Modified growth hormone polypeptides

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

 

The invention relates to fused proteins modified growth hormone and dimers containing these fused proteins, the molecules of nucleic acids encoding these proteins, and methods of treatment involving the use of these proteins.

Growth hormone (GH) is an anabolic hormone, a cytokine important for growth in length during childhood and normal to the Constitution in adults. Regulation of GH activity is complex and involves several interacting polypeptide and peptide agonists and antagonists. GH may exert their effects either directly linking the receptor for growth hormone, or indirectly by stimulating the formation of insulin-like growth factor-1 (IGF-1). Thus, the main function of GH is to stimulate the formation of IGF-1 by the liver. In addition, the secretion of GH is controlled by two peptide hormone with the opposite activity. Releasing factor, growth hormone (GHRH) is a peptide of 44 amino acids, formed by the arcuate nucleus of the hypothalamus. It functions by stimulating the formation of GH from the anterior pituitary gland. Somatostatin is a peptide hormone which counteracts the effects of GHRH, and is the result of processing a larger pre-propeptide with the formation of 14 and 28 amino acids. Neuroendocrine cells of the periventricular nucleus of the hypothalamus Secretary the somatostatin in the hypothalamic-hypophyseal system of the portal vein, connecting the hypothalamus to the anterior pituitary gland, where somatostatin inhibits the secretion of GH.

GH is consistently associated with two membrane-bound receptors of growth hormone (GHR) through two separate sites on GH, called site 1 and site 2. Site 1 is the binding site with high affinity, and site 2 is the binding site with low affinity. One GH molecule binds 1 GHR via the site 1. Then there is the involvement of the second GHR through the site 2 with the formation of the complex GHR:GH:GHR. Then there is the internalization of the complex, and it activates a cascade of signal transduction, leading to changes in gene expression. The extracellular domain of the GHR exists in the form of two related domains, each of which contains approximately 100 amino acids (SD-100), while the C-terminal domain SD-100 (b) is closest to the cell surface, and the N-terminal domain SD-100 (a) is located at the greatest distance. When the binding of the hormone occurs conformational changes in these two domains with the formation of the trimeric complex GHR-GH-GHR.

Excess GH is associated with several painful conditions, such as acromegaly and pituitary gigantism. Most cases of excess GH is the result of a tumor of pituitary somatotrophs of the anterior pituitary gland. These tumors are benign and gradually increases the t of the secretion of GH. Symptoms of excess growth hormone include thickening of the bones of the jaw, fingers and toes, pressing on a nerve/muscle and insulin resistance. The main treatment of excess GH associated with a tumor is surgical removal of pituitary tumors. Recently, the use of GH antagonists for inhibition of GH signaling is becoming the preferred treatment because of its noninvasive nature. The GH antagonists can be either a recombinant form of the somatostatin or somatostatin analogues (e.g. octreotide, lanreotide)or modified GH.

Review antagonists, which represents a modified GH shown in Kopchick (2003) European Journal of Endocrinology 148; S21-25, which describes commercially available GH antagonist, called pegvisomant, which combine the modification of human GH in position G120 attach polyethylene glycol to increase the molecular weight of the modified GH. The problem with the introduction of growth hormone, is its rapid clearance of renal filtration and/or proteolysis. Attaching polyethylene glycol to reduce these losses. However, it is known that polyethylene glycol reduces the affinity of GH in relation to the GHR, and thus, to compensate for this reduced affinity, you must enter the increased amount of the modified GH. This can lead to poboon the m effects. Would be desirable to provide an antagonist, represents a modified GH, which you can enter in a smaller dose, avoiding, thus, the problems associated with pegvisomant. This can either be in the reduction in the number or in reducing the frequency of administration.

At the same time under consideration the request of the authors of the present invention, WO 03/070765, the authors present invention described fused proteins modified GH, including modification of site 1 and site 2 in GH. These modified molecules GH merged with the extracellular domain of the GHR. Here the authors present invention disclosed fused proteins modified GH, have significantly increased the half-life in serum and forming dimers, which may be associated with improved pharmacokinetics of these fused proteins or due to reduced renal clearance, or due to the protection of the modified GH from proteolysis. Improved pharmacokinetic profiles of these fused proteins growth hormone will make possible a treatment regimen that does not require multiple injections and reducing unwanted side effects.

