New version of exendin or its conjugate

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to a conjugate of a version of exendin with a PEG molecule, and can be used in medicine. The above conjugate involves exendin with the amino acid sequence SEQ ID NO: 4 and one PEG molecule with molecular weight 21 kDa to 29 kDa conjugated with a cysteine residue in exendin. The invention also refers to a method for preparing an exendin conjugate, a pharmaceutical composition and a kit for lowering blood glucose providing using the exendin conjugate.

EFFECT: invention enables preparing the exendin conjugate with PEG with GLP-1 receptor agonist activity with maintaining the maximum effect on the stimulation of cAMP (Emax) production in PEGylation.

7 cl, 44 dwg, 5 tbl, 26 ex

 

The technical FIELD

The present invention relates to new versions of the basis, its conjugates with polymers containing pharmaceutical compositions and to the use of variants, conjugates and pharmaceutical compositions of the basis for reducing the level of glucose in the blood, especially in the treatment of diabetes (in particular type II diabetes). The present invention also relates to the use of conjugates of the basis weight of the body.

BACKGROUND of the INVENTION

All over the world as social development, increasing life expectancy and changing lifestyles diabetes has become one of the leading health problems. The incidence of diabetes is increasing rapidly in both developed and developing countries. In 2007, there were approximately 246 million patients with diabetes, and every 10 seconds in the world, one person dies from diabetes. It is projected that the population of patients with diabetes in the world will increase to 333 million by 2025. China is problematic region on diabetes. Today in China the population of patients with diabetes is 40 million, the number of diabetes patients in China with incidence of about 5%, ranks second in the world (after India). Diabetes is of two types: one is insulin-dependent diabetes mellitus (type 1 diabetes), and the second which is non-insulin dependent diabetes mellitus (type 2 diabetes). Of them type 2 diabetes accounts for over 90% of all cases of diabetes. Type 2 diabetes differs uncontrolled secretion or function of insulin, as well as dysfunction of β-cells, which leads to disturbances in the metabolism of lipids, carbohydrates and proteins. This can lead to chronic hyperglycemia and eventually cause complications in micro - and macrovascular system and various organs. Currently there are two classes of drug compounds for control of diabetes: 1) amplifiers insulin secretion, such as sulfonylurea, meglitinide and inhibitors of dipeptidyl-peptidases, analogues of GLP-1; and 2) amplifiers secretion of other compounds, in addition to insulin, such as insulin, inhibitors of α-glycosidase, a biguanide preparations, thiazolidinediones and insulin analogs. Currently, the majority of antidiabetic drugs commonly used in clinical practice, less effective against type 2 diabetes. They are unable to stop the progressive damage of β-cells, nor to lower HbA1c levels in the blood or to prevent complications caused by diabetes, such as heart disease and kidney failure. In addition, they have to a certain extent expressed in toxic side effects. Accordingly, there is a need to research new drugs cf the of funds for the treatment of type 2 diabetes.

In 1985 he opened a hormone of the gastrointestinal tract, like peptide-1 (GLP-1). GLP-1 is a product of gene expression of proglucagon after eating and is mainly secreted by L-cells of the mucosa of the gastrointestinal tract. GLP-1 stimulates insulin secretion, islet β-cells of the pancreas (J. Med. Chem., 47, 4128-4134, 2004) and plays an important role in stabilizing glucose level in the blood. In patients with diabetes mellitus type 2 the introduction of GLP-1 can stabilize the glucose content in the blood at normal levels (Diabetes Care, 15, 270-276, 1992; Lancet, 359, 824-830, 2002; Endoer. Rev, 16, 390-410, 1996; Diabetologia, 28, 565-573, 1985). In the body of GLP-1 does the following: glucosidation effect on islet β-cells of the pancreas, increasing the insulin gene transcription and biosynthesis and insulin secretion; stimulates proliferation and differentiation of β-cells and inhibits apoptosis of β-cells, increasing the number of islet β-cells of the pancreas; inhibits secretion of glucagon; increases the sensitivity of insulin receptors in peripheral cells; reduces HbA1c levels; suppresses appetite and reduces food intake; delays the feeling of emptiness in the stomach (Diabetic Med., 18, 144-149, 2001; Diabetes, 51, 1443-1452, 2002; Diabetologia, 45, 1263-1273, 2002; Diabetes, 50, 525-529, 2001; Diabetes, 50, 725, 2001; Diabetes, 52, 365-371, 2003; Recent Prog. Hormne Res. 56, 377-399, 2001; Disbetologia, 39, 1546-1553, 1996; Am. J. Physic. Edocrinol. Metab, 281, E242-247, 2001; U.S. patent 477967, 478017, 478425; Diabetes Care, 22, 403-408, 1999; J. Clin. Endocrinology and Metabolism, 88, 3082-3089, 2003; Diabetes, 44, 1295, 1995). However, in the body of GLP-1 is rapidly destroyed under the action of dipeptidylpeptidase (DPP IV), and its half-life is less than 2 minutes, and therefore, GLP-1 cannot be used as an effective antidiabetic medicines.

On the basis 4 is a polypeptide found in the saliva of Arizona adotube, poisonous lizards that live in the States of Arizona and new Mexico, USA (J. Biol. Chem., 265, 20259-20262, 1990; J. Biol. Chem., 267, 7402-7405, 1992). On the basis 4 has a high degree of homology with GLP-1 (7-36) (53%). Posted that on the basis of 4 can bind the receptor for GLP-1 and exhibit pharmacological agonistic effect similar to the effect of GLP-1, for example, increasing the synthesis of insulin, increasing glucosidation insulin secretion; stimulating the proliferation and regeneration, as well as inhibiting apoptosis of β-cells, thereby increasing their number; inhibiting the secretion of glucagon; inhibiting the formation of glycogen, without causing acute hypoglycemia; inhibiting the motility and secretion of the gastro-intestinal tract after a meal; reducing appetite and food intake; protecting nerve cells (Nat. Biotech, 23, 857-861, 2005; J. Biol. Chem., 266, 2897-2902, 1991; J. Biol. Chem., 266, 21432-21437, 1992; Diabetes, 44, 16-19, 1995; Nature, 379, 69-72, 1996). The ability of the basis 4 to potentiate the secretion of insulin and inhibiting the secretion of glucagon after meals depends on the level of glucose in the blood, which is preferable than the one used currently by the sulfonylureas, and, in particular, on the basis of 4 to a lesser extent, causes hypoglycemia and significantly reduces the frequency of glucose in the blood, and reduces the weight of the body. Preparation of basis 4 for the reception twice a day (Exenatide sold under the name Byetta), jointly developed by Amylin and Eli Lilly, has been approved for sale in the United States and Europe (U.S. patent№ 5424286, 6858576, 6872700, 6902744, 6956026, 7297761). Accordingly, this type of drugs widely used for the treatment of diabetes and obesity worldwide.

Pharmaceutical peptides require multiple injections throughout the day, because these drugs have a short half-life in the body and poor physical and chemical stability, and they are susceptible to degradation by various proteases. Eczematic is injected twice a day via subcutaneous injection, resulting in a heavy load on the body, the psyche and the financial condition of the patient, and therefore ill patients comply with the treatment regimen. Accordingly, current research in the field of antidiabetic medicines focus on the structural modification of the basis 4 and the development of new dosage forms for longer half-life of basis-4 in plasma and HC is to increase system availability of medicinal compounds.

Modification of polymers is a promising technique developed in the 1970s, with a typical modification is Pegylation (modification of molecules of polyethylene glycol, PEG). This technique is a chemical conjugation of PEG and pharmacological protein for surface modification of the protein. After PEG-modification increases the molecular weight of the protein, and the rate of its excretion by the kidneys is reduced. In addition, conjugated chain PEG sterically hinder access to the surface of the protein molecule, which leads to the reduction of its hydrolysis by proteases of the blood. Therefore, this effectively increases the lifetime of the protein in the bloodstream and leads to longer half-life in plasma and systemic availability of drugs, increasing its effectiveness.

At the moment, has already developed the second generation techniques Paglierani - site-directed Pegylation. Site-directed Pegylation can be used for specific modification of some amino acids in proteins, preventing, thus, non-specific modification. In fact, to a lesser extent affected the structure of the active centre of the protein, and some antigenic sites are violated selectively, which reduces the number of disadvantages such as decrease in biological activity and increasing heterogeneity of sleds is of non-specific Paglierani.

Scientists have conducted some research on the modification of the basis 4 polymers. Young and Prickett (CN1372570A) have shown that the metabolism of basis 4 is realized mainly through the kidneys. So they modified the basis 4 using PEG with molecular weight in the range from 500 to 20,000 daltons (Da). Wenchao Bao, Hongjing Xu, Gang Yu, Yajun Zuo (CN 101125207 A) has modified amino acids of the basis 4 using PEG with molecular weight ranging from 20 kDa to 50 kDa.

However, the currently existing basis-4 and its variants, and various modified forms have disadvantages, including high frequency introduction that leads to a severe load on the body, the psyche and the financial condition of the patient. So ill patients comply with the treatment regimen, it is impossible to widely use these medicines. Accordingly, there is still a need for new versions of the basis-4 and variants, modified polymers.

A BRIEF DESCRIPTION of the INVENTION

In one aspect the present invention relates to a variant of the basis with the activity of the agonist receptor GLP-1, in which one or more amino acid residues substituted with cysteine in comparison with the sequence of wild-type basis. Optional, variant basis of one or several amino acids can additionally b is to be removed, inserted and/or substituted and inserted or a replacement of amino acids can be a natural amino acids or analogs of amino acids.

In the embodiment of the invention a variant of basis has the amino acid sequence in which one or more amino acid residues substituted by cysteine, and, optionally, another amino acid compared with the sequence of basis 4 wild-type

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 1)

Or a sequence of basis-3 wild-type

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 2), or similar sequence. Amino acid substitutions, each independently, are located in the N-terminal, C-terminal and/or in the inner part of the amino acid sequence of the wild type.

In a preferred embodiment of the invention a variant of the basis has to cysteine substitution at least at the s-end variant. In addition, it is preferable that the last amino acid of the C-end variant of the basis was replaced by cysteine. In addition, a variant of basis, preferably, has a cysteine substitution at least at one or more positions selected from the group consisting of positions corresponding to Arg in the icii 20, Trp at position 25, Ala at position 35 and Ser at position 39 of the basis 4 or basis 3.

In an additional aspect, the present invention relates to a conjugate version of the basis in which one or more molecules of the polymer (preferably, the physiologically acceptable polymer, such as polyalkyleneglycol, more specifically, PEG) conjugated with choice of basis. In the embodiment of the invention, the conjugate contains the data of numerous molecules of the polymer, and the polymer molecule may be the same or not. Preferably, the polymer molecule was conjugated with choice of basis through one or more cysteines. In the embodiment of the invention conjugated to a polymer molecule with a variant basis through a thioester bond.

Experienced it is known that conjugation of the polymer molecule with biomolecules biological activity of conjugated biomolecules decreases exponentially with increasing molecular weight conjugating molecules (for example, starting from 4 kDa) (Bailon et al. Rational design of potent, long-lasting form of interferon: A 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C. Bioconjugate Chem., 2001, 12: 195-202; Bowen et al. Relationship between molecular weight and duration of activity of polyethylene glycol conjugated granulocyte colony-stimulating factor mutein. Experimental Hematology 1999, 27:425-32; Bailon et al. PEG-modified biopharmaceuticals. Expert Opin. Deliv., 2009, 6: 1-16). An experienced specialist from the local, what is the biological half-life and/or the half-life in plasma, as well as systemic availability of conjugated molecules extends or increases gradually with increasing molecular weight conjugating molecules.

However, the inventors have unexpectedly found that, compared with unconjugated choice of basis molecule conjugate version of the basis of the present invention still retains much of the agonist activity of receptor GLP-1 even with increasing molecular weight polymer molecules to 30 kDa. Even with increasing molecular weight of PEG molecules to 40 kDa or above conjugate version of the basis of the present invention still retains significant activity of the receptor agonist of GLP-1. In particular, when increasing the molecular weight of polymer molecules with 5 kDa to 27 kDa, the activity of the conjugate types of basis as agonist of the receptor for GLP-1, essentially unchanged compared with the unconjugated choice of basis.

Therefore, in the preferred embodiment, the present invention relates to a conjugate version of the basis with increased biological half-life and/or half-life in plasma and a significant agonist activity of receptor GLP-1.

In the embodiment of the invention the polymer is polyalkyleneglycol and VK is ucet inter aliafor example, polyethylene glycol, polypropyleneglycol. One or more molecules of the polymer can have any suitable molecular weight, for example, from 2 kDa to 50 kDa, preferably from 5 kDa to 30 kDa, more preferably from 20 kDa to 30 kDa, for example, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa and 30 kDa, as well as any value between the above values of molecular weight.

Optionally, the conjugate version of the basis can also be anywhereman with one or more of the above polymers, the same or not one or more other amino acid residues. One or more amino acid residues may each independently be located in the N-terminal, C-terminal and/or internal option part of the basis.

In the present invention, the polymer for conjugation may be in any appropriate configuration, including, for example, single-beam, dual beam, multibeam and/or branched, in which each of the rays or branches may be the same or not.

The present invention additionally relates to a method for producing a conjugate version of the basis described above, comprising contacting a variant of the basis of the polymer; for conjugation of the activated polymer with one or more cysteine residues in the variant of the basis is predpochtitelno, so at the moment of contact to the polymer was attached reactive group or it was activated. In the embodiment of the invention cysteine in the variant of the basis of specific modified PEG with different length of the polymer chain and the polymer structure by selecting a specific activating groups and the corresponding pH. In a specific embodiment, the invention-specific activating group is maleimide.

The present invention additionally relates to pharmaceutical compositions containing variant of basis and/or conjugate version of the basis of the present invention, and, optionally, pharmaceutically acceptable carrier.

The present invention additionally relates to a method for treatment of diseases involving the introduction of a therapeutically effective amount of a variant of the basis, and/or conjugate version of the basis, and/or pharmaceutical compositions of the present invention, the needy in this subject. Similarly, the present invention also relates to the use of basis, and/or conjugate version of the basis, and/or pharmaceutical compositions of the present invention in the manufacture of a medicinal product for the treatment of diseases. The disease can be selected from the group consisting of postprandial de the ping syndrome, postprandial hyperglycemia, glucose intolerance, disorders or diseases, which may facilitate the inhibition of secretion of glucagon regulation of triglycerides and/or reducing food intake, obesity, eating disorders, insulin resistance syndrome, diabetes, hyperglycemia and hypoglycemia. The preferred disease is diabetes. More preferably the disease is type 1 diabetes or type 2 diabetes, particularly type 2 diabetes.

It is well known that the basis reduces body weight in obese patients and nausea and vomiting, the mechanism of occurrence of which is associated with inhibition of food center and activation of the vomiting center in the Central nervous system (Larsen. Mechanisms behind GLP-1 induced weight loss. Br J Diabetes Vasc Dis 2008; 8: S34-S41; Schick et al. Glucagon like peptide 1 (7-36)-amide acts at lateral and medial hypothalamic sites to suppress feeding in rats. Am J Physiol Regul Integr Comp Physiol 2003; 284:R1427-35). Due to its increased weight conjugate of the basis or its variants cannot cross the blood-brain barrier and therefore causes less vomiting compared to basis. Accordingly, before performing the present invention, the authors naturally expected that the conjugate of the basis or its variants will also show weaker effects in reducing food intake and reduce body weight, TNA is radovanyi the Central nervous system. However, the authors found that the conjugate of the basis or its variants significantly increased the effects of reduced body weight and reduced food consumption, although on the basis of wild-type and unconjugated version of the basis, both reduced body weight and food consumption.

Therefore, in another aspect, the present invention relates to a method of reducing body weight by introducing a conjugate of the basis, or its variant, and/or containing pharmaceutical compositions. In addition, the present invention also relates to the use of the conjugate of the basis, or its variant, and/or containing pharmaceutical compositions in the manufacture of a medicinal product to reduce body weight. In the embodiment of the invention, the conjugate of the basis or its variant is a conjugate version of the basis of the present invention, described above.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows the mass spectrum of the compound PB-105, obtained in example 1.

Figure 2 shows the molecular structure of PEG.

Figure 3 shows the result of HPLC analysis of purity PB-110 (PEG-RV-105)obtained in example 2.

Figure 4 shows the molecular structure 5000b.

Figure 5 shows the molecular structure Pegs.

