. The results obtained for the tested compounds of the present invention and are presented in Table 1, showed that each of these by soedineniya soleus muscle

Assessment of the activity of the compounds of the present invention as Amylin agonists was performed using the soleus muscle (Soleus mucle) as follows. To maintain the weight allocated to the soleus muscle of less than 40 mg used of male rats (Harlan Sprague-Dawley) weighing approximately 200 g For 4 hours before killing by decapitate animals were not given food. The skin was removed from the lower limbs, which was then rolled on a cork Board. The Achilles tendon (tendo achilles) cut just above the calcaneus (os calcis), and the gastrocnemius muscle (gastrocnemius muscle) were exposed from the back surface of the tibia. Then freely separated soleus muscle (M. soleus) (small, 14-20 mm long, thick, flat muscle on the bone surfaces of the gastrocnemius muscle) and purified it from perimete using fine scissors and forceps. After that, the soleus muscle was divided into equal parts using the blade of a scalpel, making incisions from the front to the rear part of the abdomen muscles, which received a total of 4 muscle strips from each animal. After dissection of the muscles of the animal, it was kept in a short period of time in physiological solution. However, there is no need to endure the muscle in Chennai glucose into glycogen.

The muscles were placed in a 50 ml Erlenmeyer flask containing 10 ml of pre soda bicarbonate buffer Krebs-ringer comprising (per liter): 118,5 mm (6,93 g) NaCl, 5,94 mm (443 mg), KCl, 2.54 mm (282 mg) CaCl2, 1,19 mm (143 mg), 1,19 mm (162 mg) KH2PO4, 25 mm (2.1 g) NaHCO3, 5.5 mm (1 g) of glucose and recombinant human insulin Humulin-R, Eli Lilly (IN); test connection, as described in more detail below. pH at 37oC was maintained in the range of 7.1 to 7.4. The muscles were divided into different flasks so that the 4 piece of muscle from each animal were evenly distributed throughout the tests with different conditions. The incubation medium was slightly aziraphale equalising with Carbogen (95% O2WITH 5% CO2) above the surface, and constantly stirring, in a water bath-shaker at 37oC. After half an hour "preincubation" period in each flask was added a 0.5 MK Ku U14C-glucose, and these flasks were incubated for another 60 minutes. After that, each piece of muscle was rapidly removed, blokirovala and frozen in liquid N2then weighed and stored for subsequent determination14C-glycogen.

Definition14C-glycogen was carried out in 7-ml scintillation vial. Each of abrasion and in the conditions of mixing. Dissolved glycogen besieged in the vial by adding 3 ml of absolute ethanol and cooling at -20oC during the night. The supernatant was removed by gentle suction, glycogen again washed with ethanol was aspirated and the precipitate was dried in vacuum. In order to avoid quenching of fluorescence during the counting of the pulses of all the ethanol evaporated. The remaining glycogen was again dissolved in 1 ml of water and 4 ml of scintillation fluid, and counted14C.

The degree of incorporation of glucose into glycogen (expressed in μm/g per hour) was determined on the basis of the specific activity14C-glucose 5.5 mm glucose incubation medium and the total number of14C remaining in the glycogen extracted from each muscle. Curves dose-response built on the logical model with 4 parameters using an iterative least squares algorithm (ALLFIT, version 2.7, and NIH, MD), and resulted EC50. Because EC50have a log normal distribution, they were expressed in the logarithm of the average square deviation. Pairwise comparison was performed using standard software SYSTAT, based on the t-criteria t-test (Wilkinson, "SYSTAT: the system for statistics SYSTAT Inc., EvanstonlL (1989)).

Dose-dependent crepe connection, added in the final (nominal) concentrations 0, 1, 3, 10, 30, 100, 300 and 1000 nm. Each analysis also used an internal positive control, consisting of one party archive rat Amylin, liofilizirovannogo and stored at -70oC.

Human Amylin is a known hyperglycemic peptide; and value EC50drugs Amylin, measured in the analysis using the soleus muscles are usually between about 1-10 nm, although some commercial preparations, which have a purity of less than 90%, show higher EC50due to the presence of contaminants, resulting in lower measured activity. The results obtained for the tested compounds and are presented in Table 1 showed that all these compounds have amelineau activity.