According to one aspect of the invention proposed a nucleic acid molecule containing a nucleic acid sequence chosen from:

1) nucleic acid sequence as represented by the SEQ ID NO: 1;

2) nucleic acid sequence as shown in SEQ ID NO: 2;

3) nucleic acid sequence as shown in SEQ ID NO: 4;

4) nucleic acid sequence as shown in SEQ ID NO: 5;

5) nucleic acid sequence as shown in SEQ ID NO: 7;

6) nucleic acid sequence as shown in SEQ ID NO: 8;

7) nucleic acid sequence as shown in SEQ ID NO: 10;

8) nucleic acid sequence as shown in SEQ ID NO: 11;

9) nucleic acid sequence as shown in SEQ ID NO: 13;

10) nucleic acid sequence as shown in SEQ ID NO: 14;

11) nucleic acid sequence as shown in SEQ ID NO: 16;

12) nucleic acid sequence as shown in SEQ ID NO: 17;

13) nucleic acid sequence as shown in SEQ ID NO: 19;

14) nucleic acid sequence as shown in SEQ ID NO: 20;

15) nucleic acid sequence as shown in SEQ ID NO: 22;

16) nucleic acid sequence as shown in SEQ ID NO: 23;

17. the nucleic acid molecule containing a sequence of nucleic acid hybridization in stringent conditions of hybridization with SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22 or 23;

the encoding the polypeptide, having antagonistic activity against hormone receptor growth.

Hybridization of nucleic acid molecules occurs with the formation of multiple hydrogen bonds between two complementary nucleic acid molecules. The stringency of hybridization can be varied in accordance with the conditions of the environment of the nucleic acids, the nature of the hybridization method and the composition and length of the molecules of nucleic acids. Calculations regarding hybridization conditions required for attaining particular degrees of stringency, discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). Tmrepresents the temperature at which 50% of the chain molecules of nucleic acid hybridizes with its complementary chain. Below are the approximate number of hybridization conditions, and it is not restrictive.

Very strict conditions (allow hybridization of sequences having at least 90% identity)

Hybridization:5-fold (5x) solution of chloride and sodium citrate (SSC) at 65°C for 16 hours
Wash twice: 2x SSC at room temperature (RT) for 15 minutes for each fold
Wash twice:0,5x SSC at 65°C for 20 minutes for each fold

Strict conditions (allow hybridization of sequences having at least 80% identity)

Hybridization:5x-6x SSC at 65-70°C for 16-20 hours
Wash twice:2x SSC at RT for 5-20 minutes each times
Wash twice:1x SSC at 55-70°C for 30 minutes for each fold

The conditions of reduced stringency (allow hybridization of sequences having at least 50% identity)

Hybridization:6x SSC at RT to 55°C for 16-20 hours
Wash at least twice:2x-3x SSC at RT to 55°C for 20-30 minutes for each fold

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as PR is dostavleno in SEQ ID NO: 1.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 2.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 4.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 5.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 7.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 8.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 10.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 11.

In the preferred embodiment of the invented what I specified nucleic acid molecule contains or consists of a nucleic acid sequence, as represented in SEQ ID NO: 13.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 14.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 16.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 17.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 19.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 20.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 22.

In the preferred embodiment of the invention the specified nucleic acid molecule contains or consists of a nucleic acid sequence as shown in SEQ ID NO: 23.

According to gnome aspect of the proposed invention the polypeptide, encoded by a nucleic acid according to the invention.

According to another aspect of the invention proposed a polypeptide containing an amino acid sequence chosen from:

1) amino acid sequences as shown in SEQ ID NO: 3;

2) amino acid sequences as shown in SEQ ID NO: 6;

3) amino acid sequences as shown in SEQ ID NO: 9;

4) amino acid sequences as shown in SEQ ID NO: 12;

5) the amino acid sequence as shown in SEQ ID NO: 15;

6) amino acid sequences as shown in SEQ ID NO: 18;

7) amino acid sequence as shown in SEQ ID NO: 21;

8) amino acid sequences as shown in SEQ ID NO: 24;

9) amino acid sequences as shown in SEQ ID NO: 25;

10) amino acid sequences as shown in SEQ ID NO: 26;

11) amino acid sequences as shown in SEQ ID NO: 27;

12) the amino acid sequence as shown in SEQ ID NO: 28;

13) amino acid sequence as shown in SEQ ID NO: 29;

14) the amino acid sequence as shown in SEQ ID NO: 30;

15) the amino acid sequence as shown in SEQ ID NO: 31;

16) amino acid sequence as shown in SEQ ID NO: 32;

with agonistically activity against receptor growth hormone.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 3.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 6.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 9.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 12.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 15.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 18.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 21.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 24.

