RU2782758C2 - USE OF COMPLEX OF 9-PHENYL-2,3,4,5,6,7,8,9-OCTAHYDRO-1H-SELENOXANTHENE WITH β-CYCLODEXTRIN FOR CORRECTION OF SPERMATOGENESIS DISORDERS CAUSED BY CYTOTOXIC EFFECT - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to the field of medicine, namely to the use of a clathrate complex of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene with β-cyclodextrin as a remedy for correction of spermatogenesis disorders at a mass ratio of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene to β-cyclodextrin from 1:1 to 1:10.

EFFECT: expansion of the arsenal of remedies intended for correction of spermatogenesis disorders caused by a cytostatic effect due to enhancing processes of reparative testicular tissue regeneration with reduction in a size of a cell population of fast renewing and reserve (stem) spermatogonia.

1 cl, 4 tbl, 9 ex

Description

The invention relates to medicine, namely to andrology, and can be used in pharmacology for the correction of spermatogenesis disorders caused by cytostatic effects.

The invention also relates to veterinary medicine and can be used to treat mammals with impaired fertility.

Often used anticancer drugs (cytostatics), regardless of their mechanism of action, have a pronounced toxic effect on actively dividing normal (not damaged by the tumor) tissues of the body. The latter include the spermatogenic epithelium of the male gonads [Gershanovich M.L. Complication in chemotherapy and hormone therapy of malignant tumors. - M. Medicine, 1982, - S. 51-80], in which, due to constant cell divisions (spermatogonia and spermatocytes), the maturation of male germ cells - spermatogenesis occurs. Violation of spermatogenesis observed against the background of cytostatic exposure differs in etiology and pathogenesis from other types of pathological processes that are accompanied by inhibition of spermatogenesis (trauma, inflammation, endocrine disorders, genetic diseases).

Currently, the chemotherapeutic method for the treatment of malignant neoplasms allows in some cases to achieve not only long-term complete remissions, but even cure [Antineoplastic chemotherapy. / Ed. N.I. Translator. - M.: Medicine, 2005, - S. 27-51]. In this regard, the problem of irreversible male infertility as a possible consequence of cytostatic chemotherapy becomes relevant [Reznichenko A.G. Effect of chemotherapy and radiotherapy on spermatogenesis in cancer patients. // Problems of reproduction. - 2007, - No. 4, - S.70-75].

The basis of irreversible changes is the damaging effect on the source of the proliferative pool of spermatogenesis - stem spermatogonia. Irreversible changes in the male gonads can significantly reduce the quality of life of young patients, since the state of their reproductive function, taking into account its biological, social, psychological significance, is an important life factor.

One of the ways to solve the problem of paternity of patients who underwent cytostatic therapy and are in long-term complete remission is the cryopreservation of seminal fluid before treatment [Auger J., Kuntsmann J., Czuglik F. et al. Conservation du sperme avant traitement anticancerenx. Une mesure efficce pour preserverles chances de conception future. // Contacept.-fertil.-sex. - 1993, - Vol.21, No. 10, - P. 749-752]. However, due to the limited availability of this method for most patients, cryopreservation has not found wide application. The search for ways to combat male infertility caused by cytostatic exposure continues. There are high hopes for germ cell transplantation [Meachem S., Schonfeldt V., Schlatt S. Spermatogonia: stem cells with a great perspective. // Reprod. - 2001, - Vol.121, No. 6, - P.825-834], but it is necessary to prove that their introduction will not lead to a decrease in the therapeutic effect of the cytostatic.

A known method of reducing the damaging effects of cytotoxic drugs on the male gonads with the help of pharmacotherapy: antioxidants (leuzea soflorovidnaya) [Schemerova Yu.A. Effect of topoisomerase activity inhibitors vepezid and irinotecan on the reproductive system of male rats: Abstract of the thesis. diss. Candidate of Biol. Sciences. - Tomsk, 2006, - 23 p.]; hormones (testosterone) [Delic J., Harwood J., Stanly J. Time dependence for the protective effect of androgen from procarbazine - induced damage to rat spermatogenesis. // Cancer. - 1987, - No. 5, - P.1344-1347]; the drug phospholicin, which reduces the toxic effect on the testes of platinum cytostatic drugs by reducing their nephrotoxicity [Yoshi S., Monabe F., Hiroshi T. et al. Huxon ragary pexo tarray dsasel. // Chemotherapy. - 1990, - Vol.38, No. 7, - P.670-675]. The disadvantages of the proposed methods for reducing the damaging effect of cytotoxic drugs on the male gonads are low efficiency, limited use, the possibility of side effects and contraindications for patients with a history of cancer. In the instructions for medical use of the above medicines, there is no information on the state of spermatogenesis in the long term after the administration of the drugs (3 and 6 months), which is most relevant for patients who are in long-term complete remission after undergoing treatment. There are also no data on the effect on spermatogenic stem cells, the preservation of which prevents the development of irreversible male infertility.

