RU2318510C1 - 3,3'-diindolylmethane (metindol)-base vaginal suppository - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates to suppository used in treatment of genital condylomae and neoplastic diseases of reproductive organs. Agent for treatment of genital condylomae and neoplastic diseases of reproductive organs as a suppository comprises metindol (3,3'-diindolylmethane) as an active component, lipophilic base containing solid confectionary fat, polyvinylpyrrolidone and butylhydroxyanisole taken in the definite ratio. Proposed agent promotes to effective treatment of genital condylomae and neoplastic diseases of reproductive organs.

EFFECT: valuable medicinal properties of suppository.

2 cl, 4 dwg, 1 tbl, 5 ex

Description

The invention relates to the field of medicine and the pharmaceutical industry, and in particular relates to agents for the treatment of genital warts and neoplastic diseases of the reproductive organs.

Human papillomavirus infection (PVI) is the most common sexually transmitted infection. According to statistics, more than 50% of the sexually active population is infected with human papillomavirus (HPV) during their lifetime. The biological and medical interest in papillomaviruses is explained by the fact that they are tumorigenic, capable of causing benign hyperplasia, and some of their types can initiate the development of malignant tumors of the anogenital region: cervical, vulvar, vaginal and other cancers. Cervical intraepithelial neoplasias are also considered to be a consequence of HPV infection. (CIN) related to precancerous conditions. HPV DNA of a high degree of oncogenic risk (mainly of types 16 and 18) is found in 50-80% of samples of moderate and severe cervical squamous dysplasia and in 90% of invasive cancer.

Cervical cancer (cervical cancer) is the second most common cancer among women. About half a million new cases of the disease and 270,000 deaths caused by it are registered annually in the world. The standardized incidence and mortality rate for cervical cancer is 16.2 and 9 per 100,000 population, respectively. However, approximately 52% of all cases of cervical cancer occur in Asia. In Russia, cervical cancer (4.8% of all malignant tumors in women) annually dies more than 6 thousand patients (in women aged 20 to 40, cervical cancer is the main cause of death), the incidence rate is approximately twice as high.

An equally important medical problem is the benign manifestations of PVI, which include exophytic and endophytic lesions of various parts of the epithelium of the vulva, vagina and cervix, as well as the surrounding skin and mucous membranes, including the anus. Exophytic forms (genital warts) include genital, papillary and papule-shaped formations that are diverse in appearance and size, often complicated by the addition of a secondary bacterial infection and accompanied by not only psychological, but also physical suffering of the patient.

The frequency of genital warts (genital warts) resulting from infection with benign HPV types, according to the Ministry of Health of the Russian Federation (2001), is 26 per 100,000 population. Endophytic forms (subclinical PVI) are various morphological changes in the squamous epithelium without external growths.

Genital warts are also the cause of recurrent respiratory papillomatosis in children, which is due to the vertical transmission of the virus from the infected mother to the child (Minkina G.N., Manukhin I.B., Frank G.A., Cervical precancer, Moscow, "Airbrush-Media ", 2001).

The main methods of treating genital warts and precancerous neoplastic diseases of the reproductive organs caused by HPV are currently chemical destruction, surgical excision or ablation. These methods are often accompanied by complications, require the use of expensive medical equipment and are not always effective. At the same time, a huge number of young women with an unexpressed clinical picture of vaginal condylomatosis who are not subject to destructive treatment are carriers of infection and pose a danger to sexual partners and the unborn child.

When epithelial tissues are infected, the papillomavirus uses a whole set of mechanisms that subordinate the vital activity of the infected cell to its interests. It is known that HPV DNA encodes the synthesis of two proteins E6 and E7, inducing the transition of differentiated cells to the S phase of the cell cycle. At the stage of active reproduction of the virus, expression of the E6 and E7 genes is regulated by the protein product of the E2 gene, a repressor of the transcription of these genes. That is why, while the virus is in an episomal state, benign processes of growth of infected tissues take place. A key event in the malignancy of cells is the integration of the virus into the genome of the host cell, which is accompanied by a deletion of the E2 gene.