In the preferred embodiment of the invention indicated the p polypeptide includes or consists of the amino acid sequence, as represented in SEQ ID NO: 25.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 26.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 27.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 28.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 29.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 30.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 31.

In the preferred embodiment of the invention the specified polypeptide includes or consists of the amino acid sequence as shown in SEQ ID NO: 32.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 3.

According to another aspect of the invention offer the n-glycosilated, contains two polypeptide containing or consisting of SEQ ID NO: 6.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 9.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 12.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 15.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 18.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 21.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 24.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 25.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 26.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 27.

According to another aspect of the invention proposed glycosilated containing two polypep the IDA, containing or consisting of SEQ ID NO: 28.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 29.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 30.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 31.

According to another aspect of the invention proposed glycosilated containing two polypeptide containing or consisting of SEQ ID NO: 32.

According to another aspect of the invention proposed a vector containing the nucleic acid molecule according to the invention.

In the preferred embodiment of the invention the specified vector is an expression vector adapted for expression of the nucleic acid molecules according to the invention.

It is not necessary that the vector containing the nucleic acid (acid) according to the invention contains a promoter or other regulatory sequence, especially if the vector is intended for use for the introduction of nucleic acid into cells for recombination in the genome for stable transfection. Preferably, the nucleic acid in the vector is functionally linked to an appropriate promoter or other regulatory elements for transcription in to etka host. The vector may be a bifunctional expression vector that functions in many hosts. By "promoter" means a nucleotide sequence that is located in the opposite direction from the site of transcription initiation and containing all regulatory region necessary for transcription. Suitable promoters include constitutive, tissue-specific, inducible promoters, the promoters of the development or other promoters for expression in eukaryotic or prokaryotic cells. "Functionally linked" means associated as part of the same molecule of nucleic acid, appropriately located and oriented for transcription initiated by the promoter. DNA, functionally linked to the promoter, is under the regulation of initiation of transcription of the promoter.

In the preferred embodiment the promoter is a constitutive, inducible or regulated promoters.

According to another aspect of the invention, the proposed cell, transfusiona or transformed with the nucleic acid molecule or vector according to the invention.

Preferably, the cell is a eukaryotic cell. Alternatively, the specified cell is a prokaryotic cell.

In the preferred embodiment of the invention is shown, the cell is selected from the group consisting of cells of the fungus (e.g., Pichia spp, Saccharomyces spp, Neurospora spp), insect cells (e.g., Spodoptera spp), mammalian cells (e.g., COS cells, cells of the Chinese hamster ovary (Cho), plant cell.

According to another aspect of the invention proposed pharmaceutical composition comprising the polypeptide according to the invention, including excipient or the media.

In the preferred embodiment of the invention mentioned pharmaceutical composition can be combined with additional therapeutic agent.

With the introduction of the pharmaceutical compositions of the present invention it is administered in pharmaceutically acceptable preparations. These preparations may usually contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and possibly other therapeutic agents.

The pharmaceutical compositions according to the invention it is possible to enter any conventional method of administration, including injection. Introduction and application may, for example, be oral, intravenous, intraperitoneal, intramuscular, intra, intra-articular, subcutaneous, local (eye), skin (e.g., creamy fat-soluble tab for skin or mucous membrane), percutaneous, or intranasal.

The pharmaceutical compositions according to the invention is administered in effective amounts. "The effect is positive number" represents a number of drugs/compositions which by itself or together with further doses or synergistic drugs lead to the desired response. This may include only a temporary slowing of disease progression, however, preferably it includes persistent cessation of progression of the disease. This can be controlled by conventional means or can be controlled in accordance with the diagnostic methods.

Dose pharmaceutical compositions, administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the method of administration and the condition of the subject (e.g., age, gender). With the introduction of the pharmaceutical compositions according to the invention is used in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. When used in medicine, the salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts can easily be used to obtain their pharmaceutically acceptable salts and is not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, without limitation, salts derived from the following acids: hydrochloric, Hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic and the like. In addition, pharmaceutically acceptable salts can be obtained as salts of alkaline or alkaline earth metals, such as salts of sodium, potassium or calcium.