There is also known a method for correcting disorders of spermatogenesis caused by cytotoxic effects, using the drug "Neupogen" - recombinant human granulocyte colony-stimulating factor (G-CSF) / Patent RF2406527, publ. 20.12.2010 / This method, which consists in the use of pharmacotherapy, is the closest to the claimed and is selected as a prototype. Significant disadvantages of this method for correcting spermatogenesis disorders caused by cytotoxic effects with the help of G-CSF preparation are:

- the presence of side effects (nausea / vomiting, muscle and bone pain, dysuria, alopecia, diarrhea, skin rash, inflammation of the mucous membranes, fever, fatigue, headaches, cough, anorexia, leukocytosis, etc.);

- a complex and expensive method for its production (non-glycosylated protein, consisting of 175 amino acids, produced by a laboratory strain of the Escherichia coli bacterium, into which the human granulocyte colony-stimulating factor gene has been introduced by genetic engineering);

- an inconvenient and time-consuming method of preparation, use of the drug, which requires special storage conditions (available as an injectable solution for intravenous and subcutaneous administration; application is carried out under the close supervision of a doctor; stored at a temperature of 2-8 ° C).

The data of clinical observations indicate that the violation of spermatogenesis after the use of a number of chemotherapeutic regimens is irreversible [Gershanovich M.L. Complication in chemotherapy and hormone therapy of malignant tumors. - M. Medicine, 1982, - S. 51-80; Reznichenko A.G. Effect of chemotherapy and radiotherapy on spermatogenesis in cancer patients. // Problems of reproduction. - 2007, - No. 4, - S. 70-75]. Irreversible changes in the convoluted seminiferous tubule, leading to pathozoospermia, may be associated with the depletion of the proliferative pool of spermatogenesis, the source of which is the population of spermatogonia, namely, stem (reserve) cells [Lipatova N.A. Laboratory criteria for ejaculate fertility // Clinical laboratory diagnostics. - 1998, - V.5, - S. 11-15; Sukhorukov V.S., Shamshad D.S. Determination of the degree of maturity of the rat spermatogenic layer during its regeneration and in the process of maturation of the intact testis // Archive of Anatomy, Histology and Embryology. - 1989, - T.97, No. 9, - 89 p.]. These are progenitor cells that divide only when damaged and are the basis of the proliferative pool of spermatogenesis. For the formation of stem spermatogonia, it is necessary to have niches that form Sertoli cells (cells of the microenvironment) [Biology of stem cells and cell technologies. / Ed. M.A. Paltsev. - M.: Medicine, Shiko, 2009, - V.2, - 456 p.]. The latter lose the ability to divide in the mature convoluted seminiferous tubules [Kurnosova T.R., Verbitsky M.Sh., Sukhikh G.T., Molnar E.M. Modern ideas about the structural foundations of reparative regeneration of the testes of mammals and humans. // Bull. experimental biol. and honey. - 1994, - No. 4, - S. 396-399]. Regeneration of the seminiferous tubules as a whole is possible in only one way - through the formation of seminiferous tubules of an immature type from the regeneration zone, which contains Sertoli cells and primary sexual determinants, giving rise to new spermatogonia. Insufficiency of the regenerative potential of the testis leads to atrophy of the tissue as a whole and causes infertility.

As a result of our experimental studies, a hitherto unknown property of the pharmaceutical composition 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene with β-cyclodextrin was found to stimulate the processes of reparative regeneration of testicular tissue in reduction in the cell population of rapidly renewing and reserve (stem) spermatogonia in violation of spermatogenesis caused by cytostatic exposure.

The property of the pharmaceutical composition 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene with β-cyclodextrin to enhance the processes of reparative regeneration of testicular tissue while reducing the number of rapidly renewing and reserve (stem) cell populations spermatogonia in disorders of spermatogenesis caused by cytostatic effects is not obvious to a specialist and does not follow from the state of the art in this field, is not found in the patent and medical literature. The above property of the pharmaceutical composition of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene with β-cyclodextrin can be used in practical public health and veterinary medicine to improve the quality of treatment. Thus, the proposed technical solution meets the criteria for patentability of the invention, namely "novelty", "inventive step" and "industrial applicability".

The technical problem to be solved by the claimed invention is the expansion of the arsenal of agents intended for the correction of spermatogenesis disorders caused by cytostatic effects by enhancing the processes of reparative regeneration of testicular tissue while reducing the cell population of rapidly renewing and reserve (stem) spermatogonia. The technical result consists in the implementation of the proposed tools that provide correction of violations of spermatogenesis caused by cytostatic effects.

To solve the stated technical problem and achieve the maximum possible result, the present invention proposes the use of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene as a means for correcting spermatogenesis disorders caused by cytostatic impact, by enhancing the processes of reparative regeneration of testicular tissue.

A pharmaceutical composition is proposed for the correction of spermatogenesis disorders caused by cytotoxic effects, which contains 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene in an effective amount as an active ingredient.

The active component of the pharmaceutical composition for the correction of spermatogenesis disorders caused by cytotoxic effects, 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene, can be presented in the form of a clathrate complex with β-cyclodextrin or may additionally contain β-cyclodextrin in ratios from 1:1 to 1:10.

What is proposed is a medicinal preparation for the correction of spermatogenesis disorders caused by cytotoxic effects, which contains the specified pharmaceutical composition as an active ingredient. The specified medicinal product can be used in the form of tablets, with a content of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene from 2 to 10%, and the rest is one or more excipients ( and). While tablets can be formed by pressing or molding. The drug can also be used in the form of gelatin capsules, with the following ratio of components:

- 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene from 2 to 10%;

- filler(s) or carrier(s) - the rest.

The drug can be made in the form of solutions, emulsions, suspensions, etc., with an active ingredient content of 0.025 to 1%; the rest is filler(s) or carrier(s).

The present invention can be used as a means for correcting spermatogenesis disorders caused by cytotoxic effects. The proposed tool provides an increase in the processes of reparative regeneration of testicular tissue while reducing the cell population of rapidly renewing and reserve (stem) spermatogonia.

