RU2740377C1 - Method of treating endometriosis and accompanying adhesive process based on 3,3'-diindolylmethane in combination with b-cyclodextrin - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention refers to pharmaceutics, namely to a method of treating endometriosis and accompanying adhesive process. Method comprises administering by a patient a medicinal agent containing complex of 3,3-diindolylmethane and β-cyclodextrin, with a certain ratio of components and dosage of 3,3'-diindolylmethane 400–600 mg a day.

EFFECT: invention provides higher bioavailability and improved therapeutic effect of the medicinal agent containing 3,3-diindolylmethane for treating endometriosis, as well as providing effective treatment of accompanying adhesive process.

1 cl, 5 tbl, 6 ex

Description

Technology area

The invention relates to medicine and can be used for the treatment and prevention of diseases of the female reproductive system.

State of the art

Endometriosis is a systemic pathological process in which the cells of the endometrial layer spread outside the uterus and, growing, form foci of the uterine mucosa in other parts of the body.

The prevalence of endometriosis has skyrocketed in recent decades. If in the middle of the 20th century only specialists knew about this disease, today characteristic complaints sound at the reception of gynecologists constantly; the diagnosis itself, as they say, is more and more heard - and more and more attracts the attention of research medicine, since many questions remain unclear. There is also no precise epidemiological data: published approximate estimates range from 6-7% to 50% of the general population of women. A number of authors consider endometriosis to be one of the most common gynecological diseases today, affecting women of the most active age (usually in the interval from 20 to 40 years, although there are cases of much earlier manifestation). It is known that endometriosis is found in half of patients undergoing treatment for infertility and in most women (up to 80%) with chronic pelvic pain. The Caucasian race is susceptible to this pathology to a greater extent than the other two [L.V. Adamyan et al. Endometriosis: a guide for doctors (2006) Moscow: Medicine, Ed. 2nd, rev. and additional, 410 pages].

The most common and pronounced symptom of endometriosis is the soreness of physiological acts (urination, menstruation, coitus, defecation) and a standard gynecological examination. Sometimes there are also disorders of the cycle, menorrhagia (abnormal profusion of menstruation), persistent nausea. As mentioned above, endometriosis can cause infertility and persistent pain in the lower abdomen.

Currently, there are many hypotheses for the development of endometriosis. Immune system disorders, genetic and epigenetic factors, hormonal metabolism disorders, failures in the endometriotic cell differentiation program, inflammation and microbial factor and much more are considered.

Modern approaches to the treatment of endometriosis include conservative and surgical treatment. All modern standards of conservative treatment are aimed at regulating hormonal mechanisms of regulation of the vital activity of endometrial cells, in particular, suppression of estrogen synthesis. For the treatment of endometriosis, estrogen-suppressing drugs are widely used, including birth control pills: Depo-Provera®, Lupron® and Danazol®. They have many side effects, including depression and osteoporosis.

Another medical approach is to administer a low-dose progestin: dienogest in Visanne® tablets or levonorgestrel in the Mirena® IUD. Low doses of progestins work more slowly because they do not suppress ovulation or estrogen. Instead, it prevents the growth of endometriosis foci.

There is an active search for new non-hormonal treatments for endometriosis, including the following:

Angiogenesis inhibitors (drugs that inhibit angiogenesis or the growth of new blood vessels)

- anti-inflammatory drugs (drugs that reduce inflammation);

- immunomodulators (drugs that change the activity of the immune system) [Simone Ferrero et al. Current and emerging treatment options for endometriosis (2018) Expert Opinion on Pharmacotherapy doi: 10.1080 / 14656566.2018.1494154; E. Tafi et al. Advances in pharmacotherapy for treating Endometriosis (2015) Expert Opin. Pharmacother. 16 (16)].

Surgical treatment remains an essential element in the treatment of endometriosis. However, in a large percentage of cases, the process recurs and with each next intervention, the severity of the disease only worsens. In addition, endometriosis is often accompanied by adhesive disease, and any, even laparoscopic intervention in the pelvic area stimulates the development of adhesions, which ultimately affects the severity of the disease, causing severe pain syndrome, bowel obstruction, etc.

For this reason, fundamental research on the etiology and pathogenesis of endometriosis remains an important practical task, since they open up new mechanisms for the development of the pathological process and create the prerequisites for the creation of drugs that act on the key mechanisms of pathology without affecting the metabolism of female sex hormones.

In recent years, many fundamentally new mechanisms for the development of endometriosis have been described.