Example 20

Getting23Ser,25,28,29Pro-human Amylin.

Solid-phase synthesis of the specified analogue of human Amylin performed on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxy-ndimethylacetamide - narratologically resin (Novabiochem. 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.). In General, in the process of synthesis cycles are used is in the presence of diisopropylethylamine using N-methyl-pyrrolidone as solvent. However, in some positions the combination is less effective than expected, and required double combination. In particular remains Pro29Pro28Pro25, Asn22, Asn21Asn3and Cys2- require all dual combinations. N-end complete it using (bis-tBoc)-lysine in the loop final combination. The final removal of the protective groups in the finished peptide polymer was achieved using a mixture of triethylsilane (0.2 ml), identicial (0.2 ml), anisole (0.2 ml), water (0.2 ml) and triperoxonane acid (15 ml) using standard methods (see "Introduction to Cleavage Techniques, Applied Biosystems, Inc.). The peptide is precipitated in a mixture of ether and water (50 ml) and centrifuged. The residue is treated with glacial acetic acid and lyophilizer, and the obtained crude peptide re-dissolved in water and treated with Tris-carboethoxy-phosphine to provide a complete generation of free thiols. Treatment with potassium ferricyanide at pH of 6.5 causes cyclization to the formation of the peptide with monosulfide bridges. In the acidification and processing of anion-exchange resin Biorad AG4X4 remove all remnants of ions Fe2+and Fe3+. After lyophilization obtain the crude peptide.

On the stages of purification and analysis of use"ptx2">

The solution containing the peptide, put on a preparative column C-18 and purified (from 10% to 40% Solvent B in Solvent And more than 40 minutes). The purity of the fractions determined isocrate using analytical column C-18. Pure fractions drain, receiving the above-mentioned peptide. Analysis lifeseaving peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) allows to determine the retention time of the resulting peptide. Electrospray mass spectrometry13Ser25,28,29Pro-people-Amylin(M): computed value 3965.5.

Example 21

Getting13Thr25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems. Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then spent anaphorically And over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry13Thr25,28,29-h-Amylin(M): computed value 3979.5.

Example 22

Getting17Leu25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems. Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry17Leu25,28,29Pro-h-Amylin(M): computed value 3963.5.

Example 23

Getting17IIe25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid)-Fmoc aminoindan amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry17Leu25,28,29Pro-h-Amylin(M): computed value 3963.5.

Example 24

Getting19Thr25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems. Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then spent amatoriale And over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry19Thr25,28,29Pro-h-Amylin(M): computed value 3963.5.

Example 25

Getting20Thr25,28,29Pro-people-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry20Thr25,28,29Pro-h-Amylin(M): computed value 3963.5.

Example 26

Getting20Gln25,28,29Pro-people-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid)-Fmoc protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1 % of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry 20Gln25,28,29Pro-h-Amylin(M): computed value 3990.5.

Example 27

Getting20Asn25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then spent historiale And over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry20Asn25,28,29-people-Amylin(M): computed value 3976.5.

Example 28

Getting21Gln25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry21Gln25,28,29-people-Amylin(M): computed value 3963.5.

Example 29

Getting21His25,28,29-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid)-Fmoc eminendy amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry21His25,28,29Pro-h-Amylin(M): computed value 3973.5.

Example 30

Getting23Leu25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then spent amatoriale And over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry23Leu25,28,29Pro-people-Amylin(M): computed value 3915.5.

Example 31

Getting23Tyr25,28,29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry23Tyr25,28,29Pro-h-Amylin(M): computed value 3965.5.

Example 32

Getting25Pro26Ala28,29-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-dimethoxyphenyl the m Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25Pro26Ala28,29Pro-h-Amylin(M): computed value 3907.5.

Example 33

Getting25Pro26Leu28,29Pro - h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). which of solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25Pro26Leu28,29-h-Amylin(M): computed value 3949.5.

Example 34

Getting25Pro28Leu29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxy - ndimethylacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25Pro28Leu29Pro-h-Amylin(M): computed value 3965.5.

