Human growth hormone derivative highly resistant to proteolytic degradation, method for preparing this derivative, using it, method of treating, and pharmaceutical composition

FIELD: medicine.

SUBSTANCE: invention refers to molecular biology, medicine, biochemistry and gene engineering. Presented is a human growth hormone derivative containing an additional disulphide bond as compared to hGh defined by SEQ ID No. 1, wherein the derivative contains at least one pair of mutations described by H21C/M170C, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1, and possesses the activity of the human growth hormone, as well as a method for preparing this derivative, using it, a method of treating and a pharmaceutical composition.

EFFECT: invention possesses high stability and resistance to proteolytic degradation as a consequence of the introduction of cysteine residues.

18 cl, 3 dwg, 6 tbl, 5 ex

 

The SCOPE of the INVENTION

The present invention relates to stable compounds of growth hormone (GH) that are resistant to proteolytic destruction.

PRIOR art

Growth hormone (GH) is a polypeptide hormone secretory by adenogipofiza in mammals. Depending on the type GH is a protein consisting of approximately 190 amino acid residues, which corresponds to a molecular mass of approximately 22 kDa. GH binds to receptors on the cell surface, GH-receptor (GHR), and transmits signals through them. GH plays a key role in growth stimulation, maintenance of normal body composition, anabolism and metabolism of lipids. It also has a direct impact on intermediary metabolism, such as, for example, reduces the absorption of glucose, increases lipolysis, stimulates amino acid uptake and protein synthesis. This hormone also affects other tissues, including adipose tissue, liver tissue, intestine, kidney, skeleton, connective tissue and muscle tissue. The obtained recombinant hGH, and it is commercially available as, for example: Genotropin™ (Pharmacia decision Upjohn), Nutropin™ and Protropin™ (Genentech), Humatrope™ (Eli Lilly), Serostim™ (Serono), norditropin (Novo Nordisk), omnitrope (Sandoz), nutropin depot (Genentech and Alkermes). In addition, the market also has an analogue with an additional methionine residue at the N-to the nce, for example: Somatonorm™ (decision Upjohn Pharmacia/Pfizer).

GH has a common topology with other members of the GH family of proteins, prolactin (PRL) and placental lactogenic (PL). GH is classified as a protein having the structure chetyrekhmernogo beam (Fig.1), showing the topology of the "up-up-down-down, with two conservative disulfide bonds. Specifically, human GH (hGH) wild type consists of 191 amino acid residue and has four cysteine residue at positions 53, 165, 182 and 189, which stabilizes the spatial structure of this protein by the formation of two intramolecular disulfide bonds connecting s with S and S with S, respectively (Fig.1). The structure of hGH was experimentally determined by x-ray crystallography in the free form (Chantalet L. et al. (1995) Protein and Peptide Letters 3, 333-340) and in complex with communicating with him in protein (extracellular domain of the human GHR (hGHR)) (Devos, A. M. et al. (1992) Science 255, 306-312). These structures deposited in the Bank of protein structures (PDB) and are publicly available (access codes in PDB 1HGU and 1HWG, respectively). So, among the published structures of hGH can identify residues essential for binding with hGH hGHR. In addition, the dynamic properties of hGH investigated by nuclear magnetic resonance (NMR) (Kasimova M. R. et al. J. Mol. Biol. (2002) 318, 679-695). Combined x-ray diffraction data and the aleesa and NMR highlight areas hGH, which are well-structured and well-defined, from areas that are less structured and dynamic. It is expected that less structured and dynamic areas of hGH will be particularly sensitive to proteolytic cleavage, and proper stabilization of these areas will lead to improved proteolytic stability.

hGH has been subjected to extensive mutagenesis in an attempt to obtain analogues of hGH with the desired chemical or biological properties. In particular, the cysteine mutants, obtained for different purposes.

In the US 2003/0162949 described cysteine variants members supergene family GH. Provides a General way to create site-specific, biologically active conjugates of these proteins. This method involves adding cysteine residues to non-essential areas proteins or replacement of non-essential amino acids in proteins at cysteine residues using site-specific mutagenesis and then the implementation of covalent binding between the reactive by cysteine polymer or other reactive to cysteine group and proteins through the added cysteine residue.

In WO 02/055532 describes genetically engineered mutants of hGH containing at least one attached through a covalent bond which is not a polypeptide group, in particular Muta what you hGH, which entered the residue cysteine was used to paglierani.

In the US 5951972 describes physiologically active derivatives of natural and recombinant proteins and polypeptides of mammalian and human derived protein with different substituents at least one natural or introduced into the protein residue cysteine.

Proteolytic cleavage of hGH studied in detail. Long loop, which consists of residues 128-154, contains putative cleavage sites for various proteases, such as thrombin, plasmin, collagenase, subtilisin and chymotrypsinogen serine protease. Accordingly, it is shown that this part of the hGH is particularly sensitive to proteolytic cleavage (Lewis, U. J. Ann. Rev. Physiol. (1984) 46, 33-42). Enzymes known as responsible for the destruction of hGH include thrombin, plasmin, subtilisin, chymotrypsinogen serine proteases and kallikrein.

Investigated the destruction of hGH in the tissue of rats (Garcia-Barros et al. J. Endocrinol. Invest. (2000) 23, 748-754).

Discovered that the thyroid gland of rats chymotrypsinogen protease, preferably carrying out the splitting bulky and lipophilic amino acid residues, were first digested peptide bond between Y143 and S144 with obtaining a double-stranded molecule, and then split the connection between Y42 and S43 with the release of the N-terminal peptide F1-Y42. Subsequent processing of the split loop in such a double-stranded molecule is accomplished by splitting the connection between F146 and D147 under the action of chymotrypsinogen proteases and forth under the action of carboxypeptidase.

Describes several methods for obtaining analogs of hGH, stabilized against proteolytic destruction.

Using specific point mutations, Alam et al. (J. Biotech. 65, 183-190 (1998) have constructed mutants of hGH, resistant to thrombin and plasmin. Thrombin specifically cleaves between hGH R134 and T, and a double mutation R134D, TR resulted in obtaining hGH variant that is resistant to cleavage by thrombin, and the triple mutation R134D, TR, K140A led to resistance to plasmin. In addition, the last mutant hGH was resistant to proteolysis under the action of human blood plasma within 7 days.

In ER describes mutants of hGH, stabilized against proteolytic destruction by changing R134 by alanine, leucine, threonine, phenylalanine, Proline, or histidine.

In WO2004022593/Nautilus describes the General performance of the methods of obtaining the modified cytokines using directed evolution, including variants of GH, with increased proteolytic stability.

In WO2006048777/Nautilus specifically describes the modified hGH analogs with enhanced proteolytic stability. These analogues with the hold from one to five mutations in positions 1-55, 57, 58, 60-63, 67-87, 89-91, 93, 95-100, 102-128, 131-132, 135-139, 141, 142, 144, 148-182, 184, 185 and 187-191. The introduction of cysteine residues may potentially cause the formation of undesirable connected by disulfide bonds of the dimers, and in WO2006048777 replacement of amino acid residues in the cysteine specifically excluded from the scope of the invention; WO2006048777 (page 65) formulated: "the Replacement of amino acids at residue cysteine definitely not allowed, because such a replacement will theoretically lead to the formation of intermolecular disulfide bonds".

There is an obvious need to develop compounds hGH, which are resistant to proteolytic destruction. Such stable compounds will demonstrate increased stability to proteolytic cleavage while maintaining the desired biological properties of hGH. Such molecules GH will have increased stability, slow clearance and/or prolonged half-life existence in vivo.

In addition, it is usually necessary to protein therapeutic agent was administered intravenously or subcutaneously, because they usually do not have sufficient oral availability. Low oral bioavailability of proteins is partly due to proteolytic degradation in the gastrointestinal tract. Therefore, there is also a need to develop compounds hGH that you can enter PE is given orally for the treatment of hGH-associated disorders.

The INVENTION

The present invention relates to compounds hGH containing additional disulfide bonds. In connection hGH of the present invention is introduced at least one additional cysteine residue by changing at least one of the amino acid sequence hGH wild type cysteine. In the compounds of hGH of the present invention the mutation sites selected in such a way that (1) is added(s) residue(s) cysteine appropriately placed(are) in the spatial structure of the folded protein, to give the opportunity to develop additional disulfide linkages that are not present in the wild-type protein, (2) the native structure of hGH is not distorted, (3) connection of hGH demonstrates increased stability to proteolytic cleavage compared to wild-type hGH or other superior functional characteristics, and (4) connection of hGH retains the desired biological activity associated with wild-type hGH. Such disulfide variants compounds hGH, resistant to proteolytic degradation in the gastrointestinal tract, can be designed in the form of oral input medicines for the treatment of hGH-associated disorders.

A BRIEF DESCRIPTION of GRAPHIC MATERIALS

Fig.1

The structure of hGH in a bound state with two copy and hGH-binding protein (1HWG in PDB). Four main spiral in hGH shown in dark gray and marked as N1-N4. Direction (N→s-end) arrows. N - and C-ends of hGH marked as N and C, respectively. Two disulfide bonds connecting s with S and S with S, respectively, represented by the black sticks and balls. Also noted the remains L128 and D154, representing the first and last residues, respectively, in a long flexible loop connecting H3 and H4.

Fig.2

Amino acid sequence of wild-type hGH with four major helices (H1-H4), highlighted and marked. Also noted three loops (L1-L3) connecting the main spiral. Definition of spirals belong to hGH in complex with its binding protein (1HWG in PDB).

Fig.3

The dependence of the proteolytic cleavage of the wild-type hGH and connections hGH with additional disulfide bonds from time to time. Use the following proteases: chymotrypsin (panel A) and elastase (panel B). The analysis performed as described in Example 5. The number of intact protein (% relative to t=0) delayed depending on the time of incubation. The value of T½ (hours) obtained by approximation of data to ownexperience dependencies are given in tables.

DETAILED description of the INVENTION

The present invention relates to stable compounds hGH with additional disulfide the e connection. Disulfide bonds are formed between pairs of cysteines, of which one or both entered through point mutations in the sequence of the wild-type hGH. The sites of mutation is chosen so that the introduced cysteine residues appropriately housed in the spatial structure of the folded protein to the possible formation of disulfide bonds. If you enter only one cysteine, his partner in the formation of disulphide bonds will include one of the four cysteine residues present in wild-type hGH. A folded protein with an additional disulfide bond can be obtained by expression of the corresponding cysteine mutant of hGH in a soluble form suitable host organism or extracted from Taurus enable using standard conditions for refolding compounds of growth hormone, which are well known to the person skilled in the art (Cabrita and Bottomley, Biotechnology Annual Review 10, 31-50 (2004)). Identify provisions that are candidates for the introduction of an additional disulfide bonds, can contribute to computational methods, for example using experimentally established spatial patterns hGH (access code 1HWG in PDB) in complex with two copies of communicating with him protein. The choice of appropriate provisions for the introduction of a disulfide bond can be based on to whom icereach distance and geometry for disulfide bonds, described in A. Dombkowski, A., Bioinformatics 19, 1852-1853 (2003) and Petersen et al., Protein Eng. 12, 535-548 (1999).

Cysteine mutants chosen so that the introduced disulfide bond did not distort the native structure of this protein and have had minimal negative impact on the desired biological activity associated with hGH. Thus, the compounds of the design so that the introduced disulfide bond is not weakened interaction with hGHR. Areas in hGH that are important for interaction with the receptor, identified on the basis of 1HWG. Thus, in the selection of appropriate provisions for the introduction of disulfide bonds, neutral with respect to biological activity, can be guided by the analysis of the structure 1HWG.

You can choose these cysteine mutants that introduced disulfide bond provided increased stability to proteolytic cleavage. The susceptibility of the protein to cleavage by proteases partly determined by the primary amino acid sequence of the specified protein. The protease can be relatively are not specific or can recognize, with varying degrees of selectivity, specific motifs in the primary amino acid sequence. However, spatial structure and dynamic properties of protein molecules, acting as the substrate, a strong influence on proteolitic the stability. With great flexibility and dynamism loop structures are particularly vulnerable to catalyzed by protease cleavage, while well-structured plots, as a rule, to a lesser extent. Thus, protection against proteolytic cleavage can be obtained in the stabilization of the dynamic parts of the protein by the introduction of disulfide bonds.

One aspect of the invention relates to the compound growth hormone containing additional disulfide bonds in SEQ ID No. 1. As stated in the description below, the polypeptide compounds of growth hormone according to the invention preferably has a high level of identity with human growth hormone identified by SEQ ID No. 1 and, accordingly, the compound growth hormone contains one or more than one additional disulfide bond(s) compared to human growth hormone as defined in SEQ ID No. 1.

In line with this, according to one embodiments of the present invention proposed a stable connection GH corresponding to SEQ ID No.1, is made resistant to proteolytic destruction by introducing an additional disulfide bonds.

In one of the embodiments corresponding to the invention, the compound growth hormone contains additional disulfide bond between at least onethe pairs of amino acids at positions corresponding to R16C/L117C, AS/ES, NS/MS, D26/V102C, D26/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, RS/ES, RS/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1.

In one of the embodiments of the present invention, the compound growth hormone contains additional disulfide bond between at least one of the pairs of amino acids in positions corresponding to R16C/L117C, AS/ES, NS/MS, N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, RS/ES, RS/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1, but is not restricted by them.

In one of the embodiments of the present invention, the compound growth hormone contains additional disulfide bond between at least one of the pairs of amino acids in positions corresponding to AS/ES, NS/MS, S71C/S132C, Q84C/Y143C and R94C/D107C in SEQ ID No. 1, but is not restricted by them.

In one of the embodiments of the present invention, the compound growth hormone contains additional disulfide bond between two amino acids in positions corresponding to Q84C/Y143C in SEQ ID No. 1.