In particular, the role of intracellular signaling cascades has been established, the hyperactivity of which determines the high proliferative activity of endometrial cells [Annu Makker et al. PI3K-Akt-mTOR and MAPK signaling pathways in polycystic ovarian syndrome, uterine leiomyomas and endometriosis: an update (2012) Gynecological Endocrinology, 28 (3): 175-181; Daniela Madanes et al. PI3K / AKT pathway is altered in the endometriosis patient's endometrium and presents differences according to severity stage (2019) Gynecological Endocrinology pp 1-5; doi: 10.1080 / 09513590.2019.1680627]. Show the critical role of hypoxia and pathological angiogenesis in the development of endometriosis [Xiong et al. Hypoxia-inducible factor la-induced epithelial-mesenchymal transition of endometrial epithelial cells may contribute to the development of endometriosis (2016) Human Reproduction, Vol.0, No.0 pp.1-12; M.-H. Wu et al. Hypoxia: The force of endometriosis (2019) J. Obstet. Gynaecol. Res. doi: 10.1111 / jog.13900]. The role of epigenetic modifications in the genome of endometrial cells has been established as a result of which the genetic program of cells changes, forcing them to continuous division and invasion [Koukoura et al. DNA methylation in endometriosis (Review) (2016) Molecular Medicine Reports 13: 2939-2948; Caplakova et al. DNA Methylation Machinery in the Endometrium and Endometrial Cancer (2016) ANTICANCER RESEARCH 36: 4407-4420; Kuei-Yang Hsiao et al. Epigenetic regulation of the pathological process in endometriosis (2017) Reprod Med Biol. 16: 314-319]. It turned out that with endometriosis, the processes of epithelial-mesenchymal transition are actively triggered, which is the reason for active migration and invasion of endometrial cells into the surrounding tissues [Matsuzaki and Darcha, Epithelial to mesenchymal transitionlike and mesenchymal to epithelial transition-like processes might be involved in the pathogenesis of pelvic endometriosis (2012) Human Reproduction, Vol.0, No.0 pp. - ten].

Among the very promising molecules that are considered as potential drugs for the treatment of endometriosis, we can name a group of indole compounds, a prominent representative of which is 3,3'-diindolylmethane.

3,3'-diindol yl methane (diindolylmethane, DIM), its analogs and derivatives have a wide range of biological activities, which allows us to consider it as a very promising pharmacologically active compound. DIM is the main oligomeric product of indole-3-carbinol (I3C) for which a pronounced selective activity has been proven against transformed cells of various origins [Aggarwal BB, Ichikawa H. Molecular targets and anticancer potential of indole-3-carbinol and its derivatives. Cell Cycle 2005,4 (9) 1201-1215].

As shown by pharmacokinetic studies, when administered orally under the influence of the acidic environment of the stomach, I3C is almost instantly converted to DIM [Arneson DW, Hurwitz A, McMahon LM, Robaugh D (1999) Presence of 3,3'-diindolylmethane in human plasma after oral administration of indole -3-carbinol (abstr.) Proc. Am. Assoc. Cancer Res, 40, 2833], therefore, many authors investigating the antitumor activity of I3C are inclined to believe that most of the clinical effects observed when taking I3C are in fact due to the dimeric form of indole-3-carbinol - DIM.

It has been experimentally proven that DIM has a very broad spectrum of antiproliferative activity [Chang X, Tou JC, Hong C et al. (2005) 3,3'-Diindolylmethane inhibits angiogenesis and the growth of transplantable human breast carcinoma in athymic mice. Carcinogenesis, 264 (4), 771-778; Firestone GL, Bjeldanes LF (2003) Indole-3-Carbinol and 3,3'-Diindolylmethane anti-proliferative signaling pathways control cell cycle gene transcription in human breast cancer cells by regulating promoter-Sp1 transcription factor interactions. J. Nutr., 133, 2448S-2455S; Ge X, Yannai S, Rennert G et al. (1996) 3,3'-Diindolylmethane induces apoptosis in human cancer cells. Biochem. Biophys. Res. Commun. 228,153-158; Li Y, Li X, Sarkar FH. (2003) Gene expression profiles of I3C- and DIM-treated PC3 human prostate cancer cells determined by cDNA microarray analysis. J. Nutr., 133,1011-1019; Nachshon-Kedmi M, Yannai S, Haj A, Fares FA. (2003) Indole-3-carbinol and 3,3'-diindolylmethane induce apoptosis in human prostate cancer cells. Food Chem. Toxicol., 41, 745-752].