Cell cycle and differentiation are monitored by means of oncogenic proteins E6 and E7, which inactivate the “key” proteins that regulate cell proliferative activity — the proapoptotic p53 protein and retinoblastoma protein (pRB). It was found that the E7 protein is able to form a stable complex with the pRB protein, causing its degradation, which leads to the release of the transcription factor E2F, which stimulates the transcription of genes necessary for DNA replication in the S phase of the cell cycle. In addition, E7 affects the activity of a number of other cell cycle regulator proteins, such as A and E cyclins, cdk2 kinase, and cyclin-dependent kinase inhibitors p21 and p27.

It is known that in the pathogenesis of a viral infection, leading to an increase in the proliferative activity of infected cells of a benign nature (and sometimes with their malignant degeneration), the endocrine system plays a significant role, in particular the level and activity of female sex hormones - estrogens (Bulletin of Dermatology and Venereology, 2000 , No. 6, p. 20-23). Moreover, the relative constancy of proliferative cell activity in estrogen-sensitive tissues is controlled by special metabolic mechanisms that convert estradiol (the most active estrogen) to its biologically active derivatives. The main pool of endogenous estrogen is utilized through the P-450 monooxygenase cytochrome system localized in the liver, which catalyzes the formation of its hydroxy derivatives, which facilitates their solubility and subsequent excretion from the body through the kidneys and biliary tract. The enzymatic system of cytochromes P-450 provides the conversion of estradiol into two main metabolites: 16α-hydroxyestron (16α-HEE1) and 2-hydroxyestrone (2-OHE1) (Fig. 1).

16α-OHE1 belongs to the category of “aggressive” hormones causing a long-term carcinogenic effect (Proc. Natl. Acad. Sci. USA, 1982, v. 79, p.3047-3051). It was shown that this effect is due to the formation of strong covalent bonds of 16α-OHE1, an estrogen agonist, with nuclear estrogen receptors. 2-OHE1 has moderate functions and, in contrast to 16α-OHE1, on the contrary, normalizes cell growth. With an increase in the level of 2-OHE1, a tendency is observed for the death of tumor cells and the prevention of their further formation.

A study of the functions of these two metabolites revealed a unique relationship between the level of 16α-OHE1 and the risk of developing tumors in estrogen-dependent tissues and concluded that the ratio of 2-OHE1 to 16α-OHE1 is both a universal biomarker and a reliable diagnostic criterion for determining the risk and prognosis of estrogen development -dependent tumors.

It has long been noted that tissue changes in the cervical canal caused by HPV are localized mainly in estrogen-sensitive areas. Moreover, it was found that where active expression of HPV proteins is observed, a high level of synthesis (16α-OHE1) is observed, comparable to that in breast cancer cells.

It should be emphasized that normal cervical epithelial cells are not able to ensure the conversion of estradiol to 16α-hydroxyestrone. Thus, active reproduction of HPV induces the formation of an “aggressive” metabolite in infected cells (Kiselev V.I., Kiselev O.I. Human papillomaviruses in the development of cervical cancer. St. Petersburg. - M.: Rosa Mira, 2003).

At the same time, it was shown that HPV-infected human keratinocytes exhibit proliferative activity in vitro, but do not have a tumor phenotype under microscopic examination. Adding exogenous 16α-OHE1 to the culture medium transforms the cells into typically cancerous (Int. J. Cancer, 1991, v. 49, p.867-869).

Thus, a vicious circle is formed in which the virus, through the formation of an “aggressive” form of estradiol, creates favorable conditions for tumor development, stimulating the synthesis of E7 oncoprotein. In turn, the oncoprotein E7, on the one hand, activates the mechanisms of pathological cell proliferation, and on the other hand, blocks the antiviral immunological defense (Fig. 2).

Summarizing the above, it can be concluded that infection of HPV epithelial cells is a necessary but insufficient factor for their malignancy. The formation of irreversible neoplasia requires: 1) the induction of metabolic mechanisms for the conversion of estradiol to 16α-OHE1, which plays a key role in the cancerous degeneration of HPV-infected cells; 2) active expression of the oncogenes E6 and E7 of the virus, stimulated by the interaction of the hormone-receptor complex with the HPV DNA promoter; 3) the induction of multiple damage to chromosomal DNA in an infected cell, completing the process of tumor transformation.