Figure 6 shows (A) the image obtained by staining with iodine, and (C) the image obtained by staining Ku is Assi diamond blue Paglinawan conjugate PB-105; in the respective tracks are marked: 1) molecular weight standards; 2) PB-106 (PEG-PB-105); 3) PB-107 (PEG-PB-105); 4) PB-108 (PEG-PB-105); and 5) PB-109 (PEG×2-PB-105).

7 shows the result of HPLC analysis of purity PB-106 (PEG-RV-105)obtained in example 3.

On Fig shows the molecular structure 20000b.

Figure 9 shows the molecular structure Pegs.

Figure 10 shows the molecular structure 20000d.

Figure 11 shows the molecular structure Page.

On Fig shows the result of HPLC analysis of purity PB-112 (PEG-RV-111)obtained in example 3.

On Fig shows the result of HPLC analysis, the purity of the RV-107 (PEG-RV-105)obtained in example 4.

On Fig shows the result of HPLC analysis of purity PB-108 (PEG-RV-105)obtained in example 5.

On Fig shows the result of HPLC analysis of purity PB-114 (PEG-RV-113)obtained in example 5.

On Fig shows the molecular structure PEG×2.

On Fig shows the result of HPLC analysis of purity PB-109 (PEG×2-PB-105)obtained in example 6.

On Fig shows the molecular structure PEG×2b.

On Fig shows the molecular structure PEG×2c.

On Fig shows the molecular structure PEG×2d.

On Fig shows the result of HPLC analysis of purity PB-119 (PEG-RV-105)obtained in example 7.

On Fig shows the result of HPLC and the aleesa purity PB-120 (PEG-RV-105), obtained in example 8.

On figa shows the result of HPLC analysis PB-106 (PEG-RV-105) after storage at pH 4.5, -20°C for 60 days.

On FIGU shows the result of HPLC analysis PB-106 (PEG-RV-105) after storage at pH 7.0, 4°C for 60 days.

On figs shows the result of HPLC analysis PB-106 (PEG-RV-105) after storage at pH 7.0, -20°C for 60 days.

On Fig shows the dependence of the dose effects of PB-101 and Tr-105 on the intracellular level of camp in cells RS.

On Fig shows the effects of PB-105 and its Paglinawan conjugate on the intracellular level of camp isin vitro.

On figa shows the relationship between the molecular weight (MW) of PEG in Paglinawan the conjugate and pharmacological activityin vitro(Log EC50).

On FIGU shows the relationship between the molecular weight (MW) of PEG in Paglinawan the conjugate and the maximum pharmacological activity (Emax).

On Fig shows the effect of PB-105 and its Paglinawan conjugate on the intracellular level of camp isin vitro.

On Fig shows the time dependency of the effect of reducing the level of glucose in the blood for PB-101 and Tr-105.

On Fig shows the dependence of the dose effect of reducing the level of glucose in the blood for PB-101 and Tr-105.

On Fig shows the dependence of the dose effect of reducing the level of glucose in the blood for PB-101 and its variants PB-102.

On Fig pok is related to the time dependence of the effect of reducing the level of glucose in the blood for RV-105 and its Paglinawan conjugate.

On figa shows the relationship between the molecular weight of PEG in Paglinawan variant of the basis and the biological half-life (T1/2).

On FIGU shows the relationship between the molecular weight of PEG in Paglinawan variant of the basis and the maximum effect of reducing the level of glucose in the blood (in % of the concentration of glucose in the blood to a dose of connections).

On figs shows the relationship between the molecular weight of PEG in Paglinawan variant of the basis and the area above the curve (AAC) for the reduction curve of glucose in the blood.

On Fig shows the time dependency of the equivalent effect of reducing the level of glucose in the blood for RV-105 and its Paglinawan (PEG) conjugate, RV-107.

On Fig shows the dependence of the dose effect of reducing the level of glucose in the blood for RV-105 and its Paglinawan (PEG) conjugate, PB-106.

On Fig shows the time dependency of the equivalent effect of reducing the level of glucose in the blood for PB-105, RV-111 and Paglierani conjugates. Experimental data are presented as mean ± SEM (standard error of the mean).

On Fig shows the time dependency of the effect of reducing the level of glucose in the blood for PB-101, its variants PB-105, RV-111 and PB-113, and Paglierani conjugates, PB-106, RV-112 RV-114. Experimental data are presented as mean ± SEM.

p> On Fig shows the time dependency of the equivalent effect of reducing the level of glucose in the blood for RV-105 and its Paglinawan conjugate. Experimental data are presented as mean ± SEM. * represents statistically significant difference compared with group RV-105 (p<0,05).

On Fig shows the effects of PB-101, RV-105, RV-106 and RV-120, respectively, to delay vomiting (A) and vomiting (In) in pigeons. Experimental data are presented as mean ± SEM, (a) represents a statistically significant difference in the dosage of 3 mg/kg compared with a group of RV-105 (p<0,05); (b) represents a statistically significant difference in the dosage of 6 mg/kg as compared with the group of PB-101, and with the band PB-105 (p<0,05).

On Fig shows the effects of PB-101, RV-105 and the RV-106 allergic response in systemically immunized Guinea pigs. Experimental data are presented as mean ± SEM, (a) represents a statistically significant difference compared with the group that was injected with saline (p<0,05); (b) represents a statistically significant difference compared with the group of PB-101 or with a group of RV-105 (p<0,05).

On Fig shows the effects on the increase in body weight in Guinea pigs for (A) 0-18 days and (In) 0-4 days after the introduction of PB-101, RV-105 and the RV-106. Experimental data are presented as mean ± SEM, (a) is statistically the reliable difference compared with group, which were injected with saline (p<0,05); (b) represents a statistically significant difference compared with Ecstatica or with a group of RV-105 (p<0,05).

On Fig shows the effects on (A) body weight and (C) food intake in rats after administration of RV-105, RV-106, PB-119 and RV-120. In b and D shows the area under the curve for the curve depending on the weight of the body from time curve and the dependence of the consumption of food from time to time for the respective groups after administration of the compounds. Experimental data are presented as mean ± SEM, (a) represents a statistically significant difference compared with the group that was injected with saline (p<0,05); (b) represents a statistically significant difference compared with group RV-105 (p<0,05).

On Fig shows the dependence of concentration on time with bolus injections of Exenatide and PB-105.

On Fig shows the dependence of concentration on time with bolus injection of PB-105 and Paglinawan conjugate of the basis.

On figa shows the relationship between the MW of PEG in Paglinawan variant of the basis and the half-life in plasma.

On FIGU shows the relationship between the MW of PEG in Paglinawan variant of the basis and the area under the curve (AUC) curve for the dependence of concentration on time.

DESCRIPTION of the PREFERRED embodiments of the INVENTION

The definition is of

Used herein the term "amino acid" includes natural amino acids, unnatural amino acids and analogs of amino acids and their D - and L-stereoisomers. Unnatural amino acids include, but are not limited to this, azetidinone acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoethanol acid, 3-aminoadamantane acid, 2-aminoheptanoic acid, tert-butylglycol, 2,4-aminoadamantane acid, 2,2'-diaminoguanidine acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, gemopolis, hydroxylysine, allowedactions, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alliteration, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylmorphinan, N-methylvaline, elinination, Norvaline, norleucine, ornithine, glycinamide, 2-piperidino acid and tiopronin. Similar amino acids include natural amino acids and unnatural amino acids in which the carboxyl group at the C-end amino group at the N-end or side group reversibly or irreversibly chemically blocked or chemically modified to another functional group, such as, for example, methanesulfonic, methanesulfonic, S-(carboxymethyl)the cyst is h, S-(carboxymethyl)citiinsurance and S-(carboxymethyl)citiinsurance.

Used herein the term "polypeptide" or "protein" are used interchangeably to mean a chain of at least two amino acid residues connected to one another by covalent bonds (such as a peptide bond), which can be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide.

Used herein, the term "cysteine substitution" means the replacement of one or more other amino acid residues in the natural polypeptide (such as on the basis 4) the cysteine residue using genetic engineering or chemical synthesis.

Used herein, the terms "variant polypeptide", "variant" or "analog" refers to a polypeptide in which amino acid sequence is different due to the presence of one or more substitutions, deletions, insertions, m, shortenings, or any combination thereof. The variant polypeptide may be fully functional, or it may be missing one or more functions. Fully functional variant may contain changes, for example, only conservative changes or remains insignificant or unimportant area. The functional variant can contain a substitution of a similar amino what slotow, not producing or resulting in negligible change functions. Functionally important amino acids can be identified by methods known in this field, such as site-directed mutagenesis or glycine-scanning mutagenesis (Cunningham Β. and J. Wells, Science, 244: 1081-1085, 1989). Position, the key activity of the polypeptide can be determined, for example, by structural analysis such as crystallization, NMR or photoaffinity tagging (Smith L. et al., J. Mol. Biol., 224: 899-904, 1992; de Vos, A. et al., Science, 255: 306-312, 1992). The term "tially option" means variants of the polypeptide, in which the thiol is present in the amino acid sequence of the replacement, insertion, merge, or any combination thereof.

Used herein, the term "conjugate" refers to a product formed by covalent or non-covalent connection of the polypeptide or the variant polypeptide and the modifying group of the present invention. The modifying group includes, but is not limited to the above examples.

Used herein the term "modified polypeptide" or "modified variant polypeptide" means a polypeptide or variant polypeptide, in which one or more amino acids, chemically modified, and the modification represents a covalent or non-covalent fashion the classification of different groups, including, but not limited to this, phosphorylation, glycosylation, methylation, Pegylation, biotinylation, SUMO (accession molecule SUMO), acetylation etc

Used herein the term "alkyl" means a substituted or unsubstituted, unbranched or branched alkyl, such as C1-C30-alkyl, C1-C20-alkyl, C2-C15-alkyl or C3-C10-alkyl, optionally substituted by one or more substituents independently selected from the group consisting, for example, from galactography, amino, nitro, etc.

Used herein, the term "cycloalkyl" means substituted or unsubstituted C3-C8-cycloalkyl, optionally substituted by one or more substituents independently selected from the group consisting, for example, of C1-C10-alkyl, C2-C10-alkenyl, C2-C10-quinil, galactography, amino and nitro.

Used in this document, the term "alkenyl" means substituted or unsubstituted, unbranched or branched alkenyl, having one carbon-carbon double bond, which may consist of, for example, 2-20, 3-15, 4-10 carbon atoms, and which optionally may be substituted by one or more substituents independently selected from the group consisting, for example, from galactography, amino and nitro.

COI is Leshey herein, the term "aryl" means a C6-C10-aryl, optionally substituted by one or more substituents independently selected from the group consisting, for example, of C1-C10-alkyl, C2-C10-alkenyl, C2-C10-quinil, galactography, amino and nitro.

Used herein, the term "linker" means an organic group which attaches the PEG to the basis. In the present invention, the linker can be an alkyl, a simple ether, amidon, complex ether, thiol and the like, and the linker may contain, for example, to 30 carbon atoms, e.g., 1-25, 2-20, 3-15 or 3-10 carbon atoms.

In General, as used herein, the term "polyethylene glycol" is set, which usually understands by this term an experienced specialist in this field, and, unless otherwise indicated, includes polyethylene glycolper seand its derivatives with modifications at the ends.

In addition, for a polymer, such as polyethylene glycol, there is a set of methods for determining molecular weight. To represent the molecular weight of the polymer is typically used average molecular weight (in particular, srednetsenovoj molecular weight or srednevekovoi molecular weight), because the polymer is composed of molecules with different degrees of polymerization in the range of the distribution. Srednetsenovoj molecular weight and srednevekovoi molecular weight of usually equal to the of polimerov with a narrow range of distribution, although they may to some extent be different for large differences in the degree of polymerization of the polymers. For polymers such as polyethylene glycol, referred to herein specified molecular weight can be either srednekislovsky molecular weight, or srednevekovym molecular weight.

Variants of basis

In one aspect the present invention relates to a variant of the basis with the activity of the agonist receptor GLP-1, in which one or more (e.g., 1, 2, 3, 4, 5 or more amino acid residues substituted with cysteine in comparison with the sequence of wild-type basis. Cysteine substitutions, each independently, are N-terminal, C-terminal and/or internal parts of the sequence variant of basis. In some embodiments of the invention in the variant of the basis of at least 1, 2, 3, or 4 amino acid residue of its C-terminal part replaced by cysteine. Preferably, a variant of basis had cysteine replacement last amino acids of the C-end. In some other embodiments of the invention it is preferable that the basis had cysteine replacement 1, 2, 3, 4 or more amino acid residues C-terminal, N-terminal and/or internal parts of the sequence variant of basis.

The sequence of the wild-type basis or analogs of the Naya sequence, described in this invention can be any known sequence in this area, including their various variants or analogues, or the sequence of their agonists. Information on the basis can be found, for example, materials Eng J. et al., J. Biol. Chem., 265: 20259-62, 1990; Eng, J. et al., J. Biol. Chem., 267: 7402-05, 1992; WO 00/66629 and WO 00/41546, each of which is fully incorporated herein by reference.

Variant of basis, optionally, may also have one or more (e.g., 1, 2, 3, 4, 5 or more additional modifications of amino acids, including, for example, substitution, deletion, insertion and/or addition of amino acids. Similarly, additional amino acid modification can independently be N-terminal, C-terminal and/or internal parts of the sequence of basis. Replacement, inserted and/or added amino acid can be any natural amino acids, unnatural amino acid or similar amino acids, or D - or L-stereoisomer of the amino acids. In some embodiments of the invention additional amino acid modification is a conservative amino acid substitution, for example, the replacement between Ala/Gly, Ser/Thr, Glu/Asp, Gln/Asn, Ala/Val/Ile/Leu, Arg/Lys, Phe/Tyr, etc. Relatively conservative amino acid substitutions large amount of information present in the prototype, for example, see O/2006/083301, which is fully incorporated herein by reference.

In the present invention the basis of the wild type can be the basis of 3 or on the basis of 4. Therefore, in some embodiments implementing the present invention relates to such variants of the basis in which one or more (e.g., 1, 2, 3, 4, 5 or more amino acid residues substituted by cysteine, compared to

His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 1; the basis 4) or

His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser (SEQ ID NO: 2; basis 3) or a similar sequence. Preferably, a variant of basis had to cysteine substitution at least one or more positions selected from the group consisting of positions corresponding to Arg (arginine) at position 20, Trp (tryptophan) at position 25, Ala (alanine) at position 35 and Ser (serine) at position 39 of the sequences SEQ ID NO: 1 or SEQ ID NO: 2.

In a preferred embodiment of the invention a variant of the basis has an amino acid sequence selected from the group consisting of

As mentioned above, a variant of the basis of the present invention optionally may also have one or more (e.g., 1, 2, 3, 4, 5 or more additional modifications and is inoculat, including, for example, substitution, deletion, insertion and/or addition of amino acids. Additional amino acid modification can independently be located in N-terminal, C-terminal and/or internal parts of the sequence of basis. Replacement, inserted and/or added amino acid can be any natural amino acids, unnatural amino acid or similar amino acids, or D - or L-stereoisomer of the amino acids. In some embodiments of the invention additional amino acid modification is a conservative amino acid substitution, for example, the replacement between Ala/Gly, Ser/Thr, Glu/Asp, Gln/Asn, Ala/Val/Ile/Leu, Arg/Lys, Phe/Tyr, etc.

Variant of basis can be obtained in a number of ways known in this field, including, for example, recombinant methods of preparation, chemical synthesis, etc.

In the embodiment variant of the invention the basis of the synthesized using solid-phase peptide synthesis and then purified on a laboratory scale, for example, one-step purification on reverse-phase HPLC column, or other suitable chromatographic methods.

In another embodiment of the invention a variant of basis receive recombinant means, including, for example, the expression in appropriate prokaryotic or eukaryotic cells, and then the option accendi is and the present invention produce standard techniques. For example, the first chemically synthesized nucleotide sequence encoding the peptide, clone and sequence in an appropriate expression vector for expression under the control of an appropriate promoter. In an alternative embodiment, the nucleotide sequence encoding a variant of a basis, can be obtained from the wild-type basis using mutagenesis, such as PCR mutagenesis, and then the clone sequence in an appropriate expression vector for expression under the control of an appropriate promoter. These techniques are included in the scope of the skills of an experienced specialist in this field and in this field there are many methodological literature.

Suitable eukaryotic cell hosts include mammalian cells, such as Cho, COS, SOME 293, KSS, SK-Hep and HepG2. The cells are preferably grown in conditions that are suitable for expression of the variant of the basis of the present invention. Regarding the reagents and conditions for obtaining or releasing a variant of the basis of the present invention has no specific limitation, and can be any known or commercially available system. In a preferred embodiment of the invention a variant of the basis obtained by the method described in this field.