In one of the embodiments corresponding to the invention, the connection of HGH contains at least one pair of mutations according to the corresponding R16C/L117C, AS/ES, NS/MS, D26/V102C, D26/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, RS/ES, RS/CS, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1.

In one of the embodiments of the present invention the connection of HGH contains at least one pair of mutations corresponding to R16C/L117C, AS/ES, NS/MS, N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, RS/ES, RS/CS, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1, but is not restricted by them.

In one of the embodiments of the present invention the connection of HGH contains at least one pair of mutations corresponding to AS/ES, NS/MS, S71C/S132C, Q84C/Y143C and R94C/D107C in SEQ ID No. 1, but is not restricted by them.

In one of the embodiments of the present invention the connection of the growth hormone of the present invention contains one pair of mutations corresponding to the position Q84C/Y143C in SEQ ID No. 1.

In one of the embodiments of the present invention proteolytic stability of connection growth hormone reach by introducing a disulfide bond between a loop segment and a helical structure.

In one of the embodiments of the present invention proteolytic stability of compound g is Ramona growth reached by introduction of disulfide bonds within the hinge segment.

In one of the embodiments of the present invention proteolytic stability of connection growth hormone reach by introducing a disulfide bond between the hinge segments.

In one of the embodiments of the present invention proteolytic stability of connection growth hormone reach by introducing a disulfide bond between the spirals.

In one of the embodiments of the present invention at least one of the introduced disulfide bond links two of the cysteine residue in the compound of growth hormone and at least one of the cysteine residues is not present in wild-type hGH.

In one of the embodiments of the present invention introduced disulfide bond connections growth hormone are located between cysteine residues, which are selected based on criteria of distance and geometry described in A. Dombkowski, A., Bioinformatics 19, 1852-1853 (2003) and Petersen et al., Protein Eng. 12(7), 535-548 (1999).

In one of the embodiments of the present invention introduced disulfide bonds(and) compound growth hormone stabilizes(ut) loop connecting the NC and H4 (L3 residues 128-154), i.e. at least one of cysteines in the introduced disulfide bond is located in the segment containing residues 128-154 (Fig.1 and 2).

Table 1
The first amino acid defined by sequence alignment with SEQ ID No. 1The second amino acid defined by sequence alignment with SEQ ID No. 1United segments of secondary structureand
1.16117H1-H3
2.17174N1-N4
3.21170N1-N4
4.26102H1-L2
5.26103H1-L2
6.4750L1-L1
7.49161L1-L1
8.54143L1-L3
9.54144L1-L3
10.54146L1-L3
11.55143L1-L3

tr>
The first amino acid defined by sequence alignment with SEQ ID No. 1The second amino acid defined by sequence alignment with SEQ ID No. 1United segments of secondary structureand
12.57143L1-L3
13.58141L1-L3
14.58143L1-L3
15.58144L1-L3
16.5937 L1-L3
17.6166L1-L1
18.6167L1-L1
19.71132L1-L3
20.73132H2-L3
21.73139H2-L3
22.77138H2-L3
23.77139H2-L3
24.81141H2-L3
25.81143H2-L3
26.84143H2-L3
27. 84144H2-L3
28.85143H2-L3
29.85144H2-L3
30.89146H2-L3
31.92146H2-L3
32.92148H2-L3
33.94107H2-H3
34.102105L2-H3
35.156146H4-L3
36.156148H4-L3
37.185188Ct-Ct
a) H1-H4 refers to the spiral 1-4, L1-L3 refers to loops 1-3, a Ct refers to the C-terminal segment.

As described above, the invention relates to a compound growth hormone containing additional disulfide bonds between a loop segment and a helical segment, or within a loop segment, or between the hinge segments, or between the helical segments of the polypeptide. The location of any such additional disulfide bond described, taking into account the tasks of this application, with reference to the hGH polypeptide defined in SEQ ID No. 1.

In one of the embodiments of the compound of growth hormone according to the invention contains at least one pair of mutations corresponding to R16C/L117C, AS/ES, NS/MS, D26/V102C, D26/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, RS/ES, RS/CS, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to AS/ES, NS/MS, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No.1.

In one of the embodiments of the compound of growth hormone contains at least about the well pair mutations, relevant AS/ES, NS/MS, F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

One of the embodiments corresponding to the invention, refers to the connection of growth hormone containing additional disulfide bonds, at least one of cysteines present in L3 (AA (amino acids) 128-154 in SEQ ID No. 1) or, for example, in the middle of this loop defined by AA 135-148, or corresponding amino acid residues.

In one embodiments of the compounds of growth hormone and at least one cysteine additional disulfide bond is present in L3 in a position corresponding AA, AA, AA, AA, AA or AA, preferably AA143 or AA144 in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C and/or F92C/T148C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations, with the corresponding F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or F92C/T148C in SEQ ID No. 1.

One of the embodiments corresponding to the invention, refers to the connection of growth hormone containing additional disulfide bond connecting L3 with L1.

In one of the embodiments of the compound of growth hormone contains additional disulfide bonds linking amino acid residue corresponding to AA, AA, AA, AA, AA or AA in L3, with the amino acid corresponding to AA or AA in L1 in SEQ ID No. 1.

In one of the embodiments of the compound of growth hormone according to the invention contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C and/or S71C/S132C in SEQ ID No. 1.

In one of the embodiments of the growth hormone contains at least one pair of mutations corresponding to F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C and/or S71C/S132C in SEQ ID No. 1.

One of the embodiments corresponding to the invention, refers to the connection of growth hormone containing additional disulfide bond connecting L3 spiral segment, such as helix 2 (H2).

In one of the embodiments of the compound of growth hormone contains additional disulfide bonds linking amino acid residue corresponding to AA or AA in H2, with the amino acid corresponding to AA or AA in L3 in SEQ ID No. 1.

In one of the embodiments of the connection of the hormone R the one hundred contains at least one pair of mutations, relevant L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C and F92C/T148C in SEQ ID No 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or F92C/T148C in SEQ ID No. 1.

One of the embodiments corresponding to the invention, refers to the connection of growth hormone containing additional disulfide bond connecting L2 spiral 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to D26C/V102C or D26C/Y103C.

As for the disulfide bonds between two cysteine residues, cysteine residues can be introduced in any of the sites or any of the provisions that previously described, or to hold them replaced with cysteine residues in order to facilitate formation of one or more of the introduced disulfide bonds, as necessary. Replacement and insertion of amino acid residues can be made by standard methods known to the expert in this field.

In accordance with this invention one or more than one additional disulfide bond(s) is injected through the implementation of aminokislotnoi replacement of at least two amino acids compared to SEQ ID No. 1. In the following embodiment, the compound contains exactly one additional disulfide bonds compared to SEQ ID No. 1. In one of the embodiments of the compound according to the invention contains at least 2 amino acid substitutions compared to SEQ ID No. 1. In one of the following embodiments, the compound contains exactly 2 amino acid substitutions compared to SEQ ID No. 1. In one of the embodiments of the polypeptide compounds of growth hormone according to the invention contains at least two additional cysteines compared to human growth hormone as defined in SEQ ID No. 1. In the following embodiment, the polypeptide contains exactly two additional cysteines compared to human growth hormone as defined in SEQ ID No. 1.

In one of the embodiments of the present invention the connection of growth hormone, in addition to the formation of additional disulfide bonds, chemically modified by joining groups such as PEG, carbohydrates, albumin-binding substances, fatty acids, alkyl chain, lipophilic groups, vitamins, bile acids, but not limited to; or spacers to the side chain or main chain of the compound growth hormone.

In one of the embodiments of the present invention, the compound growth hormone is chemically modified in comparison with hGH in order to facilitate transport through the epithelium.

In one of the three embodiments of the present invention the connection of the growth hormone of the present invention is chemically modified in order to obtain prolonged duration of action in vivo.

In one of the embodiments of the present invention, the compound growth hormone is chemically modified in order to obtain prolonged duration of the half period of the functional existence in vivo.

In one of the embodiments of the present invention chemical modification of compound growth hormone can also be temporary, i.e. they can easily be removed in vivo.

In one of the embodiments of the present invention the modification of the connection of growth hormone can be implemented in any amino acid residue without affecting the binding of the compounds of growth hormone with hGHR.

In one of the embodiments of the present invention, the compound growth hormone has increased stability to proteolytic cleavage.

In one of the embodiments of the present invention, the compound growth hormone has increased stability to proteolytic destruction under the action of pancreatic proteases.

In one of the embodiments of the present invention, the compound growth hormone has increased stability to proteolytic destruction under the action of proteases present in the gastrointestinal tract.

In one of the embodiments of the present invention, the compound growth hormone has increased stability to proteolytic destruction under the action of proteases are present is in the plasma of mammals.

One of the embodiments of the present invention relates to the compound growth hormone containing one or more than one additional disulfide bond(s), which is stabilized against destruction under the action of the protease(s), such as digestive proteases, such as pepsin, trypsin, chymotrypsin, carboxypeptidase and/or elastase.

In one of the embodiments of the present invention, the compound growth hormone has increased stability to proteolytic destruction under the action of trypsin, chymotrypsin and/or elastase.

In one of the embodiments of the compound of growth hormone stable against destruction under the action of chymotrypsin and/or elastase.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to n/M, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to n/M, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C and/or S85C/Y143C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to D26/V102C, D26/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

In one of the in the of plosiny connection HGH contains at least one pair of mutations, relevant I58C/S144C, P59C/Q137C, S71C/S132C, Q84C/Y143C, S85C/Y143C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID No. 1.

In one of the embodiments of the compound of HGH contains at least one pair of mutations corresponding to Q84C/Y143C and/or S85C/Y143CB SEQ ID No. 1.

In one of the embodiments of the present invention, the compound growth hormone has an increased half-life existence in vivo.

In one of the embodiments of the present invention, the compound growth hormone has an increased shelf life.

In one of the embodiments of the present invention, the compound growth hormone can be a protein.

One of the embodiments of the present invention relates to the connection of growth hormone, a polypeptide sequence which is at least 80%, such as 90%, such as 95% identical hGH defined by SEQ ID No. 1. In the following embodiments, the polypeptide is at 96%, 97%, 98% or 99% identical hGH defined by SEQ ID No.1.

In one of the embodiments of the present invention, the compound growth hormone is a polypeptide containing an amino acid sequence identical to at least 20%, such as at least 30%, such as at least 40%, the AK for example at least 50%, for example, at least 60%, such as at least 70%, such as at least 80%, such as at least 90% identical, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% with SEQ ID No. 1, and the polypeptide has the activity measured in the analysis described in Example 3 and Example 3A (hypophysectomised rats) comprising at least 1%, such as at least 5%, such as at least 10%, such as at least 25% of the activity of hGH. For the avoidance of doubt it should be noted that the connection of growth hormone according to the invention may also have a higher activity in these assays than hGH. If the connection of growth hormone in some way modified, the activity of growth hormone in relation to hGH should be measured on an unmodified compound growth hormone, as this modification can significantly change the activity. For example, in the case of compound growth hormone modified using the modifying its properties group, which prolong the half-life of the functional existence in vivo of the compounds of the growth hormone activity of such modified compounds of growth hormone can be a lot lower than the activity of the hGH, and uh what about the decrease is compensated by prolonged retention time in the body. In one of the embodiments of the compound of growth hormone is a fragment of such a polypeptide, and in this snippet saved a significant amount of activity of growth hormone, as described above.

In one of the embodiments of the present invention, the compound growth hormone is a shortened version of hGH, that is, one or more amino acid residues from the N - and/or all of the corresponding SEQ No. 1 delegated while maintaining the specified connection is desired biological properties of wild-type hGH.

One of the embodiments of the present invention relates to the compound growth hormone containing additional disulfide bonds in SEQ ID No. 1 or containing one or more than one additional disulfide bond(s) compared to human growth hormone as defined in SEQ ID No. 1, with the specified connection has activity in vitro that is comparable to the activity in vitro hGH defined by SEQ ID No. 1. Activity in vitro of the compounds of growth hormone is preferably measured in BAF-analysis, which is described in this description in Example 3. In one of the embodiments of the compound according to the invention can have activity in vitro, different from activity in vitro hGH. As described above, the lower activity in vitro can be compensated for other functional properties in vivo. In one of the embodiments of activity in vitro which may be for example, at least 1%, such as at least 5%, such as at least 10%, such as at least 25% of the activity of hGH. In the following embodiment EU50for connection to EC50for wild-type hGH, identified by SEQ ID No. 1, is not more than 10, not more than 8, no more than 6, no more than 4, no more than 2. In one of the embodiments of the ratio EC50for the specified connection to the EU50for wild-type hGH, identified by SEQ ID No. 1 is 5-0,01 or, for example, 3-0,01, or is, for example, 2-0,01. Alternatively EC50in accordance with this invention can be measured in the analysis using surface plasma resonance (Biacore) as described in Example 4. In the respective embodiments of activity in vitro, was determined using Biacore, may be, for example, at least 1%, such as at least 5%, such as at least 10%, such as at least 25% of the activity of hGH. In the following embodiments the ratio EC50for connection to the EU50for wild-type hGH, identified by SEQ ID No. 1 determined using Biacore, is not more than 10, not more than 8, no more than 6, no more than 4, no more than 2. In one of the embodiments of the ratio EC50for the specified connection to the EU50for wild-type hGH, identified by SEQ ID No. 1 is 5-0,01, and the and, for example, 3-0,01, or, for example, 2-0,01.