In connection with the advent of the viral concept of cervical carcinogenesis, approaches to the treatment of cervical dysplastic processes have fundamentally changed. Currently, surgical methods (due to the high percentage of relapses after their use) have begun to play a secondary role. Etiopathogenetic therapy has come to the first place, having two main directions: 1) effect on the etiological factor - HPV; 2) blocking the basic mechanisms of carcinogenesis.

Unfortunately, there are currently no drugs that selectively affect HPV. Interferon (IFN), a protein produced by cells of the immune system in response to stimulation with viral antigens, is most commonly used to treat HPV infection. However, in most cases, even prolonged IFN-tolerance does not lead to clinical improvement. It was shown that the resistance to the action of IFN of HPV-infected cervical cells is determined by the increased expression level of E7 oncoprotein, which intracellularly inactivates the regulation factor of IFN, which includes transcription of genes encoding the synthesis of antiviral proteins.

Over the years, searches have been conducted for natural and synthetic antitumor compounds that can stop the development of precancerous conditions of the cervix. Finally, these searches were successful. Recently, a chemical compound with anticarcinogenic properties has been identified - indole-3-carbinol (I3C) - a phytonutrient, an isoflavonoid found in cruciferous vegetables. Numerous literature data indicate that prolonged use of this compound prevents the development of tumors of the intestine, lungs, and organs of the female reproductive system. I3C stimulates the antitumor effect of many drugs, reduces the mutagenic activity of carcinogens.

Most of the studies on I3C relate to its antitumor activity in the so-called estrogen-dependent organs and tissues (mammary glands, endometrium and cervix), which are characterized by a cyclic change in the level of cell proliferative activity. The uniqueness of the action of I3C lies in the fact that it intervenes in both of these pathways, disrupting their functioning and thus inhibiting cell proliferation. On the one hand, it has a pronounced antiestrogenic effect, stimulating the formation of antiproliferative 2-hydroxyestrone and thus improving the ratio of 2-OHE1 / 16α-OHE1 in favor of the former, and on the other hand, it prevents phosphorylation of cytoplasmic proteins - participants of cascade transmission induced by EGF. Another important mechanism of the antitumor effect of I3C is its ability to induce apoptosis - the "programmed death" of tumor cells through the bax-bcl system.

Cervical cancer associated with HPV has also been investigated as a potential target for I3C therapy. The first promising results were obtained in 1999 by a group of American researchers on a model of transgenic mice containing an integrated form of HPV-16 in the genome. It was previously established that the content of these mice on a diet that includes 17-α-estradiol leads to the development of cervical cancer in them (J. Cell. Biochem., 2000, 34 (Suppl.): 103-114). In further studies, it was possible to reliably confirm the diversity of I3C antitumor activities in HPV-transformed cells of the cervical epithelium. It was shown that in vitro and in vivo:

1) I3C removes the estradiol-dependent induction of the E7 oncogen, thus sharply reducing the expression level of the E7 oncoprotein and inhibiting the hormone-dependent proliferation of infected cells;

2) I3C normalizes the metabolism of estradiol in cells infected with HPV, preventing the formation of a carcinogenic metabolite 16α-OHE1, stimulating the expression of HPV oncogenes;

3) I3C induces apoptotic processes of HPV-infected cells, causing selective death of cells with tumor properties (J. Nutrit, 2001, 131, 3294-3302).

The antitumor activity of I3C as a means of preventing and treating cervical cancer has been confirmed in recent placebo-controlled clinical trials (Gynecol. Oncol, 78, 123-129, 2000). In patients with CIN II and III degree who daily took 200-400 mg of I3C (experimental group), complete tumor regression was observed in 47% of cases, while in the control group there was not a single case of regression.

