RU2315594C1 - Anti-estrogenic and anti-proliferative agent for treatment and prophylaxis of female reproductive system diseases - Google Patents

Abstract

FIELD: medicine, gynecology, chemical-pharmaceutical industry, pharmacy.

SUBSTANCE: invention relates to an anti-estrogenic and anti-proliferative agent used in treatment and prophylaxis of diseases of female reproductive system and represents a tablet covered with envelope and containing indole-3-carbonol and lactose, microcrystalline cellulose, butylhydroxyanisol, modified maize starch, magnesium or calcium stearate as accessory substances, and an enteric coating "AKRILIZ" as envelope. The tablet composition provides the enhanced therapeutic effect, high stability and bioavailability.

EFFECT: improved and valuable medicinal properties of agent.

6 dwg, 3 ex

Description

The invention relates to the field of medicine and the pharmaceutical industry.

Currently, phytochemicals based on indole compounds isolated from the Cruciferous family, which include all types of cabbage, Brussels sprouts, cauliflower and broccoli, as well as their synthetic analogues, are widely used in medical practice. Interest in the compounds of this group is caused, in particular, by their anti-carcinogenic and anti-estrogenic properties, and therefore they can be used in diseases of the breast, such as mastopathy, cancer.

Mastopathy (fibrocystic disease) is a dishormonal benign disease characterized by a violation of the ratio of the epithelial and connective tissue components of the mammary glands. Today it is one of the most common pathologies in women. According to statistics, various forms of mastopathy (of which about 60 are known in all) are found in every second woman older than 35 years. At the initial examination of the doctor, mastopathy is detected in 30-40% of women, and with a more detailed examination using ultrasound, mammography, radiography and histological studies, almost 80%.

It is generally recognized that mastopathy is a polyetiological or multifactorial disease. There are many internal and external causes, the effect of which leads to hormonal imbalance in the female body and, ultimately, causes the appearance of seals and nodes in the mammary glands. Among them: a genetic predisposition, unfavorable factors of a reproductive nature (late first birth, early and late menarche, lack of pregnancy and full lactation, concomitant gynecological diseases (it is known that 75% of gynecological inflammatory diseases are accompanied by changes in the mammary gland), disorders in the endocrine system, neurological disorders.

The mammary glands, due to physiological characteristics, are in a constant state of proliferation and involution processes associated with the phases of the menstrual cycle and the corresponding different levels of sex hormones - estrogen and progesterone, produced by the ovaries and adrenal cortex, as well as gonadotropic hormones of the anterior pituitary gland - follicle-stimulating hormone, luteinizing hormone and prolactin. During pregnancy, hormones produced by the placenta have a great influence. Therefore, a violation of the normal functioning of the hypothalamic-pituitary system at any level can cause hormonal imbalance. Under the influence of hormonal changes, physiological transformations in the tissues of the mammary glands are disrupted and foci of pathological proliferation of the epithelium develop. Chronic disorders in the physiology of the mammary glands can lead to the development of mastopathy, and in some cases to breast cancer (breast cancer).

In the structure of cancer incidence in women, breast cancer occupies the first place, and on average, the incidence of breast cancer increases by 1% per year. In the structure of mortality, it ranks second, while the "growth rate" in terms of mortality remains the highest (28%). According to WHO experts, about one million new cases of breast cancer are registered every year around the world, one third of which are fatal. According to some researchers, in the next decade, 5 million women worldwide will suffer from this malignant tumor.

It should be emphasized that mastopathy is not an obligatory precancerous disease, however, some of its forms can contribute to the development of breast cancer. It has been established that against the background of benign neoplasms, breast cancer is found 3-5 times more often, and with some forms of nodular mastopathy with signs of proliferation and atypia, it is 30-40 times more common than in healthy women.

All of the above makes us treat mastopathy extremely wary and encourages researchers to develop new, more advanced diagnostic methods that can detect chronic and newly emerging pathologies of the mammary glands at the earliest possible stages of development, as well as conduct appropriate treatment measures in a timely manner.

There are many classifications of various forms of mastopathy, but from a practical point of view (in particular, when analyzing data obtained using x-ray radiography (mammography) and morphological studies), the following classification is most acceptable:

I. Diffuse fibrocystic mastopathy:

- with a predominance of the glandular component (adenosis);

- with a predominance of the fibrous component (fibrosis);

- with a predominance of the cystic component (multiple cysts);

- mixed form (glandular cystic);

- sclerosing adenosis.

II. Nodular fibrocystic mastopathy:

- fibroadenoma;

- a cyst.

The severity of these processes in mammograms is conditionally determined by the ratio of the connective-tissue-glandular complex and the fat background and is classified as:

- mild mastopathy - a condition in which adipose tissue predominates over the parenchyma of the gland;

- moderate mastopathy - a condition where adipose tissue and the dense structures that form the mammary gland are represented in approximately equal proportions;

- a pronounced degree of mastopathy - a condition when the mammary gland is represented mainly by connective tissue and glandular structures, little adipose tissue.

According to the recommendations of the Problem Commission on the Morphology of Tumors of the Academy of Medical Sciences of the USSR, a classification of dishormonal dysplasias has been developed, which is based on the histological classification of WHO breast tumors (2000). In accordance with this classification, the following forms of mastopathy (or fibroadenomatosis) are distinguished:

a) non-proliferative (lobular, ductal, cystic, fibrous);

b) proliferative epithelial (solid, papillary, fibrous);

c) fibroepithelial (cystadenopapilloma);

d) myoepithelial (sclerosing adenosis) (fibroadenomas are included in the heading of benign tumors).

Despite the extremely important medical and social significance of benign diseases of the mammary glands, pathogenetically substantiated tactics of their conservative treatment have not yet been developed. In essence, it comes down to taking vitamins, iodine preparations, and medicines based on plant extracts. These palliative measures in some cases allow you to remove individual symptoms of the disease. However, due to the unclear mechanisms of action of the drugs used, the long-term results of such treatment are difficult to predict.

At the same time, taking into account the fact that some forms of mastopathy are precursors of the processes of malignancy of mammary gland tissues, their treatment should be carried out using drugs that block the main pathogenetic links of this disease.

It is known that breast cells are continuously exposed to many different factors that stimulate them to active division (proliferation) and induce the launch of specific signaling cascades. Among them, three main intracellular mechanisms leading to the activation of cell proliferation can be distinguished: 1) hormonal (or estrogen-dependent); 2) induced by growth factors and 3) activated by pro-inflammatory cytokines.

The participation of estrogens in the development of neoplastic processes in hormone-dependent tissues (mammary gland epithelium, endometrium and cervix) is currently considered universally recognized and is considered as one of the leading etiological factors in their occurrence.

As you know, monthly changes in mitotic cell activity occur in the tissues of the female reproductive system, regulated by rhythmic changes in the level of sex hormones. In the first (so-called proliferative) phase of the menstrual cycle, estrogen secreted by the ovaries induces the proliferation of endometrial cells that die during menstruation. Similarly, each menstrual cycle of estrogen stimulates the proliferation of the inner layer of epithelial cells of the ducts of the mammary glands, which subsequently also undergo apoptosis. In total, during the approximately 40-year reproductive period, several hundred such cycles are carried out in a woman's body.