Other examples of compounds of GH, which may be introduced an additional disulfide bridges include compounds optionee in WO 92/09690 (Genentech), US 6004931 (Genentech), US 6143523 (Genentech), US 613636 (Genentech), US 6057292 (Genentech), US 5849535 (Genentech), WO 97/11178 (Genentech), WO 90/04788 (Genentech), WO 02/055532 (Maxygen APS and Maxygen Holdings), US 5951972 (American Cynanamid Corporation), US 2003/0162949 (Bolder Biotechnologies, Inc.), included in this description by reference. Also included natural hGH variants, such as the variant with a molecular mass of 20 kDa, described in Masuda, N. et all., Biochim. Biophys. Acta 949 (1), 125-131 (1988).

In all embodiments described herein, there is also the possibility that the compound growth hormone has a Gly residue at the position corresponding to the position 120 in SEQ ID No. 1.

In the context of the present invention, the words "human growth hormone (hGH)", "wt hGH and hGH wild-type (wthGH)" are used interchangeably and all refer to a protein with the amino acid sequence SEQ ID No.1.

In the context of the present invention, the terms "peptide" and "polypeptide" are used interchangeably and are intended to indicate the same. Assume that the terms "peptide" or "polypeptide" refers to a sequence of two or more amino acids connected by peptide bonds, these amino acids may be natural or unnatural. WMO is Asia in the composition of amino acids can be from the group of amino acids, encoded by the genetic code, and they can be natural amino acids that are not encoded by the genetic code, as well as synthetic amino acids. Natural amino acids that are not encoded by the genetic code are, for example, Hyp (hydroxyproline), Y-carboxyglutamate, Orn (ornithine), phosphoserine, D-alanine and D-glutamine. Synthetic amino acids include amino acids obtained by chemical synthesis, such as D-isomers of amino acids encoded by the genetic code such as D-alanine and D-leucine, Aad (α-aminoadipyl acid), Aib (α-aminoadamantane acid), Abu (α-aminobutyric acid), Agl (α-aminoglycan), Asu (α-aminoarabinose acid), Cha (β-cyclopentylamine), Chg (cyclohexylglycine), Dab (α,γ diaminobutane acid), Dap (α,β-diaminopropionic acid)The NSA (homocysteine), Hpr (homopolar), Nle (norleucine), Phg (phenylglycine), Hph (homophenylalanine), 1Nal (β-(1-naphthyl-alanine)), 2Nal (β-(2-naphthyl-alanine)), 2Pal (β-(2-pyridyl)-alanine), 3Pal (β-(3-pyridyl)-alanine), Pip (4-aminopiperidin-4-carboxylic acid), Pra (propargyl-glycine), Pyr (pyroglutamyl acid), Gla (γ-carboxyglutamic acid), Hci (homotetramer), Nva (Norvaline), Tle (tert-butyl glycine, β-alanine, 3-aminomethyl-benzoic acid and orthoaminophenol acid.

This term also encompasses the term "proteins", which can consist of one polypeptide chain or two sludge is more polypeptide chains held together by non-covalent or covalent interactions, such as, for example, cysteine bridges.

It should be understood that this term also is intended to include peptides, on the basis of the obtained derivative, for example by joining groups such as PEG, carbohydrates, fatty acids, albumin-binding substances, alkyl chain, lipophilic groups, vitamins, bile acids, but not limited to; or spacers to the side chain or main chain of the peptide in addition to the additional disulfide bonds. The term "peptide" includes any suitable peptide and can be used interchangeably with the terms "polypeptide" and "protein", unless specified otherwise or there are no contradictions in context, assuming that the reader knows that each type of the corresponding molecule containing the amino acid polymer that can be associated with significant differences and thus to form an individual embodiment of the present invention (for example, such a peptide as an antibody, which consists of several polypeptide chains, substantially different, for example, single-chain antibodies, peptide immunoadhesin or single-stranded immunogenic peptide). Therefore, used in this description, the term "peptide" in General, you should understand the ü as a reference to any suitable peptide of any suitable size and composition (in relation to the number of amino acids and the number of linked chains in the protein molecule). Moreover, the peptides described herein, may contain non-natural and/or non-L amino acid residues, unless otherwise indicated or there is no contradiction in context.

The term "peptide", unless specified otherwise or there are no contradictions in context (and if treated as an individual embodiment of the term(s) "polypeptide" or "protein"), also encompasses derivatives of the peptide molecules. Derived peptide molecule is a peptide in which one or more amino acid residues of this peptide is chemically modified (for example, by alkylation, acylation, the formation of ester or education amide) or associated with one or more than one non-amino organic and/or inorganic atomic or molecular substituent (for example, the group of polyethylene glycol (PEG), a lipophilic substituent (which may be associated with the amino acid sequence of the peptide through the remainder or a spacer group, such as β-alanine, γ-aminobutyric acid (GABA), L/D-glutamic acid, succinic acid, and the like), fluorophores, Biotin, a radionuclide, and so on), and which (peptide) may also or alternatively be irrelevant, unnatural and/or non-L amino acid residues, unless otherwise indicated or there is no contradiction what about the context (however, again it should be recognized, such derivatives themselves can be considered as independent features of the present invention, and the inclusion of such molecules in the concept of the peptide most likely be done for the sake of convenience in the description of the present invention than to imply any equivalence between the unmodified peptides and derivatives such).

Non-limiting examples of such amino acid residues include, for example, residues of 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, β-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoethanol acid, 3-aminoadamantane acid, 2-aminopimelic acid, 2,4-diaminoalkanes acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, illegitimizing, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allitaliana, N-methylglycine, N-methylisoleucine, 6-N-methyllysine, N-methylvaline, Norvaline, norleucine, ornithine, propargylglycine and halogenated amino acids statins.

"Connection" described in this invention can be a "protein" or "peptide" or "polypeptide", which can be an "analog" or "derivative" or "variant", saranayde the desired biological activity, similar to those in wthGH, regardless of how it was modified.

The term "analog" or "variant" as used in this description, related polypeptide, means a modified version of the specified peptide where one or more amino acid residues of the peptide is replaced with other amino acid residues, and/or where one or more amino acid residues delegated from the peptide, and/or where one or more amino acid residues added to the peptide. Such substitution, or insertion, or deletion of amino acid residues may occur at the N-end of the peptide, and/or at the C-end of the peptide, and/or between the N - or C-ends of the peptide. It should be understood that all amino acids that do not specify an optical isomer, represent the L-isomer.

The term "disulfide bonds or disulfide bridge" are used interchangeably and are intended to indicate the same. "Disulfide bond or a disulfide bridge in proteins is formed between the thiol groups of cysteine residues.

The terms "additional cysteine" or "introduced cysteine" are used interchangeably and are intended to indicate the same. It is implied that these terms include the cysteine residue that is not present in wild-type hGH. In order to minimize structural changes, the residue(s) cysteinate introduced by replacing the amino acid residue(s), thus, the length of hGH is saved. Inserting additional Cys residue can be tolerated in the areas of the loops or in the border spirals areas, whereas the introduction of Cys residues within helices is less attractive.

The terms "additional disulfide bonds" or "introduced disulfide bond" are used interchangeably and are intended to indicate the same. It is implied that these terms include disulfide bond formed between two cysteine residues, at least one of which is not present in wild-type hGH.

The term "derivative", as used herein, refers to a peptide or polypeptide in which one or more amino acid residues of this peptide is chemically modified by the introduction of polymer, such as PEG, carbohydrate groups, albumin-binding substances, fatty acids, lipophilic groups, vitamins, bile acids or spacer in the side chain or the main chain of the compounds of growth hormone. Chemical modification can also be temporary, i.e. they can easily be removed in vivo. Chemical modifications can be introduced excision, for example with the cell, or can be a chemical modification implemented in the peptide after the expression.

The term "identity" to the th known in the art, refers to the relationship between sequences of two or more peptides, which is determined by comparing these sequences. In this area, "identity" also means the degree of similarity of the sequences of the peptides, which is determined by the number of similar pairs in circuits consisting of two or more amino acid residues. "Identity" is a measure of the percentage of identical pairs for two or more sequences using the alignment taking into account gaps (if any) and with reference to a particular mathematical model or computer program (i.e., "algorithms"). Identity of related peptides can be calculated with known methods. Such methods include, but are not limited to, the methods described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M. and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the tested sequences. Methods for determining the identity described in computer programs, which in revelation is the access. Preferred methods using computer programs to determine identity between two sequences include the GCG software package, including GAP (Devereux et al., Nucl. Acid Res. 12, 387 (1984); Genetics Computer Group (GCG), University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215. 403-410 (1990)). The BLASTX program is publicly available at the National center for biotechnology information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., above). Identity is also possible to use a well-known algorithm, Smith Waterman.

For example, using the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wis.), two of the peptide, which must be defined percent identity of the sequences align with the aim of obtaining optimal matching of their respective amino acids ("similar area", which is determined by the algorithm). With this algorithm uses the penalty to break" (which is calculated in the form multiplied by 3 average diagonal; the "average diagonal" is the average value of the diagonal matrix used in the comparison; the "diagonal" is the score or metric assigned for each full match of amino acids in accordance with the specific matrix comparison) and the penalty for the length of the gap (which is usually the submitted is a 1/10 of a share of the penalty to break), and matrix comparisons, such as RAM (protein weight matrix) 250 or BLOSUM 62. Standard matrix comparison (see Dayhoff et al., Atlas of Protein Sequence and Structure, 5, (1978) for a matrix comparing FRAMES 250; Henikoff et al., PNAS USA 89, 10915-10919 (1992) for a matrix comparison BLOSUM 62) also used this algorithm.

Preferred parameters for comparison of peptide sequences include the following:

algorithm: Needleman et al., J. Mol. Biol. 48, 443-453 (1970);

matrix comparison: BLOSUM 62 from Henikoff et al., PNAS USA 89, 10915-10919 (1992);

- the penalty for gap: 12;

- the penalty for a gap length: 4;

the threshold of similarity.

The GAP program is useful when working with the above settings. The aforementioned parameters are the default parameters for comparisons of peptides (along with no penalty for end gaps) using the GAP algorithm.

It is believed that the terms "protease or protease" includes all the enzymes, which are able to catalyze the hydrolytic cleavage of the peptide bond. The protease can be intracellular, extracellular or membrane-bound by proteases, proteinases or peptidases and include protease, located in the lumen of the intestine of mammals, and protease present in the plasma of mammals. The protease can be a protease of two types: endoprotease and ectoprocta. The protease can be a protease which of the following types: serine, cysteine, aspartate or metalloprotease, but not limited to that. Specific examples of proteases are trypsin, chymotrypsin, pepsin, elastase, factor VIIa, factor XA, proteinase K, carboxypeptidase, DPPIV (dipeptidyl peptidase-IV), neutral endopeptidase, Grasim In, Proline-endopeptidase, staphylococcal peptidase I, thermolysin, thrombin, Arg-C proteinase, Asp-N endopeptidase, caspase 1-10, clostridia, enterokinase, glutamyltranspeptidase, Grasim, LysC, LysN, Proline-endopeptidase and Staphylococcus peptidase I.

The terms "resistant to proteolytic destruction", or "increased stability to proteolytic destruction", or "increased stability to proteolytic cleavage", or "improved proteolytic stability", or "proteolytic stability" are used interchangeably and are intended to indicate the same. As applied to the connection hGH according to the invention assumes that these terms indicate that the polypeptide chain of the specified connection hGH is cleaved under the action of the protease under specific conditions with a lower rate than wild-type hGH.

The rate of proteolytic cleavage of the protein can be measured by several methods known to the person skilled in the art. An example of the analysis, which measure the rate of destruction of hGH or the unity hGH, described in Example 5.

The invention also relates to methods useful for improving the pharmacological properties of the compounds of hGH. These pharmacological properties can represent, for example, increasing the half-life of the functional existence in vivo half-life existence in plasma in vivo, the average retention time in the body, decreasing renal clearance.

The term "half-life functional existence in vivo" is used in its usual meaning, i.e. the time at which saves 50% of the biological activity of the peptide, for example compounds of growth hormone, the compound growth hormone is still present in the body/target organ, or the time at which the activity of the peptide, for example compounds of growth hormone, is 50% of its initial value. Alternatively, the determination of the half-period of the functional existence in vivo it is possible to determine the half-period of existence in plasma in vivo and duration, i.e. the time at which the blood circulates 50% of the peptide before will be displayed. It is often easier to perform the determination of the half-period of existence in the blood plasma than half of the functional existence, and usually the amount of half existence in plasma is a good indicator of the magnitude of the half-cycle functional with the existence in vivo.

The present invention also relates to pharmaceutical compositions containing compounds of growth hormone that are defined or described in this specification.

In one of the embodiments of the pharmaceutical compositions of the present invention can be introduced in various dosage forms, for example, in the form of solutions, suspensions, emulsions, microemulsions, multiple emulsion, foams, salves, pastes, plasters, ointments, tablets, coated tablets, tablets, manufactured together with enhance the absorption of compounds, means for rinsing, capsules, such as hard gelatin capsules and soft gelatin capsules, capsules, coated, suppositories, drops, gels, sprays, powders, microparticles, nanoparticles, aerosol, inhalation, injection, solutions for transformations in situ, for example gelation in situ solidification in situ deposition of in-situ crystallization in situ, solution for infusions and implants).

In one of the embodiments of the present invention the pharmaceutical compositions may be introduced via oral, subcutaneous, intramuscular, nasal and/in (intravenous) administration.

In one of the embodiments of the present invention, the oral pharmaceutical compositions can be introduced by several routes of administration, for example, lingual, sublingual, transbuccal enim, in the mouth, stomach and intestines.

In one of the embodiments of the pharmaceutical compositions of the present invention are useful in the manufacture of solids, semi-solid substances, powder and solutions for the introduction of peptide conjugate in the lungs, such as, for example, the conjugate based on GH, using, for example, the dosing inhaler, dry powder inhaler and a nebulizer, all of these devices are well known to specialists in this field of technology.