If desired, the pharmaceutical compositions can be combined with a pharmaceutically acceptable carrier. When used herein, the term "pharmaceutically acceptable carrier" means one or more than one compatible solid or liquid filler, diluent or encapsulating substance that is suitable for administration to humans. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which combine active ingredient for ease of use. The components of the pharmaceutical compositions can also be mixed with molecules of the present invention and with each other so that there will be no interaction which would substantially reduce the desired pharmaceutical efficiency.

The pharmaceutical compositions may contain suitable buffering agents, including acetic acid in the form of a salt, citric acid in the form of salts, boric acid in the form of salts and phosphoric acid in salt form.

Perhaps the pharmaceutical compositions may also contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens and thimerosal.

Pharmaceutically what songs can easily be represented in the form of standard dosage forms and can be made in any of the ways well known in the pharmaceutical field. All methods include the stage of the Association of the active agent and carrier, representing one or more than one additional ingredient. In most cases, the composition is produced by uniform and close Association the active compound with a liquid carrier, a finely ground solid carriers or both and then, if necessary, provide the product form.

Compositions suitable for oral administration may be presented as discrete units such as capsules, tablets, pellets, each of which contains a predefined number of active connections. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, an elixir, or an emulsion.

Compositions suitable for parenteral administration, are eligible include sterile aqueous or non-aqueous preparation, preferably isotonic to the blood recipient. This drug can be manufactured according to known methods using suitable dispersing or wetting agents and suspendida agents. A sterile preparation for injection may also be a sterile solution or suspension for injection in a non-toxic diluent or solvent, acceptable DL is injecting, for example, in the form of a solution in 1,3-butanediol. Among the acceptable solvents may be used water, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspendida environment traditionally use sterile non-volatile oil. This purpose can be used any soft fixed oils, including synthetic mono - or diglycerides. In addition, in preparation for injection can be used fatty acids such as oleic acid. The composition of the carrier suitable for oral, subcutaneous, intravenous, intramuscular and the like introduction can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

According to another aspect of the invention, a method of treatment of a subject-a person suffering from an excess of growth hormone, comprising introducing an effective amount of at least one polypeptide according to the invention.

In the preferred method according to the invention the specified polypeptide is injected.

In an alternative preferred method according to the invention the specified polypeptide is administered subcutaneously.

In another preferred method according to the invention the specified polypeptide administered daily or two-day intervals; preferably, this polypeptide is administered with weekly, fortnightly or monthly intervals is.

In the preferred method according to the invention the specified excess growth hormone causes acromegaly.

In the preferred method according to the invention the specified excess growth hormone causes gigantism.

According to another aspect of the invention, a method of treatment of a subject-a person suffering from cancer, comprising introducing an effective amount of at least one polypeptide according to the invention.

When used herein, the term "cancer" refers to cells having the capacity for Autonomous growth, i.e abnormal status or condition characterized by a rapid proliferating cell growth. Assume that this term includes all types of malignant growth or oncogenic processes, metastatic tissues or malignant way of transformed cells, tissues or organs, irrespective of histopathologic type or stage of invasion. The term "cancer" includes malignancies of the various organ systems, such as malignancies, affecting, for example, lung, breast, thyroid, lymphoid organs, gastro-intestinal and genito-urinary tract as well as adenocarcinomas, including malignant neoplasms, as the majority of malignant tumors of the colon, renal cell cancer, prestat the school of cancer and/or tumors of the testis, non-small cell lung cancer, cancer of the small intestine and cancer of the esophagus. The term "carcinoma" is accepted in the art and refers to malignant neoplasms of epithelial or endocrine tissues including carcinoma of the respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular, breast carcinoma, carcinoma of the prostate, carcinoma endocrine system and melanoma. Examples of carcinomas include carcinomas, originating from the tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term "cancer" also includes carcinosarcoma, for example, include malignant tumors composed of cancerous and sarcomatous tissues. The term "adenocarcinoma" refers to carcinoma, originating from glandular tissue, or tumor whose cells form recognizable glandular structures. The term "sarcoma" is accepted in the art and refers to malignant tumors of mesenchymal origin.

In the preferred method according to the invention specified cancer is a cancer of the prostate.

In the description and claims of this application, the words "include" and "contain" and variations of these words, for example "comprising" and "comprises", mean "kiuchumi, without limitation" and imply that they do not exclude other groups, add-ons, components, integers or stage.