Once in the cell, estrogen (E) activates the estrogen receptor (ER), which is in the inactive state in the cytoplasm. The interaction of the hormone with the receptor activates the latter and promotes its penetration into the nucleus. Once in the nucleus, the hormone-receptor complex stimulates the expression of the so-called estrogen-dependent genes, among which most directly or indirectly control cell proliferation, and also increases the sensitivity of cells to factors that activate hyperplastic processes (Figure 1). These are genes encoding the epidermal growth factor receptor (EGFR), keratinocyte growth factor (KGF), cell cycle regulators - cyclin proteins and cyclin-dependent kinases (CDK), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) and many other proteins.

Regularly repeated cycles of estrogen-induced cell division can have a twofold effect on the development of the tumor process in hormone-dependent tissues. First, proliferate under the influence of estrogens can already malignant (exposed to an external carcinogen or having a set of germline mutations) cells, the proliferation of which leads to the formation of a "tumor clone". However, most often there is a different situation. Periodically repeating cycles of cell division inevitably increase the frequency of the appearance of new, spontaneous mutations (especially with increasing age of the woman, as well as in the presence of other risk factors). Given the accumulation of 3 to 7 such genetic errors that are not eliminated by the immune and / or reparative cellular systems, estrogen-dependent proliferation of transformed cells will also lead to the formation of a tumor. Moreover, the initiator of the tumor process, in its traditional sense, is absent in the second case.

If the woman’s body contains an increased content of one of the estrogen derivatives, namely 16α-hydroxyestrone, pathological proliferative processes in hormone-dependent tissues are multiplied (Clemons M, Goss P. Estrogen and risk of breast cancer, N. Engl. J . Med., 2001, 344 (4), 276-285). That is why the increased content of 16α-hydroxyestrone is currently considered as a risk factor for the development of breast cancer.

However, pathological cell proliferation can occur by an estrogen-independent mechanism. In this case, signaling cascades stimulated by polypeptide growth factors and cytokines are included.

The main polypeptide factor that stimulates the growth of breast cells is epidermal growth factor (EGF). The EGF factor, through a sequence of signaling proteins, activates the nuclear transcription factor NF-κB, which stimulates the expression of a large number of genes responsible for cell survival and proliferation. Among them are genes encoding the EGF receptor (EGFR), keratinocyte growth factor (KGF), cell cycle regulators - cyclin-dependent kinases, vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF) and many other proteins.

The cytokine pathway of cell growth regulation is associated with tumor necrosis factor TNF-α. In high concentrations, this cytokine activates pro-apoptotic receptor-mediated signaling cascades, i.e. stops the processes of cell division and causes physiological cell death. However, in small doses, it also acts as a factor in the survival and proliferation of cells. At the same time, the activity of cyclooxygenase-2 (COX-2), the main enzyme involved in the prostaglandin biosynthesis and stimulating the expression of NF-κB factor, which, as we have said, includes the expression of genes that stimulate cell division, increases.

Thus, at present, the pathogenetic mechanisms of the development of hyperplastic processes in the mammary gland are quite well studied. Obviously, by blocking the main channels of signaling that stimulate the proliferation of breast cells, we can count on success in the prevention and treatment of pathological conditions arising on this basis. In other words, pharmacological correction of hyperproliferative breast diseases should be carried out at all stages and in relation to all signaling cascades mediating key pathophysiological functions.

Long-term searches for natural compounds that block the development of hyperplastic processes in hormone-dependent tissues have finally been crowned with success. One of these compounds is the phytonutrient indole-3-carbinol (I3C) contained in the vegetables of the cruciferous family (various types of cabbage), on the basis of which a new tool in tablet form was developed.

The antitumor protection provided by I3C is due to the wide range of its biological activities. One of the mechanisms of the antitumor effect of I3C is the interaction of this substance with aryl carbon receptors (AhR), which blocks the binding of AhR to carcinogens. Thus, I3C is the natural ligand AhR. When AhR is activated as a result of its interaction with I3C, the formed ligand-receptor complex penetrates the nucleus and enhances the expression of CYP1A1, an isoform of cytochrome P450, hydroxylating estrone (estrogen metabolite) in the 2nd position with the formation of 2-hydroxyestrone (2-OHE1). This metabolite was found to have pronounced antiproliferative (antiestrogenic) activity (Hong CB, Kim HA, Firestone GL, Bjeldanes LF, 3,3'-Diindolyl-methane (DIM) induces a G (1) cell cycle arrest in human breast cancer cells that is accompanied by Sp1-mediated activation of p21 (WAF1 / CIP1) expression, Carcinogenesis (Lond) 2002; 23: 1297-1305). The isoenzyme CYP1A1 is an inducible form and is activated in response to food ingredients and cigarette smoke. In the absence of I3C, the AhR receptor is activated by carcinogenic aryl hydrocarbons that enter the body from the environment or with food (especially canned food), which promotes the expression of CYPB1, an isoform of cytochrome P-450, hydroxylating estrone at 16α and 4 positions to form respectively, 16α-hydroxyestrone (16α-OHE1) and 4-hydroxyestrone (4-OHE1) (Smigel K. Breast Cancer Prevention Trial shows major benefit, some risk. J. Natl. Cancer Inst. (Bethesda), 1998, 90: 647 -648). It was shown that the main metabolite 16α-OHE1 formed during this process is able to covalently bind to estrogen receptors, nuclear histone proteins and DNA, resulting in the prolongation of estrogenic activity (estrogen-dependent proliferative signal), as well as the initiation of genotoxic DNA damage. Thus, these hydroxy derivatives of estrone are the strongest carcinogens for target cells.

Thus, estradiol can be converted into two metabolic forms - hydroxy derivatives of estrone - 2-ОНЕ1 and 16α-ОНЕ1, formed by catalysis by different isoenzymes of cytochrome P-450 and having absolutely opposite biological properties. 2-OHE1 does not affect cell proliferation, while 16α-OHE1, on the contrary, stimulates cell growth and is an estrogen agonist. A study of the functions of these two metabolites revealed an unambiguous relationship between the level of 16α-OHE1 and the risk of developing tumors in estrogen-dependent tissues. At the same time, with an increase in the level of 2-OHE1, a tendency toward the death of tumor cells and prevention of their further formation was observed.

Numerous clinical studies have shown that in order to maintain a normal hormonal balance in both pre- and postmenopausal women, it is necessary that the concentration of 2-OHE1 exceeded the concentration of 16α-OHE1 at least twice. By lowering this ratio, the risk of breast cancer is statistically significantly increased (Leong H, Firestone GL, Bjeldanes LF, Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression, Carcinogenesis (Lond) 2001: 22: 1809-1817; Patent WO 9203973, Onset ofantiestrogen resistence in breast cancer, 1992).