In addition, in one of the embodiments of the pharmaceutical composition according to the invention can be mixed with a carrier of drugs, drug delivery and advanced drug delivery or attached to them, for example through covalent, hydrophobic and electrostatic interactions, with the aim of further increasing the stability of the conjugate on the basis of GH, increase bioavailability, increase solubility, mitigation of adverse effects, achieve chronotherapy well known to specialists in this field, and to improve compliance of patients mode and a treatment regimen or any combination thereof. Examples of carriers, drug-delivery systems and advanced drug-delivery systems include, but are not limited to, polymers, for example the EP cellulose and derivatives, polysaccharides, such as dextran and derivatives, starch and its derivatives, poly(vinyl alcohol), acrylate and methacrylate polymers, polylactic and polyglycolic acid and their block copolymers, polyethylene glycols, carrier proteins, such as albumin, gels, for example thermogenerators system, such as a block copolymer system, well known to experts in this field, micelles, liposomes, microspheres, nanoparticles, liquid crystals and their dispersion, the crystal phase L2 and their dispersions are well known to experts in the field of phase behavior in systems of lipid-water, polymeric micelles, multiple emulsions, savemarriage agents, sonomicrometry agents, cyclodextrins and derivatives thereof, and dendrimers.

Various examples of delivery systems for oral compositions included in this description by reference, include nonionic surfactants, which are known to increase the penetration of hydrophilic compounds. Examples of nonionic surfactants are: katrinasacay sodium, tartaric acid, Brij56, Brij58, Brij35, Brij30, esters of sugars and of fatty acids, taurodeoxycholate sodium, sodium dodecyl sulphate, p-tert-op-polyoxyethylene-9.5 (Triton X-100), as described in Takatsuka et al., Eur. J. Pharm. Biopharm. 62, 52-58 (2006). Oral delivery system may also include in the BOJ inhibitors of proteases and mucolytic agents. Examples of protease inhibitors are soybean trypsin inhibitor, Aprotinin and hemostatis. Examples mucolytic substances are dithiotreitol and N-acetylcysteine. The increased absorption in the intestine subabsolute hydrophilic compounds is provided by the simultaneous use of a mucolytic agent and non-ionic surfactants. In addition, Emisphere has developed a 5-CNAC and similar compounds (WO2008101240, WO200811283687, WO2008027854, WO2008014430, US20080095837).

Delivery system oral compositions can also include modulators of claudino, which function as specific substances, the opening close contacts of epithelial cells. These modulators of claudino function as from time to time, and constantly and serve as a barrier to protein complexes that hold epithelial cells in close contact (Kondoh et al., Mol. Pharmacology, 67, 749-756 (2005)). Other examples of delivery systems oral compositions include mucoadhesive agents, for example, tiradera excipients (jointly included in the composition) or covalently attached side chains can improve the adhesion to the mucosa, chitosan and molecules carbomer, polyacrylates, PEG and its derivatives (Palmberger et al., Eur. J. Pharm. Biopharm. 66, 405-412 (2007); Leitner, V. M. et al., Eur. J. Pharm. Biopharm. 56, 207-214 (2003); H. L Leuβen et al., Pharm. Res. 13, 1668-1672 (1996; H. L. Leuβen et al., Int. J. Pharmaceuticals 141, 39-52 (1996); Takatsuka et al., Eur. J. Pharm. Biopharm. 62, 52-58 (2006)). Additional examples of delivery systems oral compositions include caveola/lipid rafts, SMVT (sodium-dependent Transporter for multivitamins). Other examples of compositions for oral delivery include receptor-mediated transcytosis are activated, for example with the participation of the IRF (receptor for intrinsic factor) that uses vitamin B12 (cobalamin) as substrate, FcRn (neonatal Fc receptor) and transferrin (M. Gumbleton, Adv. Drug Del. Rev. 49, 281-300 (2001); K. C. Partlow et al., Biomaterials 29, 3367-3375 (2008); Lee et al., Biotechnol. Appl. Biochem. 46, 211-217 (2007); S. Y. Chae et al., Bioconjugate Chem. 19, 334-341 (2008); Russell-Jones, G.: Chapter 17 in Membrane Transporters as Drug Targets (1999); Said and Mohammed, Curr. Opin. Gastroent. 22, 140-146 (2006); Chalasani et al., J. Con.Release 117, 421-429 (2007); H. Li & Z. M. Qian Med. Res. Rev. 22, 225-250 (2002); Liang & Yang Biochem. Biophys. Res. Comm. 225, 734-738 (2005)).

In one embodiments of the compounds GH according to the present invention show activity of growth hormone and can be used to treat diseases or conditions in which it will be useful to the increase in the number of circulating in the blood growth hormone. Such diseases or conditions include growth hormone deficiency (GHD); Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; down syndrome; chronic renal disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in children receiving treatment at p is ogramme HAART (highly active antiretroviral therapy (high active anti-rertrovirus therapy) (children diagnosed with HIV/HALS); a small (in size) of children born small for gestational age (SGA); short stature in children born with very low birth-weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature (ISS); GHD in adults; fractures in the tubular bones or bones, such as tibia, fibula, femur, humerus, radial bone, ulna, clavicle, metacarpal bone, metatarsal bone and the bones of the toes; the fractures in the cancellous bone or spongy bone, such as the skull, the base of the brush and the base of the foot; patients after surgery on the tendons or ligaments, such as the wrist, knee, or shoulder; patients having or experiencing distraction osteogenesis; patients after hip replacement or (articular) disk, recovery, meniscus, binding vertebrae or fixation of prostheses, such as the knee, hip, shoulder, elbow, wrist or jaw; patients with fixed therein a material for osteosynthesis, such as pins, screws and plates; patients with narastaniem or improper healing of fractures; patients after osteotomy, for example, the tibia or 1st toe; patients after transplantation of the graft; the degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patie the tov with Turner syndrome; osteoporosis in men; adult patients on continuous dialysis (APCD); associated with malnutrition, cardiovascular disease on the background of the APCD; reversal of cachexia in the background APCD; cancer in APCD background; chronic obstructive pulmonary disease on the background of the APCD; HIV in APCD background; old age in APCD; chronic liver disease on the background of the APCD, fatigue syndrome in APCD background; Crohn's disease; the weakening of the liver; individuals males with HIV infections; syndrome shortened small intestine; Central obesity; syndrome of HIV-associated lipodystrophy (HALS); male infertility; patients after major elective surgery, alcohol/drug detoxification or neurological trauma; aging; weakened elderly; osteoarthritis; traumatic injury of the cartilage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain damage; subarachnoid haemorrhage; very low weight at birth; metabolic syndrome; glucocorticoid myopathy; or short stature due to treatment with glucocorticoids in childhood. Growth hormone is also used to accelerate healing of muscle tissue, nervous tissue or wounds; the acceleration or improvement of blood flow to damaged tissue; or the decrease of infection in the damaged tissue.

In one of the in the of leweni the present invention relates to a method of treatment of diseases, where the activity of compounds of growth hormone can be used to treat diseases or conditions in which it will be useful to the increase in the number of circulating in the blood connection, growth hormone, and this method includes the introduction to the patient an effective amount of the pharmaceutical compositions of the compounds of growth hormone or its conjugate with SEQ ID No.1.

In one of the embodiments of the present invention relates to a method comprising the administration to a patient in need, an effective amount of a therapeutically effective amount of a compound of growth hormone according to the invention. Thus, according to the present invention, a method for treatment of these diseases or conditions, comprising the administration to a patient in need, a therapeutically effective amount of a compound of growth hormone according to the present invention.

The term "therapeutically effective amount" of the compounds according to the invention as it is used herein, refers to the amount sufficient to cure, mitigate or partial suppression of clinical symptoms of a specific disease and its complications. A quantity sufficient for this exercise, is defined as "therapeutically effective amount". The effective amount for each task will depend, for example, on the severity Zab the diseases or lesions, as well as weight, gender, age and General health of the subject. It is obvious that the determination of the appropriate dosage can be achieved using routine experimentation and is within the competence of a qualified physician or veterinarian.

In one of the embodiments of the invention the application of the compounds of growth hormone or its conjugate in the manufacture medicines used in the treatment of the aforementioned diseases or conditions.

It is expected that the compounds of growth hormone, which is defined and described in the present invention, will be used as a therapeutic protein.

Methods plucene polypeptides are well known in the art. For example, polypeptides can be obtained classical peptide synthesis, e.g. solid phase peptide synthesis, using chemistry using tert-BOC (tert-butyloxycarbonyl) or Fmoc (fluorenylmethyl-carbonyl), or other recognized methods, see, for example, Greene and Wuts, "Protective Groups in Organic Synthesis", John Wiley & Sons, 2006.

The polypeptides can also be obtained by a method which comprises culturing the host cell containing the DNA sequence encoding this polypeptide, and is able to Express the polypeptide in a suitable nutrient medium under conditions dopuskau the x expression of the peptide. With regard to polypeptides containing non-natural amino acid residues, the recombinant cell must be modified so that non-natural amino acids have been incorporated into the polypeptide, for example, by use of a mutant tRNA.

In addition, the polypeptides can be produced using cell-free systems of transcription/translation in vitro. The polypeptide containing the new unnatural amino acids can also be obtained using systems suppression of mutations shift the reading frame and nonsense mutations, for example as described in J. Am. Chem. Soc. 125, 11782-11783 (2003); Science 301, 964-967 (2003); Science 292, 498-500 (2001); Science 303, 371-373 (2004) and in the references.

The medium used for culturing the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex medium containing the appropriate supplements. Suitable environments are available from commercial suppliers or may be prepared according to the published writings (e.g., in catalogues of the American type culture collection). The peptide produced by the cells may then be recovered from the culture medium by conventional methods, including separating the host cells from the medium by centrifugation or filtration. With regard to the extracellular the products, the protein components of the supernatant allocate filtration, column chromatography or precipitation, such as microfiltration, ultrafiltration, isoelectric precipitation, purification using a variety of chromatographic procedures, e.g. ion exchange chromatography, hydrophobic interaction chromatography, gelfiltration chromatography, affinity chromatography, or the like, depending on the type of the considered polypeptide. In the case of intracellular or periplasmatic products cells isolated from the culture medium, is subjected to destruction or permeabilization and extraction to extract the product of the polypeptide or its predecessor.

The DNA sequence encoding the polypeptide, by origin may be of genomic or cDNA, for example, resulting from the preparation of genomic libraries or cDNA and screening for DNA sequences coding for the peptide in whole or in part, by hybridization using specific probes are DNA or RNA according to standard techniques (see, for example, Sambrook, J, Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the polypeptide, can also be obtained by synthesis using widely accepted the standard methods, for example phosphoramidite method described in Beaucage and Caruthers, Tetrahedron Letters 22, 1859-1869 (1981), or the method described in Matthes et al., EMBO Journal, 3, 801-805 (1984). The DNA sequence can also be obtained using the polymerase chain reaction using specific primers, for instance as described in US 4683202 or Saiki et al., Science 239, 487-491 (1988).

The DNA sequence encoding the peptide, subject expression can be integrated into any vector, which conveniently can be subjected to procedures of recombinant DNA, and the choice of vector will often depend on the host cell into which it is introduced. Thus, the vector may be a stand-alone can replicate the vector, i.e. a vector which exists in the form of extrachromosomal structural units, replication which does not depend on chromosomal replication, e.g. a plasmid. Alternatively, the vector may be such that upon introduction into the host cell integrates into the genome of the host cell and is replicated together with the chromosome(s) in which(s) he integrated.

The vector may be a expressing the vector in which the DNA sequence encoding a polypeptide that is functionally linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence, which demonstriruet the transcriptional activity in the selected host cell and may be derived from genes encoding proteins either homologous or heterologous proteins of the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide, which is necessary to Express the number of host cells are well known in this field, for comparison see, for example, Sambrook et al., above.

The DNA sequence encoding the peptide to be expressed, if necessary, can also be functionally connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. Recombinant vector according to the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell.

The vector may also contain breeding marker, e.g. a gene product that complements to the norms of a defect in the host cell or which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. In the case of large-scale production of breeding marker, for example, may not be resistant to antibiotics, such as genes for antibiotic resistance in the vector can be removed when the vector is used to coarsely Arinaga production. Methods of elimination of genes for resistance to antibiotics of vectors known in this field, see, for example, US 6358705, which is included in this description by reference.

To direct the expression of the peptide for the secretory pathway of the host cells, the recombinant vector may be provided secretory signal sequence (also known as a leader sequence, shall sequence or pre sequence). The secretory signal sequence is joined to a DNA sequence that encodes a peptide, in the correct reading frame. The secretory signal sequence is usually located in the 5'-position with respect to the DNA sequence that encodes the peptide. The secretory signal sequence may be a sequence, normally associated with a peptide, or can be a sequence from a gene encoding another secretory protein.

The procedures used for ligation of DNA sequences coding for a peptide to be expressed, the promoter and possibly a terminator and/or secretory signal sequence, respectively, and procedures for introducing them into suitable vectors containing the information necessary for replication, are well known to specialists in this field (see, for example, ambrook et al., above).

A host cell, into which is injected the DNA sequence or the recombinant vector may be any cell capable of producing the peptide, and includes bacteria, yeast, fungi, and cells of higher eukaryotes. Examples of suitable host cells are well known and used in the field, are, without limitation, E. coli, Saccharomyces cerevisiae or cell lines of mammalian BHK (kidney baby hamster) or Cho (Chinese hamster ovary). The peptide to be expressed, can also be obtained using the system of transcription/translation in vitro, well-known in this field.