When discussing the antitumor and antiviral activity of I3C, it should be borne in mind that this compound is extremely unstable and easily undergoes oligomerization, which is amplified many times in an acidic environment. The main oligomeric product of I3C is its dimeric form - 3,3'-diindolylmethane (DIM). As shown by pharmacokinetic studies, under the influence of the acidic environment of the stomach, oral I3C taken almost instantly turns into DIM (Ameson DW, Hurwitz A, McMahon LM, Robaugh D: Presence of 3,3'-diindolylmethane in human plasma after oral administration of indole-3- carbinol (abstr.) Proc. Am. Assoc. Cancer Res, 40, 2833, 1999).

It has been experimentally proven that almost all of the multiple antitumor mechanisms induced by I3C in vitro and in vivo are also characteristic of DIM.

One recent experimental study showed the ability of DIM to induce apoptosis of cervical HPV-infected human keratinocytes in vitro. Moreover, on one of the three studied cell lines of cervical cancer, DIM was several times more effective than I3C (value (LD50) was 50-60 μM for DIM and 200 μM for I3C, respectively), but the same as its metabolic precursor (I3C), did not cause apoptotic changes in normal (non-transformed) keratinocytes (J. Nutrit, 2001, 131, 3294-3302).

Several years ago, using the method of microarray analysis, it was found that the similarity of the antitumor and antiviral activity of I3C and DIM is also manifested at the submolecular (genetic) level. In cervical cancer cell lines, DIM, like I3C, constitutively altered the transcription of more than 100 genes, most of which directly or indirectly control cell proliferation (apoptosis), as well as the expression of HPV oncogenes in virus-infected cells (J. Nutr. 132, 3314 -3324, 2002).

In conclusion, it is necessary to mention another, recently described, most important property of DIM - its immunomodulatory activity. In vitro in tumor cells, DIM has been shown to stimulate IFNα-dependent signaling cascades by activating expression of IFNα receptors as well as other IFN-responsive regulatory proteins (Cells. Mol. Pharmacol, 2006, 69, 430-439). Given the high antiviral and antitumor activity of IFNα, this property can be of great clinical importance when using DIM for the treatment of cervical dysplasia due to HPV.

Summarizing all of the above, we can conclude that the drug based on DIM should have high antitumor and antiviral activity against HPV-infected cells.

However, although methindole is a more stable compound than indole-3-carbinol, it has low bioavailability, which may limit its use as a medicine.

To increase solubility and bioavailability, various dosage forms of DIM have been developed. However, they also have several disadvantages. Therefore, the search for new, more effective formulations is still relevant.

Known vaginal suppositories DIM for the treatment of leishmaniasis obtained by heating cetostearyl alcohol with the active substance, as well as with the addition of ceramides or their derivatives, followed by the introduction of the mixture with a triglyceride base and molding candles (WO 2005/107747, 2005-11-17).

Known patent US 6689387, 02/10/2004, describing various forms of diindolylmethane for the treatment of mastalgia and endometriosis. In particular, it is noted that it is possible to use in the form of suppositories with traditional binders and a carrier such as triglycerides.

Known suppositories do not provide the prolonged action of the active substance necessary for the treatment of human papillomavirus infection. In addition, the treatment of neoplastic disorders requires considerable time. At the same time, triglyceride bases with prolonged use cause ulceration of the mucous membranes.

As the closest analogue may be specified patent RU 2196568.

This solution relates to a pharmaceutical composition for the prophylaxis and treatment of tumors associated with human papilloma viruses, namely dysplasia and cancer of the cervix uteri and laryngeal papillomatosis. The invention lies in the fact that a composition based on indole-3-carbinol is proposed as an active principle, possibly in the form of vaginal suppositories, based on Vitepsol. Since the active substance is not sufficiently stable, it cannot provide the necessary treatment effectiveness.

The present invention is the development of a dosage form for more effective treatment of genital candidiasis and neoplastic diseases in the absence of side effects, stable during storage.

The problem is solved by the drug, which is vaginal suppositories (“Cervicon”) containing methindole (3,3'-diindolylmethane - DIM), a lipophilic base containing solid confectionery fat, polyvinylpyrrolidone and butylhydroxyanisole or butylhydroxyhydroxide as the active substance. .%:

Methindole 2.0-6.0 Polyvinylpyrrolidone 0.5-1.2 Butylhydroxyanisole or butylhydroxytoluene 0.3-0.5 Lipophilic base rest

Metindol is contained in an amount of 0.045-0.110 g per dose.