In randomized clinical trials conducted in the late 90's, it was found that postmenopausal women with a diagnosis of breast cancer showed a reduced ratio of 2-OHE1 / 16α-OHE1 compared with the corresponding control. Moreover, it was shown that patients with stage III and IV breast cancer had lower ratios of 2-OHE1 / 16α-OHE1 than patients with stage I and II disease (Huang CS, Chem HD, Chang KJ, et al. Breast cancer risk associated with genotype polymorphism of the estrogen-metabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility. Cancer Res, 1999, Vol. 59, N.19, p. 4870-4875).

Currently, a simple, non-invasive, reliable and relatively inexpensive enzyme-linked immunosorbent method for determining estrogen metabolites - 2-OHE1 and 16α-OHE1 - in urine (Kabat GC, Chang CJ, Sparano JA et al. Urinary estrogen metabolites and breast cancer: a case-control study. Cancer Epidemiol. Biomarkers Prev., 1997, 6, 505-509), which in the future opens up unlimited prospects for expanding the scope of prevention and treatment of preclinical forms of hormone-dependent tumors (especially among patients at risk according to diseases).

Since indole-3-carbinol directly affects the level of 2-hydroxyestrone and 16α-hydroxyestrone, measuring the ratio of these metabolites can serve as a reliable marker of its effectiveness. A stable excess of 2-hydroxyestrone more than 2 times for 1-3 months with I3C is an indicator of an adequate correction of the hormonal background and therapeutic efficacy of this compound.

Preparations based on indole-3-carbinol in the form of dietary supplements are widely used in the United States to correct dishormonal disorders. The first clinical study on this subject was intended to test the hypothesis of the possibility of using I3C to stimulate the formation of an estrogen derivative, 2-hydroxyestrone (2-OHE1) (Bradlow et al. Long-responses of women to indol-3-carbinol. Cancer Epidemiol. Biomarkers Prev., 1994, 7, 591-595).

This clinical study involved 2 groups of women with 20 patients each. The first (experimental) group received I3C at a dose of 400 mg per day for 3 months. The second (control) group received a placebo. Monthly, standard biochemical blood parameters and the ratio of estrogen metabolites 2-OHE1 / 16α-OHE1 were examined in women. In the control group, no changes in biochemical parameters were recorded. In the experimental group, the standard biochemical parameters also remained unchanged, while in 17 women there was a significant change in the ratio of estradiol metabolites in favor of 2-OHE1, and this ratio remained unchanged for 3 months after the cancellation of I3C. Three women in the experimental group did not respond to the drug. The authors concluded that I3C is an effective tool for the correction of estrogen metabolism.

In 1997, 60 female volunteers residing in the United States participated in randomized clinical trials. All participants were divided into three groups: 1) a control group receiving a placebo; 2) a group receiving low doses of I3C (50, 100 and 200 mg per day), and 3) a group receiving high doses of I3C (300 and 400 mg per day) for 4 weeks. In all women, the ratio of 2-OHE1 / 16α-OHE1 was determined as a biomarker of the effectiveness of the therapy. The results of the studies allowed the authors to draw two important conclusions:

1. In no case were any side effects observed.

2. The minimum daily dose of I3C, at which a change in the ratio of estrogen metabolites was observed, was 300 mg, on the basis of which the authors recommend 300-400 mg / day doses of I3C for the prevention of breast cancer.

A year later, the Fund for Preventive Oncology conducted clinical trials on the same issue. Female volunteers aged 35 to 47 years took I3C at a dose of 400 mg per day for 2 months. Measurement of the level of 2-hydroxyestrone (2-OHE1) showed a significant increase in this metabolite as a result of taking I3C, which indicates the high therapeutic efficacy of this drug in relation to the correction of hormonal levels.

In the course of numerous experimental studies, the versatility of the I3C antitumor activity was proved, due to the ability of this compound to block all the main transduction pathways of intracellular signals stimulating cell growth, including estrogen-independent proliferative pathways activated by growth factors and cytokines.

Growth factors, which include epidermal growth factor (EGF), fibroblastic growth factor (FGF), keratinocyte growth factor (KGF or FGF-7), insulin-like growth factor (IGF-I), etc., activate in the absence of any obstacle specific receptors with tyrosine kinase activity. Ligand-induced receptor dimerization activates a cascade of cytoplasmic signal kinases (MAPK, Akt), proto-oncogenes (fos, ras), transcription activators (NF-κB), which leads to the expression of ligand-induced genes, among which are the growth factors and their receptors mentioned above ; kinases; factors stimulating the formation of new blood vessels; heat shock proteins; as well as other proteins necessary for tumor metabolism.

Penetrating into the cell, indole-3-carbinol interferes with the phosphorylation of tyrosine kinase residues, which interferes with the cascade transmission of proliferative signals from the surface to the cell nucleus (Chinni SR, Sarkar FH. Akt Inactivation Is a Key Event in Indole-3-carbinol-induced Apoptosis in PC-3 Cells. Clin. Cancer Research, 2002, 8, 1228-1236). In addition, it inhibits the nuclear transcription factor NF-κB, which is the main activator of transcription of a large number of genes involved in proliferation and inflammation (Brignall MS. Prevention and treatment of cancer with indole-3-carbinol, Altem. Med. Rev., 2001 , 6 (6), 580-589).

Another mechanism of tumor blockade carried out by I3C is associated with the inhibition of the activity of cyclooxygenase-2 (COX-2), an enzyme involved in the synthesis of prostaglandins (PGE2). MOR-2 is activated in response to the action of pro-inflammatory cytokines - tumor necrosis factor-alpha (TNF-α) and its "partner" - interleukin-1 (IL-1). The interaction of cytokines with their receptors leads, in particular, to the activation of COX-2 and the synthesis of PGE2, followed by transcription of vascular endothelial growth factor (VEGF), the main component of tumor neoangiogenesis.

There is evidence that OS-mediated suppression of the proliferative activity of tumor cells is accompanied by an inhibition of the activity of cell cycle regulatory proteins, cyclins D1 and E and cyclin-dependent kinases (CDK2, CDK4 and CDK6), as well as stimulation of the formation of tumor growth suppressors - proteins p15, p21 and p27. In this case, the cell cycle stops in phase G1 (Aggarwal BB, Ichikawa H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle, 2005, 4 (9), 1201-1215; Cover CM, Hsieh SJ , Tran SH et al. Indole-3-carbinol Inhibits the Expression of Cyclin-dependent Kinase-6 and Induces a G1 Cell Cycle Arrest of Human Breast Cancer Cells Independent of Estrogen Receptor Signaling. Biol. Chem., 1998, 273 (7) , 3838-3847; Garcia HH, Brar GA, Nguyen DH, Bjeldanes LF, Fire-stone GL, Indole-3-carbinol (I3C) inhibits cyclin-dependent kinase-2 function in human breast cancer cells by regulating the size distribution, associated cyclin E forms, and subcellular localization of the CDK2 protein complex. J. Biol. Chem., 2005, 280 (10), 8756-8764; Kim YS, Milner JA. Targets for indole-3-carbinol in cancer prevention. J. Nutr. Biochem., 2005, 16 (2), 65-73; Matsuzaki Y, Koyama M, Hitomi T, Kawanaka M, Sakai T, Indole-3-carb inol activates the cyclin-dependent kinase inhibitor p15 (INK4b) gene. FEBS Lett., 2004, 576 (1-2), 137-140).