The essence of the invention is illustrated by examples of a specific implementation, in which:

"Complex" - clathrate complex of β-cyclodextrin with 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene in a ratio of 10:1, obtained in a standard way /patent RU2451680, C07D 345 /00, publ. May 27, 2012/.

Strengthening the processes of reparative regeneration of testicular tissue with a reduction in the cell population of rapidly renewing and reserve (stem) spermatogonia in male Wistar rats under the influence of the proposed agents for correcting spermatogenesis disorders caused by cytotoxic exposure was demonstrated in two series of experiments. In the present invention, unless otherwise indicated, the concentrations are in wt.%.

EXAMPLE. The clathrate complex of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene with β-cyclodextrin (“Complex”) as a means for correcting spermatogenesis was evaluated in experiments on Wistar rats with cytotoxic exposure to Paclitaxel.

The experiments were carried out on 50 male Wistar rats, 3 months old. Animals were kept in standard plastic cages (no more than 10 individuals in each) with small wood shavings, on a standard diet - granular food PK 120-3 (according to order No. 1179 of the USSR Ministry of Health dated 10/10/83).

Prior to the experiment, the animals were quarantined in the vivarium for 5 days. Next, the body weight of the animals and their distribution into groups were determined. "Paclitaxel" was administered to 30 male rats of the experimental groups once intravenously at the maximum allowable dose (MTD) - 5.0 mg/kg of body weight. The dose of "Paclitaxel" was chosen taking into account the experience of clinical use and was calculated for animals by the method of graphical probit analysis when observing them for 30 days.

1 month after the administration of Paclitaxel, a clathrate complex of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene with β-cyclodextrin was administered intragastrically for 10 days in the following doses : 0.3 and 1.0 mg/kg body weight. Another group of animals was injected subcutaneously for 5 days with the comparison drug "Neupogen" at a dose of 100 μg/kg 1 month after the administration of "Paclitaxel". Animals of the control group received "Paclitaxel" and distilled water in an equivalent volume. After 2 and 3 months after the administration of Paclitaxel, the body weight of rats was determined, then they were killed by dislocation of the cervical vertebrae (5 animals for each period of the experiment), the testes and epididymis were isolated. Next, we determined the total number of germ cells per epididymis, the percentage of their mobile forms [Guidelines for experimental (preclinical) study of new pharmacological substances. / Ed. Khabrieva R.U. - M.: Medicine, 2005]. When calculating the total number of spermatozoa (ACS) per epididymis, a homogenized cell suspension of the epididymis was used. The testicles were fixed for histological analysis in Carnoy's fluid. Then paraffin sections were prepared. Deparaffinized sections 5 µm thick were stained with hematoxylin-eosin. On the section of the testis, the following were determined: the number of cell population of spermatogonia, the number of Sertoli and Leydig cells [Sanotsky I.V., Fomenko V.I. Long-term consequences of the influence of chemical compounds on the body. - M.: Nauka, 1979, - 210 p.]. The endocrine activity of the testis was calculated as the ratio of the number of Leydig cells to Sertoli cells. Based on the count of Sertoli cells, the degree of maturity of the spermatogenic layer was calculated [Sukhorukov V.S., Shamshad D.S. Determination of the degree of maturity of the rat spermatogenic layer during its regeneration and in the process of maturation of the intact testis // Archive of Anatomy, Histology and Embryology. - 1989, - T.97, No. 9, - 89 p.].

When choosing the timing of the studies, the duration of spermatogenesis in rats, which is 3 months, was taken into account. The period of 2 months after the administration of the drug corresponds, in terms of the time of manifestation, to the effect on actively renewing spermatogonia, 3 months - on the stem (reserve spermatogonia, which divide when damaged).

"Paclitaxel" was administered intravenously, "Neupogen" - subcutaneously, which corresponds to the methods of their use in the clinic. "Complex" was administered intragastrically, which corresponds to the intended clinical use - ingestion.

The dose and mode of administration of the drug "Neupogen" are based on data obtained in studies on the stimulating effect of the drug on regenerative processes in the testes [Borovskaya T.G., Dygay A.M., Zhdanov V.V., Rumpel O.A., Pakhomova A. .V., Perova A.V., Zyuzkov G.N., Goldberg V.E. - Patent for the invention of the Russian Federation No. 2406527. A method for correcting spermatogenesis disorders caused by cytostatic exposure. - 2010].

Rats were randomized into groups based on their body weight:

1. Intact animals (n=10) - background;

2. Control. Rats (n=10) were administered Paclitaxel once intravenously at the maximum tolerated dose (MTD) - 5.0 mg/kg of body weight and distilled water;

3. Clathrate complex of β-cyclodextrin with 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene (“Complex”). Rats (n=10) were injected intragastrically with the test drug at a dose of 0.3 mg/kg for 10 days, starting 1 month after the administration of "Paclitaxel";

4. Clathrate complex of β-cyclodextrin with 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene (“Complex”). Rats (n=10) were injected intragastrically with the test drug at a dose of 1.0 mg/kg for 10 days, starting 1 month after the administration of "Paclitaxel";

5. Comparator drug. Rats (n=10) were injected subcutaneously with Neupogen (G-CSF) at a dose of 100 μg/kg of body weight once a day for 5 days 1 month after the administration of Paclitaxel.

