Injection drug for increasing human sperm production and method for use thereof

FIELD: chemistry.

SUBSTANCE: invention discloses an injection drug in form of a water-oil suspension of a chimeric protein with water-insoluble enzymatically inactive chloramphenicol acetyltransferase without 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and a somatostatin-14 amino acid sequence AGCFWKTFTSC in refined vegetable oil with addition of apyrogenic water for injection. The invention also relates to a method of increasing human sperm production, which comprises hypodermic injection of said drug twice with an interval of 14-18 days in amount of 50-100 mcg protein per kg body weight.

EFFECT: invention improves reproductive capacity of men by increasing ejaculate volume, sperm count, the percentage of live, active sperm when the injection drug is used with low reactogenicity of the adjuvant, which allows painless injection.

6 cl, 1 tbl

 

Scope

The invention relates to andrology, and specifically to inject the drug to increase spermophobia person and the method of its application. The invention is intended to enhance the reproductive ability of the individual, allowing you to increase the amount of ejaculate and to raise some qualitative characteristics of human sperm.

Known level

Modern medical science in several ways solves the problem of increasing ejaculate volume, sperm count in the ejaculate and improve their quality characteristics is the use of hormonal, adaptogenic, herbal preparations. As hormonal therapies aimed at increasing the number of sperm in the ejaculate, using drugs from the group of antiestrogen - clomiphene citrate and preparations based on human chorionic gonadotropin (R.V. Aivanov, NS Parfenova, became popular Kurbatov. Treatment oligospermii in men with infertility. Problems of endocrinology, 2010, No.1, p.31-34).

According to literature data, medical therapy oligospermii are not always successful. In this case, recommend alternative treatments - prescribed drugs-antioxidants, substances containing increased amounts of vitamin C, selenium, folic acid, L-carnitine. According to clinical issledovaniia substances neutralize free radicals and enhances the activity of metabolic processes in sperm a beneficial effect on male fertility. ("Andrology and genital surgery" is a scientific and medical peer-reviewed multidisciplinary journal, Hamidov SR, Tazetdinov OF Results of an open comparative study drugs "Speman and Spiractin" as a means of treatment of idiopathic photosphere. No. 2, 2011, p.61-66.)

Another method of increasing the volume of ejaculate and enhancing qualitative characteristics of human sperm based on the increase in the body concentration of endogenous growth hormone induction of the synthesis automatisation antibodies. This leads to a decrease in the concentration of endogenous somatostatin and enhance the content of the hormone that, in turn, has a stimulating effect on the growth and proliferation of cells in the gonads of the person.

Somatostatin is a biologically active tetradecapeptide produced in the hypothalamus and gastrointestinal tract of mammals. For the first time the biological activity of a substance, later defined as somatostatinoma, was discovered in 1968 by Crusecom. In subsequent from the hypothalamus of animals was selected substance representing low-molecular peptide having the ability to regulate the concentration of growth hormone in animals.

Somatostatin-14 has a strong inhibitory action is the development of a number of hormones (somatotropin, thyroid-stimulating hormone, insulin, glucagon, gastrin, pepsin), triggers the inhibition of the secretion of digestive enzymes, the pancreas, small intestine, slow motility of the gastrointestinal tract and the evacuation of its contents.

The study of amino acid composition of somatostatin various members of the animal Kingdom showed a sufficient degree of homology peptides, synthesized by living organisms at different stages of evolutionary development. Amino acid sequence of somatostatin-14 is identical to living organisms, from fish to mammals and humans.

A wide range of physiological actions of somatostatin-14 and no, unlike growth hormone, species specificity in mammals has been the basis for exploring the possibility of its use to optimize breeding and fattening, raising dairy animal productivity, and increase spermophobia manufacturers of farm animals and chickens.