To obtain a basis and/or option is you can use many expression vectors, you can choose from a group consisting of eukaryotic and prokaryotic expression vectors. Prokaryotic expression vectors may include, for example, plasmids, such as pRSET, pET and pBAD, etc. in which suitable promoters include, for example, promoters lac, trc, trp, recA or araBAD and the like Eukaryotic expression vectors include (i) the vectors used for expression in yeast, such as RAO, pPIC, pYES, RMIT, in which you can use promoters, such as OH, GAP, GAL1, AUG1, etc.; (ii) the vectors used for expression in insect cells, such as RMT, RAS[delta], plB, pMIB, RVs, etc. in which it is possible to use promoters, such as PH, P10, MT, Us, OplE2, gp64, polh, etc.; and (iii) the vectors used for expression in mammalian cells, such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retroviruses and the like, in which it is possible to use promoters, such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin. In a preferred embodiment variant of the invention Express basis in the system of prokaryotic or eukaryotic cells, and use coding sequences after optimization of codons. In a preferred embodiment of the invention, the sequence for the expression of variant, aksen the ina contains a leader peptide and/or a signal peptide to facilitate secretion of the variant of the basis of the cells in the extracellular space, followed by separation and purification. In another preferred embodiment, the sequence for expression of a variant of basis does not contain a leader and/or signal peptide. Variant of basis is not secreted in the extracellular space, and its isolation and purification is performed by lysis of the cells.

Conjugates of the choice of basis

Variant of basis can be konjugierte with one or more polymer molecules, forming a conjugate with the choice of basis. Used in the present invention the polymer is preferably physiologically acceptable and includes a polymer, soluble in an aqueous solution or suspension, and has no negative effects, such as adverse effects on mammals after the introduction of conjugate polymer with a basis in pharmaceutically effective amounts. For polymers that can be used in the present invention, there are no specific limitations. Preferably, the polymers have from 2 to about 3000 duplicate links. Polymer molecule may be selected from natural or synthetic polymers, for example, including, but not limited to these, polysaccharides, polyalkylene glycols such as polyethylene glycol (PEG), polypropyleneglycol (BCP), polyethylene oxide (PEO), a copolymer of ethylene glycol and propylene glycol, polyvinyl alcohol, and any to whom MINALI. In a preferred embodiment of the invention for modification via conjugation in the conjugate version of the basis of the present invention use one or more molecules of PEG.

In the present invention the polymer is not limited to a particular structure and can be linear (such as alkoxy-PEG-or bifunctional PEG), branched or radial (fork PEG or PEG connected with the monomer of polynuclear alcohol), dendritic or to have a degradable linkage. In addition, the internal structure of the polymer can be any type of organization, which can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers and block trimers. The polymer may optionally include polyalkylbenzene polymers, primulinum acid and poly(D,L-alanine).

In some embodiments of the invention the polymer is polyethylene glycol (PEG) or its derivatives, such as methoxypolyethyleneglycol (MPEG). Unless specified otherwise, as used in the present invention, the polyethylene glycol (PEG) includes a PEG with terminal hydroxyl groups and PEG with other terminal groups. These other end groups include, but are not limited to, alkoxy, cycloalkane, cycloalkane-, kenil-, aryloxy or arylalkylamine. Such molecules PEG is widely known in this field and are typically used in the modification of polypeptides. Side chains of PEG may be linear, branched, open-end or radiation. Various glycols can have a different length of the polymer chain and various polymeric structure.

Molecular weight of PEG used in the present invention are not specifically limited, and may be in the range from 0.1 to 200 kDa, for example, from 1 to 150 kDa, from 2 to 100 kDa, from 3 to 80 kDa, or from 4 to 50 kDa, and can be in the range from 5 to 40 kDa. Particularly suitable PEG has a molecular weight in the range from 5 to 30 kDa. Some other suitable molecules include PEG PEG disclosed, for example, in WO 03/040211, US 6566506, US 6864350 and US 6455639. In particular, the PEG has a General formula BUT-CH2CH2O-(CH2CH2O)n-CH2CH2HE, in which n is in the range from 5 to 4000. As described above, the PEG used in the present invention, includes PEG with other terminal groups, for example, methoxide, branched PEG and fork PEG. The corresponding branched PEG can be obtained, as described in U.S. patent No. 5932462, the full contents of which are incorporated herein by reference. Branched PEG is a PEG, which is branched on the phase is near the end of the polymer chain, and the main chain of the branched PEG may be linear or branched.

Experienced specialist in this field it is known that in the case of biologically active molecules, conjugated with molecules of polymers, biological activity of conjugated molecules decreases with increasing molecular weight polymer molecules (Bailon et al. Rational design of potent, long-lasting form of interferon: A 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C. Bioconjugate Chem. 2001; 12:195-202; Bowen et al. Relationship between molecular weight and duration of activity of polyethylene glycol conjugated granulocyte colony-stimulating factor mutein. Experimental Hematology 1999; 27:425-32; Bailon et al. PEG-modified biopharmaceuticals. Expert Opin Deliv. 2009; 6:1-16). Also experienced known that the biological half-life and/or the half-life in plasma increased with increasing molecular weight polymer molecules.

However, it has been unexpectedly found that the present invention is a conjugate version of the basis of the present invention still retains much of the agonist activity of receptor GLP-1 (for example,in vivoactivity) compared to unconjugated choice of basis even when increasing the molecular weight of the polymer molecule (such as PEG) to 30 kDa. Even with increasing molecular weight of PEG molecules to 40 kDa or more conjugate version of the basis of the present invention still retains significant activity of the agonist is of receptor GLP-1 (for example, in vivoactivity). In particular, when increasing the molecular weight of polymer molecules with 5 kDa to 27 kDa, the activity of the conjugate types of basis as agonist of the receptor for GLP-1, essentially remains the same as for unconjugated version of the basis.

To ensure a stable therapeutic effect for a long time and reduce the frequency of dosing to improve patients ' adherence to treatment, it is desirable to maximize the biological half-life of the conjugate version of the basis, while maintaining significant agonist activity of receptor GLP-1. Accordingly, in the embodiment, the present invention relates to a conjugate version of the basis with increased biological half-life and significant activity of the receptor agonist of GLP-1.

In a specific embodiment of the invention the molecular weight of one or more polymer molecules (such as PEG) conjugate version of the basis is from 2 kDa to 50 kDa, preferably from 3 kDa to 40 kDa, more preferably from 4 kDa to 35 kDa, even more preferably from 5 kDa to 30 kDa, for example, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 30 kDa and 40 kDa, as well as any value between the above values of molecular weight. It should be noted that unless otherwise stated, the molecular weight of the polymer molecules, the con is girobank with a conjugate version of the basis, calculated as the total molecular weight of all conjugated polymer molecules in the conjugate, if the conjugate contains more than one conjugated polymer molecules.

In a preferred embodiment of the invention the molecular weight of one or more polymer molecules (such as PEG) conjugate version of the basis is from 20 kDa to 30 kDa, preferably from 21 kDa to 29 kDa, more preferably from 23 kDa to 27 kDa, for example, 20 kDa, 21 kDa, 22 kDa, 23 kDa, 24 kDa, 25 kDa, 26 kDa, 27 kDa, 28 kDa, 29 kDa, 30 kDa, and any value between the above values of molecular weight.

In this area it is known that the polymer used in the present invention, can be obtained by various means, including using a commercial firms, such as CarboMer Ink., J.T. Baker, The Dow Chemical Company, etc.; alternatively it can be obtained by methods known in this field, such as described in ER. The present invention is not limited to polymers, obtained any specific methods.

In the conjugate of the present invention at least one polymer may be attached to the basis through the amino group, carboxyl group, hydroxyl group and/or Tilney group, etc. basis. Such groups are often located in α-amino, α-carboxy and side groups of the amino acid astatke is, such as lysine, aspartate, glutamate, cysteine, etc.

In some embodiments of the invention, one or more polymer molecules are connected with the choice of basis through Tilney group of the cysteine variant of basis. Modification of the polymer, such as polyethylene glycol, thiol of the cysteine residue in proteins can increase the selectivity of the modification process, because there are a number of reagents that are specific reactive Tilney group, and in the protein suitable for modification tirinya groups are much less common free amino groups, for example, lysine residues.

Accordingly, in a preferred embodiment of the invention, one or more molecules of the polymer can be conjugated to a cysteine residue in the variant of the basis, more preferably conjugated to a cysteine residue in the variant of the basis through a thioester bond.

Preferably, the basis had one or more cysteine substitutions at positions corresponding to position 20, position 25, position 35 and/or position 39 basis 3 or basis-4 wild-type and variant of basis was attached to the polymer, such as polyethylene, through Tilney group. More preferably, the variant of basis had to cysteine substitution in the position corresponding to position 35 and/or position 39 basis 3 or accendi the a-4 wild type. Most preferably, the variant of basis had to cysteine substitution at position corresponding to position 39 of basis 3 or basis-4 wild-type.

In a specific embodiment of the invention the polymer molecule conjugated to a cysteine residue in the variant of the basis of the present invention through a thioester bond. For example, in the case of a molecule of polyethylene glycol with maleimides activating group of the thioester bond can be formed by alkenyl of maleimide and the thiol of cysteine for conjugation of one or more molecules of polyethylene glycol with a cysteine residue in the variant of the basis of the present invention. In some other embodiments of the invention Pegylation of cysteine residues can be performed, for example, using PEG-vinylsulfonic, PEG-iodated or PEG-peridiniales. In this area there are a number of ways conjugation of polymer molecules, such as PEG, polypeptides, and all of these methods can be used in the present invention.

Used herein, the terms "Targeted on the basis", "PEG-modified on the basis of" or "PEG-conjugate version of the basis" include the option of basis, conjugated with one or more molecules of PEG. Used in this document the term "Pegylation" or "PEG-modification on the given conjugation of one or more molecules of PEG with basis. A suitable method of Paglierani disclosed, for example, in US 5122614 and US 5539063, in which all of the disclosed methods Paglierani fully incorporated herein by reference.

In some embodiments of the invention, the conjugate has a structure of the following formula I

in which the basis is a variant of the basis of the present invention, Y is N or RPEG-X-, each of X and Z, independently, represent a linker, PEG represents -(co2CH2)n-, n is a positive integer, R represents an end group of PEG, preferably, each R is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, cycloalkenyl, alkenyl, aryl or arylalkyl.

In some specific embodiments of the invention the alkyl can be C1-C6-alkyl, preferably C1-C4-alkyl, such as methyl, ethyl, N-propyl, isopropyl, butyl, isobutyl and tert-butyl; cycloalkyl can be C3-C7-cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl; cycloalkenyl can be cycloalkyl-C1-C4-alkyl, such as cycloalkenyl and cycloalkylation, including cyclohexylmethyl and cyclohexylethyl, and the like; aryl can be phenyl, and were naphthyl, etc.; arylalkyl can be vinylmation, phenylethyl is m and naphthylmethyl, negotiation, etc. In this area known molecules of PEG with different terminal groups, and optionally, you can choose them or other molecules of PEG. The PEG molecules with the desired end group can be synthesized by known methods.

In some embodiments of the invention each element RPEG-" in the above formula I independently has the following structure:

in which k and n are integers, k=0, 1, 2, 3, 4, 5 or 6; n=40, 41,..., 45, 46, 47, 48,......, 1200.

In some other preferred embodiments of the invention each element RPEG-" in the formula (I) independently has the following structure:

in which k and n are integers, k=0, 1, 2 or 3; n=40, 41,..., 45, 46, 47, 48,......, 1200.

In some preferred embodiments of the invention each item "-X" in the formula (I) independently has the following structure:

in which p and m are integers, p=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; m=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

In some other embodiments of the invention each item "-X" in the formula (I) independently has the following structure:

in which p and m are integers, p= 0, 1, 2, 3, 4 or 5; m = 0, 1, 2, 3, or 4.

In some what that other embodiments of the invention, each element is a Z-basis" in the formula (I) independently has the following structure:

Preferably "-Z-basis" in the formula (I) has the following structure:

in which i, j, q, w are integers, i=0 or 1; j=1, 2, 3, 4, 5 or 6; q=1, 2, 3, 4, 5 or 6; w=1, 2, 3, 4, 5 or 6.

Conjugates version of the basis of the present invention can be synthesized by any suitable method. In this area, the number of known methods of conjugation of polymers to proteins or peptides, including incubation polymers (preferably, activated polymers) with a variant of the basis of the present invention. In the embodiment of the invention the polymer is polyethylene glycol, which can be activated and konjugierte with choice of basis, for example, cyanopolyyne way, carbonyldiimidazole way, N-hydroxysuccinimide method, cyanuramide way, etc. In the alternative, the PEG can be specific to konjugierte with the thiol of the cysteine residue in the variant of the basis, using PEG-vinylsulfonic, PEG-iodated or PEG-pyridinylmethyl.

In some specific embodiments the invention, the activated PEG can be incubated with a variant of the basis of the present invention under the following conditions: reaction about the W ill result for 0.5-12 h at pH 5.0-7.0, a molar ratio of PEG to the peptide 1-10, temperature 0-50°C, for example, 2-40°C or 4-37°C.

After the reaction conjugation conjugate can be distinguished by suitable methods. Suitable methods include, for example, ultrafiltration, dialysis, or chromatography, and the like, all of these methods are included in the scope of the skills of an experienced specialist in this field.

The pharmaceutical composition

Variant of basis and/or conjugate version of the basis of the present invention can be used for various purposes, including, for example, lowering the level of glucose in the blood. Therefore, the present invention additionally relates to pharmaceutical compositions for reducing blood glucose comprising a therapeutically effective amount of a variant of the basis and/or conjugate version of the basis of the present invention, and, optionally, pharmaceutically acceptable carrier. Preferably, the pharmaceutical composition was suitable for the treatment of diabetes, more preferably 1 diabetes and/or type 2, in particular, preferably, for the treatment of type 2 diabetes.

A therapeutically effective amount of a variant of the basis and/or conjugate version of the basis of the present invention depends on the route of administration, the type of subject and physiological parameters specific question mammal. About atomu specialist well known for these factors and the relationship between them, as well as a specific amount in a specific case. The amount and route of administration can be selected for optimum effect, resulting in the delivery of peptides to a subject. However, it all depends on known factors, such as body weight, diet, concurrent medication and other factors well known to the experienced Clinician.

The pharmaceutical composition can be entered in the combined treatment, i.e. the pharmaceutical composition is administered in combination with one or more other drugs, and they are administered together or sequentially. In other embodiments of the invention these other medicines can be entered before the introduction, during or after administration of one or more of the choices of basis and/or conjugate version of the basis of the present invention or other pharmaceutical compositions. These other medicines are suitable in the present invention include, for example, drugs to reduce the level of glucose in the blood, such as insulin, insulin analogues, agonists dextrin, cholecystokinin, and/or other compounds or compositions that are used for the treatment of diseases. Preferably, the combined introduction gave a combined or even blue the power of the effect.

Used herein, the terms "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" can be used interchangeably, and they include one or more of all without exception physiologically compatible salts, solvents, dispersion media, coated, antibacterial agents and antifungal agents, isotonic agents and inhibits the absorption of agents, etc. In some embodiments of the invention, the carrier is suitable for intravenous, intramuscular, subcutaneous, spinal or dermal administration (e.g. by injection or infusion). Depending on the route of administration of therapeutic agent can be coated with some material to protect therapeutic agent from acids or other natural environment, which can inactivate therapeutic agent.

With the introduction of the pharmaceutical composition of the present invention is administered in pharmaceutical compositions in pharmaceutically acceptable amounts. The term "pharmaceutically acceptable" means a non-toxic substances that do not disrupt the biological activity of the active components. The structure always contains salts, buffers, preservatives, compatible carrier and, optionally, other therapeutic agents, such as additional amplifiers immune is than, including adjuvants, chemokines and cytokines. For use in drug salt should be pharmaceutically acceptable, salts, which are not suitable for pharmaceutical applications, can be used to obtain pharmaceutically acceptable salt, so they are not excluded from the scope of the present invention.

If necessary, the option of basis or conjugate version of the basis of the present invention can be combined with a pharmaceutically acceptable carrier. Used herein, the term "pharmaceutically acceptable carrier" means one or more compatible solid or liquid fillers, diluents or packaging materials that are suitable for use in mammals, for example humans. The term "media" is organic or inorganic, natural or synthetic components, which are combined with the existing components to facilitate application. The components of the pharmaceutical compositions can also be mixed in the form, in which there is no interaction, significantly violate the required efficiency of a drug.