Example 35

Getting25Pro28IIe29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25Pro28IIe29Pro-h-Amylin(M): computed value 3965.5.

Example 36

Getting25Pro28Thr29Pro-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). ZAT is ritala B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25Pro28Thr29Pro-h-Amylin (M): computed value 3953.5.

Example 37

Getting25,28Pro29Thr-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl - phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem. 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems. Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of policerelated.Elektrorazpredelenie spectrometry25,28Pro29Thr-h-Amylin(M): computed value 3953.5.

Example 38

Getting25,28,29Pro31Asn-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid)-Fmoc asimenia amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then conducted an analysis of lyophilized peptide using HPLC with reversed phase (gradient from 30% to 60% Solvent B in Solvent a over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25,28,29Pro31Asn-h-Amylin(M): computed value 3950.5.

Example 39

Getting25,28,29Pro31Gln-h-Amylin

Solid-phase synthesis of the specified analogue of human Amylin carried out on 4-(2'-4'-acid) the Fmoc aminomethyl-phenoxyacetamide-norleucine-methylbenzhydrylamine resin (Novabiochem, 0.55 mmol/g) using Fmoc-protected amino acids (Applied Biosystems, Inc.), derived from polymer, in respect of which spent the removal of protective groups and purification in the same manner as described in Example 20. When conducting analyses used Solvent A (0.1% of triperoxonane acid in water) and Solvent B (0.1% triperoxonane acid in ACN). Then spent amatoriale And over 30 minutes) to determine the retention time of the resulting peptide. Electrospray mass spectrometry25,28,29Pro31Gln-people-Amylin(M): computed value 3963.5.

1. Similar-Amylin agonist having the amino acid sequence of General formula I:

'A1-X-Asn-Thr-5Ala-Thr-y-Ala-Thr-10Gln-Arg-Leu-B1-Asn-15Phe-Leu-C1-D1-E1-20F1-G1-Asn-H1-Gly-25J1-J1-Leu-K1-L1-30Thr-M1-Val-Gly-Ser-35Asn-Thr-Tyr-Z

A1represents Lys, Ala, Ser or hydrogen;

B1represents Ala, Ser or Thr;

C1represents Val, Leu or Jle;

D1represents His or Arg;

E1represents Ser or Thr;

F1represents Ser, Thr, Gln or Asn;

G1represents Asn, Gln or His;

H1represents Phe, Leu or Tyr;

J1represents Ala or Pro;

J1represents Jle, Val, Ala or Leu;

K1represents Ser, Pro, Leu, Jle, or Thr;

L1represents Ser, Pro or Thr

M1represents Asn, Asp, Gln

where at least two of J1, K1, L1are Pro X and Y are independently selected amino acid residues having side zip pocket wit represent an amino group, provided that when

a) A1= Lys, B1=Ala, C1=Val, D1=Arg, E1=Ser, F1=Ser, G1=Asn, H1=Leu, J1= Pro J1=Val, K1=Pro, L1=Pro, M1=Asn or

b) A1= Lys, B1=Ala, C1=Val, D1=His, E1=Ser, F1=Asn, G1=Asn, H1=Leu, J1= Pro J1=Val, K1=Ser, L1=Pro, M1=Asn,

then one or more of A1-M1not an L-amino acid.

2. Similar-Amylin agonist under item 1, where X and Y represent the Cys - residues linked by a disulfide bond.

3. Similar-Amylin agonist under item 1 or 2 where J1, K1and L1are a Pro.

4. Similar-Amylin agonist on PP.1, 2 or 3, where D1=Arg.

5. Similar-Amylin agonist on PP.1, 2 or 3, where J1=Val.

6. Similar-Amylin agonist on PP.4 or 5, where A1=Lys.

7. Similar-Amylin agonist under item 4, where A1-hydrogen.

8. Similar-Amylin agonist under item 1, which is a18Arg25,28Pro - h-Amylin, des1Lys18Arg25,28Pro - h-Amylin,25,28,29Pro - h-Amylin,18Arg25,28,29Pro - h-Amylin, des1Lys25,28,29Pro - h-Amylin, des1Lys18Arg25,28,29Pro - h-Amylin, 25Pro26Val

10. Composition exhibiting the property of the Amylin agonist containing the active ingredient and pharmaceutically acceptable carrier, wherein the active ingredient contains an analog-Amylin agonist of formula I under item 1 in an effective amount.