All references, including publications, patent applications, and patents, cited herein, have been included thus in its entirety by reference, and to the same extent as if each reference was specifically and individually indicated to include by reference, and was described in this description in its entirety (the maximum possible extent permitted by law).

All headings and subheadings used in this description for convenience only, and should not be construed as in any way limiting this invention.

The use of any and all examples, or typical language style (e.g., "such as"), adopted herein, is provided mainly on the I am the best lighting of the invention and is not intended to limit the scope of the invention, unless approved otherwise. Any wording in this description should not be construed as indicating any non-claimed element that is essential for the application of the invention in practice.

Citation and inclusion in the description of the patent documents is made for convenience only and do not reflect any opinion as to the validity, patentability and/or legal support these patent documents.

This invention includes all modifications and equivalents of the subject matter shown in the accompanying description, the claims, as permitted by the proper law.

The invention described herein, without restrictions on this document, hereinafter described in the following embodiments.

Embodiment 1. Compound growth hormone containing additional disulfide bonds in SEQ ID No. 1.

The embodiment 2. Compound growth hormone, corresponding to the embodiment 1, containing additional disulfide bond between at least one of the pairs of amino acids in positions corresponding to R16C/L117C, AS/ES, NS/MS, N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, RS/ES, RS/CS, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1.

Embodiment 3. The connection gormo the and growth, corresponding to embodiment 2, where the compound growth hormone contains additional disulfide bond between at least one of the pairs of amino acids in positions corresponding to AS/ES, NS/MS, Q84C/Y143C, S71C/S132C, and/or R94C/D107C in SEQ ID No. 1.

Embodiment 4. Compound growth hormone, corresponding to the embodiment 1, where the connection HGH contains at least one pair of mutations corresponding to R16C/L117C, AS/ES, NS/MS, N47C/T50C, Q49C/G161C, F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, RS/ES, RS/T67C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1.

Embodiment 5. Compound growth hormone, corresponding to the embodiment 4, where the connection HGH contains at least one pair of mutations corresponding to AS/ES, NS/MS, S71C/S132C, Q84C/Y143C and R94C/D107C in SEQ ID No. 1.

Embodiment 6. Compound growth hormone containing one or more than one additional disulfide bond(s) compared to human growth hormone as defined in SEQ ID No. 1.

Embodiment 7. Compound growth hormone, corresponding to any of the preceding embodiments, where the compound growth hormone contains additional disulfide bond between a loop segment and a helical segment, or within a loop segment, or between the hinge segments, or between sleep the social segments.

Embodiment 8. Compound growth hormone corresponding to embodiment 6 or 7, where the compound contains at least one pair of mutations corresponding to R16C/L117C, AS/ES, NS/MS, D26/V102C, D26/Y103C, N47C/T50C, Q49C/G161C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, RS/ES, RS/CS, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C, F92C/T148C, R94C/D107C, V102C/A105C, L156C/F146C, L156C/T148C and/or V185C/S188C in SEQ ID No. 1.

Embodiment 9. Compound growth hormone, corresponding to the embodiment 8, wherein the compound contains at least one pair of mutations corresponding to AS/ES, NS/MS, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

The embodiment 10. Compound growth hormone, corresponding to the embodiment 9, where the compound contains at least one pair of mutations corresponding to AS/ES, NS/MS, F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

Embodiment 11. Compound growth hormone, corresponding to any of the preceding embodiments, where the compound growth hormone contains additional disulfide bond, and at least one of cysteines present in L3 corresponding to AA 128-154 in SEQ ID No. 1, or, for example, in the area corresponding to AA 135-148 in SEQ ID No. 1.

Embodiment 12.Compound growth hormone, corresponding to embodiment 11, where at least one of cysteine additional disulfide bond is present in L3 in a position corresponding AA, AA, AA, AA, AA or AA, preferably AA or AA in SEQ ID No. 1.

Embodiment 13. Compound growth hormone, corresponding to the embodiment 12, where the compound contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C and/or F92C/T148C in SEQ ID No. 1.

Embodiment 14. Compound growth hormone, corresponding to the embodiment 13, where the compound contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID No. 1.

Embodiment 15. Compound growth hormone, corresponding to the embodiment 14, where the compound contains at least one pair of mutations corresponding to F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or F92C/T148C in SEQ ID No.1.

Embodiment 16. The connection of growth hormone according to any one of the preceding paragraphs, where the compound growth hormone contains additional disulfide bond connecting L3 with L1.

Embodiment 17. Compound growth hormone, corresponding to the embodiment 16, where the compound contains complement the function disulfide bonds, linking amino acid residue corresponding to AA, AA, AA, AA, AA or AA in L3, with the amino acid corresponding to AA or AA in L1, SEQ ID No. 1.

Embodiment 18. Compound growth hormone, corresponding to the embodiment 16, where the compound contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, F54C/F146C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C and/or S71C/S132C in SEQ ID No. 1.

Embodiment 19. Compound growth hormone, corresponding to the embodiment 18, where the compound contains at least one pair of mutations corresponding to F54C/S144C, F54C/F146C, I58C/Q141C, I58C/S144C, P59C/Q137C and/or S71C/S132C in SEQ ID No. 1.

The embodiment 20. Compound growth hormone, corresponding to any one of embodiments 1-15, where the compound growth hormone contains additional disulfide bond connecting L3 spiral segment.

Embodiment 21. Compound growth hormone, corresponding to the embodiment 20, where the compound growth hormone contains additional disulfide bond connecting L3 spiral 2.

Embodiment 22. Compound growth hormone, corresponding to the embodiment 21, where the compound contains an additional disulfide bonds linking amino acid residue corresponding to AA or AA in H2, with the amino acid corresponding to AA or AA in L3, SEQ ID No. 1.

Embodiment 23. Compound growth hormone, corresponding to the embodiment 21, where the link which contains at least one pair of mutations, relevant L73C/S132C, L73C/F139C, R77C/I138C, R77C/F139C, L81C/Q141C, L81C/Y143C, Q84C/Y143C, Q84C/S144C, S85C/Y143C, S85C/S144C, P89C/F146C, F92C/F146C and F92C/T148C in SEQ ID No. 1.

Embodiment 24. Compound growth hormone, corresponding to the embodiment 23, where the compound contains at least one pair of mutations corresponding to L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID No. 1.

Embodiment 25. Compound growth hormone, corresponding to the embodiment 24, where the compound contains at least one pair of mutations corresponding to L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or F92C/T148C in SEQ ID No.1.

Embodiment 26. Compound growth hormone, corresponding to any one of embodiments 1-10, where the compound growth hormone contains additional disulfide bond connecting L2 spiral 1.

Embodiment 27. Compound growth hormone, corresponding to the embodiment 26, where the compound contains at least one pair of mutations corresponding to D26C/V102C or D26C/Y103C.

Embodiment 28. Compound growth hormone corresponding to any of the previous embodiments where the polypeptide sequence at least 80%, for example 90%, such as 95%, such as 96%, such as 97%, for example 98% or, for example 99% identical hGH defined by SEQ ID No. 1.

Embodiment 29. Compound growth hormone corresponding to any of the preceding embodiments, where the activity in vitro of the compounds is at m is re 5% of the activity of wild-type hGH, defined by SEQ ID No. 1.

The embodiment 30. Compound growth hormone, corresponding to any of the preceding embodiments, where the bottom half of the functional existence of the polypeptide in vivo in 2 or more times higher compared to human growth hormone.

Embodiment 31. Compound growth hormone, corresponding to any of the preceding embodiments, where the bottom half of the functional existence of the polypeptide in vivo 2-10 times higher in comparison with human growth hormone.

Embodiment 32. Compound growth hormone corresponding to any of the preceding embodiments, where the compound growth hormone is stabilized against destruction under the action of the protease(s), such as digestive proteases, such as pepsin, trypsin, chymotrypsin, carboxypeptidase and/or elastase.

Embodiment 33. Compound growth hormone corresponding to embodiment 32, where the connection is stable against decay under the action of chymotrypsin and/or elastase.

Embodiment 34. Compound growth hormone corresponding to embodiment 33, where the compound contains at least one pair of mutations corresponding to n/M, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID No. 1.

Embodiment 35. Compound growth hormone corresponding to embodiment 33, where the connection of the content is it at least one pair of mutations, relevant n/M, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C and/or S85C/Y143C in SEQ ID No. 1.

Embodiment 36. Compound growth hormone corresponding to embodiment 33, where the compound contains at least one pair of mutations corresponding to D26/V102C, D26/Y103C, S57C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

Embodiment 37. Compound growth hormone corresponding to embodiment 33, where the compound contains at least one pair of mutations corresponding to I58C/S144C, P59C/Q137C, S71C/S132C, Q84C/Y143C, S85C/Y143C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1.

Embodiment 38. Compound growth hormone corresponding to embodiment 33, where the compound contains at least one pair of mutations corresponding to S57C/Y143C, Q84C/Y143C, S85C/Y143C and/or S85C/S144C in SEQ ID No.1.

Embodiment 39. Compound growth hormone corresponding to embodiment 33, where the compound contains at least one pair of mutations corresponding to Q84C/Y143C and/or S85C/Y143CB SEQ ID No. 1.

Embodiment 40. Compound growth hormone, corresponding to any of the preceding embodiments, where one or more than one additional disulfide bonds to obtain amino acid substitution at least two amino acids compared to SEQ ID No. 1.

Embodiment 41. Compound growth hormone, corresponding to any of the preceding embodiments, where the compound contains exactly one additional the bacterial disulfide bonds compared to SEQ ID No. 1.

Embodiment 42. Compound growth hormone, corresponding to any of the preceding embodiments, where the compound contains exactly 2 amino acid substitutions compared to SEQ ID No. 1.

Embodiment 43. Compound growth hormone, corresponding to any of the preceding embodiments, contains at least two additional cysteines compared to human growth hormone as defined in SEQ ID No.1.

Embodiment 44. Compound growth hormone, corresponding to any of the preceding embodiments containing exactly two additional cysteines compared to human growth hormone as defined in SEQ ID No. 1.

Embodiment 45. Compound growth hormone corresponding to any of the preceding embodiments, where the compound growth hormone is chemically modified by paglierani by attaching polymers, such as sugar groups, fatty acids, lipophilic groups, the albumin-binding substances, vitamins, bile acids, but not limited to, spacers to the side chains or main chain of the peptide.

Embodiment 46. Compound growth hormone corresponding to embodiment 45 where the chemical modification of compound growth hormone is temporary, i.e. they can easily be removed in vivo.

Embodiment 47. Compound growth hormone corresponding to any of the in the of plosiny 45-46, where chemical modification of amino acid residues may be N-end of the peptide on the C-end of the peptide and/or between the N - and C-ends of the peptide.

Embodiment 48. Compound growth hormone, corresponding to any one of embodiments 45 to 47, where the chemical modification is carried out on amino acid residues Phe1, Gln40, Gln141 or Phe191.

Embodiment 49. Compound growth hormone, corresponding to any of the embodiments 45-48, where the chemical modification is performed with the use of PEG and where the molecular weight of the PEG is from 500 Da to 50 kDa.

The embodiment 50. Compound growth hormone, corresponding to any of the embodiments 1-49, where the compound growth hormone is chemically modified to facilitate transport through the epithelium.

Embodiment 51. Compound growth hormone, corresponding to any of the embodiments 1-49, where the compound growth hormone is chemically modified to facilitate transport through the epithelium compared with wthGH.

Embodiment 52. Compound growth hormone, corresponding to any of the embodiments 1-49, where the compound growth hormone is chemically modified in order to obtain prolonged duration of the half period of the functional existence in vivo compared with wthGH.

Embodiment 53. Compound growth hormone corresponding to embodiment 52, where half of the functional existence in vivo specified the CSOs compounds of growth hormone in 2 or more times higher compared to hGH.

Embodiment 54. Compound growth hormone corresponding to embodiment 53, where half of the functional existence in vivo 2-10 times higher compared to hGH.

Embodiment 55. Compound growth hormone, corresponding to any of the embodiments 45-49, where the chemical modification is carried out on amino acid residues without affecting the binding of the compounds of growth hormone with hGHR.

Embodiment 56. Compound growth hormone, corresponding to any of the embodiments 49-55, where the compound growth hormone is stabilized against proteolytic destruction under the action of the protease(s), such as digestive proteases, such as pepsin, trypsin, chymotrypsin, carboxypeptidase and/or elastase.

Embodiment 57. The method of obtaining compounds of growth hormone with increased stability to proteolytic destruction, including the state

a) the introduction of an additional disulfide bonds in hGH.

Embodiment 58. The method of obtaining compounds of growth hormone with increased stability to proteolytic destruction, corresponding to any one of embodiments 1-14, including the state

a) the introduction of an additional disulfide bonds in hGH by replacing one or more amino acid residues in hGH on one or more than one cysteine.

Embodiment 59. The method of obtaining compounds of growth hormone with increased stability to proteolytic R is zrusenie, corresponding to the embodiments 1 to 14, including the state

a) the introduction of an additional disulfide bonds in hGH by adding one or more cysteine residues.

The embodiment 60. A pharmaceutical composition comprising a compound growth hormone, corresponding to any one of embodiments 1-56, and pharmaceutically acceptable carrier(s).

Embodiment 61. The pharmaceutical composition corresponding to the embodiment 60, where the specified composition can be administered to the patients lingual, sublingual, transbuccal, in the oral cavity, oral, stomach and intestines, nasal, lung, epidermal, dermal, transdermal and parenteral.

Embodiment 62. The pharmaceutical composition corresponding to the embodiment 60 or 61, where this composition is an oral input composition.

Embodiment 63. A method of manufacturing a pharmaceutical composition, where this composition contains a compound growth hormone, corresponding to any one of embodiments 1-56, and pharmaceutically acceptable carrier(s).