Preferably, the pharmaceutical composition of the present invention can contain a buffer system, and, preferably, the buffer system was acetate buffer is the first solution with a pH of from about 3.0 to about 6,0, or phosphate buffer solution with a pH from about 5.0 to about 9.0 in. In some certain embodiments of the invention suitable buffer includes acetate, citrate, borate, phosphate.

Optionally, the pharmaceutical composition may also contain suitable preservatives, such as benzalkonium chloride; chloride tert-butyl alcohol; parabens and thimerosal.

The pharmaceutical composition can typically be a standard dose, and it can be obtained by any method well known in the pharmaceutical field. All methods include the stage of the Union of the acting agent of the carrier, and the carrier contains one or more auxiliary components. In General, an active agent and a liquid carrier, a finely powdered solid carrier, or both media are thoroughly mixed to obtain a composition and, if necessary, then give the product form.

Pharmaceutical composition suitable for parenteral administration, can be an aseptic aqueous or non-aqueous composition that contains one or more variants of the basis or conjugates of the choice of basis. In some embodiments of the invention the composition is isotonic relative to the blood of the subjects. To obtain the composition according to the known methods can be used suitable dispersion agent Il is a moisturizing agent and suspendisse agent. Aseptic composition for injection may also be an aseptic aseptic solution or suspension for injection in a non-toxic suitable for parenteral administration in a diluent or solvent, for example, a solution in 1,3-butanediol. Acceptable carriers and solvents that are suitable for use include water, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspension medium is often used aseptic non-volatile oil. Accordingly, it is possible to use any light non-volatile oil, including synthetic monoglycerides or diglycerides. In addition, the composition for injection can be used fatty acids such as oleic acid. The composition of carriers suitable for oral, subcutaneous, intravenous, intramuscular, etc. introduction can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

Variant of basis or conjugate version of the basis of the present invention can be obtained together with a carrier, such as the composition of the controlled release of compounds (including implants, transdermal patches, and system delivery in microcapsules, which protects variant of basis or conjugate version of the basis from rapid degradation. May be suitable biodegradable, biocompatible poly is a career, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. In this area, the number of known methods for obtaining such compounds, see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978, etc.

The pharmaceutical composition of the present invention can be entered by any convenient route, including injection or by gradual infusion. For example, the introduction can be performed oral, intravenous, intraperitoneal, intramuscular, intracavitary, intratumoral, or transdermal means.

The pharmaceutical composition of the present invention is administered in an effective amount. The effective amount is the amount of any variant of the basis or conjugate version of the basis given in this document, which causes the desired response (e.g., reduces the level of glucose in blood of the subject separately or in conjunction with another therapeutic agent. This effect may include only a temporary delay development of diabetes, and alternatively, in some embodiments of the invention, this effect can include a complete halt in the development of diabetes.

This number of course depends on the specific disease against which provides treatment, severity of the disease, the Persian is the national parameters of the patient (including age, physiological status, height and weight), duration of treatment, the properties held at the same time of treatment (if any), certain routes of administration and other factors known to medical practitioners. These factors are well known, experienced, and easy to obtain using conventional experiments. In General, it is preferable to use the maximum dose of each component or combination thereof, that is, the highest safe dose, defined in accordance with the medical appropriateness. However, the skilled person will understand that the patient may require a lower dose or permissible dose for medical, physiological, or any other reason.

Preferably, the pharmaceutical composition used in the above methods, was aseptic and contain an effective amount of a variant of the basis or conjugate version of the basis, by weight or volume suitable for administration to patients, which separately or combined with another composition causes the body to a desired response, such as lowering the level of glucose in the blood.

Input entities dose version of the basis or conjugate version of the basis can be chosen in accordance with different parameters, in particular, in accordance with the route of administration and the state subjectthe factors include the desired duration of treatment. If the answer of the subject is insufficient when used initial dose, it is possible to use a higher dose in the range, the acceptable degree of tolerance of the patient (or you can increase the effective dose due to the different route of administration (local)).

In some embodiments of the invention the pharmaceutical composition contains 0.20 mg/ml ~ 5 mg/ml version of the basis and/or contains 4 mg/ml ~ 40 mg/ml conjugate version of the basis, preferably 0.20 mg/ml ~ 5 mg/ml version of the basis and/or 4 mg/ml ~ 40 mg/ml conjugate version of the basis, more preferably 0.5 mg/ml ~ 2 mg/ml version of the basis and/or 10 mg/ml ~ 20 mg/ml conjugate version of the basis. In General, the dosage range, the choices of basis or conjugate version of the basis of the present invention may be from about 10 μg/kg body weight of the patient to about 100,000 μg/kg body weight of the patient. In some embodiments of the invention, the range of dosages can be from about 0.1 mg/kg to about 20 mg/kg, In other embodiments of the invention, the range of dosages can be from about 0.1 mg/kg to about 5 mg/kg, 0.1 mg/kg to 10 mg/kg or 0.1 mg/kg to 15 mg/kg, In other embodiments of the invention, the range of dosages can be from about 1 mg/kg to 5 mg/kg, 5 mg/kg to 10 mg/kg, 10 mg/kg to 15 mg/kg or 5 mg/kg to 20 mg/kg In other embodiments of the invention, the dosage may be about 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 17 mg/kg, 20 mg/kg, 25 mg/kg or 30 mg/kg In another embodiment of the invention, the dosage may be about 1 mg/kg, 3 mg/kg, 5 mg/kg or 6 mg/kg depending on the composition of the dosage can be entered continuously (for example, using a continuous pump) or periodically. In some embodiments of the invention with intravenous dosage options of basis or conjugate version of the basis of the present invention may be from 0.1 to 20 mg/kg, or can be set to any value in this interval. Experienced specialist without conducting additional experiments can determine the desired interval between the repeated introduction of a specific composition. Experienced other known schemes compositions described herein, in which the dose, map, place, route of administration, etc. may differ from those described above. In the embodiment of the invention, the dosage administered intravenously. In another embodiment of the invention the Protocol introduction provides an introduction to dosing with intravenous bolus.

Also in the scope of the present invention includes a kit containing a variant of the basis is or conjugate version of the basis (for example, in the pharmaceutical composition and instructions. The kit may optionally contain at least one other reagent, for example, one or more drugs to reduce the level of glucose in the blood. In another embodiment of the invention, the kit may contain the base separated for thorough fixation of the container or set of containers, such as vials, tubes, vials, bottles, syringes and the like). The components of the kit can be Packed in water or can be in lyophilized form.

The composition described in this invention may be dried or may be located in the aquatic environment.

Preferably, the subject was vertebrate. More preferably, the subject was a mammal. Most preferably, the subject was a man. However, the subject may be an animal such as an animal companion (e.g., dog, cat etc), pet (for example, cattle, sheep, pigs, horses and the like) or a laboratory animal (e.g., monkey, rat, mouse, rabbit, Guinea pig etc).

Variant of basis and/or conjugate version of the basis of the present invention can be applied individually. However, they are preferably applied in the form of pharmaceutical compositions, which always contains a suitable pharmaceutical is vspomogateljno substance, the diluent or carrier selected depending on the intended route of administration. They can be applied to patients/subjects in need of it, by any suitable means. The exact dosage will depend on various factors, including the exact characteristics of a variant of the basis and conjugate version of the basis.

Some suitable routes of administration include, but are not limited to) oral, rectal, nasal, local (including buccal and sublingual), subcutaneous, vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.

In some embodiments of the invention the pharmaceutical composition according to the present invention contains an isotonic agent and/or preservative, preferably isotonic were one connection or more of sucrose, mannitol, sodium chloride and glycerin and preservative was selected from the group consisting of m-cresol, benzyl alcohol, methyl-p-hydroxybenzotriazol, ethyl-p-hydroxybenzoate, propyl-p-hydroxybenzoate and butyl-p-hydroxybenzoate. Experienced specialist in this field are able to get suitable solution choices of basis or conjugate version of the basis of the present invention, using, for example, isotonic auxil atelinae substance, such as saline, ringer's solution or ringer's solution-lactate, etc. If necessary, you can add a stabilizing agent, a buffering agent, an antioxidant and/or other additive. Pharmaceutical composition for oral administration may be in the form of tablets, capsules, powder or liquid, etc. the Tablet may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical composition always contains a carrier liquid, such as water, petroleum jelly, butter or vegetable oil, mineral oil or synthetic oil. It may also contain saline, glucose or other carbohydrates, or diatomic alcohols, such as ethylene glycol, propylene glycol or polyethylene glycol. In some embodiments of the invention the pharmaceutical composition is a liquid composition and/or lyophilized composition. Preferably, the freeze-dried composition contains a protective agent during lyophilization. More preferably, the protective agent for lyophilization was selected from the group consisting of sucrose, lactose, mannitol, trehalose and other carbohydrates.

Variant of basis and/or conjugate version of the basis is preferable to introduce the subjects in the "therapeutically effective amount" or "effective amount"Kompoziciu preferably entities in a "therapeutically effective amount", a "therapeutically effective amount" or "effective amount" is sufficient to effect the composition of the subjects. The actual number of input composition and the speed and process of the introduction will depend on the condition and severity of the disease entities that will be treated. Treatment (for example, determining the dosage and so on) is assigned to medical professionals, taking into account the diseases against which it is supposed to carry out treatment, individual patient, the introduction of the compound, the route of administration and other factors known to the doctor.

In some embodiments of the invention, the range of dosages for the variant of basis and/or conjugate version of the basis can range from 30 mg/kg of body weight per day to 0,00001 mg/kg of body weight per day, or from 3 mg/kg/day to 0.0001 mg/kg/day or 0.3 mg/kg/day 0.01 mg/kg/day.

The present invention additionally relates to a method of treating a disease, comprising introducing a therapeutically effective amount of a variant of the basis and/or conjugate version of the basis to the needy in this subjects. In some embodiments of the invention the disease is selected from the group consisting of postprandial dumping syndrome, postprandial hyperglycemia, glucose intolerance, obesity, eating disorders,insulin resistance syndrome, diabetes and hyperglycemia. In a preferred embodiment of the invention the disease is type 2 diabetes.

As is well known, the basis can reduce the body weight of obese patients and may cause nausea and vomiting, and the mechanism of action of the basis associated with inhibition of food center and activation of the vomiting center in the Central nervous system (Larsen. Mechanisms behind GLP-1 induced weight loss. Br. J Diabetes Vasc. Dis. 2008; 8: S34-S41; Schick et al. Glucagonlike peptide 1 (7-36)-amide acts at lateral and medial hypothalamic sites to suppress feeding in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 284: R1427-35). Due to the increased molecular weight of the conjugates of the basis or its variants cannot cross the blood-brain barrier and therefore less vomiting compared to basis. Accordingly, before the present invention, the author expected that the conjugates of the basis or its variants will also reduce the effects of basis in respect of reduction in food intake and body weight, mediated by the Central nervous system. However, the author found that the conjugates of the basis or its variants significantly increased the effects of reduced body weight and food consumption, despite the fact that on the basis of wild-type and unconjugated version of the basis, both reduced body weight and food consumption.

Therefore, in another aspect, the present invention relates to a method of the reduction of body weight using the conjugates of the basis or its variants, and/or containing pharmaceutical compositions. In addition, the present invention also relates to the use of conjugates of basis or its variants, and/or containing pharmaceutical compositions in the manufacture of a medicinal product to reduce body weight. In the embodiment of the invention, the conjugates of the basis or its variants represent a conjugate version of the basis of the present invention, described above.

The present invention is further illustrated by examples, which in no way should be considered restrictive. All cited in this application references (including documents issued patents, published patent applications and patent applications, at the same time under consideration) are clearly included in this document in full. Reagents and materials used in the following examples are commercially available products, which at least have analytical purity (or a similar level of purity).

EXAMPLES

Sequence number and a brief description used in the examples of Akindinov, options Akindinov and their conjugates in the table below to facilitate understanding of the technical solutions in the following examples.

The sequence number Short description
PB-101on the basis of 4 wild type, also referred to as Exenatide
PB-102on the basis of 4, with the substitution of amino acids in the 35th position in the Cys
RV-103on the basis of 4, with the substitution of amino acids in the 30th position in the Cys
PB-104on the basis of 4, with the substitution of amino acids in the 25th position on Cys
PB-105on the basis of 4, with the substitution of amino acids in the 39th position in the Cys
RV-106PB-105, conjugated with PEG
RV-106bPB-105, conjugated with PEG, dual beam b
RV-106cPB-105, conjugated with PEG, two-beam Pegs
RV-106dPB-105, conjugated with PEG, dual beam d
RV-106ePB-105, conjugated with PEG, dual beam-Peg
RV-107PB-105, conjugated with PEG
RV-108PB-105, conjugated with PEG
PB-109PB-105, conjugated with PEG×2, dual beam PEG
RV-109bPB-105, conjugated with PEG×2, dual beam b
RV-109cPB-105, conjugated with PEG×2, two-beam Pegs
RV-109dPB-105, conjugated with PEG×2, dual beam d
PB-110PB-105, conjugated with PEG
RV-110bPB-105, conjugated with 5000b
RV-110cPB-105, conjugated with Pegs
RV-111PB-105, Tyr added on To the end
RV-112RV-111 conjugated with PEG
PB-113PB-105, Gly at position 2 substituted with dAla
PB-114PB-113 conjugated with PEG
PB-119PB-105, conjugated with PEG
RV-120PB-105, conjugated with PEG

Example 1. TBE is diesny synthesis of basis-4 and its variants

Polypeptide synthesis is a standard technique in the field of biochemistry and pharmacy. Different types of polypeptide synthesizers are commercially available from a number of commercial organizations (such as GE HealthCare, Applied Biosystems, and the like), and many commercial organizations (such as Sangon Biotech (Shanghai) Co., Ltd., Shanghai Biocolor BioScience&Technology Company) provides services on the synthesis of polypeptides. For example, the polypeptides with a specific sequence can be synthesized by polypeptide synthesizer, using the following procedure.

The basis-4 and its variants with thiol was synthesized using to protect the amino group with Fmoc-strategy, and as a solid substrate resin Fmoc-Rinker Amide MBHA. First stage: carried out the reaction with Fmoc-protected amino acid in the solvent N,N-methylformamide (DMF) for 1-5 h using HBTU/DIPEA as a condensing agent, and the completeness of the completion of the reaction was monitored using the ninhydrin test. Second stage: the reaction was carried out in DMF using 10-30%piperidine as an agent for removing protection for 10-30 min, and completeness of removing the protection of the amino group was controlled by ninhydrin test. Third stage: repeating the first and the second stage using the amino acids corresponding to the target polypeptide sequence before joining the last amino acids in the placenta is successive. Fourth stage: for cleavage of the polypeptide from the solid substrate and simultaneously to remove the protection reaction was performed using TFA as a chip off the agent, within 1-5 hours Fifth stage: after removal of the solution of the polypeptide was besieged by using ethyl ether and was filtered. Collected filtrate. Used chromatography on a C18 column for elution with a solution of 0.1% TFA/acetonitrile in water as mobile phase, to obtain the product was collected and liofilizirovanny faction. Sixth stage: the purity of the product was determined by HPLC and the structure of the product was identified by amino acid sequencing and mass spectrometry.

The author claims ordered in biomedical research company Chengdu Kaijie synthesis of polypeptides by the above method with the sequence below:

PB-101: on the basis of 4 wild type, with the following amino acid sequence:

PB-102: variant of basis 4 with the following amino acid sequence in which at position 35 C-end is Cys:

PB-105: variant of basis 4 with the following amino acid sequence in which at position 39-the end is Cys:

RV-103: variant of basis 4 with the following amino acid sequence in which at position 30 With-the end is Cys:

PB-104: variant of basis 4 with the following amino acid sequence in which at position 25-the end is Cys:

RV-111: variant of basis 4 with the following amino acid sequence in which the position of the 39-end of the substituted Cys and connected with Tyr:

PB-113: variant of basis 4 with the following amino acid sequence in which Gly at position 2 of the N-end replaced by dAla, and the position of the 39-end substituted for Cys:

As an example, figure 1 shows the results of mass spectrometric analysis of the PB-105: peak M+1 (mass + 1) for PB-105 is 4203,3 Yes, some using MALDI-TOF-mass spectrometry, with theoretical mass (4202,8).

Example 2A. Receipt and analysis of the PB-110 (PEG-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 5 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 5000 indicates that the molecular weight of the PEG is about 5 kDa, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding hut is ka solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for further purification.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (column HK/20, macroCap SP packing, GE Inc.), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The analysis was performed using analytical HPLC (Agilent 1200)equipped with a reversed-phase analytical column C4 with pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes When analyzing the sample using an analytical HPLC (Agilent 1200) retention time was 10.4 min, purity 100% (see figure 3). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three columns SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was 97%.