The experimental data obtained were processed statistically using the nonparametric Wilcoxon-Mann-Whitney test [Gubler E.V. Computational methods for the analysis and recognition of pathological processes. - M.: Medicine, 1978. - S. 72-75].

First series of experiment

Assessment of the state of the reproductive system of male rats 2 months after the administration of "Paclitaxel"

The results of assessing the body weight of rats, the total number of germ cells per epididymis, the percentage of motile forms of mature spermatozoa 2 months after the administration of "Paclitaxel" and 1 month after treatment with the drug "Complex" are presented in Table 1. It was found that the body weight of animals at the beginning experiment and 2 months after the administration of the cytostatic drug in all compared groups did not differ (P>0.05). The data obtained indicate that at the beginning of the experiment, a qualitative randomization of animals by body weight was carried out, and also that during the 2 months of the experiment, all pharmacological effects did not lead to a change in the body weight of the animals.

In intact animals, the total number of germ cells (attributable to the epididymis) and the percentage of their mobile forms were within the age norm (the age of rats is 5-6 months) [Borovskaya T.G., Goldberg V.E., Shemerova Yu.A., Poluektova M.E., Timina E.A., Perova A.V. Influence of topoisomerase activity inhibitors on morphological and functional parameters of rat spermatogenesis // Bull. experimental biol. and honey. - 2005. - App. No. 1. - S. 28-30]. 2 months after the administration of Paclitaxel to male rats, a statistically significant decrease in the total number of cells per epididymis, as well as the percentage of their mobile forms, was noted.

Table 1 - Morphological and functional parameters of mature spermatozoa and testes of rats 2 months after the administration of the cytostatic "Paclitaxel"

Indicators Background Control "Complex" 0.3 mg/kg "Complex" 1.0 mg/kg G-CSF Animal body weight
at the beginning of the experiment (d) 352.6±19.8 352.4±2.58 353.6±18.78 353.4±5.55 354.4±23.06 Animal body weight
at the end of the experiment (d) 445.2±10.7 456.6±9.87 464.8±23.64 445.4±9.33 457.6±40.24 Number of mobile
spermatozoa (%) 84.8±1.69 63.7±1.36 ^# 75.8±4.82 ^* 69.2±3.60 ^# 81.7±3.30 ^* Total
spermatozoa (ACS) (abs. units) 55.0±1.82 43.80±2.22 ^# 51.6±2.87 ^* 52.4±2.42 ^* 76.8±6.16 ^#*

Note: here and below

# - statistical significance of differences compared to background

* - statistical significance of differences compared to control ("Paclitaxel").

The decrease in the total number of germ cells and their motility observed during these periods of study in groups of rats treated with Paclitaxel indicates (taking into account the duration of spermatogenesis in rats) the toxic effect of the drug on semi-stem spermatogonia. Literature data confirm the fact that spermatogonia are the target of Paclitaxel [Sukhacheva T.V., Bogush T.A., Kolomiets O.L. The damaging effect of taxol on mouse spermatogenesis. // Bull. experimental biol. and medical - 2001.- T.132, No. 11. - S. 554-560].

ACS in the groups of rats treated with the drug "Complex" in both studied doses (0.3 and 1.0 mg/kg) increased statistically significantly (by 18-19.6%, respectively) compared with the control ("Paclitaxel"), and reached background values. The introduction of the reference drug (G-CSF) also led to an increase (by 75%) of this indicator compared to the control, exceeding the background values.

The percentage of mobile forms in the group of rats, which after "Paclitaxel" was injected with the drug "Complex" at a dose of 0.3 mg/kg, increased compared with the control ("Paclitaxel") and practically did not differ from the background. With the introduction of the drug "Complex" at a dose of 1.0 mg/kg, the value of this indicator was below the background. The introduction of the reference drug G-CSF led to a significant increase in the percentage of mobile forms in comparison with the control to the level of background values.

Thus, 2 months after the administration of Paclitaxel, testicular insufficiency is detected, the signs of which are oligospermia and asthenospermia. Judging by the timing of manifestation, these disorders are due to the toxic effect on dividing spermatogonia (half-stem cells). The introduction of the drug "Complex" had a positive effect on the state of spermatogenesis in rats exposed to "Paclitaxel", as evidenced by an increase in the productivity of spermatogenesis (an increase in ACS in both doses, an increase in the proportion of mobile forms in the "Complex" group, a dose of 0.3 mg / kg ).

It was established (Table 2) that 2 months after the administration of Paclitaxel, the number of the cell population of spermatogonia was reduced (it was 69.7% of the background values). With the introduction of the drug "Complex" at a dose of 0.3 mg/kg 1 month after the administration of "Paclitaxel", the number of spermatogonia was also statistically significantly reduced compared to the background and practically did not differ from the control values. In rats treated with "Complex" at a dose of 1.0 mg/kg, this indicator was statistically significantly higher (by 17%) compared with the control, although it did not reach the values of intact animals. Similar data were obtained with the introduction of the reference drug.

Determining the number of Leydig cells (testosterone-producing cells) showed that in all experimental groups it was similar to that in intact animals (Table 2).

When calculating the number of microenvironment cells (Sertoli cells), their statistically significant decrease was revealed in the groups of rats exposed to Paclitaxel. In animals treated with the drug "Complex" in both doses used, this indicator increased compared to the control. A statistically significant increase in the number of Sertoli cells compared with the control was also revealed with the introduction of the reference drug. This indicator turned out to be higher than the background values.