 

Same patents:
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The invention relates to certain Aza cyclopentapeptide compounds having a nitrogen atom attached to cyclohexadienone ring on the 5th carbon atom of the component 4-hydroxy-ornithine (C-5-orn"), formula I, where R1Is H or OH; R2- H, CH3or OH; R3- H, CH3CH2CN, CH2CH2NH2or CH2CONH2; RI- C9-C21-alkyl, C9-C21alkenyl, C1-C10-alkoxyphenyl or C1-C10alkoxyaryl; RII- H, C1-C4-alkyl, C3-C4alkenyl, (CH2)2-4OH, CO(CH2)1-4NH2, (CH2)2-4NRIVRV; RIIand RIIItaken together, -(CH2)4-, -(CH2)5-, -(CH2)2O(CH2)2-, -(CH2)2NH(CH2)2-; RIV- H or alkyl; RVIs H or alkyl and salt additive

The invention relates to the field of medicine

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The invention relates to new polypeptide compound and its pharmaceutical acceptable salts

The invention relates to polypeptides or their salts, with a strong tool to lipopolysaccharides, particularly endotoxins

FIELD: organic chemistry, biochemistry, medicine, pharmacy.

SUBSTANCE: invention relates to macrocyclic peptides of the general formula (I): wherein W means nitrogen atom (N); R21 means hydrogen atom (H), (C1-C6)-alkoxy-, hydroxy-group or N-(C1-C6-alkyl)2; R22 means hydrogen atom (H), (C1-C6)-alkyl, CF3, (C1-C6)-alkoxy-group, (C2-C7)-alkoxyalkyl, C6-aryl or Het wherein het means five- or six-membered saturated or unsaturated heterocycle comprising two heteroatoms taken among nitrogen, oxygen or sulfur atom and wherein indicated Het is substituted with radical R24 wherein R23 means hydrogen atom (H), -NH-C(O)-R26, OR26, -NHC(O)-NH-R26, -NHC(O)-OR26 wherein R26 means hydrogen atom, (C1-C6)-alkyl; R3 means hydroxy-group or group of the formula -NH-R31 wherein R31 means -C(O)-R32, -C(O)-NHR32 or -C(O)-OR32 wherein R32 means (C1-C6)-alkyl or (C3-C6)-cycloalkyl; D means a saturated or unsaturated alkylene chain comprising of 5-10 carbon atoms and comprising optionally one-three heteroatoms taken independently of one another among oxygen (O), sulfur (S) atom, or N-R41 wherein R41 means hydrogen atom (H), -C(O)-R42 wherein R42 means (C1-C6)-alkyl, C6-aryl; R4 means hydrogen atom (H) or one-three substitutes at any carbon atom in chain D wherein substitutes are taken independently of one another from group comprising (C1-C6)-alkyl, hydroxyl; A means carboxylic acid or its alkyl esters or their derivatives. Invention relates to pharmaceutical compositions containing indicated compounds and eliciting activity with respect to hepatitis C virus and these peptides inhibit activity of NS3-protease specifically but don't elicit significant inhibitory activity with respect to other serine proteases.

EFFECT: valuable biochemical and medicinal properties of peptides.

106 cl, 9 tbl, 61 ex

FIELD: medicine, pharmacology, biochemistry.

SUBSTANCE: invention relates to the highly purified enzyme α-galactosidase A (α-Gal A) and to different methods for its purification. Invention relates to the development of α-Gal A preparation with changed charge and to methods for preparing such preparations. Also, invention relates to α-Gal A preparations with prolonged half-time life in blood current of mammal-host, to methods for their preparing, to methods and doses in administration of α-Gal A preparations in patient. Invention provides expanding assortment used for treatment of patients suffering with α-galactosidase A insufficiency.

EFFECT: improved method for treatment, valuable medicinal properties of preparations.

15 cl, 9 tbl, 10 dwg, 5 ex

FIELD: biochemistry.