Another unique feature of I3C is its ability to selectively induce programmed cell death processes in tumor cells - apoptosis (Powles T.J. Efficacy of tamoxifen as treatment of breast cancer. Semin. Oncol., 24 (Suppl.1): S1-48- S1-54, 1997). It is known that the translocation of the proapoptotic protein Bax from the cytoplasm to mitochondria is a critical event in the development of the mitochondrial apoptosis pathway. I3C stimulates this process, which is accompanied by a decrease in mitochondrial potential, release of cytochrome c from mitochondria, activation of proapoptotic caspases (-3 and -9) and, in fact, the onset of apoptosis. At the same time, along with the manifestation of apoptotic activity of I3C, inhibition of the activity of anti-apoptotic proteins is observed - Bcl-2, Bcl-xL, survivin, LAP (inhibitor-of-apoptosis protein), XIAP (X chromosome-linked IAP) and FLIP (Fas-associated death domain protein-like interleukin-1-beta-converting enzyme inhibitory protein).

In addition, there is evidence of the antioxidant properties of I3C, as well as its ability to inhibit angiogenic, invasive, and migratory processes during carcinogenesis in the mammary glands (McCarty MF, Block KI. Multifocal Angiostatic Therapy: An Update, Integrat. Cancer Ther., 2005 4, 301-314; Meng Q, Goldberg ID, Rosen EM, Fan S. Inhibitory effects of Indole-3-carbinol on invasion and migration in human breast cancer cells, Breast Cancer Res Treat, 2000, 63 (2), 147 -152; Wu HT, Lin SH, Chen YH. Inhibition of cell proliferation and in vitro markers of angiogenesis by indole-3-carbinol, a major indole metabolite present in cruciferous vegetables. J. Agric. Food Chem., 2005, 53 ( 13), 5164-5169).

Thus, I3C is a universal corrector of pathological proliferative processes in the organs of the female reproductive system. The universality of the antitumor effect of I3C is due to the ability of this compound to block all the main (hormone-dependent and hormone-independent) transduction pathways of intracellular signals that stimulate cell growth, as well as induce selective apoptosis (programmed cell death) of tumor cells. I3C is a truly unique compound, since it can suppress the development of the tumor process at almost all its levels: submolecular (activation of tumor suppressor genes and suppression of genes that stimulate oncogenesis), molecular (blocking proliferative signals throughout their length - from receptors to nuclear proteins and DNA ) and supramolecular (inhibition of cell migration and invasion).

The combination of antiestrogenic and antiproliferative activities of I3C makes it indispensable in the treatment and prevention of hyperplastic processes in the organs of the reproductive system. It has been shown that mammary gland, myometrium, endometrium, and other hormone-dependent tissues can be target organs of I3C. The high clinical effectiveness of I3C as a pharmacological corrector of hyperplastic processes in hormone-dependent tissues has been confirmed in clinical studies conducted on models of diffuse mastopathy, uterine fibroids and cervical dysplasia. This property of I3C is of great importance in clinical practice, as mastopathy is often combined with pathologies of other hormone-dependent organs and tissues and develop against the background of dishormonal disorders.

The listed pharmacological properties of indole-3-carbinol allow us to consider this substance as a very promising candidate for creating drugs based on it. However, I3C is a very unstable compound and, when it enters the acidic environment of the stomach, very quickly turns into several oligomeric derivatives, the main of which is its dimeric form - 3,3'-diindolylmethane (DIM) (Figure 2) (Arneson DW, Hurwitz A, McMahon LM et al. Presence of 3,3'-diindolylmethane in human plasma after oral administration of indole-3-carbinol [abstract 2833], Proc. Am. Assoc. Cancer Res. 1999; 40, 429; Grose KR, et al Oligomerization of indole-3-carbinol in aqueous acid. Chem Res Toxicol, 1992, 5, 188-193). That is why, despite the numerous effects of I3C in vivo, there is still no complete clarity regarding their true source, i.e. the question remains open whether the biological properties of I3C are directly related to its own effect on cellular targets or is it the result of the activity of the products of condensation and oxidation of indole-3-carbinol, in particular diindolylmethane.

At the same time, it is known that the clinical effect of I3C in various pathologies depends on the individual characteristics of the metabolism of patients taking this drug, in particular, on the ability to convert I3C into its various derivatives, between which, in turn, synergistic and / or antagonistic interactions (Dalessandri KM, Firestone GL et al. Pilot Study: Effect of 3-Diindolylmethane Supplements on Urinary hormone metabolites in Postmenopausal women with a history of early-stage breast cancer. Nutrition and cancer, 2004, 50 (2), 161-167). At the same time, there are experimental data indicating the independent biological activity of I3C, independent of the biological activity of metabolites formed as a result of its oligomerization (Anderton MJ, Manson MM, Verschoyle RD, Gescher A, Lamb JH, Farmer PB, Steward WP, Williams MIL. Pharmacokinetics and Tissue Disposition of Indole-3-carbinol and Its Acid Condensation Products after Oral Administration to Mice. Clin. Cancer Research, 2004, 10, 5233-5241; Hsu JC, Zhang J, Dev A, Wing A, Bjeldanes LF, Firestone GL Indole-3-carbinol inhibition of androgen receptor expression and downregulation of androgen responsiveness in human prostate cancer cells. Carcinogenesis, 2005, 26 (11), 1896-1904).

One of the fundamental criteria that any chemical compound that claims to be the basis for creating a drug must meet is storage stability. Indole-3-carbinol is an extremely labile compound, rapidly decomposing in the light and in the presence of oxygen. It is also poorly soluble and, accordingly, has low bioavailability, which may limit its use as a medicine.

Researchers have attempted to create forms with increased absorption of diindolylmethane.

A number of solutions are known in the art for dosage forms of I3C (indole-3-carbinol).

In particular, US Pat. No. 6,689,387, publ. 02/10/2004, which describes microparticles of I3C or 3,3'-diindolylmethane in a starch matrix, including in the form of solid dosage forms for oral administration. These forms contain 30-70% starch. Which, increasing the solubility of the active substance, does not provide sufficient stability during storage.

Known patent US 639964, publ. 2002, describing a tool for the treatment of neoplastic diseases based on indole-3-carbinol and / or diindolylmethane. This tool can be made in the form of a capsule or tablet containing as auxiliary substances lactose, starch, cellulose derivatives and the like. This composition is not stable.

Also known is US patent 2004156910, which can be indicated as a prototype, describing various dosage forms of an agent for treating mastopathy and endometriosis based on indole derivatives with a high degree of absorption, including in the form of tablets, for example, containing a binder, surfactant and water. The tablets may be coated. The active substance is present in microcrystalline form. The developed dosage forms according to this source do not provide improved water solubility of the active substance, they are dispersion systems that have a more heterogeneous structure.