In the description and claims of this application only includes the plural, unless the context otherwise requires. In particular, using terms that indicate an indefinite number, a description should be understood as implying a lot, as well as the singular, unless the context requires otherwise.

Properties, integers, characteristics, compounds, chemical group or groups described in connection with a particular aspect, embodiment or example of the invention, should be understood as applicable to any other aspect, embodiment or example described here, if they are compatible with it.

The embodiment of the invention will now be described only by means of examples and with reference to the following graphic materials.

Table 1 shows the analysis by Bradford fractions 1B8v2.

Figa: 1B8v0 consists of GH (contains the mutation site 1)associated with the extracellular part of the GHR (domains 1 and 2) through a linker G4Sx4; this structure contains the restriction sites near the linker region and the 3'-end; Figb presents the encoded amino acid sequence.

Figa: 1B8v1. This molecule has the origin from 1B8v0, but does not contain extraneous sequence at the 5'- and 3'-Kon the Ah and contains the linker G4Sx4; on Figb presents the encoded amino acid sequence.

Figa: 1B8v2. This molecule has the origin from 1B8v0, but does not contain extraneous sequence and contains the linker G4Sx5; Figb presents the encoded amino acid sequence.

Figa: 1B8v3. This molecule has the origin from 1B8v0, but does not contain extraneous sequence and does not contain a linker; Figb presents the encoded amino acid sequence.

Figa: 1B9v0 consists of GH (contains the mutation site 1 and site 2)associated with GHR (domains 1 and 2) through a linker G4Sx4; this structure contains the restriction sites near the linker region and the 3'-end; Figb presents the encoded amino acid sequence.

Figa: 1B9v1. This molecule has the origin from 1B9v0, but does not contain extraneous sequence at the 5'- and 3'-ends and contains a linker G4Sx4; Figb presents the encoded amino acid sequence.

Figa: 1B9v2. This molecule has the origin from 1B9v0, but does not contain extraneous sequence and contains the linker G4Sx5; Figb presents the encoded amino acid sequence.

Figa: 1B9v3. This molecule has the origin from 1B9v0, but does not contain extraneous sequence and does not contain a linker; Figb presents encoded by amino the PCI-e slot sequence.

Figure 9 shows the basic method of ligating to sublimirovanny molecules G120R in plasmid expression mammals.

Figure 10 shows the design 1B9v0.

Figure 11 shows Nar1-Avrll fragment V v2 (524 base pairs (BP)). New linker region is shown in bold, the restriction site is underlined. This fragment is ligated into a plasmid pGHsecTag-1B8v1 education

plasmids pGHsecTag-1B8v2.

On Fig shown Nar1-Avrll fragment V v2 (524 base pairs (BP)). New linker region is shown in bold, the restriction site is underlined. This fragment is ligated into a plasmid pGHsecTag-1B9v1 education

plasmids pGHsecTag-1B9v2.

On Fig shows a Western blot using GH-specific antibodies to detect expression as 1B8v2 (lanes 1 and 2)and 1B9v2 (lane 3) from the culture media of a stable Cho cell line Flp-ln. Samples have corresponding expected for each protein size (~75 kDa) and does not show signs of degradation.

On Fig shown that in the presence (+) of 0.5 nm rhGH samples of media from stable cell lines 1B8v2, and stable cell lines 1B9v2 able to anlagenservice effects of rhGH. In the absence (-)of 0.5 nm GH none of the molecules does not show biological activity. Shows the calibration curve GH (0-5 nm).

On Figa shows polyacrylamide gel electrophoresis (PAGE) with dodecenal the volume of sodium (SDS) (SDS-PAGE analysis of purified protein fractions with staining of Kumasi. The images shows that the purified protein (1B8v2) has a corresponding expected size (~75 kDa), along with no visible degradation products with a lower molecular weight; Figb shows SDS-PAGE analysis 1B9v2.

Figa. Peak levels of protein in the serum 24 hours after injection after subcutaneous (SC) injection V. U still can be detected 10 days after injection. Figb. Peak levels of protein in the serum within 1 hour after injection, followed by a sharp decline after intravenous (IV) injection W.