When studying the endocrine activity of the testis, which is calculated by the ratio of the number of Leydig cells to Sertoli cells, it was found that it was similar in almost all the studied groups of animals, with the exception of rats treated with Paclitaxel and G-CSF. Animals of this group were characterized by reduced endocrine activity, both in comparison with the control and background. The latter is apparently due to the fact that the number of Sertoli cells significantly increased against the background of the introduction of G-CSF.

Table 2 - Morphological and functional parameters of rat testicles 2 months after the administration of the drug "Paclitaxel"

Indicators Background Control "Complex" 0.3 mg/kg "Complex" 1.0 mg/kg G-CSF Number of spermatogonia (abs. units) 18.03±0.32 12.57±0.46 ^# 13.49±0.51 ^# 14.70±0.36 ^{# *} 16.41±0.10 ^#* Number of Leydig cells (abs. units) 15.75±0.36 15.65±1.24 15.92±0.62 15.84±0.68 15.12±0.58 Number of Sertoli cells (abs. units) 2.75±0.02 2.58±0.06 ^# 2.94±0.07 ^* 2.82±0.08 ^* 4.89±0.18 ^#* Endocrine activity (arb. units) 5.73±0.12 6.10±0.56 5.42±0.22 5.64±0.32 3.11±0.19 ^#* Degree of maturity of the spermatogenic layer 6.07±0.00 5.49±0.01 ^# 5.42±0.01 ^{# *} 5.44±0.01 ^{# *} 5.07±0.03 ^#*

When determining the degree of maturity of the spermatogenic layer, which is the main indicator characterizing the severity of tissue repair processes, it turned out that with the introduction of both one cytostatic drug and all the studied compounds, its values significantly decreased compared to the background. This indicates that in the testes of all animals treated with the cytostatic drug, processes of reparative regeneration of testicular tissue occurred due to the formation of new tubules. However, with the introduction of the drug "Complex" (in both doses used) and the comparison drug 1 month after the administration of "Paclitaxel", the degree of maturity of the spermatogenic layer was lower than the control values (by 10.8%; 10.4% and 23.0%, respectively). . The data obtained indicate that the "Complex" drug, like the reference drug, has the ability to stimulate the processes of reparative regeneration of spermatogenic tissue. Of course, this should lead to an increase in the productivity of spermatogenesis 3 months after the administration of the cytostatic drug. Judging by the percentage of the background, the degree of maturity of the spermatogenic layer in rats treated with G-CSF turned out to be higher compared to that when using the "Complex" preparation. Since this indicator is calculated based on the number of Sertoli cells, the noted effect of G-CSF is the result of a significant increase in their number compared to the background.

Thus, 2 months after the cytostatic effect of "Paclitaxel" on the testicular tissue, in the testes of rats of all groups, tissue regeneration processes were launched, however, in the groups of therapy with the studied drug and the comparison drug, the renewal of testicular tissue, judging by the degree of maturity of the spermatogenic layer, occurred more intensively. The effectiveness of the reference drug turned out to be higher in this indicator than that of the "Complex" (both doses), which, obviously, is due to a significant increase in the number of Sertoli cells. Against the background of the introduction of the “complex” drug (at a dose of 1.0 mg / kg), 1 month after the administration of “Paclitaxel”, there is an increase, as with the use of G-CSF, in the number of spermatogonia, which is apparently a consequence of increased intensity reparative processes.

Second series of experiment

Assessment of the state of the reproductive system of male rats 3 months after exposure to "Paclitaxel"

The results of assessing the body weight of rats, the number of total germ cells per epididymis, the percentage of motile forms of mature spermatozoa 3 months after the administration of "Paclitaxel" and 2 months after treatment with the drug "Complex" are presented in Table 3. It was found that the body weight of animals at the beginning experiment and 3 months after the administration of the cytostatic drug in all compared groups did not differ (P>0.05). The data obtained indicate that at the beginning of the experiment, a qualitative randomization of animals by body weight was carried out, and also that during the 3rd month of the experiment, all the pharmacological effects provided did not lead to a significant decrease in the body weight of the animals.

In rats treated with Paclitaxel, ACS attributable to the epididymis remained statistically significantly reduced (57% of baseline values). This indicates a pronounced insufficiency of spermatogenesis at a time corresponding to the manifestation of the effect on stem spermatogonia. The values of ACS in the group of rats treated with the drug "Complex" in both studied doses (0.3 and 1.0 mg/kg) 1 month after the administration of "Paclitaxel" were higher, respectively, by 96 and 69% compared with the control ("Paclitaxel"). ACS with the introduction of the drug "Complex" at a dose of 1.0 mg/kg did not statistically significantly differ from the background values. The introduction of the reference drug (G-CSF) also led to an increase in this indicator (by 72%), compared with the control, and did not differ from the background indicators. Attention is drawn to the fact that in the group of rats treated with "Complex" at a dose of 0.3 mg/kg, ACS statistically significantly exceeded the background values.

The percentage of mobile forms in the group of rats treated with the drug "Complex" in both studied doses (0.3 and 1.0 mg/kg) 1 month after the administration of "Paclitaxel" was at the level of control values. The introduction of the comparator drug (G-CSF) led to a statistically significant increase in the percentage of mobile forms compared to the control, reaching background values. The data obtained indicate that the drug "Complex" led to an increase in the productivity of spermatogenesis, the effectiveness of which, judging by the ACS, was higher than that of the reference drug. In terms of sperm motility, however, the comparison drug, G-CSF, proved to be more effective.