Embodiment 64. The method of treatment of a disease, where the activity of growth hormone can be used to treat diseases or conditions in which it will be useful to the increase in the number of circulating in the blood connection, growth hormone, where the method includes the introduction of effective patient the number of connections growth hormone, corresponding to any one of embodiments 1-56, or pharmaceutical compositions corresponding to any of the embodiments 60-62.

Embodiment 65. The method of treatment of a disease corresponding to the embodiments of 63 or 64, where the disease is selected from growth hormone deficiency (GHD); Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; down syndrome; chronic renal disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in children receiving treatment program HAART (children diagnosed with HIV/HALS); small children born small for gestational age (SGA); short stature in children born with very low birth-weight (VLBW) but SGA; dysplasia of the skeleton; hypochondroplasia; achondroplasia; idiopathic short stature (ISS); GHD in adults; fractures in the tubular bones or bones, such as tibia, fibula, femur, humerus, radial bone, ulna, clavicle, metacarpal bone, metatarsal bone and the bones of the toes; fractures in the cancellous bone or spongy bone, such as the skull, the base of the brush and the base of the foot; patients after surgery on the tendons or ligaments, such as the wrist, knee, or shoulder; patients having or experiencing distraction osteogenesis; patients after replacement of the hip joint or articular disk, restore the Oia meniscus, the binding of the vertebrae or fixation of prostheses, such as knee, hip, shoulder, elbow, wrist or jaw; patients with fixed therein a material for osteosynthesis, such as pins, screws and plates; patients with narastaniem or improper healing of fractures; patients after osteotomy, such as the tibia or 1st toe; patients after transplantation of the graft; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on continuous dialysis (APCD); associated with malnutrition, cardiovascular disease on the background of the APCD; reversal of cachexia in the background APCD; cancer in APCD background; chronic obstructive pulmonary disease on the background of the APCD; HIV in APCD background; old age in APCD; chronic liver disease on the background of the APCD, fatigue syndrome in APCD background; Crohn's disease; weakening of the liver; individuals males with HIV infections; syndrome shortened small intestine; Central obesity; syndrome of HIV-associated lipodystrophy (HALS); male infertility; patients after major elective surgery, alcohol/drug detoxification or neurological trauma; aging; weakened elderly; osteoarthritis; traumatic injuries of the cartilage is; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid haemorrhage; very low weight at birth; metabolic syndrome; glucocorticoid myopathy; or short stature due to treatment with glucocorticoids in children.

Embodiment 66. Growth hormone, corresponding to any one of embodiments 1-56, for use as pharmaceuticals.

Embodiment 67. The use of growth hormone, corresponding to any one of embodiments 1-56, as a medicine.

Embodiment 68. The use of compounds of growth hormone, corresponding to any one of embodiments 1-56, in the treatment of disease.

Embodiment 69. The application corresponding to the embodiment of embodiment 67 or 68, where the disease is selected from growth hormone deficiency (GHD); Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; down syndrome; chronic renal disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in children receiving treatment program HAART (children diagnosed with HIV/HALS); small children born small for gestational age (SGA); short stature in children born with very low birth-weight (VLBW) but SGA; dysplasia of the skeleton; hypochondroplasia; achondroplasia; idiopathic short stature (ISS); GHD in adults; fractures of the tubular bones or bones, such as tibia, fibula, femur, humerus, radial bone, ulna, clavicle, metacarpal bone, metatarsal bone and the bones of the toes; fractures in the cancellous bone or spongy bone, such as the skull, the base of the brush and the base of the foot; patients after surgery on the tendons or ligaments, such as the wrist, knee, or shoulder; patients having or experiencing distraction osteogenesis; patients after hip replacement or articular disk, recovery, meniscus, binding vertebrae or fixation of prostheses, such as knee, hip, shoulder, elbow, wrist or jaw; patients with fixed therein a material for osteosynthesis, such as pins, screws and plates; patients with narastaniem or improper healing of fractures; patients after osteotomy, such as the tibia or 1st toe; patients after transplantation of the graft; degeneration of articular cartilage in the knee caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients on continuous dialysis (APCD); associated with malnutrition, cardiovascular disease in APCD background; reversal of cachexia in the background APCD; cancer in APCD background; chronic obstructive pulmonary disease on the background of the PCD; HIV background APCD; elderly in APCD; chronic liver disease on the background of the APCD, fatigue syndrome in APCD background; Crohn's disease; weakening of the liver; individuals males with HIV infections; syndrome shortened small intestine; Central obesity; syndrome of HIV-associated lipodystrophy (HALS); male infertility; patients after major elective surgery, alcohol/drug detoxification or neurological trauma; aging; weakened elderly; osteoarthritis; traumatic injuries of the cartilage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid haemorrhage; very low weight at birth; metabolic syndrome; glucocorticoid myopathy; or short stature due to treatment with glucocorticoids in children.

The embodiment 70. The use of compounds of growth hormone, corresponding to any one of embodiments 1-56, in the manufacture of a medicinal product which will be used in the treatment of growth hormone deficiency (GHD); Turner syndrome; Prader-Willi syndrome (PWS); Noonan syndrome; down syndrome; chronic renal disease, juvenile rheumatoid arthritis; cystic fibrosis, HIV-infection in children receiving treatment program HAART (children diagnosed with HIV/HALS); small children, Ojdanic small for gestational age (SGA); short stature in children born with very low birth-weight (VLBW) but SGA; skeletal dysplasia; hypochondroplasia; achondroplasia; idiopathic short stature (ISS); GHD in adults; fractures in the tubular bones or bones, such as tibia, fibula, femur, humerus, radial bone, ulna, clavicle, metacarpal bone, metatarsal bone and the bones of the toes; fractures in the cancellous bone or spongy bone, such as the skull, the base of the brush and the base of the foot; patients after surgery on the tendons or ligaments, for example, hand, knee, or shoulder; patients having or experiencing distraction osteogenesis; patients after hip replacement or articular disk, recovery, meniscus, binding vertebrae or fixation of prostheses, such as knee, hip, shoulder, elbow, wrist or jaw; patients with fixed therein a material for osteosynthesis, such as pins, screws and plates; patients with narastaniem or improper healing of fractures; patients after osteotomy, such as the tibia or 1st toe; patients after transplantation of the graft; degeneration of articular cartilage in the knee, caused by trauma or arthritis; osteoporosis in patients with Turner syndrome; osteoporosis in men; adult patients, n is held constant dialysis (APCD); associated with malnutrition, cardiovascular disease on the background of the APCD; reversal of cachexia in the background APCD; cancer in APCD background; chronic obstructive pulmonary disease on the background of the APCD; HIV in APCD background; old age in APCD; chronic liver disease on the background of the APCD, fatigue syndrome in APCD background; Crohn's disease; weakening of the liver; individuals males with HIV infections; syndrome shortened small intestine; Central obesity; syndrome of HIV-associated lipodystrophy (HALS); male infertility; patients after major elective surgery, alcohol/drug detoxification or neurological trauma; aging; weakened elderly; osteoarthritis; traumatic injuries of the cartilage; erectile dysfunction; fibromyalgia; memory disorders; depression; traumatic brain injury; subarachnoid haemorrhage; very low weight at birth; metabolic syndrome; glucocorticoid myopathy; or short stature due to treatment with glucocorticoids in children.

Examples

The invention will be further defined by reference to the following examples, which describe the fabrication and characterization of the various compounds described herein, and methods of analysis of their biological activity. Specialists in this field will show is about, what in practice can be applied to many of the modifications that apply to materials and methods, without deviating from the scope of the invention.

Used in the examples of These is a microbial transglutaminase from Streptoverticillium mobaraense according US5156956.

Example 1. General method of preparing compounds hGH

The gene encoding connection growth hormone, was built by recombinant method in the plasmid vector. Cysteine mutations were introduced using the QuikChange kit for site-specific mutagenesis (Stratagene). Then a suitable strain of E. coli was subjected to transformation with this plasmid vector. Protein is expressed as a soluble protein with N-terminal is rich in histidine peptide tag, suitable for purification by affinity chromatography with immobilized metals.

Preparing a concentrated cell suspension in 50% glycerol and stored at -80°C. the Concentrated strain in glycerol was inoculable Cup with LBA (apparitional medium Luria-Bertani (LB)) and then incubated at 37°C over night. The contents of each Cup was washed with LB medium and diluted in 500 ml of LB medium+AMR (ampicillin) to implement the expression. Cultures were incubated at 37°C with shaking at 220 rpm until reaching an optical density OD600equal to 0.6. Subsequent induction was performed using 0.2 mm IPTG (from ropyl-β-D-thiogalactopyranoside), at 30°C for 6 hours, getting in the end OD6002,0. Finally the cells were collected by centrifugation.

After that, cells are suspended in 20 mm Tris-HCl, pH 8.5, and destroyed using cell disintegrator, at 30 EC per square inch (207 MPa). The supernatant was collected by centrifugation and then subjected to chromatographic purification.

Purification was performed using affinity chromatography with immobilized metals as the capture stage, and then deleted the peptide tag using diaminopimelate from Unizyme. Final purification was achieved using ion-exchange chromatography. Purification can also be achieved using, but not limited to, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, steric exclusion chromatography and separation techniques using membranes, well-known specialist in this field.

Group PEG was attached to the N-end, interacting compounds hGH with 2 equivalents, for example, PEG-5000-aldehyde (RAPP Polymere, 12 5000-6). The reaction was initiated by addition of NaCNBH3in 0.5 ml MeCN for 10 stages. The reaction mixture was set aside 20 hours

Group PEG was attached to Q40, initially interacting compounds hGH with 1,3-diamino-2-propanol (Fluka 33262) using microbial transglutaminase in which the quality of the catalyst.

The reaction mix transaminasemia and oxidized compounds hGH (I) with a group of MPEG

Preparing the following solutions:

buffer a: triethanolamine (119 mg; 0.8 mmol) was dissolved in water (40 ml) and the pH was brought to 8.5;

buffer B: 20 mm triethanolamine; 0.2 M NaCl.

(A) Transamination hGH (III) using 1,3-diamino-2-propanol

hGH (8,64 g) was dissolved in buffer A (500 ml) under stirring. To this solution was slowly added to a mixture of 1,3-diamino-2-propanol (DAP) (8,1 g; Fluka 33262) in buffer A (50 ml). the pH of the mixture was brought to 8.5 by adding aq. HCl. Added microbial Thatu (2,8 ml; 1.3 mg/ml) under stirring. The final mixture was stirred over night at RT (room temperature).

The reaction mixture was diluted with buffer A (1.2 l) and the product was purified by ion exchange chromatography. 100 ml/min - 200 ml/fraction.

Phase: buffer In - 40%; gradient 40-100% buffer B in 15 CV (column volumes) for 225 minutes

(B) Oxidation transaminasemia hGH

Buffer a: triethanolamine (119 mg; 0.8 mmol) was dissolved in water (40 ml) and the pH was brought to 8.5.

Buffer: 3-methylthio-1-propanol (725 mg, 7.1 mmol) was dissolved in buffer A (10 ml).

Buffer: HEPES (N-2-hydroxyethyl-piperazine-N-2-econsultancy acid) (5,96 g) was dissolved in water (1.0 l) and the pH was brought to 7.0.

Periodat: NaIO4(48,1 mg; 0,225 mmol) was dissolved in water (1.0 ml).

To a solution of hGH after interaction with DAP(10 mg; 0.5 mmol) was added a buffer (0.2 ml), then the solution periodate (0,03 ml). After 20 min of innovatsii on cold mixture were dialyzed 4 times using buffer C. the Residue was concentrated to 1 ml

(C) Reductive amination of oxidized hGH using reagent PEG-based

The final solution from stage (B) (1 ml; 0.45 mmol) was mixed with a solution of PEG-amine (2 ml; 0.3 mmol) in 25 mm HEPES-buffer, pH 7.0, and the resulting mixture was slowly stirred at room temperature for 1 h After 1 h portions (10×) was added NaCNBH3(100 μl of a solution NaCNBH3(20 mg) in water (0.5 ml)). The mixture was left at room temperature in the dark for 18 to 24 hours. The mixture was purified on a MonoQ replaced the buffer and concentrated. Reagents MPEG-amine are available for sale.

Example 2. Chemical characterization of the purified protein compounds growth hormone

Source purified protein was analyzed using MALDI-MS (mass spectrometry with ionization by laser desorption of ions from the matrix). Detected mass corresponded to theoretical mass of the established on the basis of amino acid sequence.

The expected formation of three disulfide bonds in each compound was demonstrated using peptide mapping using splitting under the action of trypsin and AspN with subsequent MALDI-MS analysis of the hydrolyzate before and the settlement of the e recovery disulfide bonds with DTT (dithiothreitol).

Example 3. Analysis of biological activity of the purified compounds of HGH

Biological activity of compounds hGH was measured in the analysis of the effectiveness of their interaction with receptor-based cell proliferation, namely BAF-analysis. This method is recognized for connections hGH.

Cells BAF-3 (line b-lymphoid precursor cells of mice or rats, isolated from bone marrow) for their growth and survival requires the presence of IL-3 (interleukin-3). IL-3 activates JAK-2 (Janus kinase 2) and STAT (transcription factor). signal transducer and activator of reduced), which are of the same mediators of GH, which are activated under the action of GH as a result of stimulation.

Cells BAF-3 was transfusional a plasmid containing the hGH receptor. The clones are capable of proliferation after stimulation of hGH, turned into hGH-dependent cell line, hereinafter referred to as BAF3-GHR. Cell lines gave dose-dependent response to GH, and therefore they can be used to assess the effect of various compounds hGH in the analysis of cell proliferation.