Figa. Western blot of native PAGE sample. 1: 1B7v0, protein native GH, 2: 1B7v1, native GH, 3: 1B7v2, native GH, 4: 1B7v3, native GH, 5: V, protein modified GH. All samples show two distinct bands that are characteristic of the monomer and the formation of the dimer. On Figb presents equivalent colored Kumasi gel showing the formation of dimer.

On Fig shows the % increase of body weight in NZ white rabbits for 12 days for comparison 5 input doses of pegvisomant with single doses IB8 and IB9.

On Fig shows the pharmacokinetics (PK) IB8 white rabbits NZ for 250 hours.

Materials and methods

The design of the molecule antagonist W

Designed molecule with a mutation of the amino acid glycine at position 120 for arginine at site 2 (site with low affinity) of the GH molecule (G10R). The binding molecule of GH with GH receptor through site 1 with high affinity is not affected, however, the binding with the receptor through site 2 GH ingibirovalo surround the side group of a molecule of arginine.

Previously, to obtain the GH molecule containing a mutation G120R, was applied PCR method, and using appropriate restriction enzymes cut sites made possible the cloning of this fragment into a plasmid expression pTrc-His education clone pTrc-His-1A7 (G120R associated with the extracellular domain B GHR).

Then vector cut Bsu36l-Not1 fragment length of 300 BP and rigorously it in plasmid expression mammals pGHsecTag-1B7 (GH associated with extracellular domains a and b GHR) with the formation of pGHsecTag-V (secretory expression controls secretory signal sequence GH). Cm. Fig.9.

The design of the molecule antagonist 1B9v0

Designed molecule with a mutation of the amino acids as in site 1 and site 2 of the GH molecule. The binding molecule of GH with GH receptor through site 1 with high affinity strengthened by these mutations, whereas binding to the receptor through site 2 GH ingibirovalo one substitution of glycine for arginine.

The method of site-directed mutagenesis using single-stranded DNA used to obtain the GH molecule containing a mutation as site 1 and site 2. The use of suitable restriction enzymes cut sites made possible the cloning of this fragment into the expression plasmids pTrc-His and Rita (+). With the use of PCR has been clone, which contained the signal sequence of GH (GHss) with flanking sites Nhe1 and Not1. His ligated into a plasmid expression mammals pGHsecTag-1B8v0 (GH associated with extracellular domains a and b GHR) with the formation of pGHsecTag-1B9v0 (secretory expression controls secretory signal sequence GH). Cm. Figure 10.

The design of the clones-options as 1B8v0 and 1B9v0

Plasmid pGHsecTag-1B7v3 were digested using restricted HindIII-EcoRV and the fragment ligated into the plasmid pGHsecTag-1B8v0 and 1B9v0 for constructing plasmids pGHsecTag-1B8v1 and 1B9v1 (these molecules do not contain any erroneous sequence on the 3'-end). The next stage was to remove restriction sites near the linker region with the formation of the plasmid pGHseTag-1B8v2 and 1B9v2. This was carried out using gene synthesis, in which the original linker was replaced by a linker G4Sx5.

Synthesis genes were designed following fragments with flanking restriction sites Narl and Avrll and ligated either pGHsecTag-1B8v1 or 1B9v1; see 11 and 12.

The biological activity of molecules options antagonists in vitro

The biological activity of each of the chimeric molecule in vitro were studied by means of analysis with GH-specific luciferase reporter. Essentially, cell line, originating from a person consistently has transfusional man is Kim GH receptor and then transtorno was transfusional luciferase reporter signal. This analysis revealed a physiological levels of GH, see Fig.

Purification of molecules antagonists

Cho cell line Flp-In expressing how 1B8v2 and 1B9v2 in the form of secreted product, were grown in free protein environments. Before affinity purification medium was collected, concentrated and osvetleni. To clear prepared with 20 ml of the activated N-hydroxysuccinimide (NHS) resin Sepharose 4 Fast Flow associated with a monoclonal antibody A to human GH (hGH). Typically, the sample media were concentrated ten times and diluted in the ratio 1:1 with binding buffer (25 mm Tris-HCl/150 mm NaCl, pH 7,4) before cleaning.

The material was brought on the column with a flow rate of 2 ml/min After washing, bound protein was suirable at a flow rate of 1 ml/min 200 mm glycine, pH 2.7, and with subsequent neutralization of 1 M Tris-HCl, pH to 9.0. The samples were analyzed by SDS-PAGE (see Figa and 15B). On Figa and 17B shows the formation of dimers IB9 compared to the chimeric molecules of native human growth hormone.