Example 2b. Obtaining and analysis of PB-110b (5000b-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 5 mg 5000b (supplied PegBio Co., Ltd. (Suzhou), and the molecular structure is shown in figure 4) weighed the accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 2A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min), and purity was determined as 96,7%.

Example 2C. Obtaining and analysis of PB-110S (PAGS-RV-102)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 5 mg Pegs (supplied PegBio Co., Ltd. (Suzhou), and the molecular structure is shown in figure 5) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 2A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min), and purity was determined as of 97.8%.

Example 3A. Obtaining and analysis of PB-106 (PEG-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 5 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates the molecular weight of the PEG 20 kDa, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 21 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (column HK/20, macroCap SP packing, GE Inc.), equilibrated with five column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5) and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected solution was analyzed by electrophoresis in LTO-SDS page and staining Kumasi diamond blue and iodine (see figa and 6B). The elution was performed on a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes sample Analysis was performed using analytical HPLC (Agilent 1200), and retention time was 11.5 min, purity 100% (see Fig.7). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with the tandem W hen speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 98,9%.

Example 3b. Obtaining and analysis of PB-106b (20000b-RV-105)

1.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 2.5 mg 20000b (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates that the molecular weight of the PEG 20 kDa, and the molecular structure is shown in Fig) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 3A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as of 98.2%.

Example 3C. Obtaining and analysis of PB-s (Pegs-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5)and 20 mg Pegs (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates that the molecular weight of the PEG 20 kDa, and the molecular structure is shown in Fig.9) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 3A. In the end used the GPC analysis (chromatog is AFE SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 97,2%.

Example 3d. Obtaining and analysis of PB-106d (20000d-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5)and 20 mg Pegs (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates that the molecular weight of the PEG 20 kDa, and the molecular structure is shown in figure 10) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 3A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 97,5%.

Example 3E. Obtaining and analysis of PB-e (Page-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5)and 20 mg Page (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates that the molecular weight of the PEG 20 kDa, and the molecular structure is shown figure 11) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 3A. In what once used GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as the 95.8%.

Example 3f. Obtaining and analysis of PB-112 (PEG-RV-111)

2.0 mg PB-111 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 19 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates the molecular weight of the PEG 20 kDa, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was conducted for 1 hour at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected solution was suirable on reversed-phase analytical C4 column with a pore size 300 E (upiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes sample Analysis was performed using analytical HPLC, and the retention time was 10.6 min, and the purity of 98.5% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 98,7%.

Example 4. Obtaining and analysis of RV-107 (PEG-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 30 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 30000 indicates molecular weight PEG-30 kDa, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced two what hemami column 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected solution was analyzed using gel electrophoresis in the LTO-SDS page and staining Kumasi diamond blue and iodine (see figa and 6B). The elution was performed on a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes sample Analysis was performed using analytical HPLC, and the retention time was 11.5 min, and the purity of 97.3% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 98,7%.

Example 5A. Obtaining and analysis of PB-108 (PEG-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 40 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 40000 indicates molecular weight PEG-40 kDa, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixtures and with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected solution was analyzed using gel electrophoresis in the LTO-SDS page and staining Kumasi diamond blue and iodine (see figa and 6B). The elution was performed on a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes sample Analysis was performed using analytical HPLC (Agilent 1200), and retention time was 11.4 min, purity 100% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed or 1.0 ml/min, and the purity was determined as 97,2%.

Example 5b. Obtaining and analysis of PB-114 (PEG-RV-113)

2.0 mg PB-113 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 40 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 40000 indicates molecular weight PEG-40 kDa, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected elution solution was performed using a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in the MCA and 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes Sample analysis was performed using analytical HPLC, and the retention time was 12.6 min, and clean - 97,9% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as to 98.4%.

Example 6A. Obtaining and analysis of PB-109 (PEG×2(dual beam PEG)-PB-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 40 mg PEG×2 (supplied PegBio Co., Ltd. (Suzhou), 20000 indicates molecular weight PEG 20 kDa single beam, and the molecular structure is shown in Fig) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly men is whether to 100%buffer B (50 mm sodium acetate buffer (pH 4.5), containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected solution was analyzed using gel electrophoresis in the LTO-SDS page and staining Kumasi diamond blue and iodine (see figa and 6B). The elution was performed on a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes When analyzing the sample using an analytical HPLC (Agilent 1200) retention time was 11.5 min, purity 100% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 99,3%.

Example 6b. Obtaining and analysis of PB-109b (PEG×2(dual beam PEG)b-PB-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 40 mg PEG×2b (supplied PegBio Co., Ltd. (Suzhou), 20000×2 indicates the molecular weight of the PEG 20×2 kDa, and the molecular structure is shown in Fig) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 99,2%.

Example 6C. Obtaining and analysis of PB-s (PEG×2(dual beam PEG)-PB-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 40 mg PEG×2C (supplied PegBio Co., Ltd. (Suzhou), 20000×2 indicates the molecular weight of the PEG 20×2 kDa, and the molecular structure is shown in Fig) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 6A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 98,8%.

Example 6d. Obtaining and analysis of PB-109d (PEG×2(two-PEG-d-PB-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 40 mg PEG×2d (supplied PegBio Co., Ltd. (Suzhou), 20000×2 indicates the molecular weight of the PEG 20×2 kDa, and the molecular structure is shown in Fig) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above Rast is ROS. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. Other stages were similar to example 6A. In the end used the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate, the elution was performed at a speed of 1.0 ml/min, and the purity was determined as 99,2%.

Example 7. Obtaining and analysis of PB-119 (PEG-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 22 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 23000 represents a molecular weight of 23 kDa single beam in the PEG, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°C for cleaning.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly m is applicable to 100%buffer B (50 mm sodium acetate buffer (pH 4.5), containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected elution solution was performed using a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes sample Analysis was performed using analytical HPLC (Agilent 1200), and retention time was $ 11.6 min, and clean - 96,0% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as to 97.1%.

Example 8. Obtaining and analysis of PB-120 (PEG-RV-105)

2.0 mg PB-105 was dissolved in 1 ml of 20 mm phosphate buffer (pH 6.5), and 24 mg PEG (supplied PegBio Co., Ltd. (Suzhou), 27000 represents a molecular weight of 27 kDa single beam in the PEG, and the molecular structure is shown in figure 2) were weighed in accordance with the molar ratio of PEG to the peptide as 2:1 and added to the above solution. The solution, respectively shaking to dissolve the PEG and the formation of homogeneous mixture with the peptide. The reaction was carried out for 1 h at 20°C and stopped by adding an excess of a solution of cysteine (0.1 ml of 0.5 M solution of cysteine) and after kept at -20°is to be cleared.

The sample was diluted 5-fold in 50 mm sodium acetate buffer (pH 4.5) and was applied to a SP-ion exchange chromatographic column (GE, column HK/20, macroCap SP packing), equilibrated with 5 column volumes of 50 mm sodium acetate buffer (pH 4.5). After the sediment column was balanced by two column volumes of 50 mm sodium acetate buffer (pH 4.5), and the buffer is then linearly changed to 100%buffer B (50 mm sodium acetate buffer (pH 4.5)containing 1 M NaCl in 20 column volumes. Eluruumis peak was collected AKTA Purifier. It was determined that from about 1 mg of peptide.

The collected elution solution was performed using a reversed-phase analytical C4 column with a pore size 300 E (Jupiter C4 E, a 4.6×250 mm) in a mixture of 0.1%aqueous TFA and 0.1%TFA solution in acetonitrile with a gradient from 61/39 to 54/46 for 10 minutes sample Analysis was performed using analytical HPLC (Agilent 1200), and retention time was 12.1 min, and the purity of 97.7% (see Fig). When using the GPC analysis (in the chromatograph SHIMADZU LC-20AD with three tandem speakers SB-802 HQ/SB-803 HQ/SB-804 HQ) in 0.1 M solution of sodium nitrate the elution was performed at a speed of 1.0 ml/min, and the purity was determined as to 98.4%.

Example 9. A stability test for PE conjugates PB-105

PB-110 (PEG-RV-105), PB-106 (PEG-RV-105), RV-107 (PEG-RV-105), PB-108 (PEG-RV-105) and PB-109 (PEG×2-PB-105) were placed respectively in the sodium-azet the private buffer (pH 4.5) and phosphate buffer (pH 7.0) at 4°C and -20°C, and evaluate their stability. The samples were taken for HPLC analysis on the 7th, 15th, 30th and 60th day. The results for the 60-day showed that the samples were stable at pH 4.5 and -20°C (figa) and at pH 7.0 and 4°C or -20°C (FIGU, 23C).

Example 10.In vitrothe effect of PB-101 and Tr-105 on the level of activity of intracellular camp

Cells RS was trypsinization, were sown on 24-well plate with a density of 105cells/ml and incubated for 48 h (to confluently 60-70%). Culture medium was removed and cells were washed twice in phosphate-saline buffer (PBS). Added 1 ml of PBS containing 1% BSA (bovine serum albumin). Variants of basis-4, RV-101 and Tr-105 (10-11, 10-10, 10-9, 10-8, 10-7and 10-6M) with cysteine at position 39 With the end incubated with 3-isobutyl-1-methylxanthine (IBMX, final concentration 100 μm) for 30 minutes Removed environment for incubation. Added 500 μl of HCl (0.1 M) for stopping the breakdown of cyclic amp enzyme. The cells were collected and literally ultrasound. The protein content in the cells was determined by the ICA method. The construction of the standard curve was performed using a series of groups of standards with Razlog concentration using an instruction set for camp-solid-phase immuno-enzymatic analysis (U.S. RD System corporation). After the reaction, the absorption was determined at 450 nm in a tablet spectrophotometer (U.S. Thermo Fisher Scientific Corporation). Con is entrely camp was obtained from the standard curve, using these absorption values. Then calculate the concentration of camp in the samples. Dose-dependent effect of PB-101 and Tr-105 for increasing the amount of intracellular camp were calculated using the software Graphpad Prizm.

The results of the experiment shown in Fig. PB-101 increased the content of camp in the cells RS dose-dependent manner. The maximum increase of camp (Emax) was 133,2±7.2 pmol/100 μg (protein), and the EU501.9×10-9M RV-105 had a similarin vitrothe effect on the level of camp in cells RS compared with PB-101. The maximum increase of camp (Emaxfor PB-105 was 129,4±6.8 pmol/100 μg (protein) (PB-105 relative to PB-101; P>0,05); EU50was 2.5×10-9M. Additional analysis showed that the values of log EC50for PB-101 and Tr-105 respectively -8,71±0.15 and -8,61±0,15 (PB-105 relative to PB-101; P>0,05). This indicates that the biological activity of PB-101 is not changed as a result of the introduction of a cysteine (thiol) at position 39-end.

Example 11.In vitrothe effect of PB-105 and its Paglierani conjugates on the level of activity of intracellular camp

Cells RS was trypsinization, were sown on 24-well plate with a density of 105cells/ml and incubated for 48 h (to confluently 60-70%). Culture medium was removed and cells were washed twice in phosphate-saline buffer (PBS). obavljale 1 ml PBS, containing 1% BSA. PB-105, Targeted (PEG) conjugate PB-105 (RV-110), Targeted (PEG) conjugate PB-105 (PB-106), Targeted (PEG) conjugate PB-105 (RV-107), Targeted (PEG) conjugate PB-105 (PB-108) and Targeted (PEG×2, with two rays) conjugate PB-105 (PB-109) (at a concentration of 10-11, 10-10, 10-9, 10-8, 10-7, 10-6and 10-5M) were incubated with 3-isobutyl-1-methylxanthine (IBMX, final concentration 100 μm) for 30 minutes Removed environment for incubation. Added 500 μl of HCl (0.1 M) for stopping the breakdown of cyclic amp enzyme. The cells were collected and literally ultrasound. The protein content in the cells was determined by the ICA method. The construction of the standard curve was performed using a series of groups of standards with different concentrations, using the instruction set for camp-enzyme-linked immunosorbent assay (U.S. RD System corporation). After the reaction, the absorption was determined at 450 nm in a tablet spectrophotometer (U.S. Thermo Fisher Scientific Corporation). The concentration camp was obtained from a standard curve using these absorption values. Then calculate the concentration of camp in the samples. Dose-dependent effect of RV-106, RV-107, RV-108, PB-109 and RV-110 for increasing the amount of intracellular camp were calculated using the software Graphpad Prizm.

The results of the experiment indicate that the PB-105 increases included the E. of camp in cells RS dose-dependent manner; the maximum increase of camp (Emax) was to 103.9±1.5 pmol/100 μg (protein), and the EU50was 1.3×10-9M Pegylation of the protein was shifted parallel to the right curve, depending on the dose, regardless of molecular weight (5-40 kDa) and reduced biological activity of PB-105 (Fig). EU50for RS-110, RS-106, RV-107, RV-108 and PB-109 respectively of 1.1×10-9, of 1.1×10-9that 1.2×10-8, 9,7×10-8and 1.3×10-7. The molecule pegylation with PEG 5 kDa (RV-110) and 20 kDa (PB-106) practically did not influence the activity of PB-105 (activity were respectively 115% of the activity of PB-105), and modification of molecules of PEG-30 kDa (RV-107) and 40 kDa (including linear PEG and PEG with two rays - RV-108 and PB-109), respectively, reduced the activity of PB-105 is approximately 90% and 99%. The correlation between the molecular weight of PEG in Paglinawan the conjugate and the activity of these drug compounds (log EC50shown in figa (including EU50for PB-105 on Fig).

Example 12.In vitrothe effect of PB-105 and its Paglierani conjugates on the level of activity of intracellular camp

Cells RS was trypsinization, were sown on 24-well plate with a density of 105cells/ml and incubated for 48 h (to confluently 60-70%). Culture medium was removed and cells were washed twice in phosphate-saline buffer (PBS). Added 1 ml of PBS containing 1% BSA. PB-105, Egulirovannyh (PEG) conjugate PB-105 (PB-119) and Targeted (PEG) conjugate PB-105 (PB-120) (at a concentration of 10 -11, 10-10, 10-9, 3×10-9, 10-8and 10-7M) were incubated with 3-isobutyl-1-methylxanthine (IBMX, final concentration 100 μm) for 30 minutes Removed environment for incubation. Added 500 μl of HCl (0.1 M) for stopping the breakdown of cyclic amp enzyme. The cells were collected and literally ultrasound. The protein content in the cells was determined by the ICA method. The construction of the standard curve was performed using a series of groups of standards with different concentrations, using the instruction set for camp-enzyme-linked immunosorbent assay (U.S. RD System corporation). After the reaction, the absorption was determined at 450 nm in a tablet-the ELISA reader (U.S. Thermo Fisher Scientific Corporation). The concentration camp was obtained from a standard curve using these absorption values. Then calculate the concentration of camp in the samples. Dose-dependent effect of PB-105, PB-119 and RV-120 for increasing the amount of intracellular camp were calculated using the software Graphpad Prizm.

The results of the experiment indicate that the PB-105 increases the amount of camp in the cells RS dose-dependent manner, and the EU50was 2.7×10-9M. Modification in PB-106 (PEG) and PB-119 (PEG) had no effect on the activity of PB-105 in relation to the camp, and modification in the RV-120 (PEG) moved the curve depending on the dose, parallel to the right and reduced the biological activity of PB-105 in relation to camp approx the RNO 50% (Fig). Size EU50for PB-106, PB-119 and RV-120 respectively of 1.5×10-9and 2.5×10-9and 5.4×10-9.

Usually it is believed that the biological activity of conjugated biomolecules decreases exponentially with increasing molecular weight conjugating group (for example, from 4 kDa) (Bailon et al. Rational design of potent, long-lasting form of interferon: A 40 kDa branched polyethylene glycol-conjugated interferon α-2a for the treatment of hepatitis C. Bioconjugate Chem. 2001; 12: 195-202; Bowen et al. Relationship between molecular weight and duration of activity of polyethylene glycol conjugated granulocyte colony-stimulating factor mutein. Experimental Hematology 1999; 27: 425-32; Bailon et al. PEG-modified biopharmaceuticals. Expert Opin Deliv. 2009; 6: l-16). However, the inventor has unexpectedly discovered (shown in Fig and 27)that the relationship between molecular weight conjugating polymer group and the ability of the conjugate version of the basis to promotein vitrothe level of camp does not meet this assumption. At least for the variant of the basis conjugated to PEG with molecular weight up to 23 kDa Pegylation had no effect on the ability of the conjugate version of the basis to promotein vitroproduction of camp in the cells (and when the molecular weight PEG has reached 27 kDa, was observed only a weak effect). In contrast, Pegylation had no effect on the maximum effect of PB-105 in relation to stimulating the production of camp (Emax). Emaxfor 110 PB, PB-106, RV-107, RV-108 and PB-109 pillar is and accordingly 102,1±1,8, 111,9±2,1, 126,2±3,4, 100,4±1,7 and of 115.5±3.5 pmol/100 μg protein. Molecular weight of PEG in Paglierani the conjugates did not correlate with Emaxthese medicinal compounds (see figv) (including Emaxfor PB-105 on Fig).