Although the known patents mention the possibility of obtaining tablets with various additives known in the field of pharmacy, they are not posed and are not supposed to create such a form that it would allow the therapeutic effects of I3C and its metabolite to be dissociated when a concentration of 2-OHE1 exceeds the concentration of 16α-OHE1 at least twice, which provides the maximum preventive and therapeutic effect.

The objective of the invention is the following:

1) to narrow as much as possible the spectrum of its molecular intracellular targets and thus make the action of this drug as predictable as possible;

2) separate the therapeutic effects of I3C and DIM.

With this objective in mind, an antiestrogenic and antiproliferative agent was developed for the treatment and prevention of diseases of the female reproductive system, which is an enteric-coated tablet containing indole-3-carbinol, as well as excipients that increase the stability of the drug during storage and improve its solubility and bioavailability .

The proposed tool contains the following components in g:

Composition per tablet: Indole-3-carbinol 0.1-0.2 Lactose 0.10-0.15 Butylhydroxyanisole 0.0001-0.0002 Microcrystalline cellulose 0.10-0.150 Modified corn starch 0.03-0.05 Magnesium Stearate or Calcium Stearate 0.002-0.004 Enteric ACRYLIZ coating 0.03-0.05

It was found that the proposed composition in the most effective way avoids contact with the acidic environment of gastric juice when I3C is taken, resulting in the formation of metabolically active derivatives of this compound.

As a shell, Akriliz coating (Akryl-eze), (ND 42-13039-04), including:

Methacrylic Acid Copolymer ethyl acrylate 1: 1 40% Titanium dioxide fifteen% Talc 37.25% Triethyl acetate 4.8% Silicon Colloidal Anhydrous Oxide 1.25% Sodium bicarbonate 1.2% Sodium Lauryl Sulfate 0.5%

Preferably, the shell weight should be about 10% of the weight of the tablet core.

The invention can be illustrated by the following examples.

Example 1

The proposed tool. Enteric-coated tablets containing 0.1 g of indole-3-carbinol

Composition per tablet: Indole-3-carbinol - 0.1 g Lactose (for direct compression 80 MES) - 0.1058 g (GOST 4963-85, GF X) (80 mesh, 200 mesh) Butylhydroxyanisole - 0,0002 g (Heb. Pharm. 2004) Microcrystalline cellulose - 0.150 g (Vivapur 12, Heb. Pharm. 2004) Modified corn starch - 0,040 g (Starch 1500, ND 42-11973-01) Magnesium stearate - 0.004 g (TU 6-09-16-1533-90, "h") Tablet core mass - 0.400 g Enteric coating ACRYLIZE (COLORED) - 0.040 g (10%) (ND 42-13039-04) Coated Tablet Weight - 0.440 g

Example 2

The proposed tool. Enteric-coated tablets containing 0.2 g of indole-3-carbinol

Indole-3-carbinol - 0.2 g Lactose (for direct compression 80 MES) - 0.15 g (GOST 4963-85, GF X) (80 mesh, 200 mesh) Butylhydroxyanisole - 0,0002 g (Heb. Pharm. 2004) Microcrystalline cellulose - 0.1 g (Vivapur 12, Heb. Pharm. 2004) Modified corn starch - 0.046 g (Starch 1500, ND 42-11973-01) Calcium stearate - 0.0038 g Tablet core mass - 0.500 g Enteric coating ACRYLIZE (COLORED) - 0.050 g (10%) (ND 42-13039-04) Coated Tablet Weight - 0.550 g

Example 3

An experimental study of the specific pharmacological activity of the proposed drug in vitro and in vivo.

Materials and methods

The cultivation of cells. In in vitro and in vivo experiments, human breast adenocarcinoma cells of the estrogen-sensitive (ER +) line MCF-7 and the estrogen-insensitive (ER-) line MDA-MB-468 were used. Cell cultures obtained from the ATCC American Type Cell Culture Collection (Bethesda, MD, USA) were cultured in plastic culture bottles (Costar, USA) in DMEM / F-12 growth medium (1: 1) supplemented with 10% bovine embryonic serum, 4.0 g / l glucose, 3.7 g / l sodium bicarbonate, 2 mM L-glutamine, 100 u / ml penicillin and 100 μg / ml streptomycin ("GibcoBRL") at 37 ° C in a humidified atmosphere containing 5% CO ₂ .

Animals and their contents. In vivo experiments were performed on adult female nude (nu / nu) C57Black / 6 linear mice (7 weeks old) obtained from Charis Rivers Co (USA).

The duration of the quarantine (acclimatization period) for all animals was 14 days. Cages with animals were placed in separate rooms. The animals were kept under a 12-hour lighting regime, a temperature of 20-25 ° C, a relative humidity of 50-70% and free access to food and water.

Determination of the survival of tumor cells of the breast when exposed to the proposed tool.

MCF-7 and MDA-MB-468 human breast adenocarcinoma cells were scattered in 96-well microtiter plates (Costar, USA) at a concentration of 5 × 104 cells per cell. 24 hours after the cells were attached to the surface and a monolayer was reached, the culture growth medium was replaced with the medium containing the proposed agent (it was dissolved in dimethyl sulfoxide (DMSO) at a 1000-fold concentration and added to the culture medium so that the final concentration of I3C contained in it was 100 μM (680 μmol of I3C is contained in one tablet with a dosage of 0.1 g of I3C, and 1360 μmol of I3C with a dosage of 0.2 g). Growth medium containing the corresponding amount of DMSO was added to control samples.

In all experiments, 1 μl of a DMSO solution containing preparations was added per 1 ml of culture fluid.

Tumor cell survival was evaluated using an MTT test based on the formation of formazan from tetrazolium salt by living cells 24, 48, 72 and 96 hours after the start of incubation of the above compounds with cells. To this end, 50 μl (1 mg / ml) of a solution of MTT (3- [4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide) in culture medium was added to each well 2-4 hours before the end of incubation. After color development, the medium was removed, formazan crystals were dissolved in 150 μl of DMSO, and the color intensity was measured by absorption at 540 nm on a multichannel spectrophotometer (Labsystem). Cell survival was assessed as a percentage of the corresponding control (Arneson DW, Hurwitz A, McMahon LM et al. Presence of 3,3'-diindolylmethane in human plasma after oral administration of indole-3-carbinol [abstract 2833], Proc. Am. Assoc. Cancer Res. 1999; 40, 429; Mosman, T. Rapid Colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Meth., 1983, V.65, P.55-63).

The study of the antitumor efficacy of the proposed tool in experiments in vivo.

One week prior to the inoculation of tumor cells, female nude / nu linear C57Blask / 6 mice were implanted in the subscapular region with a pill containing 0.72 mg of estradiol, from which the hormone was released within 60 days.

To induce solid tumors, MCF-7 human breast adenocarcinoma tumor cells were collected using a 0.05% Trypsin-EDTA solution (Sigma, USA), washed three times with sterile phosphate-buffered saline (PBS), and then in an amount of 3 million cells in 0.1 ml of physiological saline was injected subcutaneously to each experimental animal in the lateral region (the number of living cells was counted using trypan blue dye (0.1%) and a light microscope).