Pharmacokinetic studies IB8

6 normal healthy rats with a single injection was administered 1 nmol (75 µg) of protein or subcutaneously (SC, Figa)or intravenously (IV, Figb). Control rats were injected only filler. Samples were taken with time intervals during the 10-day period and analyzed for the presence V using tverdofaznogo the enzyme-linked immunosorbent assay (ELISA) on GH its own design.

Table 1A
Protein concentration (µg/ml)Volume (ml)Total protein (mg)
Making 1B8v229310631,1
Unbound22712027,2
Flushing-120-
Elution 1-5-
Elution 260,750,3
Elution 363,650,32
Elution 484,750,42
Elution 518,55 0,09
Elution 610,050,05
30 kDa F/T8,6450a 3.9

Table 1B
Protein concentration (µg/ml)Volume (ml)Total protein (mg)
Making 1B9v231111034,2
Unbound24612530,8
Flushing-150-
Elution 19,45-
Elution 232,450,16
Elution 31295 0,65
Elution 480,150,4
Elution 537,150,19
Elution 69,450,05
30 kDa F/T9,4450

1. The nucleic acid molecule consisting of a nucleic acid sequence chosen from:
1) nucleic acid sequence as shown in SEQ ID NO: 22, or
2) nucleic acid sequence as shown in SEQ ID NO: 23,
characterized in that the nucleic acid molecule encodes a polypeptide that has an antagonistic activity against hormone receptor growth.

2. The polypeptide consisting of the amino acid sequence selected from: amino acid sequence as shown in SEQ ID NO: 24 or amino acid sequences as shown in SEQ ID NO: 32; where the specified polypeptide has an antagonistic activity against hormone receptor growth.

3. The use of the polypeptide according to claim 2 is the treatment of excess growth hormone.

4. The use according to claim 3, where the specified polypeptide is administered with weekly intervals.

5. The use according to claim 3, where the specified polypeptide is administered with a two-week intervals.

6. The use according to claim 3, where the specified polypeptide is administered at monthly intervals.

7. The use according to claim 3, where excess growth hormone is a gigantism or acromegaly.

8. Glycosilated, consisting of two polypeptides consisting of SEQ ID NO: 24 or SEQ ID NO: 32, where the specified glycosilated has antagonistic activity against receptor growth hormone.

9. The use of glycosilated of claim 8 in treatment of excess growth hormone.

10. The use according to claim 9, where the excess growth hormone is a gigantism or acromegaly.

11. Pharmaceutical composition for the treatment of excess growth hormone containing polypeptide according to claim 2, including excipient or media.



 

Same patents:

FIELD: medicine.

SUBSTANCE: what is presented is recombinant plasmid DNA pPBS-St9 coding polypeptide having a sequence of the human growth hormone somatotropin of molecular weight 4.1 MDa (6266 base pairs), as well as the Saccharomyces cerevisiae strain BY4739 [leu2 ura3 lys2 prcl::LEU]/pPBS-St9 containing recombinant plasmid DNA pPBS-St9 that is a recombinant somatotropin producer.

EFFECT: invention may be used for producing the recombinant human growth hormone in treating dwarfism.

2 cl, 5 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: what is produced is the yeast strain Saccharomyces cerevisiae able to produce secreted human somatotropin. Said strain contains a promoter contolled DNA sequence coding mature human somatotropin fused with a leader peptide in the same reading frame. The leader peptide includes a double pro-site of α-factor of yeast Saccharomyces cerevisiae. It also can contain a triple pro-site of α-factor of yeast Saccharomyces cerevisiae, or a double pro-site of HSP150 protein of yeast Saccharomyces cerevisiae, or a combination of said pro-sites. A method for producing human somatotropin provides cultivation of the human somatotropin producer strain.

EFFECT: use of the invention provides higher end product yield.

2 cl, 1 dwg, 8 ex

FIELD: medicine.

SUBSTANCE: hybridoma strain is produced by immunization of BALB/c mice. The mice are immunised by common technique by double subcutaneous introduction of LPS Francisella tularensis 15/10. On the third day following the last booster injection, splenocytes of the immune mice are hybridised (1×108 cells) and mice myeloma cells RZ-X63 Ag/8-653 (1×107 cells). Polyethylene glycol (Sigma, the USA) is used as a fusion agent. The hybridisation is followed by hybridoma selection, screening, cloning and cryopreservation. The hybridoma 11D6 under the invention produces monoclonal antibodies (MCA) to LPS F. tularensis with titre making in a culture fluid 1:1000, in immunoascitic fluid 1:100000.