Method somatostatinomas immune deprived of many of the disadvantages arising from the use of anabolic hormones or recombinant somatotropin. The mechanism of action is based on the temporary binding of endogenous somatostatin-14 specific antisomatostatin antibodies and increase concentric and endogenous growth hormone in physiological limits. However, wide application of the method of active immunization of animals against endogenous somatostatin-14 a long time it was impossible due to its high cost as the main way of obtaining peptide was chemically synthesized, which is economically not allowed to implement this approach in practice. Because of the small size of the somatostatin-14 does not allow its direct microbial synthesis using recombinant DNA technology described several methods of its synthesis in the form of chimeric proteins with subsequent isolation of the target product, not given satisfactory results. The main disadvantage of the above methods is extremely low immunogenicity obtained drugs against somatostatin, due to its masking of the molecule in the chimeric protein, therefore, these methods of producing chimeric proteins did not find wide practical use (R. Itakura et al., 1977. Expression in E. Coli of a chemically synthesized unit gene of hormone somatostatin. Science, 1986, 1056-1063; A.A. Shishkin and other Synthesis fragment somatostatin genes. Chemistry of natural compounds, 1988, No. 6, s-615).

The known method of constructing chimeric somatostatinergic proteins using amino acid spacer containing arginine and Proline, causing localization of somatostatin on the surface of carrier protein and thereby high immuno is a property of the drug (RU 2031121 C1, IPC C12N 15/12, 1995). The design consists of a water-insoluble protein carrier (fragment of bacterial chloramphenicolchloramphenicol without the 10 C-terminal amino acids), tetramer spacer and C-terminal somatostatin-14. The molecular weight of the chimeric protein is 28 kDa. This chimeric protein is expressed by Escherichia coli B-6519 transformed by the plasmid pC(Sp)4 S. Strain deposited in Russian national collection of industrial microorganisms under the number B-6519. Chimeric protein with the exposed somatostatin is a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acid residues, which through spacer elements sequence attached to the amino acid sequence of somatostatin-14. Method antisomatostatin immunization of animals with the use of this chimeric somatostatinergic protein is used in the cattle industry (EN 2034457 C1 IPC A01K 67/02, 1995).

The invention

The objective of the invention is to improve the reproductive ability of men by increasing ejaculate volume, sperm count, proportion of live, active motile sperm using the drug for injection with low reactogenicity adjuvant that allows injection without painful the feelings.

The solution to this problem provides an injectable drug to increase spermophobia person in the form of water-in-oil suspensions of the chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC refined vegetable oil with the addition of pyrogen-free water for injection.

The mechanism of drug action based on the temporary blocking activity of endogenous somatostatin person, increased concentrations of somatotropic hormone and increased activity of spermatogenesis.

In the best embodiment, injecting the drug to increase spermophobia person contains a chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC based 200-400 mg of the indicated chimeric protein per 100 ml of refined vegetable oil, comprising 5.0 to 10.0 wt.% pyrogen-free water for injection.

The drug is to increase spermophobia person can contain refined cottonseed oil or refined peanut oil.

The way to increase sperm the production person includes two subcutaneous injections with an interval of 14-18 days the injection of the drug in the form of water-in-oil suspensions of the chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC refined vegetable oil with the addition of pyrogen-free water for injection at the rate of 50-100 μg of protein per 1 kg of body weight.

In the best embodiment of the invention using injectable drug in suspension chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC in the rate of 200-400 mg of the indicated chimeric protein per 100 ml of refined vegetable oil, comprising 5.0 to 10.0 wt.% pyrogen-free water for injection. In the particular case used the drug may contain refined cottonseed oil or refined peanut oil.

The implementation of the invention and efficacy

The possibility of carrying out the invention is illustrated by the example of obtaining injectable drug to increase sperm of a man.

Obtained and purified from the impurities of the drug is protein dissolved in buffer 0.2 M Tris-HCL pH 8.0, containing 6 M guanidinium and 2 M of META (M-EDTA). Add 50-fold molar excess of β-mercaptoethanol in the calculation of the number of S-S GRU is p chimeric protein and the solution is quickly diluted 10-fold volume of buffer without handinhand. The resulting hybrid protein precipitate is separated by centrifugation for 15 minutes at 12000 g and 4°C and freeze-dried for storage or cooking oil-water suspension.