SUBSTANCE: method relates to new cyclopeptides of general formula cyclo(R1-Arg-Ile-Lys-Pro-His-R2) selected from group containing: P11: cyclo(DPhe-Pro-Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly-Glu) (SEQ ID NO:5), P16: cyclo(Arg-Ile-Lys-Pro-His-Gln-Gly (SEQ ID NO:8), P17: cyclo(Pro-Arg-Ile-Lys-Pro-His-Gln-Gly) (SEQ ID NO:9), P19: cyclo(Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly-Glu) (SEQ ID NO:10), P20: cyclo(Dphe-Pro-Gln-Ile-Met-Arg-Ile-Lys-Pro-His-Gln-Gly-Gln-His-Ile-Gly) (SEQ ID NO:11), P23: cyclo(DPhe-Pro-Arg-Ile-Lys-Pro-His-Gln) (SEQ ID NO:13), P24: cyclo(Gly-Arg-Ile-Lys-Pro-His) (SEQ ID NO:25), as well as P11, P20 and P23 with DPhe substituted by DTyr. Cyclopeptides are useful in systems for angiogenesis inhibition. System includes substrate with cyclopeptides attached by organic spacer arm optionally containing group cleavable by any fermentation system.

EFFECT: angiogenesis inhibiting cyclopeptides.

23 cl, 4 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: preparation comprises echinocandine substance of formula I or its pharmaceutically permissible salt, pharmaceutically permissible micelle-forming surface-active agent and non-toxic aqueous solvent and stabilizing agent.

EFFECT: improved stability and bioaccessibility properties.

48 cl, 4 tbl

FIELD: chemico-pharmaceutical industry.

SUBSTANCE: the present innovation deals with new stabilized pharmaceutical composition in its lyophilized form including the compound of formula I

as an active ingredient and lactose disaccharide as a stabilizing agent. The present pharmaceutical compositions are of high stability at storage. As for active ingredient it is not destroyed in the course of time.

EFFECT: higher efficiency.

10 cl, 15 ex, 6 tbl

FIELD: organic chemistry, amino acids.

SUBSTANCE: invention proposes agonists of somatostatin of the formula (I): X-A1-cyclo-(D-Cys-A3-A4-Lys-A6-A7)-A8-Y or its pharmaceutically acceptable salt wherein X represents hydrogen atom (H); A1 represents L-Cpa, L-Phe, L-Trp or L-Nal; A3 represents L-3-Pal or L-4-Pal; A4 represents D-Trp; A6 represents -NH-(CHR1)n-CO- wherein n = 2, 3 or 4; A7 represents L- or D-Cys; A8 represents D- or L-isomer of amino acid taken among the group consisting of Nal, Phe, Cpa and Trp; Y represents NH2; R1 represents hydrogen atom (H), and Cys in A3 is bound by disulfide bong in A wherein this disulfide bond is formed by thiol groups of each Cys residue.

EFFECT: valuable biological properties of compounds.

9 cl, 2 ex

FIELD: organic chemistry, polypeptides.

SUBSTANCE: invention relates to new antagonists of urotensin-II. Invention represents group of cyclic polypeptides of the general formula: (R1)a-AA1-cyclo-[AA2-AA3-AA4-AA5-AA6-Cys]-AA7-R2 wherein AA1 means L-isomer of aromatic amino acid; AA2 means L- or D-isomer of Cys; AA3 means L-isomer of aromatic amino acid; AA4 means L- or D-isomer of Trp; AA5 means L- or D-isomer of Lys, N-Me-Lys or Orn; AA6 means L- or D-isomer of Val, Thr, Leu, Ile, Tle, Nle or aromatic amino acid; AA7 means L- or D-isomer of Val, Thr, Leu, Ile, Tle, Abu, Nle or aromatic amino acid; R1 means hydrogen atom (H), lower alkyl, lower alkanoyl or lower acyl; a = 1 or 2; R2 means hydroxyl; group (OH), OR3, N(R3)2 or NHR3 wherein R3 means H, lower alkyl or arylalkyl. These cyclic polypeptides are used as antagonists of urotensin-II.