EFFECT: hybridoma produced MCAs are high specific to LPS Francisella tularensis and applicable for engineering test systems for rabbit disease agent detection.

7 dwg, 6 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, more specifically to producing a neutralising epitopes for an anti-GdF-8 antibody, and can be used in medicine. Produced are peptide versions of the area of 327 to 346 amino acid of the sequence SEQ ID NO: 1 which is specifically bound with rat's monoclonal antibody 788. Peptides are produced by a recombinant method with using a host cell transformed by an expression vector with nucleic acid coding said peptide version. The produced peptides are used to prepare vaccine compositions which are used to elicit an anti-GdF-8 immune response and to enable GDF-8 activity down-regulation in an animal. Also, the produced peptides can be used in a method of anti- antibodies selection screening.

EFFECT: produced peptides exhibit the anti-GdF-8 antibody binding activity thereby applicable for the decreased activity of a growth and differentiation factor 8.

17 cl, 2 dwg, 6 tbl, 5 ex

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: medicine.

SUBSTANCE: invention concerns a new F3H10 hybrid cell clone producing monoclonal diphtheria toxin antibody in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 with popliteal lymph node cells of BALB/c mice immunised with a preparation of diphtheria toxin (manufactured by State Institution Gamaleya's Epidemiology and Microbiology Research Institute) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises monoclonal antibodies specifically reacting with diphtheria toxin and belonging to the IgG class as determined by IEA data with typifying antiserums against certain murine Ig classes. Antibody titre reaches in the culture fluid 1:10000 1:20000, and in the ascitic 1:1000000.

EFFECT: antibodies can be used for designing immunobiological test systems of diphtheria toxin detection exceeding available analogues in sensitivity and reproducibility.

2 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to the preparation of genetically engineered construct for transgene expression in mammal cells in vitro and in vivo. The modified sequence of gene of angiopoietin-1 (Angl) is offered. The sequence is designed for transfection of mammal cells for expression of angiopoietin-1 therein. Production of angiopoietin-1 in case of using the optimised gene AGPopt is 1.6 times more than when using the natural gene hAGP. Herewith, the content of angiopoietin-1 in a culture medium of transfected cells makes not less than 0.5 mcg/ml.

EFFECT: invention can be used in medicine.

3 dwg, 1 tbl, 1 ex

FIELD: biotechnologies.

SUBSTANCE: plants are transformed with application of nucleic-acid constructs, which contain the first nucleotide sequence that codes γ-sein, or its fragment, which is able to direct and retain protein in endoplasmic reticulum of plant cell, the second sequence of nucleic acid, which codes aminoacid sequence, which is specifically split by ferment or chemical compounds, and the third sequence of nucleic acid, which codes target peptide or protein.

EFFECT: transformation of plant by such constructs makes it possible to produce fused proteins accumulated in endoplasmic reticulum of cells in the form of protein bodies, from which target proteins may be extracted, in particular calcitonin.

47 cl, 19 dwg, 1 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

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: medicine.

SUBSTANCE: invention provides using a prepared compound of a ghrelin splicing version for preparing an effective drug preparation activating body weight and food intake gain and/or stimulating growth hormone release, as well as for treating or preventing cachexia, lipodystrophy and muscle atrophy.

EFFECT: higher clinical effectiveness.

6 cl, 6 dwg, 13 ex

FIELD: chemistry.

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

EFFECT: high stability.

22 cl, 3 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of nonapeptide ethylamide, having strong LH-RH/FSH-RH activity, of formula pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·2AcOH (I), and intermediate compounds for synthesis thereof. The nonapeptide ethylamide is obtained via condensation of a C-terminal tetrapeptide of formula H-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (II) with a dipeptide of formula: X-Ser-Tyr-OH (IV), where X is a protective group. The obtained N-substituted hexapeptide ethylamide of formula X-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (III) is treated with an unblocking agent to remove the N-protective group, and then condensed with a tripeptide of formula pGlu-His-Trp-OH·HCl (V) and the end product is purified through chromatography and extracted in form of a monoacetate salt.

EFFECT: high output.

4 cl, 1 ex

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