The nuclear transcription factor NF-κB is the most important molecular target, the blocking of the activity of which is directed by the action of modern targeted drugs (drugs of targeted action) being developed and introduced into clinical practice. It has been proven that this factor mediates the inflammatory response, and also plays an important role in the regulation of proliferative (antiapoptotic), angiogenic, migratory and invasive cellular activities, carrying out the final stage of signaling cascades induced by growth factors and cytokines. In this case, the key point is the translocation of the active factor into the nucleus and the activation of the transcription of the genes responsible for these processes. It has been established that in vitro DIM [Rahman KM, Ali S, Aboukameel A et al. (2007) Inactivation of NF-kappaB by 3,3'-diindolylmethane contributes to increased apoptosis induced by chemotherapeutic agent in breast cancer cells. Mol. Cancer Ther., 6 (10), 2757-2765; Rahman KM, Sarkar FH. Inhibition of nuclear translocation of Nuclear Factor-κB contributes to 3,3'-diindolylmethane-induced apoptosis in breast cancer cells (2005) Cancer Res., 65, 364-371], as well as its metabolic precursor I3C, effectively suppress nuclear translocation and activity factor NF-κB. This means that in addition to the antiproliferative and antiangiogenic effects, the drug produced on the basis of DIM is capable of suppressing local inflammatory reactions that often accompany hyper- and neoplastic processes in hormone-dependent organs and tissues [Gonza'lez-Ramos et al. Nuclear factor-kappa B is constitutively activated in peritoneal endometriosis (2007) Molecular Human Reproduction Vol. 13, No. 7 pp. 503-509].

As mentioned earlier, in endometriosis, an increased activity of signaling cascades that stimulate the proliferation of endometrial cells was found. Of particular importance is the PI3K / Akt / mTOR / NF-κB signaling pathway, which is inhibited by DIM, which may be of great practical importance in the treatment of endometriosis with drugs based on indole derivatives [Popolo et al. Two likely targets for the anti-cancer effect of indole derivatives from cruciferous vegetables: PI3K / Akt / mTOR signaling pathway and the aryl hydrocarbon receptor (2017) Seminars in Cancer Biology V 46, Pages 132-137; Ahmad et al. Targeted Regulation of PI3K / Akt / mTOR / NF-κB Signaling by Indole Compounds and their Derivatives: Mechanistic Details and Biological Implications for Cancer Therapy (2013) Anticancer Agents Med Chem. 13 (7): 1002-1013]. An important property of DIM is its ability to inhibit the processes of epithelial-mesenchymal transition (EMT), which determine the migration and invasive ability of cells. It should be noted here that EMF processes play an important role in the development of adhesive disease. Thus, acting on these mechanisms, DIM is theoretically capable of not only suppressing migration and invasion of endometrial cells, but also preventing the development of adhesions [P Sandoval et al. Mesothelial-to-mesenchymal transition in the pathogenesis of post-surgical peritoneal adhesions (2016) Journal of Pathology 239: 48-59].

There is a known method for the treatment of endometriosis using indole, including 3,3'-diindolylmethane (DIM), in which the patient takes orally a drug that is DIM in combination with substances that increase its bioavailability, distributed in a carrier in the form of a starch matrix, in a dosage from 30 to 500 mg per day described in US patent 7384971 B2, 10.06.2008. The same patent indicates that the compositions of such DIM complexes and the method for their preparation are described in US Pat. No. 6,086,915 A, 07/11/2000. In particular, these can be DIM complexes comprising one or more solubilizing emulsifiers selected from the group consisting of tocopherol polyethylene glycol succinate (TPGS), polyethylene glycol 1000, polyvinylpyrrolidone, polyoxyethylene stearate, sodium cholate, deoxycholate and also one or more taurocholate, ao active phospholipids selected from the group consisting of phosphatidylcholine (PC), dioleoyl phosphatidylcholine, phosphatidylglycerol, and the like.

This method is taken as a prototype.

Disclosure of invention

The oral dosing method for DIM-based drugs is the most preferred because it contains a number of advantages over other dosing methods, such as the greatest comfort for the patient, flexible treatment regimens, and low cost of treatment. However, the oral bioavailability of DIM is severely limited due to its very low solubility and low absorption efficiency in the small intestine. DIM usually exhibits low solubility in physiological fluids and has a very limited ability to penetrate barrier membranes, which does not allow reaching the required concentrations of the active substance in the lesions.

Our studies of the bioavailability of DIM as part of the DIM + TPGS + PS complex according to the prototype (Bioresponse DIM® drug) in humans showed that such a complex only doubles the bioavailability.

Considering this, as well as the aforementioned molecular mechanisms of action of DIM, namely, restoration of apoptosis processes, antiproliferative, antitumor and antiangiogenic activity, inhibition of EMF and epigenetic activity, the technical problem of this invention was the development of a method for the treatment of endometriosis and associated adhesions using a drug with increased bioavailability ...

The technical problem is solved by a method for the treatment of endometriosis and concomitant adhesions, which consists in oral administration of a drug containing 3,3-diindolylmethane, which is characterized by the fact that a complex of 3,3'-diindolylmethane and β-cyclodextrin is used as a drug at a dosage of 400- 600 mg of 3,3'-diindolylmethane per day.

In addition, a drug is used with the following preferred ratio of components, wt%:

3,3-diindolylmethane 10-20 β-cyclodextrin 80-90

It is also preferable to use the drug in tablet form.