Following the experiments, the inventor conducted on the basis of this unexpected result. Because the medium containing serum, in which there are many proteases, are not used as the reaction system in this example, and the reaction solution did not add any proteases, enzymatic destruction of choices of basis and its conjugates was significantly reduced compared with the situationin vivo. In other words, in cases where a longer testin vivothe degree of decrease in the biological activity of conjugates version of the basis of the present invention will be less relative to the unconjugated basis of wild-type or variant of basis, even if the biological activity of conjugates version of the basis of the present invention is higher. In another aspect, the polymer molecule with a weight in excess of 23 kDa, can be used for conjugation with the choice of basis, substantially without affecting its biological activityin vivo. Although this theory does not limit the invention, this hypothesis is confirmed by the following examples.

Example 13. C the dependence of hypoglycemic effect of PB-101 and Tr-105 from time

Used Kunming male mice (body weight 27-32 g). Before the experiment, the mouse was not a lack of food or water. Mice were divided randomly in three groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of equal volumes of saline (10 ml/kg), PB-101 (10 mg/kg) and choices of basis RV-105 with cysteine at position 39-end (10 µg/kg). Through 0, 1, 2, 4, 8, 12 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of time-delayed values of glucose in the blood at various time points along the y-axis, and time points on the x-axis and calculated biological half the duration of the hypoglycemic effect of PB-101 and Tr-105. The results are shown in Fig. The half-life version of the basis-4, substituted by cysteine, and basis 4 respectively of 4.7±0.2 h and 4.4±0.2 h (PB-105 relative to PB-101; P>0,05). This indicates that the version of the basis-4, substituted by cysteine at position 39-end, and on the basis of 4 have similar half-life.

Example 14. The dependence of the hypoglycemic effect of PB-101 and Tr-105 dose

Used Kunming male mice (body weight 23-27 g). Preexperiment mouse received no food for 3 hours, and water was given freely. Mice were divided randomly in three groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of equal volumes of saline (10 ml/kg), PB-101 (0,01, 0,1, 0,3, 1, 3, 10, 100 µg/kg) and choices of basis RV-105 with cysteine at position 39-end(0,01, 0,1, 0,3, 1, 3, 10, 100 µg/kg). After 1 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of dose was delayed values of glucose levels in the blood on the y-axis, and used a dose of x-axis (Fig). Then calculate the parameters of the dependence of the effect of dose for PB-101 and Tr-105 (Emaxand ED50using the software Graphpad Prizm. The results indicated that the maximum hypoglycemic efficacy in bolus injection of PB-101 and Tr-105 respectively 32,2% and 36.1%, and the values of the ED50were respectively 0.6 and 1.2 µg/kg Additional analysis showed that log ED50for PB-101 and Tr-105 respectively -0,25±0,17 and 0.08±0,20 (PB-105 relative to PB-101; P>0,05). The results of the experiment indicated that the version of the basis-4, RV-105, with the cysteine at position 39-end, essentially no different from the basis 4 in which the compared hypoglycemic effect.

Example 15. The dependence of the hypoglycemic effect of PB-101 and its variants (PB-102) from dose

Used Kunming male mice (body weight 22-26 g). Before the experiment, mice received no food for 3 hours, and water was given freely. Mice were divided randomly in 18 groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of equal volumes of saline (10 ml/kg), PB-101 (0,01, 0,1, 1, 10, 100 µg/kg) and variant basis of PB-102 with cysteine at position 35-end(0,01, 0,1, 1, 10, 100 µg/kg). After 1 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of dose was delayed values of glucose levels in the blood on the y-axis, and used a dose of x-axis (Fig). Then calculate the parameters of the dependence of the effect of dose for PB-101 and Tr-102 (Emaxand ED50using the software Graphpad Prizm. The results indicated that the maximum hypoglycemic efficacy for bolus injection of PB-101 and Tr-102 respectively 39.8% and 32.8 per cent, but the ED50were respectively 0.5 and 2.5 µg/kg Additional analysis showed that log ED50for PB-101 and Tr-105 respectively -0,2867±0,272 and 0,4015±0,2946 (PB-102 relative to PB-101, P>0,05). The results of the experiment indicated that the version of the basis-4, PB-102, with the cysteine at position 35 With the end, essentially no different from the basis-4 in respect of hypoglycemic effect.

Example 16. The dependence of the hypoglycemic effect of equal amounts of PB-105 and Paglierani conjugates from time

Used Kunming male mice (body weight 22-26 g). Before the experiment, the mouse was not a lack of water and food. Mice were assigned randomly to six groups of 12 mice in each group. Each mouse was injected subcutaneous bolus injection of equal volumes of PB-105 (10 µg/kg), Paglinawan (PEG) conjugate PB-105 (RV-110) (10 µg/kg), Paglinawan (PEG) conjugate PB-105 (PB-106) (10 µg/kg), Paglinawan (PEG) conjugate PB-105 (RV-107) (10 µg/kg), Paglinawan (PEG) conjugate PB-105 (PB-108) (10 µg/kg) and Paglinawan (PEG×2, with two rays) conjugate PB-105 (PB-109) (10 µg/kg). Through 0, 1, 2, 4, 8, 12, 18, 24, 36 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of time-delayed values of glucose in the blood at various time points along the y-axis, and time points on the x-axis (Fig) and was calculated biologist who for the half-life and maximum hypoglycemic effect of PB-105 and its Paglierani conjugates, as well as the area above the curve hypoglycemic effect (table 1). As can be seen from table 1, each conjugate in this experiment had a similar or even significantly higher (PB-106) total hypoglycemic effect (see the area above the curve), and these conjugates choices of basis had significantly greater biological half-life compared to unconjugated PB-105. Analysis of the molecular weight of the PEG relative to the biological half-life, maximum hypoglycemic effect and the area above the curve hypoglycemic effect indicates that Paglierani conjugates PB-105 - PB-106, RV-107, RV-108 and PB-109 - all significantly increase the duration of hypoglycemic effect of PB-105 (half-life, t1/2). However, when the molecular weight of PEG in the range of 5-20 kDa increase in biological half-life is proportional to molecular weight, and when the molecular weight of the PEG above 20 kDa duration of hypoglycemic effect (biological half-life) remains unchanged (figa). Paglierani conjugates have the same maximum hypoglycemic effect when the molecular weight of PEG in the range of 5-20 kDa, but when the molecular weight of the PEG is greater than 20 kDa maximum hypoglycemic effect decreases with increasing molecular weight of PEG (pigv). The ratio of the area under the curve of the ISU the glycemic effect, only PB-106 (PEG 20 kDa) had significantly higher total hypoglycemic effect with respect to PB-105, and other conjugates had similar or slightly smaller effect (figs). As can be seen from the above experiments, site-specific modification by Pegylation (RV-110 RV-106) does not significantly affect the maximum hypoglycemic effect of conjugates of choices of basis (respectively, 96% of the PB-105) at least when the molecular weight of the PEG does not exceed 20 kDa. Modification by Pegylation significantly reduces the maximum hypoglycemic effect when the molecular weight of the PEG 30 kDa or even higher (RV-107, RV-108 and PB-109), although these conjugates are still relatively similar total hypoglycemic effect and a much longer biological half-life. These results indicate that conjugating a molecule with higher molecular weight may be suitable for the production of conjugates choice of basis. In fact achieved a significant increase in biological half-life and a more lenient regulation, as well as a more stable level of glucose in the blood to prevent too sharp drop in glucose level for a short period of time and prevent fluctuations of glucose levels in a large range. These data are consistent with the results of the, obtained inin vitrothe experiments measuring the level of camp, described above (see Fig).

Table 1
The biological half-life, maximum hypoglycemic effect and the area above the curve is a reduced level of glucose in the blood (AAS) for PB-105 and its Paglierani conjugates (12 mice in each group)
Conjugates of basisT1/2(watch)The maximum hypoglycemic effect (% of value up to a dose of connections)The area under the curve of glucose in the blood (AAS, mmol·h/l)
PB-1054,9±0,138,0±4,332,8±5,4
PB-1107,0±2,036,3±1,2of 31.8±4,6
PB-10613,4±0,5*36,5±3,253,9±4,3*
PB-10712,5±2,0*24,3±2,8*30,0±7,2
PB-108 10,8±2,0*22,6±2,9*25,3±5,7
PB-1099,2±2,2*20,2±3,2*21,6±4,8
*P<0,05 (relative to PB-105)

Example 17. The time dependent hypoglycemic effect equivalent quantities of PB-105 and its Paglinawan (PEG) conjugate (RV-107)

Since conjugate with PEG (RV-107)in vitroreduced biological activity of PB-105 is approximately 90% (Fig), andin vivoreduced biological activity of PB-105 approximately 50% (Fig), the dose of PB-107 increased to study the biological half-life of PB-107 in terms of its equivalence with the PB-105. Used Kunming male mice (body weight 22-25 g). Before the experiment, the mouse had no lack of food or water. Mice were divided randomly in two groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of PB-105 (10 µg/kg) and Targeted (PEG) conjugate PB-105 (RV-107) (100 μg/kg, this dose was given about 100% of the hypoglycemic effect with respect to 10 mcg/kg PB-105). Through 0, 1, 2, 4, 8, 12, 18, 24, 36, 48, 72 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device the TSS test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of time-delayed values of glucose in the blood at various time points along the y-axis, and time points on the x-axis, and calculated biological half-life of PB-105 (10 µg/kg) and PB-107 (100 µg/kg). The results are shown in Fig, biological half-life of PB-105 (10 µg/kg) and PB-107 (100 µg/kg), respectively 4,5 ħ 0,4 h and 44.6±4.5 hours (P>0,05, RV-107 relative to the PB-105). The study of the dependence effect from time to equivalent doses indicates that Pegylation (PEG) (RV-107) increases the duration of hypoglycemic effect of PB-105 ten times.

Example 18. The dependence of the hypoglycemic effect of PB-105 and its Paglinawan (PEG) conjugate (PB-106) from dose

Used Kunming male mice (body weight 20 to 24 g). Before the experiment, mice received no food for 3 hours, but water was given freely. Mice were divided randomly in 13 groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of equal volumes of saline (10 ml/kg), PB-105 (0,1, 0,3, 1, 3, 10, 30 µg/kg) and an equal dose Paglinawan (PEG) conjugate PB-106 (0,1, 0,3, 1, 3, 10, 30 µg/kg). After 1 h after injection for the RV-105 group (time point of the peak reduction of glucose, see Fig and 31) and after 4 h after injection for the RV-106 group (temporary point is and the maximum reduction of glucose, see Fig) collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of dose was delayed values of glucose levels in the blood on the y-axis, and used a dose of x-axis (Fig). Then calculate the parameters of the dependence of the effect of dose for RV-105 and the RV-106 (EminEmaxand ED50using the software Graphpad Prizm. Eminfor bolus injection of PB-105 and the RV-106, respectively of 8.3±0.2 and an 8.4±0.3 mmol/l; and Emaxrespectively 6,0±0,3 and 5.5±0.6 mmol/l (maximum hypoglycemic efficacy, respectively, was 27.8% and 34,5%). The values of the ED50were respectively 1.2 and 3.3 µg/kg Additional analysis showed that log ED50for PB-105 and the RV-106 respectively 0,07±1.2 and 0,5±0,2 (PB-106 relative to the PB-105; P>0,05). The results of the experiment indicate that Paglierani (PEG) conjugates PB-105 (PB-106), essentially no different from the PB-105 against hypoglycemic effect (including Emaxand ED50).

Example 19. The time dependent hypoglycemic effect of PB-105, RV-111 and Paglierani conjugates (PB-106 and RV-112) in normal mice

Used Kunming male mice (body weight 24-30 is). Before the experiment, the mouse was not a lack of water and food. Mice were divided randomly in four groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of PB-105 (10 µg/kg), Targeted (PEG) conjugate PB-105 (PB-106) (10 µg/kg), PB-111 (10 µg/kg) and Targeted (PEG) conjugate RV-111 (PB-112) (10 µg/kg). RV-111 is a derivative of PB-105, in which the cysteine at position 39 With the end connected with tyrosine. Through 0, 0,5, 1, 2, 4, 8, 12, 24, 48 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of time-delayed values of glucose in the blood at various time points along the y-axis, and time points on the x-axis, and calculated biological half-life and maximum hypoglycemic effect of PB-105, RV-111 and Paglierani conjugates. The results are shown in Fig, reducing random glucose level in the blood under the action of PB-105, RV-106, RV-111 and RV-112 depended on time. The biological half-life of PB-105 and the RV-111 respectively of 4.6 h and 6.0 h, and the maximum hypoglycemic effect was accounted for 44.7 per cent and 36.4 per cent. The biological half-life of PB-106 and RV-112 was appropriate to estwenno 19,6 h and 21.7 h, and the maximum hypoglycemic effect was accounted for 37.3% and 34.9%, respectively. The results indicate that the attachment of tyrosine to the 39th position-the end has no effect on the hypoglycemic effect and the biological half-life of PB-105 and its Paglinawan conjugate (PB-106).

Example 20. The time dependent hypoglycemic effect of PB-101 and options PB-105, RV-111 and PB-113, and Paglierani conjugates RV-106, RV-112 RV-114 in mice with STZ-induced diabetes

Before induction of diabetes in mice fasted for 14 hours Selected sample of blood from the tip of the tail to determine the baseline level of glucose in the blood. Then were injected subcutaneous bolus injection of freshly prepared STZ (streptozotocin) (120 mg/10 ml/kg, freshly dissolved in 0.1 M nitrate solution, pH 4.5). The level of glucose in blood was determined after 3 days. If mice random glucose level in the blood exceeded at 16.7 mmol/l, the induction of diabetes was considered successful. Before the experiment the mouse had no lack of food or water. The mice were divided into 7 groups of 4-6 mice in each group. The level of glucose in the blood at the initial time point (0 h) was determined in a blood sample from the tail vein. Each mouse was injected subcutaneous bolus injection of equal volumes of PB-101 (10 mg/10 ml/kg), PB-105 (10 µg/kg), PB-106 (10 µg/kg), PB-111 (10 µg/kg), PB-112 (10 µg/kg), PB-11 (10 mg/kg) and PB-114(10 µg/kg). RV-111 is a derivative of PB-105, in which the cysteine at position 39 With the end connected with tyrosine, PB-113 is a derivative of PB-105, in which the glycine at position 2 of the N-end substituted D-alanine, and PB-114 is a Targeted (PEG) conjugate PB-113. Blood samples were taken at time points: 0, 0,5, 1, 2, 4, 8, 12, 24, 48 h after injection and was determined by the glucose in the blood. Curve based hypoglycemic effect of time for PB-101, RV-105, RV-106, RV-111, RV-112, PB-113 and PB-114 constructed by plotting values of glucose levels at different time points along the y-axis, and used time point along the axis X. the curve tracing was performed using the software GraphPad Prizm 5 Demo (Prizm v5, GraphPad Software, Inc., San Diego, CA), and was calculated and compared the biological half-life (t1/2for PB-101, RV-105, RV-106, RV-111, RV-112, PB-113 and PB-114 using methods of mathematical statistics.

As shown in Fig, reducing random glucose level in the blood in mice with STZ-induced diabetes in the bolus injection of PB-101, RV-105, RV-106, RV-111, RV-112, PB-113 and PB-114 depended on time. The maximum efficiency was, respectively, 47,5%, 57,6%, 69,8%, 54,4%, 59,7%, 49,2% and 17.9% (38% for PB-101). The biological half-life for PB-101, RV-105, RV-106, RV-111, RV-112 RV-113 respectively 5,5, 5,5, 21,8, 5,0, 20,3 and 5.9 h and biological engineering who nd the half-life for PB-114 was impossible to calculate due to hypoglycemic effects. The experimental results indicate that the addition of tyrosine to the position of the 39-end, or replacement of glycine, D-alanine at position 2, N end do not reduce protiwaritmicescuu activity PB-105, Pegylation (PEG) does not reduce activity of PB-105 and its derivatives, as Pegylation (PEG) significantly reduces the activity of PB-105 and its derivatives.