Table 3 - Morphological and functional parameters of mature rat spermatozoa 3 months after the administration of the cytostatic "Paclitaxel"

Indicators Background Control "Complex" 0.3 mg/kg "Complex" 1.0 mg/kg G-CSF Animal weight
at the beginning of the experiment (d) 352.2±14.87 352.6±4.06 352.8±14.12 352.6±13.94 352.2±21.36 Animal weight
at the end of the experiment (d) 488.0±17.47 490.0±10.17 491.0±5.55 490.2±6.18 489.8±8.67 Number of mobile
spermatozoa (%) 84.0±1.41 59.7±1.78 ^# 66.3±3.73 ^# 64.3±2.20 ^# 78.0±2.55 ^* Total
spermatozoa (abs. units) 77.0±5.17 44.0±2.07 ^# 86.2±3.17 ^*# 74.3±2.75 ^* 75.6±2.11 ^*

Three months after the administration of Paclitaxel, the spermatogenic epithelium of the animals looked sparse compared to that of intact rats. A small number of Leydig cells were located between the tubules.

It was established (Table 4) that 3 months after the administration of Paclitaxel, the number of the cell population of spermatogonia remained reduced and amounted to 67% of the background values. These data indicate that the number of spermatogonia does not increase over time. The source of replenishment of this cell population is stem spermatogonia, which divide when damaged. Given the duration of spermatogenesis, the period of manifestation of their damage is 3 months. In this regard, the fact obtained indicates a reduction in the number of stem spermatogonia under the action of Paclitaxel.

3 months after the start of the experiment, in the groups of "Complex" administration, the spermatogenic epithelium looked less sparse than in the control group (the group of administration of one cytostatic drug). Between the tubules, Leydig cells were clearly detected, in an amount close to normal.

With the introduction of the drug "Complex" at a dose of 0.3 mg/kg, the number of spermatogonia was statistically significantly reduced compared to the background and did not differ from the control values. In rats treated with "Complex" at a dose of 1.0 mg/kg, this indicator was statistically significantly higher than the control values (by 22%). Similar data were obtained in the comparator group.

When counting testosterone-producing cells (Leydig cells), it was found that during this period of observation, their number in the control turned out to be reduced compared to the background (Table 4), which may be the result of the toxic effect of the drug on gonadotropic pituitary cells. In rats that were injected with "Complex" (in both doses) and G-CSF, the number of Leydig cells did not differ from the background values. The value of this indicator in rats treated with the drug "Complex" exceeded that in the control.

When calculating the number of microenvironment cells (Sertoli cells), their statistically significant decrease was revealed in the group of rats treated with Paclitaxel. In animals treated with "Complex" in both doses used, as well as the reference drug, this indicator exceeded the control values. With the introduction of the drug "Complex" in both doses, the number of Sertoli cells exceeded the background values.

When determining the degree of maturity of the spermatogenic layer, it was found that with the introduction of a cytostatic drug, the values of this parameter statistically significantly decreased compared to the background. In the group of rats that received the studied corrective preparations, this indicator was also below the background values. The noted facts indicate that in the testes of the animals of the experimental groups, the processes of reparative regeneration of testicular tissue took place. It is noteworthy that with the introduction of the drug "Complex" (in both doses used), as well as the comparison drug, the degree of maturity of the spermatogenic layer turned out to be lower than the control values (by 13, 14 and 10%, respectively), which may indicate that the drug " Complex”, as well as the reference drug, has the ability to stimulate the formation of stem spermatogonia. It should be noted that the lowest values of this indicator were found in the group of rats treated with "Complex" at a dose of 1.0 mg/kg.

Table 4 - Morphological and functional parameters of rat testicles 3 months after exposure to "Paclitaxel"

Indicators Background Control "Complex" 0.3 mg/kg "Complex" 1.0 mg/kg G-CSF Number of spermatogonia (abs. units) 18.44±0.27 12.35±0.17 ^# 13.18±0.36 ^# 15.02±0.44 ^#* 16.72±0.14 ^{# *} Number of Leydig cells (abs. units) 15.45±0.53 12.68±0.55 ^# 15.51±0.50 ^* 14.47±0.72 ^* 14.45±0.92 Number of Sertoli cells (abs. units) 2.51±0.18 2.10±0.09 ^# 3.46±0.05 ^*# 3.70±0.23 ^*# 2.53±0.13 ^* Endocrine activity (arb. units) 7.16±0.78 7.06±0.34 4.65±0.08 ^#* 4.16±0.38 ^#* 6.23±0.41 ^* Degree of maturity of the spermatogenic layer 6.12±0.04 5.57±0.02 ^# 5.33±0.01 ^*# 5.28±0.04 ^*# 5.49±0.02 ^{# *}

The introduction of the cytostatic drug "Paclitaxel" simulates testicular insufficiency in the testes of rats, due to the depletion of the spermatogonia population (a period of 2 months - insufficiency of rapidly renewing spermatogonia, a period of 3 months - insufficiency of stem spermatogonia). Testicular insufficiency is indicated by a decrease in the productivity of spermatogenesis (indicator - ACS). The proof that this is due to the depletion of the sources of the proliferative pool of spermatogenesis is the decrease in the number of spermatogonia 2 and 3 months after the cytostatic effect. Given that stem cells replenish the number of spermatogonia in case of damage, the data obtained indicate a partial death of stem spermatogonia under the action of Paclitaxel, which is confirmed by the literature data [Sukhacheva T.V., Bogush T.A., Kolomiets O.L. The damaging effect of taxol on mouse spermatogenesis. // Bull. experimental biol. and medical - 2001.- T.132, No. 11. - S. 554-560].