24 hours after inoculation of xenogenic mammary tumor cells, animals from the experimental group (10 animals in the group) were started to intragastrically (using a probe) the proposed drug in an amount equivalent to 10 mg I3C / kg (therapeutic dose) with a frequency of 5 times a week. The control animals were injected with saline.

The size of solid tumors was measured 1 time in 2-3 days after its appearance. Tumor volume was calculated by the formula:

V = π / 6 × L (mm) × W2 (mm ² ),

where L is long, and W is the short diameter of the tumor (Swaneck GE, Fishman J. Covalent binding of the endogenous estrogen 16 alphahydroxyestrone to estradiol receptor in human breast cancer cells: characterization and intranuclear localization. Proc Natl Acad Sci USA, 1988, 85: 7831-7835).

Quantitative determination of the level of I3C and its main metabolite, DIM, by high performance liquid chromatography in blood plasma in C57Black / 6 mice after oral administration of the proposed agent and individual I3C substance.

Quantitative determination of the level of I3C and its DIM metabolite in blood plasma in C57Black / 6 mice was carried out by HPLC (HPLC) on a System Gold liquid chromatograph (Beckman, USA) with a variable wavelength UV detector.

To obtain blood samples, animals of the experimental group were injected intragastrically (using a probe) with the proposed agent (according to examples 1 and 2) in an amount equivalent to 250 mg I3C / kg (36 animals in the group in total) and an individual substance I3C in an amount of 250 mg I3C / kg (30 animals per group). The control animals were injected with saline. After 0.25; 0.5; 0.75; 1.0; 1.5; 2.0; 4.0; 6.0; 12.0; 18.0; 24.0 and 36.0 hours after the introduction of the proposed funds and after 0.25; 0.5; 0.75; 1.0; 1.5; 2.0; 4.0; 6.0; 12.0 and 24.0 hours after the administration of the individual I3C substance in experimental animals (3 animals for each time point), peripheral blood was taken from the tail vein with a heparinized syringe. Each blood sample was placed in a test tube containing heparin. Blood samples were centrifuged (10,000 × g, 5 min), after which 1.5–2.0 ml of plasma was taken, frozen and stored at –20 ° С.

Immediately prior to the HPLC analysis, an internal standard of 4-methoxy-indole (IS) (2.5 μl of a 0.4 mg / ml concentration solution) was added to test samples of blood plasma with a volume of 250 μl, mixed with Vortex and left at room temperature for 30 min, after which the samples were extracted twice with tert-butyl methyl ether (750 μl).

In each sample, the organic phase was separated from the aqueous by centrifugation (2800 × g, 10 min) and transferred to a new 4 ml tube. The organic layers of the samples for each experimental point were combined, the ether was evaporated in a stream of nitrogen, 150 μl of eluent (acetonitrile / 50 mM Hepes buffer (volume ratio 40:60; pH 7.4) was added to the precipitate, and 50 μl of the obtained sample was introduced into the chromatograph .

Similar procedures were carried out with control plasma samples, into which then the specified amounts of indole-3-carbinol and diindolyl-methane were added in the concentration range: for I3C: 0.1-15.0 μg / ml; for DIM: 0.05-10.0 μg / ml.

Quantitative determination of indole-3-carbinol and diindolylmethane in blood plasma was carried out on a System Gold liquid chromatograph (Beckman, USA) with a UV detector at a wavelength of 280 nm.

Liquid chromatography was performed at room temperature (22-24 ° C) on a Nucleosil column, C 18.5 μm (4.6 × 50 mm). The eluent (mobile phase) consisted of water and acetonitrile. Before chromatography, the mobile phase was degassed and filtered. Elution was carried out with a concentration gradient of acetonitrile (A) according to the following scheme: 1) from 15% to 60% A during the first 20 minutes; 2) linear gradient A from 60% to 65% from 20 to 40 minutes; 3) linear gradient A from 65% to 85% from 40 to 65 minutes; 4) re-balancing the 15% A column for 5 minutes The total elution duration was 70 min; the elution rate was 1 ml / min.

The concentration of I3C and DIM in experimental samples was determined from calibration plots reflecting the relationship between the concentrations of these substances in the sample and the areas of chromatographic peaks.

The minimum sensitivity of the HPLC analysis method was 0.05 μg / ml.

experimental part

The study of the impact of the proposed tool on the survival of estrogen-dependent and estrogen-independent cells of human breast adenocarcinoma in vitro

In this study, the effect of the proposed tool on the survival of human breast adenocarcinoma cells of the estrogen-sensitive line MCF-7 and the estrogen-resistant line MDA-MB-468 was evaluated using the MTT test.

A solution of the proposed agent in DMSO was added to the cells in such an amount that the final concentration of indole-3-carbinol (I3C) contained in it was 100 μM.

As can be seen from Figure 3, the proposed tool had a pronounced inhibitory effect on the survival of human mammary adenocarcinoma cells of both the estrogen-sensitive line MCF-7 and the estrogen-resistant line MDA-MB-468. Moreover, this inhibitory effect depended on the time of incubation of cells with the drug.

In other in vitro experiments, it was shown that the inhibitory effect of the proposed drug depends on its dose in the incubation sample, i.e. on the amount of the drug added to the breast tumor cells. In this case, the concentration range of the indole-3-carbinol preparation included in the composition was 10-400 μM, and the maximum inhibitory effect of I3C was observed at its final concentration of 200 μM (not shown in Figure 3).

It should be emphasized that the proposed tool approximately equally effectively suppressed the survival of hormone-sensitive and hormone-insensitive breast cancer cells. After 24 hours of incubation of tumor cells with the drug, the percentage of live hormone-sensitive and hormone-resistant cells was 55% and 66%, respectively, and after 48 hours of incubation, 24% and 43%.

According to modern ideas, the inhibition of the growth of breast tumor cells under the action of indole-3-carbinol is due to the stop of the cell cycle (in phase G1), as well as their selective aloptotic death. There is every reason to believe that in our case, a pronounced suppression of the survival of the studied cell lines is due to the same molecular mechanisms.

The study of the antitumor efficacy of the proposed tool in experiments in vivo

To study the antitumor effect of the proposed agent, it was administered intragastrically to female C57Blask / 6 athymic mice, which were previously inoculated with xenogenic tumor cells of the human breast adenocarcinoma MCF-7 line. The proposed tool was administered in an amount equivalent to 10 mg I3C / kg with a frequency of 5 times a week.

The antitumor efficacy of these compounds was evaluated by determining the volume of a solid tumor in the experimental and control groups of animals.

As can be seen from Figure 4, starting from approximately 12 days from the start of the experiment in animals of the control group who did not receive the proposed funds, there was an intensive growth of solid tumors. Over the next 20 days (from 14 to 34 days of the experiment), the average tumor size induced by MCF-7 human adenocarcinoma cells increased ~ 10 times. At the same time, the average tumor volume in animals treated with the proposed tool for the same period of time increased by only 5 times.

Hence we can conclude that the proposed tool has a pronounced antitumor effect against breast cancer, not only in cell culture in vitro, but also in vivo in an animal xenograft model.