As a lipophilic base, it is preferable to use solid confectionery fat, such as Type A or B solid fat, but it is also possible to introduce additional emulsifiers, phthalic acid esters and alcohols, hydrogenated vegetable oils, waxes, paraffin, lanolin, cooking oil, cocoa butter.

It is known that solid confectionery fats have a fine-grained structure that melts in a narrow range, have good ductility and mix easily with many substances. The confectionery fats produced are cooked on a palm-kernel basis and on the basis of salomas.

In addition, it was found that when using this base with PVP and an antioxidant, there was practically no undesirable effect on the mucosa, as in the case of bases consisting mainly of triglycerides, such as vitepsol, estarinum, which are widely used in pharmaceutical practice.

Butylhydroxyanisole and butylhydroxytoluene are synthetic products related to phenol derivatives. They have an effective antioxidant effect and are known preservatives. Their introduction into a suppository mass gives an additional effect of exposure to the affected mucosa.

As polyvinylpyrrolidone, it is preferable to use low-molecular-weight PVP, for example, the Kollidon CL, CL-M- brands with a molecular weight of 12,600 ± 2,700 or Povidone with a mass of 8,000 ± 2,000.

A method of preparing suppositories is that the components of the lipophilic base are melted, part of it is separately triturated with the active substance to obtain a homogeneous suspension, polyvinylpyrrolidone and an antioxidant are added to the cooled remaining part of the base, and then the resulting suspension is mixed until homogeneous and the candles are formed by casting into molds given sizes.

The invention is illustrated by the following graphic materials:

Figure 1 reflects the metabolism of estradiol.

Figure 2 reflects the role of estrogen in the carcinogenesis of cervical epithelial cells infected with HPV.

Figure 3 shows the transcription of the E7 gene in CaSki cells in the presence of estradiol (Fig. 3A - 1 - before adding estradiol, 2 - incubation for 4 hours, 3 - 24 hours, 4 - 48 hours) and in the presence of estradiol + Cervicon ( Fig.3B - 1 - before adding estradiol + Cervicon, 2 - incubation for 4 hours, 3 - 24 hours, 4 - 48 hours).

Figure 4 shows the induction of apoptosis in the presence of Cervicon (Figure 4A - chromatin condensation is visible in the cells, indicating cell death).

The invention is illustrated by the following examples.

Example 1

Vaginal suppositories containing

diindolylmethane 0.05 g 3,3'-Diindolylmethane 0.05 g Solid fat type “A” 2.02 g Collidon CL 0.02 g Butylhydroxyanisole 0.01 g

TOTAL 2.1 g

Example 2

Vaginal suppositories containing diindolylmethane 1.0 g 3,3'-Diindolylmethane 0.1 g Solid fat type “A” 1.97 g Collidon CL 0.02 g butylhydroxytoluene 0.01 g

TOTAL 2.1 g

Example 3

Materials and methods.

Experimental model. The CaSki cervical carcinoma cell line containing multiple copies of the 16th type integrated HPV genome, the C33-A cell line (not containing HPV DNA) and cultured epithelial cells obtained from the cervical region in patients diagnosed with HPV-induced dysplasia were used as a model. "

Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. The suppositories of Example 1 were dissolved in an aqueous-alcoholic solution containing 20% ethanol and added to the cells so that the final concentration of the 3,3'-diindolylmethane contained in it was 50 μM.

RNA isolation. The cell suspension was mixed with 0.5 ml of a lysing solution containing 4M guanidinethiocin, 0.2% SDS, 25mM sodium citrate (p7.0), suspended in Vortex for 10 s and lysed in an ice bath for 15 minutes.

After that, 50 μl of 2M sodium acetate (pH 4.0) and 500 μl of phenol were added to the tube, the mixture was vigorously shaken, centrifuged, and the aqueous phase was taken. The procedure was repeated twice and then an equal volume of isopropanol was added to the aqueous phase. After 2 hours of incubation in a refrigerator at 20 ° C, the precipitate was collected, centrifuged and dissolved in 50 μl of TE buffer.