BAF-3GHR cells grow on minimal medium (culture medium without GH) for 24 hours at 37°C, 5% CO2. Cells are centrifuged, the medium is removed and cells resuspended in a minimal environment to 2.22×105cells/ml portions of 90 μl of cell supernatant scatter in the title of the ion microplates (96-well, NUNC-clone). To cells add connection growth hormone in various concentrations and tablets incubated for 72 hours at 37°C, 5% CO2.

AlamarBlue is an indicator of the redox potential of cells, AlamarBlue® (catalog No. BioSource Dal 1025), which is restored in the reactions characteristic of cellular metabolism, and thus provides the possibility of indirectly measuring the number of viable cells. AlamarBlue® was diluted 6 times (5 μl AlamarBlue® + 25 µl minimum protection) and to each well was added 30 µl of the diluted AlamarBlue®. The cells are then incubated in the next 4 hours. Finally, the metabolic activity of cells was measured in a fluorescent plate reader using excitation filter 544 nm and emission filter of 590 nm.

The result for the specified connection is expressed as the ratio between EC50for the specified connection and EC50for wthGH obtained in a parallel experiment. Other results are shown below in Table 6.

Table 2
The relationship between EU50for connections hGH and EC50for wthGH
Cys-containing compoundAverage ve is hichina Deviation
AS-IS0,60,1
NS-MS0,40,3
R94C-D107C0,80,4
Q84C-Y143C0,30,1
S71C-S132C0,240,02

All tested compounds hGH were equally effective or more effective than wthGH.

Example 3A. Study of dependence "dose-response" in vivo on rats Sprague Dawley remote pituitary (analysis 3A)

Dependence "dose-response in vivo was studied on male rats Sprague Dawley remote pituitary gland. Rat with a remote pituitary gland is a well known and recognized animal model of growth hormone deficiency, when after surgical removal of the pituitary gland no education growth hormone does not occur. It also leads to low levels of insulin-like growth factor-1 (IGF-1) in the blood, another important clinical sign of growth hormone deficiency in humans.

Removal of the pituitary gland was carried out on male rats aged 4 weeks weighing 90-100 g Mass of animals taken for research 3-4 weeks after surgery, was 100-110, Animals with increased body mass by more than 10% within 3-4 weeks after surgery in this study did not use.

Seventy rats Sprague Dawley remote pituitary were divided randomly into seven groups for drug injection, ten animals in each group. One group received only the diluent and served as not being treated control group. Three groups received the test compound (hGH Q84C, Y143C) number 33, and 0.33 to 3.3 nmol, respectively, and three groups received hGH in the ideal drug comparison in the amount of 50, 5.0 and 0.5 nmol, respectively. And connections, and the diluent was administered as a single subcutaneous dose in the neck area. Body weight was measured every day in between 8-10 am for one week.

As hGH Q84C, Y143C and hGH are both induced a dose-dependent increase in body weight when the weight is 0 days was compared with that on the 7th day.

Sigmoidal equation based dose-response was fitted to the experimental data (increase of body weight in the period 0-7 days), using nonlinear regression analysis to estimate values of parameters EmaxE0and ED50. This equation was represented by a sigmoidal equation of dependence "dose-response" built into the program GraphPad Prism, version 4.00 for Windows (GraphPad Software Inc., San Diego, USA). Data, including estimates of parameter values and 95% confidence intervals, are presented in Table 3.

No differences in the estimates of the parameter values E0and Emaxnot observed for hGH Q84C, Y143C and hGH. However, the value ED5o was significantly lower for hGH Q84C, Y143C than hGH, indicating increased efficiency hGHQ84C, Y143C in vivo.

Table 2A
The relationship between EC50for connections hGH with a group of PEG and EC50for wthGH
CONNECTIONThe ratio of (compound EC50(BAF) hGH/hGH wt)
HGH1,0
HGH (Q84c, Y143C) 040-PEG0,75
HGH (Q84c, Y143C) PEG from N-Terminusvalue (0.475)
HGH (Q84c, Y143C) PEG from N-Terminus1,1475
Table 3
The results of fitting reply in the form of increased body mass on the 7th day compared with 0 suck the mi to a sigmoidal equation of dependence "dose-response" for the evaluation of E maxE0and ED50
hGH Q84C, Y143ChGHwt
E0(g)0,0 (-1,9-1,8)0,2 (-1,7-2,0)
ED50(nmol)0,29 (0,20-0,41)0,70 (0,50-0,99)
Emax(g)26,3 (24,8-27,9)27,5 (25,9-29,1)
The mean value (95% confidence interval).

Example 4. The study of the interaction with the receptor in the analysis using the method of surface plasmon resonance

The interaction of compounds with hGH receptor were analyzed using analysis using surface plasmon resonance. This is a common method for compounds of hGH, and it is demonstrated on the example of the connection hGH Q84C/Y143C.

The interaction between hGH and analogues with hGH-binding protein (hGHBP) was studied using surface plasmon resonance using a Biacore instrument KZT100 (GE Healthcare, Sweden). mAb (monoclonal antibody) against hGH (Fitzgerald Industries International, USA, No. 10G05B) was immobilized on a CM-5 chip in accordance with the manufacturer's instructions, usually at the level of 5000 RU (units resonance). wthGH or analogues was captured in concentration the AI 10-25 µg/ml working buffer (10 mm HEPES, 0.15 M NaCl, 30 mm EDTA (ethylenediaminetetraacetic acid), a 0.05% surfactant P20, pH 7.4), which resulted in the receipt of 250-400 EN for the captured ligand. Then hGHBP in concentration 0-800 nm was passed over the surface at a rate of 30 µl/min Surface with immobilized mAb against hGH, but without the captured hGH, used as a comparison.

Kinetic data were analyzed using analysis software (Evaluation Software) 2,0 Biacore™ using model binding 1:1 Langmuir.

The analysis showed (table 4) that the connection hGH Q84C, Y143C had similar or slightly higher affinity to a protein that binds growth hormone than wthGH.

Table 4
ka (1/Mc)kd (1/c)KD (nm)
hGH wtof 1.9×105of 6.1×10-43,3
hGH Q84C, Y143Cof 2.0×1054,8×10-42,5

The analysis showed (table 4A) that the connection hGH Q84C, Y143C was equally effective compared to wild-type hGH within the error ek is periment.

Table 4A
Ec50±std dev. (nm)
hGH wt0,7±0,3
hGH Q84C, Y143C0,3±0,1
std dev. = standard deviation.

Example 5. Analysis of the fracture velocity hGH wild-type and connection hGH under the action of proteases

Interest compound is subjected to digestion of the relevant protease (trypsin, chymotrypsin, pepsin, elastases, factor VIIa, factor XA, proteinase K, carboxypeptidase, DPPIV, neutral endopeptidase, grannymom In, Proline-endopeptidase, staphylococcal peptidase I, thermolysin, thrombin, Arg, C-proteinase, Asp-N endopeptidase, caspase 1-10, Clostridium, enterokinase, glutamyl-endopeptidase, grannymom, LysC, LysN, Proline-endopeptidase and staphylococcal peptidase I or tissue extracts) in a suitable buffer (for example, PBS (phosphate buffered saline solution) or ammonium bicarbonate) at 37°C for up to 24 hours. Proteolytic destruction assessed by analysis using HPLC (high performance liquid chromatography).

General methods the

Proteolytic cleavage:

100 μl of a solution of test compound in a concentration of 1 mg/ml in ammonium bicarbonate buffer is subjected to the destructive action of the enzyme for up to 24 hours at 37°C. at different points In time selected samples and the proteolysis reaction is stopped by acidification of the sample, diluting it 10 times in 1% TFA (triperoxonane acid). These diluted samples analyzed by reversed-phase HPLC to assess the extent of proteolytic cleavage.

Method using HPLC:

10 μl of the above solution is injected into the column Vydac C4 (2×150 mm) for reversed-phase chromatography, loireau linear gradient from 0.1% TFA in water to 100% acetonitrile containing 0.1% TFA, over 30 min at a flow rate of 0.2 ml/min Detection peaks carried out using absorption in the UV (ultraviolet) at 214 nm. % intact connections at time t=T is calculated from the peak area at time t=T (AT) and the peak area at time t=0 (A0) in the form (AT/A0)×100%. The results given in this description below in Table 6, were obtained after 4 hours (in the above equation T=4).

Curve based % intact connections from time build, using the software GraphPad Prims, version 5,01. T½ expect from recession phase distorting the th using the software GraphPad Prims.

In this example, use the enzymes elastase (Sigma, from the pancreas of pigs) and chymotrypsin (Roche, degree of purity for sequencing). As buffer using 50 mm ammonium bicarbonate pH 8.5.

Example 5.1

100 mcg wthGH incubated in 100 μl of buffer with 13 ng chymotrypsin. T½=3.6 hours.

Example 5.2

100 mcg wthGH incubated in 100 μl of buffer with 135 ng elastase. T=1.6 hours.

Example 5.3

100 μg hGH Q84C, Y143C incubated in 100 μl of buffer with 135 ng elastase. T½=6.2 hours.

Example 5.4

100 μg hGH AS,IS incubated in 100 μl of buffer with 13 ng chymotrypsin. T½=7.5 hours.

Example 5.5

100 μg hGH H21C, M170C incubated in 100 μl of buffer with 13 ng chymotrypsin. T=22 hours.

Example 5.6

100 μg hGH R94C, D107C incubated in 100 μl of buffer with 13 ng chymotrypsin. T=2.5 hours.

Example 5.7

100 μg hGH Q84C, Y143C incubated in 100 ál of 50 mm ammonium bicarbonate buffer pH 8.5 with 13 ng chymotrypsin. T cannot be calculated because no destruction does not occur.

Table 4
Value T (hours) for the destruction of wild-type hGH and compounds under the action of chymotrypsin
ConnectionT
hGH wt 3,6
hGH AS, ES7,5
hGH H21C, M170C22
hGH R94C, D107C2,5
hGH Q84, Y143CN/A
N/A = data not available (from the English. not available).

Table 5
The value of T½ (hours) for the destruction of wild-type hGH and connections hGH Q84C, Y143C under the action of elastase
ConnectionT
hGH wt1,6
hGH Q84C, Y143C6,2

Example 6. Analysis of selected compounds using BAF-method and proteolytic cleavage, as described in Example 3 and Example 5

Table 6
Analysis of compounds of growth hormone containing additional disulfide bonds
ConnectionThe ratio EC50(BAF) (connection hGH/hGH) Stability chymotrypsin, % intact connectionStability elastase, % intact connectionThe domains associated more. by a disulfide bond
HGH1,04225
HGH (A17C, E174C)0,64510N1-N4
HGH (H21C, M170C)0,57210N1-N4
HGH (D26C, V102C)0,55565H1-L2
HGH (D26C, Y103C)0,55545H1-L2
HGH (F54C, Y143C)0,65520L1-L3
HGH (F54C, S144C)0,56020 L1-L3
HGH (F54C, F146C)0,64025L1-L3
HGH (S55C, Y143C)0,59025L1-L3
HGH (S57C, Y143C)0,37550L1-L3
HGH (I58C, Q141C)0,77025L1-L3
HGH (I58C, Y143C)0,65520L1-L3
HGH (I58C, S144C)1,26530L1-L3
HGH (P59C, Q137C)0,77235L1-L3
HGH (S71C, S132C)0,29045L1-L3
HGH (L81C, Y14C) 0,78515H2-L3
HGH (Q84C, Y143C)0,510080H2-L3
HGH (S85C, Y143C)0,58070H2-L3
HGH (S85C, S144C)0,78160H2-L3
HGH (F92C, T148C)0,64055H2-L3
HGH (R94C, D107C)0,83870H2-H3

1. Derived human growth hormone containing additional disulfide bonds in comparison with hGh, defined by SEQ ID No. 1, where the derivative contains at least one pair of mutations corresponding to NS/MS, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1, and having the activity of growth hormone h the rights.

2. Derived growth hormone under item 1, where the polypeptide sequence at least 95% identical hGH defined by SEQ ID No. 1.

3. Derived growth hormone under item 1, where the derivative of growth hormone contains additional disulfide bond, and at least one of cysteines present in loop 3 (L3) corresponding to AA 128-154 in SEQ ID No. 1.

4. A derivative of growth hormone on p. 3, where the derivative of growth hormone contains additional disulfide bond, and at least one of cysteines present in the area corresponding to AA 135-148 in SEQ ID No. 1.

5. A derivative of growth hormone on p. 3, where the derivative of growth hormone contains additional disulfide bond connecting L3 with the helix 2 (H2) or loop 1 (L1).

6. A derivative of growth hormone on p. 5, where the derivative contains at least one pair of mutations corresponding to F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L81C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C and/or F92C/T148C in SEQ ID No. 1.

7. Derived growth hormone under item 1, where the derivative of growth hormone stable against destruction under the action of the protease(s) selected(s) from the digestive proteases.

8. Derived growth hormone under item 7, where the derivative of growth hormone stable against destruction under the action of the protease(s) selected(s) from: pepsin, trypsin, chymotrypsin, carb is xipamide and/or elastase.

9. A derivative of growth hormone on p. 1 containing exactly two additional cysteines compared to human growth hormone as defined in SEQ ID No. 1.

10. Derived growth hormone under item 1, where the derivative of growth hormone is a hybrid protein.

11. Derived growth hormone under item 1, where the derivative of growth hormone chemically modified.

12. A derivative of growth hormone on p. 11, where the derivative of growth hormone is chemically modified by joining groups, such as PEG, carbohydrates, albumin-binding group, a fatty acid, alkyl chain, lipophilic groups, vitamins, bile acids or spacers joining the side or main chain derived growth hormone.

13. The derivative of PP.1-12 for use as a medicine.

14. A derivative of growth hormone on PP.1-12 for use in the treatment of diseases or conditions in which the patient increase the number of circulating derived growth hormone.