A histological examination of the tissues of animals treated with the proposed tool (killed under ether anesthesia at the end of the experiment) showed that this drug in the doses used does not cause any changes in the cellular morphology of the liver, kidneys, and other functionally important organs, and also does not affect weight experimental animals.

Quantitative determination of the level of I3C and its main metabolite, DIM, by high performance liquid chromatography in blood plasma in C57Black / 6 mice after the psoral administration of the proposed agent and individual I3C substance.

The level of I3C and its main metabolite, DIM, in blood plasma in C57Black / 6 mice was determined by high performance liquid chromatography (HPLC). To obtain blood samples, animals of the experimental group were injected intragastrically (using a probe) with the proposed agent in an amount equivalent to 250 mg I3C / kg and an individual substance I3C in an amount of 250 mg I3C / kg. The control animals were injected with saline.

After 0.25; 0.5; 0.75; 1.0; 1.5; 2.0; 4.0; 6.0; 12.0; 18.0; 24.0 and 36.0 hours after the introduction of the proposed funds and after 0.25; 0.5; 0.75; 1.0; 1.5; 2.0; 4.0; 6.0; 12.0 and 24.0 hours after the administration of the individual I3C substance in experimental animals (3 animals for each time point), peripheral blood was taken from the tail vein and experimental plasma samples were prepared for subsequent HPLC analysis.

It should be noted that, in contrast to preclinical studies on pharmacokinetics, where the doses of the drug administered to animals corresponded to therapeutic, in this experiment, the doses of the proposed drug and the individual I3C substance administered to the animals were 20-40 times higher than therapeutic (250 mg I3C / kg of animal weight).

This choice was made consciously taking into account the well-known fact that the dose of the substance administered does not significantly affect the temporal pharmacokinetic parameters (maximum time, half-life), as well as the volume of distribution and clearance, while the maximum concentration of detectable (in plasma) substances is linearly with the dose of the compound administered.

Our goal was to study the stability of the proposed product in comparison with individual indole-3-carbinol. Obviously, determining the level of metabolites formed from I3C (in particular, diindolylmethane), the maximum concentrations of which in any case will be significantly lower than the concentration of the starting material, is more convenient at the highest possible dose of the latter.

Figure 5 presents chromatograms of plasma samples of control mice (not receiving the proposed tool and I3C) (Fig. 5A), as well as mice receiving the proposed tool (Fig. 5B, D) and the individual substance I3C (Fig. 5B, D) . In Fig.6 the dynamics of changes in the level of I3C and DIM in the blood plasma of experimental animals after the introduction of individual I3C and the proposed funds is displayed in diagrams.

The peak IS in all chromatograms corresponds to the internal standard 4-methoxyindole.

It was found that already 15 minutes after oral administration of individual I3C (in the amount of 250 mg I3C / kg) to the animals, the plasma concentration of I3C, which was rapidly absorbed through the gastrointestinal tract, reached a maximum value of 4.1 μg / ml. Over the next 60 minutes, the plasma level of I3C decreased rather quickly, so that 30 minutes after the introduction of individual I3C, its plasma concentration was already 2.2 μg / ml, after 45 minutes - 0.12 μg / ml, and after 60 min - generally not reliably determined, i.e. was below the detection limit (0.05 μg / ml).

In parallel with the decrease in time of the active concentration of I3C in the same animals, the plasma level of its main biologically active metabolite - DIM, on the contrary, steadily increased (Fig. 5G; Fig. 6B), reaching a maximum (0.70 μg / ml) after 2 hours from the moment of administration of I3C to animals.

4 hours after taking I3C, the almost twice-dropped active DIM concentration was still quite high (0.42 μg / ml) μg / ml) and remained almost at the same level (0.34 μg / ml) for the next 2 hours. By the end of the first day of the experiment, the DIM formed from indole-3-carbinol was completely excreted. It follows that orally administered I3C to animals under the influence of the acidic environment of the stomach almost immediately begins to turn into a dimeric form - diindolylmethane (DIM). At the same time, the intensity of this process increases in time, passing through a 2-hour maximum.

The proposed agent administered orally to animals (in an amount equivalent to 250 mg I3C / kg body weight) is somewhat slower than the individual I3C, but much more effectively absorbed in the gastrointestinal tract.

After the introduction of the proposed drug (which is a tablet dosage form, covered with an enteric-soluble coating), the I3C contained in it, which successfully passed the stomach and got into the intestine in an unsplit form, began to penetrate into the systemic circulation after about 15 minutes, reaching an average maximum level (~ 5.0 μg / ml) to 1.5 hours (Figure 5B), and then over the next 34.5 hours (36 hours from the start of the experiment) was gradually excreted (Figure 6A). Note that although the maximum value of the concentration of I3C (active active substance of the drug) in the plasma in this case was achieved not in 15 minutes, as in the experiment with individual I3C, but in 1.5 hours, the maximum effective concentration of I3C in the plasma (5.0 μg / ml) was almost a quarter more than in the experiment with individual I3C (4.1 μg / ml). Moreover, the peak corresponding to the diindolylmethane formed from I3C was absent in the chromatograms at the indicated time points. The contribution of other I3C metabolites to overall biological activity has also been minimized.

The increased concentration of I3C detected in plasma in comparison with the previous experiment is explained by the increased stability of the proposed product compared to the individual I3C substance. As follows from Fig.5B, 15 minutes after the introduction of individual I3C in animals, a whole spectrum of its metabolites is detected in plasma, i.e. the process of converting I3C into metabolically active derivatives under the influence of the acidic environment of the stomach is in full swing at this point, and part of the initial I3C pool has already been used up. In the case of the proposed tool, the metabolic process of the constituent I3C is blocked, which means that the total pool of the constituent I3C remains unchanged for a long time. Thus, the maximum level of I3C in plasma when taking the proposed drug should be higher than the maximum level of I3C when taking an individual I3C. However, the further pharmacokinetics of the proposed drug was fundamentally different from the pharmacokinetics of the individual substance I3C. After 2 hours from the start of the DIM experiment in the blood plasma of animals receiving the proposed drug, was still not reliably determined, while I3C remained at a very high level (4.25 μg / ml). Only 4 hours after the introduction of the proposed drug into the animals, it was possible to reliably detect DIM in plasma, the concentration of which, however, was only 0.06 μg / ml, i.e. was at the lower limit of the sensitivity of the method. At the same time, the plasma level at this time point of indole-3-carbinol (3.75 μg / ml) was 75% of the maximum (recall that in plasma samples obtained from animals after administration of individual I3C, the plasma DIM reached maximum values after 2 hours from the start of the experiment (Fig.6B). After another 2 hours after the introduction of the proposed drug to animals (6 hours from the start of the experiment), the plasma DIM concentration reached 0.10 μg / ml, while the concentration in I3C plasma was still quite high at this point (3 , 25 μg / ml or 65% of the maximum). Only 12 hours after the introduction of the proposed drug, the plasma level of DIM reached its maximum value (0.15 μg / ml). Thus, some part of the proposed tool indole-3 -carbinol, once in the intestine, managed to turn into diindolylmethane and other derivatives of I3C (which corresponds to published data on the slow metabolism of indole-3-carbinol at elevated pH), but the absolute value of this maximum was almost 5 times lower compared to that When turning in the acidic environment of the stomach of the individual I3C. 24-36 hours after the administration of the proposed agent to the animals, the diindolylmethane formed from I3C was almost completely eliminated from the body.