The formation of new spermatogonia in the convoluted seminiferous tubule is limited by the number of microenvironment cells that do not multiply in mature tubules. Reparative regeneration of spermatogenic tissue proceeds only through the formation of new convoluted seminiferous tubules from the regenerative zone containing Sertoli cells and primary sexual determinants, which subsequently become spermatogonia. The newly formed seminiferous tubules are characterized by an increased content of Sertoli cells, which does not limit the reproduction of spermatogonia. The criterion for the effectiveness of reparative tissue regeneration is, first of all, the degree of maturity of the spermatogenic layer.

The results of the studies showed that the degree of maturity of the spermatogenic layer against the background of the administration of Paclitaxel alone decreases compared to the background values, both 2 and 3 months after the administration of the cytostatic drug. Thus, after cytostatic damage in the testicular tissue, natural processes of reparative regeneration take place. Against the background of the introduction of the drug "Complex" (in both doses), this indicator also decreased, but to a greater extent, which indicates more intensive processes of reparative regeneration. An effect similar in severity was observed with the introduction of the reference drug. The lowest values of this indicator were detected 2 months after the administration of Paclitaxel in the reference drug group, and 3 months after the administration of Paclitaxel in the Complex group at a dose of 1.0 mg/kg.

In the spermatogenic layer of a lower degree of maturity, there are more cells of the microenvironment. This is confirmed by the results obtained: after 2 months, their number was higher against the background of the introduction of G-CSF, after 3 months - against the background of the administration of the drug "Complex" (in both doses), where this parameter was higher than that of the reference drug G-CSF and above background values. In rats treated with G-CSF, the number of Sertoli cells did not exceed the background values.

The growth of Sertoli cells does not limit the reproduction of spermatogonia. Their number on the background of the introduction of the drug "Complex" in both doses increased (a period of 2 months - compared with the control; a period of -3 months - compared with the control and the background). The reference drug led to a significant increase in this indicator compared to the background only 2 months after the administration of Paclitaxel.

An increase in the number of spermatogonia should lead to an increase in the productivity of spermatogenesis (an increase in the total number of spermatozoa). And indeed, this indicator in the experimental groups increased compared to the control: against the background of the administration of the preparation "Complex" (for a period of 2 and 3 months - doses of 0.3 and 1.0 mg/kg); against the background of the introduction of G-CSF, an increase in the productivity of spermatogenesis was also noted (for a period of 2 and 3 months). 2 months after the start of the experiment, the effectiveness of the reference drug in this indicator was higher. The greatest increase in ACS (above the background level) was detected with the introduction of the drug "Complex" at a dose of 0.3 mg/kg 3 months after the start of the experiment. This may be due not only to the stimulation of reparative regeneration processes, but also to the normalization of the number of testosterone-synthesizing cells (in this group, their number practically did not differ from the background values). Literature data indicate the extreme resistance of Leydig cells to pharmacological effects [Sukhorukov V.S., Shamshad D.S. Determination of the degree of maturity of the rat spermatogenic layer during its regeneration and in the process of maturation of the intact testicle. Archives of Anatomy, Histology and Embryology. - 1989. - T.97, No. 9. - 89 p.]. Meanwhile, their role in the hormonal regulation of spermatogenesis is undeniable.

On the model of testicular insufficiency associated with the depletion of the proliferative pool of spermatogenesis (semi- and stem spermatogonia), caused by the cytotoxic effect of "Paclitaxel", it was found that the drug "Complex" (in both doses) has the ability to correct violations of spermatogenesis, by enhancing the processes of reparative regeneration of the testicular fabrics. The most pronounced therapeutic effect on stem spermatogonia was found in the drug at a dose of 1.0 mg/kg. The effectiveness of the reference drug, as a stimulator of the formation of stem spermatogonia, was comparable to that of the "Complex" drug. At the same time, under the influence of the "Complex" preparation, a greater number of Sertoli cells was revealed than with the introduction of G-CSF. This fact indicates a greater regenerative potential of the testes against the background of the administration of the preparation "Complex" than G-CSF.

The conducted studies revealed the ability of the "Complex" drug to normalize the level of testosterone-synthesizing cells, which was not detected when the reference drug was administered. This may also be one of the benefits of the drug being tested. The drug "Complex" (dose 0.3 mg/kg) stimulated spermatogenesis to a greater extent than the reference drug, as evidenced by the productivity of spermatogenesis 3 months after the start of the experiment.

Thus, the corrective effect of the "Complex" on the male sex glands in case of cytotoxic damage is due to the stimulation of the mechanisms of reparative regeneration of the testicular tissue as a whole, leading to the formation of new seminiferous tubules containing the necessary amount of spermatogonia to ensure a normal level of spermatogenesis.

The present invention allows for effective correction of spermatogenesis disorders caused by cytotoxic exposure using the "Complex" by stimulating the reparative processes of the testicular tissue as a whole, leading to the formation of new sources of the proliferative pool of spermatogenesis.

The proposed drug expands the arsenal of tools intended in andrology for the correction of spermatogenesis disorders caused by cytotoxic effects, by enhancing the processes of reparative regeneration of testicular tissue.

Examples of solid dosage forms include, for example, tablets, pills, gelatin capsules, etc.

Examples of liquid dosage forms for injection and parenteral administration include solutions, emulsions, suspensions, etc. Compositions are generally prepared by standard procedures involving mixing the active compound with a liquid or finely divided solid carrier.