EFFECT: valuable biological properties of compounds.

24 cl, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: preparation belongs to decapeptides considered to be an analog of receptor-binding fragment of TGFα from 22 to 31 amino acids in which the eighth amino acid residue Lys is substituted with Ser residue. The analog is bindable to wide range of cytotoxic agents and operate as vector in directed delivery of antitumor agents to tumor cells. The preparation also comprises decapeptide conjugate with a cytotoxic agent showing selective action with respect to tumors and capable of reducing tumor cells resistivity to cytotoxic agents. Conjugated parts are bound by a bond scissile with respect to acid hydrolysis. Another embodiment of the invention is related to pharmaceutical composition containing effective quantity of conjugate and carrier applicable as intravenous injections.

EFFECT: enhanced effectiveness of treatment; high antitumor action selectivity.

6 cl, 2 dwg, 2 tbl

FIELD: molecular pharmacology, in particular human endostatin preparation and method for production thereof.

SUBSTANCE: claimed preparation is isolated after cell incubation E.coli BL21 (DE3) in cultural medium up to dense level of 0.4-0.6 OD6-00. Cell contains plasmide vector pBSH-ED15 or pBSH-ED16 having size of 6675 n.p. and gene of canamicine resistance gene; vector contains replicative sites pUS ori and M13 and gene encoding lac-repressor controlled by T7-promoter, and finished product has purity of 99 % and apyretic properties. Claimed preparation is obtained by expression of endostatin gene using plasmide DNA containing in E.coli cells; product refolding and purification, wherein expression is carried out in E.coli BL21 (DE3) cells, transformed by recombinant plasmide DNA pBSH-ED15 or pBSH-ED16. Cells are cultivated in cultural medium up to dense level of 0.4-0.6 OD6-00, then 0.1-0.3 mM of isopropyltiogalactoside is added into medium and cultivation is carried out for 2-3 h up to product accumulation. Further cells is exposed with ultrasound for 50-70 s at 0°C in presence of 0.1 % sodium deoxycholate. Bodies are dissolved in presence of 1 % sodium N-lauryl sarcosine. Suspension is multiply washed and centrifuged at 5000 g. In refolding process oxidation with air oxygen is carried out for 35 h, and target product is purified using chromatography depending on used incorporated plasmide.

EFFECT: apyretic product of high purity.

6 cl, 9 ex, 7 dwg

FIELD: molecular biopharmacology.

SUBSTANCE: method for production of stress protein (heat shock protein) preparations includes such protein gene expression in E.coli cells followed by isolation, refolding and purification. Gene expression is carried out in structure of vector cDNA containing T7 promoter, kanamycin resistance gene, replicative pUC ori origin, and gene encoding lac-inductor. Vector DNA is cultured in structure of E.coli cells BL21 in nutrient medium with addition of kanamycin (100 mug/ml) and glucose (0.2 %) up to turbidity level of 0.5-0.6 OD600. Then 0.02mM isopropyl beta-D-thiogalactoside is added into medium and culturing is carried out for 1-3 h to accumulate finish product. Target product is purified by anion exchange chromatography. In said method vector DNA encoding proteins: HSP 100-200; HSP 100; HSP 90; Lon; HSP 70; HSP 60; TF 55; HSP 40; FKBPl cyclophylin; HSP 2-30; ClpP; GrpE; HSP 10, etc. of any gene are used. Invention also related to stress protein (heat shock protein) preparation obtained by abovementioned method. Protein represents recombinant HSP protein having molecular weight of about 70 kDa, which is isolated after incubation of E.coli cells in growth medium LB for 12 h at 37°C with addition of kanamycin (100 mug/ml) and glucose (0.2 %) up to turbidity level of 0.5-0.6 OD600, wherein transformed cell contains pBSH2-HSP70 plasmide DNA, carrying cDNA of heat shock protein gene, whish has size of 5118 n.p. and represents pBSH2 promoter containing T7 promoter, kanamycin resistance gene, replicative pUC ori origin, and gene encoding lac-inductor. Finished protein product has purity of at least 98 %.

EFFECT: stress proteins of high activity and high purity.

8 cl, 5 ex, 5 dwg

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