15. The method of obtaining the derivative of growth hormone with increased stability to proteolytic destruction, including the stage of the introduction of an additional disulfide bond by including at least one pair of mutations corresponding to n/M, D26/V102C, D26/Y103C, F54C/Y143C, F54C/S144C, S55C/Y143C, S57C/Y143C, I58C/Q141C, I58C/Y143C, I58C/S144C, P59C/Q137C, S71C/S132C, L8C/Y143C, Q84C/Y143C, S85C/Y143C, S85C/S144C, F92C/T148C and/or R94C/D107C in SEQ ID No. 1, a derivative of growth hormone, obtaining a derivative of growth hormone, containing additional disulfide bonds in comparison with hGH, which is defined in SEQ ID No. 1.

16. Pharmaceutical composition having activity of the growth hormone-containing derivative of growth hormone according to any one of paragraphs.1-12 in an effective amount and a pharmaceutically acceptable carrier(s).

17. The use of a derivative of growth hormone on PP.1-12 in an effective amount for the preparation of pharmaceutical compositions for the treatment of diseases or conditions in which the patient increase the number of circulating growth hormone.

18. The method of treatment of diseases, where the activity of growth hormone can be used to treat diseases or conditions in which patient will benefit from increasing the amount of circulating blood-derived growth hormone, comprising the administration to a patient an effective amount of a derivative of growth hormone according to any one of paragraphs.1-12 or pharmaceutical composition according to p. 16.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a polypeptide and its homodimer which possess agonist activity with respect to a growth hormone receptor, a nucleic acid molecule coding them, a vector which involves said nucleic acid, a cell of expression of said polypeptide, a pharmaceutical composition and a method with the use of said polypeptides for treating growth hormone deficiency. The polypeptide has an amino acid sequence presented in SEQ ID NO: 11 or 12. The homodimer consists of two polypeptides consisting of SEQ ID NO: 11 or 12. The nucleic acid molecule consists of a nucleic acid sequence presented in SEQ ID NO: 4. The pharmaceutical composition to be used in treating growth hormone deficiency contains said polypeptide or homodimer and an excipient or a carrier. The method of treating growth hormone deficiency involves introducing an effective amount of said polypeptide or homodimer.

EFFECT: invention provides creating the polypeptide which possess agonist activity on growth hormone receptor, and it is effective in treating growth hormone deficiency.

17 cl, 9 dwg, 1 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, particularly to obtaining a modified growth hormone, and can be used in medicine. By recombination, a polypeptide is obtained, which has antagonistic effect on the growth hormone receptor.

EFFECT: invention enables to obtain a polypeptide which is effective when treating conditions caused by excess growth hormone in the body of the patient.

11 cl, 19 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: there is offered a method of engineering yeast strains - stable human growth hormone (somatotropin) producers. Also, there are offered two strains Y-3506 of Russian National Collection of Industrial Microorganisms and Y-3507 of Russian National Collection of Industrial Microorganisms - stable human somatotropin producers. The producer strains have been prepared by sequential integration of expression plasmid into a recipient stain genome. Each plasmid carries in its structure a somatotropin gene (GH1); fused with a leader sequence controlled by the GAL1 promotor, as well as one of the genes URA3, LEU2, TRP1 and HIS3 complementing auxotrophic recipient strain mutations.

EFFECT: efficacy of the produced strains is 100-130 mg of somatotropin per 1 l of the medium.

3 cl, 7 dwg, 6 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: proposed are human growth hormone conjugates, obtained by removing a hydrogen atom from -NH2 in the Gln side chain which is formed from the human growth hormone or a human growth hormone compound.

EFFECT: design of an efficient method of producing human growth hormone conjugates.

6 cl, 14 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to genetic engineering, more specifically to chimeric polypeptides, containing an antagonist of growth hormone receptor. The invention can be used in medicine. The binding domain of the growth hormone is modified by substituting glycine amino acid residue in position 120 and is further modified in site 1, where at least one amino acid residue is substituted, which increases affinity of the growth hormone to its binding domain on the growth hormone receptor. The amino acid residue is then conjugated with the ligand-binding domain of the growth hormone receptor, through a peptide linker.

EFFECT: obtaining a highly effective antagonist of the growth hormone receptor with longer half-life, reduced immunogenesity and nontoxicity, compared to known mutant forms.

35 cl, 16 dwg, 1 tbl

The invention relates to biotechnology and can be used to obtain a properly curled, containing the precursor of insulin chimeric protein

The invention relates to a peptide that includes the analogue of carboxykinase sequence of growth hormone, where carboxykinase sequence contains amino acid residues 177-191 human growth hormone: Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe, or a corresponding sequence of growth hormone, non-human mammal; where in the specified similar amino acids at positions 182 and 189 hGH are connected by a communication in order to facilitate the formation of cyclic conformation, and/or amino acids at positions 183 and 186 hGH are connected by a salt bridge or a covalent bond; or its salts with organic or inorganic acid

The invention relates to an improved method for producing adducted end of the condensation products, which are Schiff bases, components of which include a protein having useful activity in animals, and aromatic o-hydroxyaldehyde, which connect the above components in an aqueous medium at pH 7.0 or higher to form a reaction mixture under conditions effective to conduct specified the condensation reaction essentially to completion by using the stage of fast compared to drying in ambient conditions removal 97,0 to 99.9% by weight, preferably approximately 98,0 - 99,0% by weight of water, already present or formed during this reaction, condensation, consistent with maintaining the integrity of reagents condensation and adductor final product

FIELD: chemistry.

SUBSTANCE: invention relates to the field of biochemistry, in particular to recombinant factor VIII, which contains one or more mutations, resulting in an increased stability of both the factor VIII and factor VIIIa, as well as to a pharmaceutical composition for treating haemophilia containing it. Also described is a molecule of nucleic acid, coding the said recombinant factor VIII, and an expression vector and host-cells, containing the said molecule of nucleic acid. The invention also relates to a method of obtaining the said factor VIII, as well as to its application in the method of treating haemophilia A in an animal.

EFFECT: invention makes it possible to obtain a biologically active factor VIII with an increased stability.

50 cl, 12 dwg, 5 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to medicine, namely to pharmacology and describes a histidine-free pharmaceutical composition containing high-purity factor VIII; arginine and saccharose, a surfactant for the prevention or at least the inhibition of a surface adsorption of factor VIII; 0.5 to 10 mM calcium chloride for the specific stabilisation of factor VIII, and sodium citrate or maleic acid as a pH buffer.

EFFECT: invention provides the protective function for preserve high-yield factor VIII over the whole cycle of pharmaceutical processing, long storage and end recovery and administration into the patient.

18 cl, 16 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention represents method of stabilisation of liquid solution of coagulation and/or clotting factors for storing in frozen state, which contains: providing liquid solution of coagulation and/or clotting factor, where said solution has concentration NaCl and/or KCl, at least, 100 mM; addition of carbohydrate to said solution until said solution reaches in freezing temperature of vitrifying -56°C or higher; and freezing of said solution for storage.

EFFECT: invention ensures stabilisation of liquid solution of coagulation or clotting factors for storing in frozen state.

20 cl, 1 tbl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and describes a method for recovery of factor VIII from human blood plasma not identified by related analysis of hepatitis and HIV1/2 viruses consisting in sequential cryoprecipitation, dissolution in an aqueous solution of heparin and solubilisation of a cryoprecipitate, sorption of a prothrombin-converting complex factor by aluminium hydrate, removal of fibrinogen, fibronectin and associated protein by polyethylene glycol-4000, viral inactivation with solvent detergents and preliminary filtration, anion-exchange chromatography, preferentially with EDM-TMAE Fractogel, with elution by a sodium chloride buffer, stabilisation by albumine solution, sterile filtration in membrane filters of pore diameter 0.22 mcm, bottling (200-300 IU/bottle), lyophilisation and second thermal viral inactivation with purification using the aqueous solution of unfractionated heparin of the concentrations equal to 5-100 international units (IU)/ml, preferentially 10-25 IU/ml, polyethylene glycol-4000 in the final concentration 3.5% and acidification of the medium, preferentially to pH 6.6, strong TMAE anion exchangers.

EFFECT: method substantially provides higher effectiveness of purification and specific activity of factor VIII.

1 tbl, 4 dwg, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns area of molecular biology and biochemistry, and can be used in medicine. There is offered mutein conjugate of the blood coagulation factor VIII (FVIII) wherein a residue not being cysteine in position 41, 129, 377, 388, 468, 491, 556, 1804, 1808, 1810, 1812, 1813, 1815 and/or 2118 is substituted by a cysteine residue with polyethylene glycol (PEG) where a PEG molecule is bound with a polypeptide in a mutant cysteine residue.

EFFECT: improved pharmacokinetic properties of the FVIII as an ingredient of the conjugate under the invention with preserved a procoagulant factor activity allows presenting new FVIII PEG-muteins for producing of a pharmaceutical compositions for treating hemophilia.

12 cl, 38 dwg, 8 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared invention refers to chemical-pharmaceutical industry. A method involves the following stages: preparing a solution of von Willebrand factor or von Willebrand factor/factor VIII complex which contains VWF in concentration up to 12 IU VWF:RCoAui and has the von Willebrand factor/factor VIII ratio equal to 0.4 or more; nanofiltering through a filter of pore size less than 35 nanometres at pressure less than or equal to 0.5 bar and in the presence of 0.05 to 0.2 M of calcium ions.

EFFECT: development of the effective method for producing concentrated von Willebrand factor or factor VIII/von Willebrand factor complex to be applied for treating hemophilia And or von Willebrand disease.

11 cl, 8 ex, 6 tbl, 1 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to obtaining versions of glycoprotein IV alpha polypeptide of human thrombocytes (GPIbalpha) and can be used in medicine to treat vascular disorders. Using a recombinant technique, a polypeptide is obtained, which contains substitutes in SEQ ID NO:2 selected from: Y276F K237V C65S; K237V C65S; Y276F C65S; or Y276F Y278F Y279F K237V C65S. The obtained polypeptide is used to inhibit bonding of leucocytes to biological tissue or for treating disorders associated with activation of thrombocytes.

EFFECT: invention enables to obtain GPIbalpha polypeptide which bonds with von Willebrand factor with affinity which is at least 10 times higher than in natural GPIbα polypeptide, and also has low affinity for bonding with alpha-thrombin, lower aggregation and/or high resistance to proteolysis relative the polypeptide with SEQ ID NO:2.

41 cl, 3 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: invention claims compositions which can include one or several mammary gland tumour proteins, their immunogenic parts or polynucleotides encoding such parts. Alternatively the therapeutic composition can include antigen-presenting cell expressing mammary gland tumour protein, or T-cell specific to cells expressing such protein. These compositions can be applied in prevention and treatment of such diseases as mammary gland cancer. Invention also claims diagnostic methods based on determination of mammary gland tumour protein or mRNA encoding such protein in sample.

EFFECT: use of peptides obtained from protein expressed from mammary gland by tumour in diagnostics and therapy of mammary gland cancer.

37 cl, 6 ex, 1 dwg

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to method of human blood coagulation VIII factor production and related product. Method includes blood serum as cryoprecipitate, heparine added, PEG-4000, centrifugation, supernatant is added with tributyl phosphate and Twin-80, repeated centrifugation, sediment washed with sodium chloride, then it is dissolved in tris-HCl buffer with additives, let through column filled with gel and attached antibodies to Willebrand's factor, factor VIII elution, dialysis. Produced concentrate does not contain Willebrand's factor and has activity not less than 300 ME/mg of protein with purity not less 98% and contains albumin of concentration of 0.1%. Product is lyophilized with further processing.

EFFECT: product does not display any toxicity and cause allergic reaction.

6 cl, 2 ex

FIELD: technological processes.

SUBSTANCE: preparation of human blood coagulation factor VIII is made by producing cryoprecipitate out of blood plasma, virus inactivating treatment, purification of cryoprecipitate solution, concentration, sterilizing filtration, bottling and drying. At the same time cryoprecipitate is produced with the help of running water with the temperature of (4±2)°C during unfreezing of fresh frozen plasma, virus inactivating treatment is carried out by means of solvent-detergent method, chromatographic purification of virus inactivated cryoprecipitate solution is carried out with application of sorbent with fractionation interval up to 20,000 kilodaltons, at the flow rate of (20±15) cm/hr, with further concentration by means of ultrafiltration on hollow fibers. Stabilizers are added, such as albumin in final concentration of (5±3) g/l, sugars and amino acids up to final concentration of (10±3) g/l. During drying initial preparation temperature is (25±15)°C below zero, final temperature is (25±8)°C, total duration of preparation drying process is (45±7) hours.

EFFECT: coagulation factor yield stability is increased and technological production design is simplified.

2 ex

FIELD: medicine, hematology, pharmacy.

SUBSTANCE: invention relates to the composition of factor VIII composed without addition of albumin and comprising the following excipients of composition in addition to factor VIII: from 4% to 10% of filling agent taken among group consisting of mannitol, glycine and alanine; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance about between 6 and 8. Alternatively, the composition can comprise from 2% to 6% of hydroxyethylstarch; from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose, arginine; from 1 mM to 5 mM of calcium salt, from 100 mM to 300 mM of NaCl, and buffer agent for pH value maintenance between 6 and 8. In additional variant of realization of invention the composition can comprise: from 300 mM to 500 mM of NaCl, from 1% to 4% of stabilizing agent taken among group consisting of sucrose, trehalose, raffinose and arginine; from 1 mM to 5 mM of calcium salt, and buffer agent. The composition provides stability in the absence of albumin or other proteins.

EFFECT: valuable properties of compositions.

35 cl, 11 tbl, 7 ex

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