The finished medicinal product according to the invention in the form of tablets contains from 2 to 10% of the active compound and the excipient(s) or carrier(s). The following are used as such for tablets: a) diluents: beet sugar, lactose, glucose, sodium chloride, sorbitol, mannitol, glycol, dibasic calcium phosphate; b) binders: magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, carboxymethylcellulose and polyvinyl pyrrole and don; c) disintegrants: dextrose, agar, alginic acid or its salts, starch, tween.

EXAMPLE 1.1

100 mg tablets containing 2 mg of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 2 mg

Lactose - 53.0 mg

Alginic acid - 20.0 mg

Citric acid - 5.0 mg

Tragacanth - 20.0 mg

EXAMPLE 1.2

100 mg tablets containing 5 mg of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 5 mg

Lactose - 50.0 mg

Alginic acid - 20.0 mg.

Citric acid - 5.0 mg

Tragacanth - 20.0 mg.

EXAMPLE 1.3

100 mg tablets containing 10 mg of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 10 mg

Lactose - 45.0 mg

Alginic acid - 20.0 mg

Citric acid - 5.0 mg

Tragacanth - 20.0 mg.

A tablet may be formed by compressing or molding the active ingredient with one or more additional ingredients. Obtaining pressed tablets is carried out on a special installation. The active ingredient in free form, such as powder or granules, in quantities of 20 g or 50 g or 100 g (the amount of substance required to obtain 10,000 tablets) is mixed with a binder - tragacanth (200 g), mixed with a diluent - lactose ( 530 g or 500 g or 450 g), a leavening agent - alginic acid (200 g) and a fragrance - citric acid (50 g) are added to the mixture.

For gelatin capsules, additional colors and stabilizers are usually used. As dyes are used: tetrazine, indigo; as stabilizers can be presented: sodium metabisulphite, sodium benzoate. The proposed gelatin capsules contain from 2 to 10% of the active ingredient.

EXAMPLE 2.1

1 capsule 2 mg contains:

Active substance:

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 2 mg in terms of anhydrous substance

Excipients:

Betadex (β-cyclodextrin) up to 190 mg content weight

Hard gelatin capsules:

Gelatin

Titanium dioxide (E171).

EXAMPLE 2.2

1 capsule 5 mg contains:

Active substance:

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 5 mg in terms of anhydrous substance

Excipients:

Betadex (β-cyclodextrin) up to 190 mg content weight

Hard gelatin capsules:

Gelatin

Titanium dioxide (E171).

EXAMPLE 2.3

1 capsule 10 mg contains:

Active substance:

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 10 mg in terms of anhydrous substance

Excipients:

Betadex (β-cyclodextrin) up to 190 mg content weight

Hard gelatin capsules:

Gelatin

Titanium dioxide (E171).

The active ingredient in free form, such as powder or granules, in quantities of 20 g or 50 g or 100 g (the amount of substance required to obtain 10,000 capsules) is mixed with the substance β-cyclodextrin (Betadex) (1880 g or 1850 g or 1800 G).

Injectable forms of the composition are preferably isotonic and oily solutions, suspensions and contain from 0.025 to 0.1% of the active ingredient. The above forms can be sterilized and contain additives such as preservatives: sodium metabisulphite, benzoic acid, sodium benzoate, a mixture of methylparaben and propylparaben; stabilizers: apricot and gum arabic, dextrin, starch paste, methylcellulose, tween; salts that regulate osmotic pressure (sodium chloride), or buffers. In addition, they may contain other therapeutically useful substances.

EXAMPLE 3.1

2 ml ampoules containing 0.5 mg of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 0.5 mg

Hydrogenated soybean oil - 2 ml.

EXAMPLE 3.2

2 ml ampoules containing 1.0 mg of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 1.0 mg

Hydrogenated soybean oil - 2 ml.

EXAMPLE 3.3

2 ml ampoules containing 2.0 mg of 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene

9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene - 2.0 mg

Hydrogenated soybean oil - 2 ml.

For the preparation of injectable forms, the active compound (0.5 g or 1 g or 2 g; quantities needed to make 1000 ampoules) is finely ground and mixed in a blender with hydrogenated soybean oil (2000 ml). The resulting solution is packed in ampoules of 2 ml and steam sterilized for 30 minutes.

The dose of the active ingredient prescribed for administration (9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene of formula 1 or its mixture with β-cyclodextrin or clathrate complex with β-cyclodextrin) varies depending on many factors, such as age, sex, body weight of the patient, symptoms and severity of the disease, the specific compound administered, the method of administration, the form of preparation in which the active compound is administered. Usually, the total prescribed dose is from 1 to 20 mg per day. The total dose may be divided into several doses, for example, for administration from 1 to 4 times a day. For oral administration, the range of total doses of the active substance is from 1 to 20 mg per day, preferably from 1 to 10 mg. For parenteral administration, the dosage range is 0.5 to 5 mg per day, preferably 0.5 to 2 mg. An effective therapeutic dose of the drug can be selected by the attending physician.

Claims (1)

The use of the 9-phenyl-2,3,4,5,6,7,8,9-octahydro-1H-selenoxanthene clathrate complex with β-cyclodextrin as a means for correcting spermatogenesis disorders at a mass ratio of 9-phenyl-2.3, 4,5,6,7,8,9-octahydro-1H-selenoxanthene to β-cyclodextrin from 1:1 to 1:10.