HPV E7 gene transcription analysis of type 16. The transcription of the E7 gene was studied by Northern hybridization. For this, 10 μg of total RNA isolated from the cell suspension was separated by agarose gel containing formaldehyde by electrophoresis and transferred to nitrocellulose filters. A hybridization sample was prepared by amplification of an E7 gene fragment in the presence of labeled dTTP (p32). As the matrix for amplification, the plasmid rTHE716 containing the oncogen E7 HPV type 16 was used.

Mass spectrometric (GC-MS) analysis of estradiol metabolites. Samples were prepared from supernatants of cells treated with estradiol and the claimed composition in concentrations of 1 and 50 μM, respectively. Deuterated estradiol was added to the supernatant (5 ml) as a standard and applied to a Sepa-Pak C 18 column (Water Associates, Milford, MA).

Steroid elution was performed with methanol / ascorbic acid solution. The total volume of the eluate was adjusted to 0.5 ml by gentle heating at 50 ° C under vacuum, and the remaining sample volume was dried in liquid nitrogen. Each sample was dissolved in 10 μl of pyridine and 50 μl of bis- (trimethylsilyl) trifluoro-octamide (BSTFA) and left overnight at room temperature. 2 μl of each sample was analyzed on a gas mass spectrometer. Analysis results were expressed in ng / mg cell protein.

Estrogen biotransformation test. Biotransformation analysis was carried out by a radiometric method based on the formation of 3H2O due to proton transfer from specifically labeled estradiol, as described in (Telang NT, Bradlow HL and Osborne MP "In vitro biotransformation of estradiol by explant cultures of murine mammary tissues" Breast Cancer Res. Treat . 1989, 13, 173-181).

Labeled estrogen was added to the cells of the monolayer in a solution of 0.1% propylene glycol. After 24 hours of incubation, the radioactivity in the culture fluid was counted. From the obtained values, the background biotransformation level in the cell-free culture medium was subtracted and converted to mg of protein determined by the Lowry method.

Experimental part.

The study of the expression and oncogenes of human papilloma viruses in transformed cells when exposed to indinol. The expression of the HPV-16 E7 gene was examined on a CaSki cell line containing up to 500 copies of the HPV-16 genome in an integrated state. Estradiol (final concentration 1 μM) and the composition of Example 1 (final concentration DIM 50 μM) were added to the culture medium, incubation was continued for 4, 24 and 48 hours. At the indicated time, cells were harvested, total RNA was isolated, and the expression level of the E7 gene was determined by Northern hybridization. The research results are presented in figure 3.

In this experiment, the level of expression of the E7 gene in CaSki cells was studied when estradiol was added to the culture medium.

From the results presented in Fig.3, it is seen that estradiol significantly induces the synthesis of E7 gene mRNA, and this effect increases over time (Fig.3A), and the addition of the claimed composition almost completely eliminates this effect (Fig.3B).

Studying the peculiarities of estradiol metabolism in epithelial cells infected with HPV. As was established earlier, in cells transformed with high-risk HPV, there is a change in the metabolism of estradiol towards the formation of an “aggressive” 16α-hydroxyestrone. This metabolite has carcinogenic properties and is able to form covalent complexes with estrogen receptors, causing prolonged hormone-dependent effects, including stimulating the expression of HPV oncogenes. We have studied the effect of indinol on estradiol metabolism, in particular, the formation of 16α-hydroxyestrone in cells transformed with HPV (see table).

The formation of 16α-hydroxyestrone in cells transformed with HPV type 16 №№ Cell type % 16α - hydroxyestrone per μg cell protein Estradiol Estradiol + Cervicon one CaSki HPV (+) 16.0 ± 0.1 2.2 ± 0.5 2 C 33-A HPV (-) 0.08 ± 0.03 0.08 ± 0.08 3 Cervical epithelium 0.7 ± 0 0.5 ± 0.1 four Cervical epithelium from transformed zones 12.6 ± 0.5 2.1 ± 0.3

Example 4

The study of the induction of apoptosis of cells transformed with HPV

in the presence of the preparation of example 2.