To prepare the finished form of injection drug use refined vegetable oil and pyrogen-free water for injection. In vegetable oil, water is added in amounts of 5-10%. Then add a portion of the dry protein at the rate of 200 mg per 100 ml of water-in-oil suspension. The suspension is homogenized for 1-2 minutes and transfer to the filling equipment. The prepared suspension of the drug Packed in containers (e.g., disposable syringes). Finished dosage form of the preparation is sterilized with ionizing radiation at a total dose of 6 kGy.

When different concentrations of the components of the injectable preparation is prepared similarly. Preferred are the following contents of the components in 95 ml of refined vegetable oil: 200 mg of chimeric protein - 5.0 wt.% apirogennoy water for injection; 400 mg of chimeric protein - 10 wt.% pyrogen-free water for injection.

Use as an adjuvant for such preparation of vegetable oil, fully metabolized in the body, greatly reduce the level of reactogenicity adjuvant isoset necessary conditions for the gradual admission of the active substance in the human body (depot preparation). Can be used on cotton and peanut butter. The presence of pyrogen-free water at the end of the recipe of preparation is justified by the need to obtain a water-in-oil emulsion of the drug.

Efficacy of injectable drug to increase sperm of a man is illustrated by the following example.

In the experimental group included men aged 30-53 years with signs of oligospermia (concentration of spermatozoa in 1 ml of ejaculate ranged from 11 to 15 million cells), ejaculate volume equal to 1.5 and 2.1 ml, the number of actively motile (a) spermatozoa equal to 18-23%. The number of live sperm was on the bottom of the norms and who was 48-53%. Patients did not suffer from diabetes mellitus, hypothyroidism, hyperthyroidism, hypercortisolism, cancer, acute or chronic liver diseases, urological and/or infectious and inflammatory diseases in the acute stage, varicocele.

The generalized results of the laboratory analyses are presented in table 1.

semen after
Table 1
ValueValues
indicators
Estimated figuresspermthe specified number of days after
spermbefore the introduction ofthe first injection of the drug,
medication(min-max)
(min-max)30 days60 days90 days
Ejaculate volume, ml1,5-2,12,6-2,9of 3.0-3.52,8-3,7
Consistencyviscousviscousviscousviscous
pH7,2-8,07,2-8,07,2-8,07,2-8,0
The number of spermatozoa in 111-1514-2118-28 21-34
ml of ejaculate, million/ml
The share of active mobile18-2321-2823-3222-41
sperm (a), %
The percentage of live spermatozoa, %48-5352-6154-6854-72

All patients were introduced subcutaneously in the shoulder area of the water-oil suspension somatostatinergic drug in the dose of 50 mg/kg of body weight twice with an interval between injections 14-18 days.

After 30, 60, 90 days after the first injection the patients were put sperm samples and analyzed laboratory values.

Examples of the invention are not exhaustive. Possible other embodiments of the invention, corresponding to the volume of patent claims.

1. Injecting the drug to increase spermophobia person in the form of water-oil is Uspenie chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC refined vegetable oil with the addition of pyrogen-free water for injection.

2. The preparation according to claim 1, characterized in that it contains a chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC based 200-400 mg of the indicated chimeric protein per 100 ml of refined vegetable oil, comprising 5.0 to 10.0 wt.% pyrogen-free water for injection.

3. The preparation according to claim 1 or 2, characterized in that it contains refined cottonseed oil or refined peanut oil.

4. The way to increase spermophobia person, including two subcutaneous injections with an interval of 14-18 days the injection of the drug in the form of water-in-oil suspensions of the chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC refined vegetable oil with the addition of pyrogen-free water for injection at the rate of 50-100 μg of protein per 1 kg of body weight.

5. The method according to claim 4, Otley is audica fact, what use injectable drug in suspension chimeric protein with a water-insoluble enzyme inactive chloramphenicolchloramphenicol without the 10 C-terminal amino acids, amino acid spacer (Sp)n, where n=1, 2, 4, 8, and somatostatin-14 amino acid sequence AGCFWKTFTSC in the rate of 200-400 mg of the indicated chimeric protein per 100 ml of refined vegetable oil, comprising 5.0 to 10.0 wt.% pyrogen-free water for injection.

6. The method according to claim 4 or 5, characterized in that use injectable preparation containing refined cottonseed oil or refined peanut oil.



 

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