It follows from the experiments that the orally administered drug (and, in fact, the same individual I3C, but in a form protected from the acidic environment of the stomach), at least for several hours (which is more than enough for the manifestation of its therapeutic activity) exclusively as I3C, without the additional contribution of the biological activities of its metabolic derivatives, in particular DIM. Since the doses of the proposed drug introduced by us exceeded the therapeutic ones by more than an order of magnitude, there is every reason to believe that when the therapeutic doses of this drug are taken orally, the maximum plasma concentration of diindolylmethane formed from I3C will be many times lower than in our experiment, i.e. e. the presence of DIM will be of little clinical significance.

Thus, in the case of using the proposed drug as a therapeutic, the biological activities of I3C and DIM are completely disconnected (or rather, the contribution of DIM activity to the total activity of the drug over the study period is minimized), and therefore, this drug becomes completely predictable with respect to the mechanism action and spectrum of molecular targets.

The invention is illustrated by the following graphic materials:

Figure 1 - Hormonal regulation of gene expression.

Figure 2 - The structure of I3C and its metabolites detected in the blood plasma and tissues of experimental animals after oral administration of I3C.

I3C - indole-3-carbinol; DIM - 3,3'-diindolylmethane;

I3CA - indole-3-carboxylic acid;

I3A - indole-3-carboxaldehyde;

LTr _l is a linear trimer of [2- (indol-3-yl-methyl) -indol-3-yl] indol-3-ylmethane;

ICZ is indolo [3,2b] carbazole;

HI-IM - [1- (3-hydroxymethyl) -indolyl-3-indolylmethane].

Figure 3 - The effect of the proposed tool on the survival of tumor cells of the mammary gland of the hormone-sensitive line MCF-7 and the hormone-resistant line MDA-MB-468. The drug was added to the cells so that the final concentration of indole-3-carbinol included in its composition was 100 μM. Cell survival was evaluated as a percentage of the corresponding control and expressed as mean ± SEM (standard mathematical deviation) of three independent experiments. In each experiment, one experimental point is the average of three parallel measurements.

) и после внутрижелудочного введения животным предлагаемого средства (

).Figure 4 - Growth Dynamics of the grafted tumor of MCF-7 in athymic (nu / nu) C57Blask / 6 mice in control (

) and after intragastric administration to animals of the proposed agent (

)

Figure 5 - Chromatograms obtained by high performance liquid chromatography of blood plasma samples of C57Blask / 6 mice in control (A), 15 minutes (B) and 1 hour after oral administration of individual I3C (250 mg / kg) to animals (G ), as well as 1.5 hours later (B) and 4 hours later (D) after oral administration of the proposed product to the animals (250 mg I3C / kg). Peaks correspond to individual substances: I3C - indole-3-carbinol; DIM - 3,3'-diindolylmethane; I3CA - indole-3-carboxylic acid; I3A - indole-3-carboxaldehyde; LTr _l is a linear trimer of [2- (indol-3-yl-methyl) -indol-3-yl] indol-3-ylmethane; HI-IM - [1- (3-hydroxymethyl) -indolyl-3-indolylmethane]; IS - 4-methoxy-indole (internal standard).

6 - Dynamics of changes in the level of I3C (A) and DIM (B) in the blood plasma of experimental animals after the introduction of individual I3C and the proposed funds.

findings

Our studies have allowed us to establish that the proposed tool (tablets containing 0.1 g and 0.2 g of indole-3-carbinol, coated with an enteric coating) has a high specific antitumor activity against breast cancer cells in vitro and in vivo, namely:

1. The proposed tool (added to tumor cells so that the final concentration of I3C included in its composition was 100 μM) had a pronounced inhibitory effect on the survival of hormone-sensitive (ER +) breast cancer cells of the MCF-7 line and hormone-resistant (ER- ) breast tumor cells of the MDA-MB-486 line. Moreover, this inhibitory effect depended both on the dose of the drug and on the time of incubation of cell lines with these substances.

2. The introduction of the proposed agent to nude / nu C57Black / 6 mice (10 mg I3C / kg animal weight) significantly inhibited the growth of solid tumors induced by inoculation of MCF-7 human mammary tumor cells into animals. Moreover, the proposed tool in the doses used did not cause any changes in the cellular morphology of the liver, kidneys, and other functionally important organs, and also did not affect the weight of the experimental animals.

3. The conversion in the body of indole-3-carbinol, which is part of the proposed product, into other biologically active derivatives is minimized due to the protection of I3C from exposure to the acidic environment of the stomach. Thus, the orally administered agent proposed actually functions in the body exclusively as indole-3-carbinol without the additional contribution of the activities of diindolylmethane and other derivatives of I3C, and therefore becomes completely predictable with respect to the mechanism of action and the spectrum of molecular targets.

As we noted above, the mechanisms of regulation and functioning of the mammary glands and the endometrium (myometrium) of the uterus as the most important hormone-dependent (estrogen-dependent) tissues are similar in many respects. Since the organs of the female reproductive system function in close interaction with each other, often in medical practice mammological and gynecological diseases are found in the history of the same patient. In addition, there is direct literature data confirming the ability of I3C and its derivatives to suppress pathological proliferative processes not only in the mammary gland, but also in the endometrium (Kojima T, Tanaka T, Mori H. Chemoprevention of spontaneous endometrial cancer in female Donryu rats by dietary indole -3-carbmol. Cancer Res., 1994, 54, 1446-1449; Leong H, Firestone GL, Bjeldanes LF, Cytostatic effects of 3,3'-diindolylmethane in human endometrial cancer cells result from an estrogen receptor-mediated increase in transforming growth factor-alpha expression, Carcinogenesis (Lond) 2001: 22: 1809-1817). Given all of the above, as well as the natural origin and absolute safety of I3C - the main active substance of the studied drug, the proposed tool (tablets, enteric-coated tablets containing the active substance indole-3-carbinol 100 mg and 200 mg) can be recommended for complex treatment of benign proliferative diseases of the mammary gland and uterus, namely, various forms of mastopathy and fibroids.

Claims (1)

Antiestrogenic and antiproliferative agent for the treatment and prevention of diseases of the female reproductive system, which is a coated tablet containing indole-3-carbinol, as well as excipients, characterized in that it contains lactose, microcrystalline cellulose, butylhydroxyanisole, starch as excipients modified corn, magnesium stearate or calcium stearate, and as a shell enteric coating Acrylis with the following components, g: indole-3-carbinol 0.1-0.2 lactose 0.10-0.15 butylhydroxyanisole 0.0001-0.0002 microcrystalline cellulose 0.10-0.150 modified corn starch 0.03-0.05 magnesium stearate or calcium stearate 0.002-0.004 enteric coating 0.03-0.05

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