We have studied the ability of the drug to induce apoptosis of cells infected with human papillomavirus.

A combination of two dyes, ethidium bromide, which penetrates cells with a damaged membrane (i.e., dead and necrotic cells) and stains their nucleus with a bright orange, almost red color, was used to quantify the survival of cells and the proportion of cells in apotosis. acridine orange, which stains living cells in a bright green color and allows you to observe different phases of chromatin condensation and membrane blebbing. In cells that are in the late stages of apoptosis, bright orange regions of condensed chromatin in the nucleus are observed, which distinguishes them from necrotic cells, which in this case turn out to be uniformly colored in orange. The proportion of cells in the state of apoptosis was calculated as a percentage of their total number.

CaSki and C 33-A cells were incubated with a Hoechst 33342 fluorescent dye (Sigma) in a culture medium at a concentration of 5 μl / ml at 37 ° C for 30 min, then fixed in 2% paraformaldehyde at room temperature for 10 min, the PBS was washed once and analyzed with using an Opton fluorescence microscope.

As experiments showed, after a 24-hour incubation of CaSki cells in a culture medium containing the claimed preparation (final concentration of DIM 50 μM), more than 50% of apoptotic cells were observed in the field of view (see Fig. 4A), whereas in cells line C 33-A under the same conditions, the percentage of cells in the state of apoptosis was not more than 5% (pigv).

Example 5. Studies on the effects on genital warts.

Studies have also been conducted on patients with a diagnosis of genital genital warts.

Diagnosis was based on cytological, histological examination of biopsy samples, determination of antibodies to HPV and detection of HPV DNA and E7 oncoprotein.

Of the 25 patients in the group, genital warts were associated with other urogenital infections in approximately 90% of cases. About 10% were previously treated by electrocoagulation.

15 patients were given 2 times a day suppositories according to example 2 and 10 patients received suppositories according to example 1 for 10 weeks - 12 weeks before genital warts resolved. During the follow-up examination after a year and a half, there was no relapse of genital warts in the main group, and 3 patients had relapse. An additional treatment was prescribed with an increase in dose.

findings

Our studies have established that the new dosage form of Cervicon (vaginal suppositories containing the substance methindole (3,3'-diindolylmethane - DIM) has a high specific antitumor activity against human epithelial cells infected with HPV, which is realized through the following mechanisms:

1. Cervicon removes the estradiol-dependent induction of the oncogen E7 HPV-16, thus inhibiting the hormone-dependent proliferation of infected cells.

гидроксиэстрона, стимулирующего экспрессию онкогенов ВПЧ.2. Cervicon normalizes the metabolism of estradiol in cells infected with HPV, preventing the formation of a carcinogenic metabolite

hydroxyestrone, stimulating the expression of HPV oncogenes.

3. Cervicon induces apoptotic processes of HPV-infected cells, causing selective death of cells with tumor properties.

There is every reason to believe that a new drug topically applied in the form of vaginal suppositories has greater antitumor and antiviral activity against HPV-infected cervical cells compared to the orally administered DIM (at the same final concentration) and its metabolic precursor, I3C, converted to DIM in the gastrointestinal tract.

Thus, the drug can be recommended in the treatment of cervical dysplastic processes caused by human papilloma viruses.

In addition, suppositories provide the necessary time with high bioavailability of the active substance, which allows them to be used for such intractable pathologies as genital candidiasis.

Claims (2)

1. An agent for treating genital warts and neoplastic diseases of the reproductive organs in the form of a vaginal suppository, characterized in that it contains methindole (3,3'-diindolylmethane), a lipophilic base containing solid confectionery fat, polyvinylpyrrolidone and butylhydroxyanisole as the active substance in the following components, wt.%: Methindole 2.0-6.0 Polyvinylpyrrolidone 0.5-1.2 Butylhydroxyanisole or butylhydroxytoluene 0.3-0.5 Lipophilic base rest 2. The tool according to claim 1, in which as a lipophilic base contains solid fat type "A".

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