Example 21. The dependence of the hypoglycemic effect from time to equivalent amounts of PB-105 and the RV-106, PB-119 and RV

Used Kunming male mice (body weight 24-30 g). Before the experiment, the mouse was not a lack of water and food. Mice were divided randomly in four groups with 6 mice in each group. Each mouse was injected subcutaneous bolus injection of PB-105 (10 µg/kg), Targeted (PEG) conjugate PB-105 (PB-106) (10 µg/kg), Targeted (PEG) conjugate PB-105 (PB-119) (10 µg/kg) and Targeted (PEG) conjugate PB-105 (PB-120) (10 µg/kg). Through 0, 0,5, 1, 2, 4, 8, 12, 24, 48 h after injection of collected blood samples from the tail. The glucose content in the blood was determined using the OneTouch glucometer and attached to the device test strips (U.S. Johnson & Johnson). To build a curve of hypoglycemic effect of time-delayed values of glucose in the blood at various time points along the y-axis, and time points on the x-axis, and calculated biological Perry is on half-life and maximum hypoglycemic effect for the RV-105 and its Paglierani conjugates. The results are shown in Fig, reducing random glucose level in the blood in mice as a result of PB-105, RV-106, PB-119 and RV-120 depended on time. Their biological half-life was, respectively, 6,0, 21,7, at 24.1 and 26.1 h, and the biological half-life increased with the increase of molecular weight of PEG. Peak time hypoglycemic effect was approximately 1 h (PB-105) and 4 h (PB-106, PB-119 and RV-120). Peak hypoglycemic effect was accordingly 35,0%, up 50.9% 48.2% and 42.2 per cent (figa). On the calculated area under the curve hypoglycemic effect from time RV-106, PB-119 and RV-120 all significantly increased hypoglycemic effect of PB-105 (3-4 times). And the total effect was increased with increasing molecular weight (pigv). The results of the experiment indicate that Pegylation does not reduce the hypoglycemic activity of PB-105 and significantly increases the duration of hypoglycemic effect and increases the total hypoglycemic effect in molecular weight in the range of 20-27 kDa. The results show that when the molecular weight of the PEG is in the range 20-27 kDa conjugates variants of the basis of the present invention, the RV-106, PB-119 and RV-120, have a higher peak hypoglycemic effect compared to unconjugated PB-105. In addition, these to Nyugati have much more prolonged hypoglycemic effect, and the total hypoglycemic effect increases with increasing molecular weight of PEG, which provides a better combination of hypoglycemic effect.

Example 22. Testing of PB-101, RV-105, RV-106 and RV-120 on the ability to induce vomiting in pigeons

Healthy pigeons, females and males were divided into 7 groups of 4-8 pigeons in each group. PB-101 (3 mg/kg, N=4 or 6 mg/kg, N=8), PB-105 (3 mg/kg, N=4 or 6 mg/kg, N=8), PB-106 (3 mg/kg, N=4 or 6 mg/kg, N=8) and RV-120 (6 mg/kg, N=8) were injected separately using bolus injections subcutaneously. The duration of vomiting and delayed vomiting (interval from dose to first vomiting) were observed and recorded using the electronic monitoring system within 24 h after injection of the compounds. Time vomiting was determined, starting with stretching of the neck, mouth opening, shake the limbs, cuts peritoneum and to soothe or finish vomiting. From previous experiments it was known that vomiting does not occur in normal, for example, without the introduction of compounds, and the introduction of PB-101 (3 and 6 mg/kg) and PB-105 (3 and 6 mg/kg) causes a significant vomiting depending on the dose. For PB-106 (3 and 6 mg/kg) and PB-120 (3 and 6 mg/kg) latency vomiting increased significantly (approximately 5-18 times, see figa), and the vomiting was significantly reduced (reduced by approximately 50-70%, see figv) compared to groups that were introduced to meet the appropriate dose of PB-101 and Tr-105. For dosage of 6 mg/kg of the differences were statistically significant (p<0,05). The results indicate that Pegylation significantly reduces vomiting caused by basis and its options.

Example 23. Systemic allergic reaction and effects on body weight, called PB-101, RV-105 and the RV-106 in Guinea pigs

Used 44 male Guinea pigs, and the body weight of each Guinea pig was approximately 300 g were divided into 5 groups, namely the group that was injected with saline (N=10), PB-101 (N=10), PB-105 (N=10), PB-106 (N=10) and group, which was administered albumin (N=4). Saline (1 ml/kg), PB-101 (100 µg/kg), PB-105 (100 µg/kg), PB-106 (100 µg/kg) and chicken ovalbumin (80 µg/kg, respectively) were injected via subcutaneous injection three times a day. 14 days after last injection stimulating amount of saline (1 ml/kg), PB-101 (300 µg/kg), PB-105 (300 µg/kg), PB-106 (300 µg/kg) and chicken ovalbumin (240 µg/kg), respectively, were administered by injection into a vein of the toe. Immediately after injection was observed response in animals within 0-3 h after stimulation. Symptoms of an allergic reaction in animals were recorded, as shown in table 2, and evaluated, as shown in table 3 (semi-quantitative).

Symptoms of an allergic reaction in animals
Table 2
0 - Norm7 - Shortness of breath14 - allure
1 - Concern8 - Urination15 - Jump
2 - stirs9 - Defecation16 - Temporary relief
3 - Shake10 - Tears17 - Spasms
4 - Cards nose11 - Shortness of breath18, Whirl
5 - Sneezing12 - Rales19 - shallow breathing
6 - Cough13 - Purpura20 - Death

Table 3
Assessment of the degree of systemic allergic reactions in animals and semiquantitative standard
0-0Negative allergic reaction
Symptoms 1-4+1A weak negative allergic reaction
Symptoms 5-10++2Positive allergic reaction
Symptoms 11-19+++3Strong positive allergic reaction
20++++4A very strong positive allergic reaction

The curve constructed by plotting the time points on the x-axis and the degree of allergic response (semi-quantitative assessment) on the y-axis, as shown in Fig. The pigs of groups, which were injected with saline did not experience any allergic reaction, that is, they showed a negative allergic reaction. Pigs of groups, which were injected albumin, died immediately after stimulation (for a period of time less than 2 min), that is, they had a very strong positive allergic reaction. In Guinea pigs of groups, which were injected PB-101 and Tr-105, was observed in the immune response, i.e. a positive allergic reaction. PB-101 and Tr-105, not only at the Ute polypeptides with 39 amino acid residues in length, which may cause the production of antibodiesin vivoafter the introduction of compounds in a long time (Buse et al. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes.Diabetes Care. 2004; 27: 2628-2635). And Targeted (PEG) conjugate PB-105 (PB-106) resulted in weak negative allergic reactions in Guinea pigs. This result indicates that Pegylation (PB-106) can significantly reduce the immunogenicity of PB-101 or RV-105 and allergic reaction.

Curve body weight of Guinea pigs (y-axis) against time (x axis) (Fig) indicates that the body weight of the Guinea pigs in the group, which was administered saline was continuously increased during sensitization (body weight increased by about 38% over the 18-day experiment). Body weight did not decrease after 4 days after two consecutive doses of albumin (compared with the group that was injected with saline). However, the body weight of the pigs, respectively, were decreased by approximately 8% and 11% (p<0.05) after 4 days after two consecutive doses of PB-101 (100 µg/kg) or RV-105 (100 µg/kg). The body weight of Guinea pigs was lower after administration of an equivalent amount of PB-106 (compared to PB-101 or RV-105). And the dose of PB-106 led to additional decrease, respectively, by approximately 8% and 11% (p<0,05) compared the structure with the introduction of PB-101 and Tr-105. The body weight of the Guinea pigs in the groups treated with PB-101, RV-105 and the RV-106, recovered 12 days after discontinuation of compounds to values comparable with the weight of animals in the group treated with saline.

Example 24. The effect of PB-105, RV-106, PB-119 and RV-120 on the body weight of the rats and the amount of food consumed

In the experiment used 24 healthy male SD rats, weighing approximately 200 g Rats were divided into 5 groups: saline (1 ml/kg, N=4), PB-105 (100 µg/kg, N=5), PB-106 (100 µg/kg, N=5), PB-119 (100 µg/kg, N=5) and RV-120 (100 µg/kg, N=5). Saline and drug compounds were administered subcutaneously by injection every other day three times. The body weight of rats and food consumption were observed every day.

Curve body weight (y-axis) against time (x axis) (figa) showed that the body weight of rats in the group that was injected with saline solution, continuously increased during the introduction (body weight increased by 33.4% over the 9 days of the experiment). Compared with the group that was injected with saline, body weight (an indicator of body weight was the area under the curve (AUC) was decreased by approximately 5.3% (p<0.05) after 9 days after successive three times the introduction of PB-105 (100 µg/kg). The introduction of an equivalent amount of PB-106, PB-119 and RS-120 has led to greater weight reduction relative to PB-105. Body weight (a measure of weight t is La was AUC), respectively, were decreased by about 7%, 8% and 8% (p<0.05) in the case of the introduction of PB-106, PB-119 and RV-120 compared with the introduction of PB-105.

The curve on the x-axis which delayed time points, and the y - axis the amount of food consumed (pigv)showed that food intake in rats, which were injected with saline, remained the same. Compared with physiological solution, the amount of food consumed (an indicator of food consumed was AUC) was decreased by approximately 16% after three successive introduction of PB-105 (100 µg/kg). The introduction of an equivalent amount of PB-106, PB-119 and RS-120 has led to greater reduction in food intake relative to PB-105. A further reduction of 18%, 19% and 19% (p<0,05) was obtained in the case of the introduction of PB-106, PB-119 and RV-120 compared with the introduction of PB-105.

It is well known that exenatide can reduce body weight in obese patients and cause nausea and vomiting, and the mechanism of their action is associated with inhibition of food center and activation of the vomiting center in the Central nervous system (Larsen. Mechanisms behind GLP-1 induced weight loss, Br. J Diabetes Vasc. Dis. 2008; 8 (Suppl 2): S34-S41; Schick et al. Glucagonlike peptide 1 (7-36)-amide acts at lateral and medial hypothalamic sites to suppress feeding in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 284: R1427-35). However, for conjugates of basis or its variants difficult to overcome the blood-brain barrier due to the increased molecular ve the and, and so they are less vomiting compared with PB-101 (see example 22). Accordingly, before the experiment, the author expected that the conjugates of the basis and its variants will reduce the effect of basis on food consumption and body weight, which is mediated by the Central nervous system. However, the inventor surprisingly found that PEG-conjugates of basis and its variants had a significantly more pronounced effect of reducing body weight and food consumption, despite the fact that on the basis of wild-type and unconjugated version of the basis, both reduced body weight and food consumption.

Example 25. Pharmacokinetic studies PB-101 and Tr-105

Male SD rats (body weight 250-300 g, were purchased in Shanghai Lab Animal Center, Chinese Science Academy) was anestesiologi 30%chloralhydrate (300 mg/kg, intraperitoneally). Catheterization of the femoral artery and vein were performed by making an incision in the right upper edge of the inguinal region and natsuka the femoral artery and vein (used a plastic tube RE, U.S. Beckton Dickinson Corporation). The catheter in the right artery was used for collection of blood samples, and the catheter in the right Vienna used for the introduction of compounds. RE-tube sent from the scruff of the neck under the skin along the back. A catheter filled with heparin solution (200 U/ml) and the incision was sutured. After surgery the rats were kept in individual p is involved in individual cells, and allowed to recover for more than 12 hours In cells of rats with catheters are limited in mobility and food. Rats were divided into groups PB-101 and Tr-105 (3-6 rats in each group). 5 mcg/kg agent was injected bolus injection in the right femoral vein. Blood samples respectively were selected through 0,08, 0,25, 0,5, 1, 1,5, 2, 2,5, 3, 4, 5, 6 hours after administration of the compound. The blood samples were placed in microcentrifuge Eppendorf tube and centrifuged to obtain plasma (5000 rpm, 5 min) and stored at -20°C until use. The concentration of drug compounds in the samples was determined using a set of Exenatide EIA Kit (Phoenix Pharmaceuticals, Inc., USA) after receipt of samples in each group. Curves of the concentration of the medicinal product PB-101 and Tr-105 from time constructed by plotting plasma concentration on the y-axis and time axis X. the Results showed that in rats RV-105 and the RV-101 had similar profiles, distribution and excretion.

Parametric statistical analysis method without using chamber model was performed using the software Kinetica 5.0 ( Thermo Fisher Scientific Inc., USA) to calculate the pharmacokinetic parameters of PB-101 and Tr-105 (CmaxAUC0-tAUC0-∞, t1/2, MRT, CL and Vssand so on). The results are shown in table 4. The half-life in plasma for PB-101 and Tr-105, respectively 4,8±07 and 4.9±1.4 CH (RV-105 relative to PB-101, P>0,05). The area under the curve of drug concentration with time were, respectively, 45,4±1,6 and 47.9±19,0 ng·h/ml (PB-105 relative to PB-101; P>0,05). Experimental results showed that PB-105 and the RV-101 had a similar pharmacokinetic properties.

Table 4
Pharmacokinetic parameters of PB-101 and Tr-105 (3-6 rats in each group)
NCmax(ng/ml)AUC0-t(ng·h/ml)AUC0-∞(ng·h/ml)
PB-101333,0±2,045,4±1,6to 85.2±4,4
PB-105636,8±10,647,9±19,0to 103.8±51,1

T1/2(h)MRT (h)CL (ml/(h·kg)Vss(ml/kg)
PB-1014,8±0,7 6,9±0,759,0±3,1403,2±21,3
PB-1054,9±1,47,2±2,2117,7±58,5563,8±186,1

Example 26. Pharmacokinetic studies PB-105 and its Paglierani conjugates

Male SD rats (body weight 250-300 g, were purchased in Shanghai Lab Animal Center, Chinese Science Academy) was anestesiologi 30%chloralhydrate (300 mg/kg, intraperitoneally). Catheterization of the femoral artery and vein were performed by making an incision in the right upper edge of the inguinal region and natsuka the femoral artery and vein (used a plastic tube RE, U.S. Beckton Dickinson Corporation). The catheter in the right artery was used for collection of blood samples, and the catheter in the right Vienna used for the introduction of compounds. RE-tube sent from the scruff of the neck under the skin along the back. A catheter filled with heparin solution (200 Units/ml) and the incision was sutured. After surgery the rats were kept separately in individual cages and allowed to recover for more than 12 hours In cells of rats with catheters are limited in mobility and food. Rats were divided into 6 groups (3 rats in each group): PB-105, RV-110, RV-106, RV-107, RV-108 and PB-109. 5 mg/kg each agent was administered bolus injection in the right femoral vein and blood samples (0.2 ml) were collected at different time points. During the first 48 h after injection of the compounds blood samples were collected using a tube RE, and after 48 h, blood samples were collected from the tail vein. Specifically, for a group of RV-105 samples were selected through 0,08, 0,25, 0,5, 1, 1,5, 2, 2,5, 3, 4, 5, 6 h after injection connection for a group of RV-110 - through 0,08, 0,25, 0,5, 1, 2, 3, 4, 5, 6, 8, 10 h, for a group RV-106 - through 0,08, 0,25, 0,5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96 h, for a group of RV-107 - 0,08, 0,25, 0,5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144 h, for groups RV-108 and PB-109 samples were selected through 0,08, 0,25, 0,5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, 144, 156, 168 h after injection of the compounds. The blood samples were placed in microcentrifuge Eppendorf tube and centrifuged to obtain plasma (5000 rpm, 5 min) and stored at -20°C until use. The concentration of drug in the samples was determined using a set of Exenatide EIA Kit (Phoenix Pharmaceuticals, Inc., USA) after obtaining plasma samples of each group. Curves concentration Paglierani conjugates from time constructed by plotting plasma concentration on the y-axis and time axis X. the Results showed that PB-105 and its Paglierani conjugates rapidly distributed to the tissues and slowly withdrawn (see Fig).

Parametric statistical analysis method without using chamber model was performed using the software Kinetica 5.0 (Thermo Fisher Scientific Inc., USA) for calc is of pharmacokinetic parameters PB-105 and its Paglierani conjugates (C maxAUC0-1AUC0-∞, t1/2, MRT, CL and Vssetc) the Results are shown in table 5. The half-life in plasma for RV-105 was 2.9±0,1 h, and the half-life in plasma Paglierani conjugates increased with increasing molecular weight of PEG. The area under the curve of the dependence of concentration on time for the RV-105 was 18.2±1.9 ng·h/ml and the area under the curve of the dependence of concentration on time for Paglierani conjugates increased with increasing molecular weight of PEG. On figa and V shown, respectively, the relationship between the molecular weight of PEG in Paglierani the conjugate and the half-life in plasma and area under the curve of drug concentration with time.

76,1±14,5
Table 5
Pharmacokinetic parameters of PB-105 and its Paglierani conjugates (3 rats in each group)
Cmax(ng/ml)AUC0-t(ng·h/ml)AUC0-∞(ng·h/ml)
PB-1052,30±2,818,2±1,924,5±2,2
PB-110101,0±25,8165,5±45,3
RV-106149,2±5,7942,5±84,61146,0±65,2
RV-107128,2±15,51485,2±123,01879,3±82,1
RV-108148,1±24,51780,7±279,92202,5±318,8
PB-109240,1±20,95478,8±654,37033,4±861,6

T1/2(h)MRT (h)CL (ml/h·kg)Vss(ml/kg)
PB-1052,9±0,14,1±0,2207,8±20,9846,3±79,0
PB-1106,1±0,89,6±1,037,5±13,5340,1±92,9
RV-10642,6±8,147,3±11,6 4,4±0,3209,0±57,3
RV-10770,5±2,675,3±9,52,7±0,1201,6±27,3
RV-10874,8±4,590,2±10,22,3±0,3193,6±50,0
PB-109103,6±2,4102,0±6,80,7±0,174,8±10,4

Variants of the basis of the present invention have improved pharmacokinetic properties, significantly reduce the level of glucose in the blood and have a comparable or better biological activity. Conjugates of the choice of basis, the resulting site-specific attachment of polymers through tirinya group cysteine significantly increase the half-life choices of basis and retain high biological activity.

The present invention is illustrated with specific examples. However, for an experienced specialist will be clear that the present invention is not limited to the specific examples, and experienced can make the scope of the present invention some changes or modifications without going beyond the nature and volume of the mA of the present invention. These changes and modifications are included in the scope of the present invention.

1. Conjugate version of the basis with the activity of the agonist receptor GLP-1, in which one molecule of the polymer conjugated with cysteine residue in the variant of the basis and in which the polymer molecule has a molecular weight of 21 kDa to 29 kDa,
in which one molecule of the polymer is a polyethylene glycol, and
which version of the basis has the following sequence:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Cys (SEQ ID NO: 4).

2. The conjugate according to claim 1, in which the polymer molecule has a molecular weight of 23 kDa to 27 kDa.

3. Conjugate version of the basis of claim 1 or 2, designed to reduce body weight.

4. A method of obtaining a conjugate according to claim 1 or 2, comprising contacting a variant of the basis of the polymer, where at the moment of contact to the polymer or attached reactive group, or activate to konjugierte activated polymer with a cysteine residue in the basis.

5. The pharmaceutical composition intended to reduce the level of glucose in the blood, containing an effective amount of the conjugate according to claim 1 or 2 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, intended for the treatment of diabetes, preferably for the treatment of dia is ETA 1 and/or type 2, more preferably for the treatment of type 2 diabetes.

7. The set, designed to reduce the level of glucose in the blood containing the conjugate according to claim 1 or 2, and instructions for use.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. What is presented is PEG-modified exendin or an exendin analogue containing one or more PEG derivatives (versions). One or more PEG derivatives have a branched structure (I) or (III) as specified in formula. The molar weight of one or more PEG derivatives mentioned above makes 5000 Da to 40000 Da. There are also presented a composition and a method of treating diabetes mellitus with using above PEG-modified exendin or exendin analogue.

EFFECT: presented PEG-modified exendin or exendin analogue has a prolonged blood half-life, high bioactivity and low immunogenicity.

25 cl, 10 dwg, 3 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention relates to production of peptide derivative mimetic of EPO, with the formula: R1-R2-(CH2)n1-R3-(CH2)n2-R4-R5 (I) and its pharmaceutical salts, where R1, R5 are selected from cyclic peptides with sequences SEQ ID NO:5, 6, 7 and 8; n1, n2 represent integer numbers independently selected from 0-10; R2, R4 are selected from -CO or -CH2; R3 is selected from O, S, CH2, N(CH2)n3NHR6, NCO(CH2)n4NHR6, CHOCONH(CH2)n5NHR6, CHSCON(CH2)n5NHR6 or CHNHCON(CH2)n5NHR6; where n3 represents an integer number selected from 1-10, n4 is an integer number selected from 2-10, n5 represents an integer number selected from 2-10, R6 is selected from H or derivatives of metoxyethylene glycol. The produced peptide or its pharmaceutically acceptable salt is used within a pharmaceutical composition for treatment of disorders characterised with EPO deficit, low or defective population of erythrocytes.

EFFECT: invention makes it possible to produce an agonist of EPO receptor having higher biological activity compared to existing EPO mimetics.

15 cl, 2 dwg, 5 tbl, 19 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the technology of producing biological preparations and can be used in the pharmaceutical industry. The method of preparing a pegylated human growth hormone involves pegylation of said protein with double-stranded polyethylene glycol in one site at pH not lower than 6.0. The obtained pegylated growth hormone is subjected to electrophoresis in polyacrylamide gel in non-reducing conditions, and separating the end product from a band which corresponds to a lower apparent molecular weight. The method, which involves pegylation of the human growth hormone at pH not lower than 8.0 and separating the obtained product, enables to obtain a preparation mainly containing pegylated human growth hormone with a lower apparent molecular weight. The pegylated growth hormone with a lower apparent molecular weight and a preparation rich in said hormone are used to prepare a medicament for treating diseases that require treatment with a growth hormone.

EFFECT: use of the invention increases biological activity of the growth hormone and prolongs its metabolic half-life.

19 cl, 24 dwg, 3 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to biochemistry. What is presented is pegylated interferon-α2b (IFN-α2b) having a structure as shown in the patent claim. Pegylated interferon-α2b is prepared by combining IFN-α2b with branched Y-polyethylene glycol (YPEG), wherein YPEG is bound to IFN-α2b by an amide bond formed by ε-amino group of a side of Lys residue in IFN-α2b in the position of 134 in SEQ ID No. 1. A method for preparing and purifying pegylated IFN-α2b involves the stages as follows. In an alkaline medium at pH 9.0, branched Y-polyethylene glycol is reacted with IFN-α2b to prepare pegylated IFN-α2b. The prepared reaction products are recovered by an anion exchange resin, and the given products are eluted by an anion gradient to prepare modified products. The anion exchange resin is Q Sepharose FF, and the anion gradient is a chloride ion gradient. Further, the modified products are eluted by a cation exchange resin with a cation gradient. The cation exchange resin is SP Sepharose FF, and the cation gradient is a sodium ion gradient. Thereafter, each peak is collected separately. The product activity of each peak is determined to choose a peak corresponding to the reaction product having the highest activity. What is also presented is a formulation for treating a disease requiring IFN-α2b to be used, which consists of a pharmaceutically effective amount of said pegylated IFN-α2b and a pharmaceutically acceptable carrier, or an inactive substance. Pegylated IFN-α2b or the above formulation is also used for preparing a medicine for treating various diseases requiring IFN-α2b to be used.

EFFECT: presented pegylated IFN-α2b has a higher specific activity of 2,65±0,185×106 IU/mg and a prolonged serum half-life.

26 cl, 11 dwg, 5 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention may be used for directed delivery of genetic structures into stem and malignant tumor cells in order to correct gene defects and to prevent diseases. Method includes preparation of genetic structure carriers, by inclusion of carrier molecules, being a DNA-binding sequence of eight remains of lysine amino acid - KKKKKKKK, of signal sequences. Connection of signal sequence to DNA-binding sequence is carried out with the help of linker section of two molecules of ε-aminohexanoic acid. Afterwards complexes of DNA/carrier are formed. Then in vitro transfection is carried out.

EFFECT: increased efficiency of gene of interest delivery into tumor and stem cells.

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FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a method of producing three-dimensional matrices for tissue-like structures from animal cells. The method involves covalent bonding of histons with the surface of pre-activated biocompatible polymer microspheres made from crystalline dextran. The microspheres with covalently bonded histons are then deposited by centrifuging. Microspheres containing 160-200 mcg protein per 1.0 g are deposited on a substrate surface in amount of 0.5-1.0 mg per 1.0 cm2 and then dried at room temperature. Further, the substrate is washed with a solution at pH 7.5 to remove material which is not bonded to the substrate. The layer of microspheres obtained on the surface of the substrate on which cells are deposited is used as a base for obtaining tissue-like cell structures.

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6 cl, 11 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to chemistry of polymers, biochemistry and medicine, and specifically to a method of preparing glucose-sensitive polymer hydrogels used as carriers for controlled secretion of insulin. The glucose-sensitive polymer hydrogels are obtained from reaction of a glucose derivative with concanavalin A. The reaction is carried out through copolymerisation in an aqueous solution under the effect of a redox initiator of 0.1-2.0 wt % N-(2-D-glucose)acrylamide, 1-5 wt % acrylamide and 0.01-0.075 wt % N,N-methylenebisacrylamide in the presence of 5-20 wt % concanavalin A.

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2 tbl

FIELD: medicine.

SUBSTANCE: offered is method of blood substitute production and related installation for method implementation. Method of blood substitute production includes production of deoxygenated haemoglobin, its polymerisation and purification. Production of deoxygenated haemoglobin includes haemolysis of water addition to erythrocytic mass, stroma separation, non-heme protein precipitation and removal from produced haemoglobin solution. Polymerisation includes processing of produced deoxygenated haemoglobin with modified glutaric aldehyde and restoration with sodium borane, with purification including ultra filtration. Deoxygenated haemoglobin is produced using leukocyte-free erythrocytic mixture. Non-heme proteins are precipitated by concentrated sodium chloride solution added to haemoglobin solution. Removal of non-heme proteins is followed with ultra filtration concentration of haemoglobin solution. Haemoglobin is produced in polymeric disposable containers, while deoxygenation and polymerisation are carried out in gas vortex reactor with nitrogen atmosphere within 1-6 hours each. Diafiltration purification is performed in polymeric disposable containers on shutoff dampers to produce end product molecular weight within 100 kDa to 450 kDa. Method allows for simplified production of polyhaemoglobin with lowered cost and higher outcome. Related installation for method implementation includes series haemoglobin production area, haemoglobin polymerisation reactor and end-product purification system. Haemoglobin production area contains series haemolysis tank with filtration manifold for stroma separation, non-heme protein precipitation tank with filtration manifold for removal of precipitated non-heme proteins. End product purification system contains ultra filtration tanks and units with shutoff dampers. All tanks within haemoglobin production area are polymeric disposable containers. Non-heme protein precipitation tank is connected to the tank for concentrated solution of sodium chloride. Polymerisation reactor is designed as gas vortex unit. End product purification system tanks are polymeric disposable containers. Haemoglobin production area, haemoglobin polymerisation reactor and end product purification system, as well as all tanks and units are interconnected by means of sterile rapid-action coupling.

EFFECT: allows for reduced material consumption of installation with higher productivity, sterile conditions of technological process.

6 cl, 1 dwg

FIELD: biochemistry, biophysic, medicine diagnosis.

SUBSTANCE: claimed method includes application of protein films on solid substrate and is based on using of cellulose mixed ester, namely cellulose acetopyvalinate as immobilizing layer for protein molecules which increases ordering ratio of protein molecules and enhances protein film filling density. Said films are useful as model of cell membrane in drug investigations.

EFFECT: method of increased efficiency.

4 ex, 5 dwg

FIELD: molecular biology, bioorganic chemistry.

SUBSTANCE: invention relates to development of a method for preparing gel biochips with backing from unmodified polymeric materials. Invention proposes using some unmodified polymeric materials used without their preliminary modification for preparing biochips backing that is designated for immobilization of hydrogels on its surface. Also, invention proposes a biochip prepared on backing made of unmodified polymeric materials, method for preparing biochip and method for immobilization of hydrogels on backings made of unmodified polymeric materials.

EFFECT: improved preparing method.

55 cl, 2 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of biotechnology and genetic engineering and can be used in veterinary for creation of vaccines regulating the sexual function in animals. A gene of a hybrid protein GHbc is obtained by the PCR method and is inserted into a polylinker region of a plasmid vector pUC9.

EFFECT: claimed is recombinant DNA, coding the hybrid vaccine protein GHbc, consisting of a nucleocapsid protein of human hepatitis B virus, fused with gonadoliberin.

2 cl, 4 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and specifically to production of human pancreatic polypeptide analogues and can be used in medicine. The disclosed analogues differ from native human pancreatic polypeptide by replacement of amino acids on one or more residues, wherein position 0 is provided with an additional amino acid which is Gly.

EFFECT: obtained analogues provide effective treatment and prevention of obesity or diabetes in a patient, and can also be effective in reducing appetite, reducing food intake or reducing calorie intake in a patient.

26 cl, 2 dwg, 2 tbl,1206 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to a derivative of peptides PYY or PP or their equivalent modified with one or more side chains bound with serum albumin and containing distal tetrazole group or carboxylic acid group. Besides, the invention refers to their compositions and treatment methods of states susceptible to modulation of Y receptors.

EFFECT: improvement of composition properties.

13 cl, 12 dwg, 15 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. What is presented is PEG-modified exendin or an exendin analogue containing one or more PEG derivatives (versions). One or more PEG derivatives have a branched structure (I) or (III) as specified in formula. The molar weight of one or more PEG derivatives mentioned above makes 5000 Da to 40000 Da. There are also presented a composition and a method of treating diabetes mellitus with using above PEG-modified exendin or exendin analogue.

EFFECT: presented PEG-modified exendin or exendin analogue has a prolonged blood half-life, high bioactivity and low immunogenicity.

25 cl, 10 dwg, 3 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: present invention provides a method of controlling conditions for site-specific binding of a polypeptide and a non-peptide polymer by controlling pH and alcohol content of the reaction medium.

EFFECT: method is intended to prevent formation of secondary conjugates, wherein a non-peptide polymer binds with a physiologically vital amino acid residue.

15 cl, 36 dwg, 25 ex

FIELD: biotechnologies.

SUBSTANCE: peptide derivatives of exendin -3 (1-39) are described, having activity of glucagon-like peptides (GLP-1), modified near C-end with amine and capable of binding with their N-end to GLP-1-receptors. Application of peptide derivatives of exendin-3 is disclosed for production of an agent for diagnostics and therapy of diseases, where expression of GLP-1 receptors is important, to define density of insulin-producing cells in a tissue, in order to define expression of GLP-1 -receptors or their density.

EFFECT: invention may be used to produce an agent for diagnostics and therapy of diseases, where expression of GLP-1 receptors is important.

21 cl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to molecular biology. There are presented peptides being GLP-1 derivatives, binding to GLP-1 receptors and used either marked, or unmarked for preparing a product for diagnosing and treating benign and malignant diseases, wherein GLP-1 receptor expression is of importance.

EFFECT: preparing the product for diagnosing and treating the benign and malignant diseases, wherein GLP-1 receptor expression is of importance.

23 cl, 4 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to peptidyl analogues of ghrelin having greater stability which are active with respect to the GHS receptor, having the formula given below: (R2)-A2-A3-A4-A5-A6-A7-A8-A9-A10-A11-A12-A13-A14-A15-A16-A17-A18-A19-A20-A21-A22-A23-A24-A25-A26-A27-A28-Rl, where values of A1-A28, R1 and R2 are given in the description, pharmaceutically acceptable salts thereof and pharmaceutical compositions containing an effective amount of said compound, as well as therapeutic and non-therapeutic applications thereof.

EFFECT: high stability.

22 cl, 3 tbl, 11 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and chemical technology. What is offered is a method for producing the peptide Exenatide. The peptide Exenatide prepared by synthesis may be used for producing drugs for type 2 diabetes mellitus.

EFFECT: higher yield of the unpurified peptide (42%-60%), as well as higher purity (58%-75%) and content of the end product in a mixture (33%-42%); besides, a cheaper condensing agent (DIC) is used at all the condensation stages, while the synthesis of more than one short fragments simultaneously enables faster end peptide process.

3 cl, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention describes neuropeptide-2 receptor agonists of formula and also their pharmaceutically acceptable salts.

EFFECT: possibility of using the compounds for treating diseases, such as, eg obesity and diabetes.

11 cl, 25 dwg, 1 tbl, 44 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to quinazolinone compounds of formula (I) and its pharmaceutically acceptable salts, wherein n is equal to 0 to 3, and R1 is defined as stated in the patent claim. The above compounds are prolyl hydroxylase inhibitors and can be used in pharmaceutical compositions and methods of treating pathological conditions, disorders and conditions mediated by prolyl hydroxylase activity.

EFFECT: compounds can be administered into the patient for treating, eg anaemia, vascular diseases, metabolic disorders, as well as for wound healing.

22 cl, 2 tbl, 211 ex

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