The technical result of the invention is to ensure the use of a drug with a higher bioavailability compared to the prototype, which increases the therapeutic effect of DIM, which has a dose-dependent nature, as well as to provide an effective treatment for the associated adhesions.

Examples of implementation of the invention

Example 1

A method for preparing a dosage form of a complex of 3,3'-diindolylmethane and β-cyclodextrin (DIM + βCD)

The β-cyclodextrin powder in an amount of 5600 g (moisture content 12%) was dissolved in 22.6 liters of water, gradually stirring. 713 g of 3,3'-diindolylmethane were added to the β-cyclodextrin solution. The suspension was stirred for 4 hours. The resulting suspension was fed into a spray dryer at a gas temperature of 180 ° C. The yield of the obtained complex 3,3-diindolylmethane-β-cyclodextrin (DIM + βCD) was 5350 g. The dry product was sieved through a sieve with a sieve element opening of 1 mm.

The ratio of DIM and dry β-cyclodextrin in the resulting product was 12.6 wt% and 87.4 wt%, respectively, which corresponds to the molar ratio of these components in the complex.

The resulting complex dissolves completely in water.

The DIM + βCD complex solution was subjected to chromatographic analysis. The results of physicochemical analysis showed almost complete agreement between the chromatograms of pure 3,3'-diindolimethane and the DIM + βCD complex.

The complex was also prepared with the following ratios of DIM and dry β-cyclodextrin: DIM from 10 to 20 wt%, PCD from 80 to 90 wt%. The deviation of the ratio from the molar ratio within the indicated limits and a slight excess of one or another component in the resulting product does not affect the properties of the drug.

Example 2

Study of the concentration of 3,3'-diindolimethane in the blood plasma of patients

Clinical studies have compared the efficacy of a water-soluble DIM + βCD complex and crystalline 3,3'-diindolylmethane in equal dosages. In all the studied groups of patients, a pronounced positive trend was established when taking the DIM + βCD complex and the absence of such when taking crystalline 3,3'-diindolylmethane. The results of measuring the concentration of 3,3'-diindolylmethane in the plasma of patients are presented in Table 1.

The test results showed that stable bioavailability parameters were achieved using the water-soluble DIM + βCD complex at a dosage of 100 mg per day. At the same time, the concentration of DIM in plasma when taking 100 mg of water-soluble complexes was more than 300 ng / ml. The test results also show that the bioavailability when using the DIM + βCD complex is much higher than the bioavailability of the DIM + TPGS + PS complex in the prototype.

Example 3

Composition of the preparation

Oral tablets, composition:

DIM + βCD complex - 71%

Microcrystalline cellulose - 21%;

Sodium starch glycolate - 7%;

Magnesium stearate - 1%

Tablet options:

1) For resorption or chewable, without shell

2) Sustained or conventional release, both uncoated and with enteric coating.

Example 4

Study of the medicinal properties of the DIM + βCD complex in a rat endometriosis model.

Materials and methods: The experiment was carried out on sexually mature female rats (60 animals). Animals were divided into groups according to pathology modeling and therapy. Group 1 - sham-operated animals receiving starch solution, group 2 - sham-operated animals receiving the DIM + βCD complex at a dosage of 5 mg / kg, in groups 3-5, endometriosis was simulated by autologous transplantation of endometrial tissue into the abdominal cavity. Animals of group 3 received 1% starch solution, in group 4 they received an active drug at a dosage of 1 mg / kg, animals of group 5 received an active drug at a dosage of 5 mg / kg. The drug was administered in the form of an aqueous suspension. On days 30 and 60, a histological evaluation of the implants was performed. Table 2 shows the morphometric characteristics (volume, mm ³ ) of endometriomas, M ± m.

Results: According to the results of the histological assessment, a tendency towards a decrease in the degree of endometrial hyperplasia was revealed in the groups of females receiving the active drug, compared with the control groups.

Conclusions: The study showed that diindol or methane in combination with cyclodextrin, when administered to animals, had a therapeutic effect and effectively reduced the size of the endometrioma and the severity of endometrioid heterotopies.

Example 5

Treatment of women diagnosed with endometrioid ovarian cyst, stage 2, using an oral dosage form based on the DIM + βCD complex.

For the clinical study, women were selected at the age of 30-35 years, with complaints of pain in the lower abdomen, irregularity and soreness of the menstrual cycle. On the basis of ultrasound and laparoscopic examination, the diagnosis was made: endometrioid cyst of one of the ovaries, stage 2.

The women were divided into two groups of 5 people. The observation was carried out for 3 months. The patients received oral tablet form DIM + βCD. Based on the content of pure diindolylmethane in the preparation: the 1st group took 200 mg -2 times a day, the 2nd group took 200 mg 3 times a day. Two clinical parameters were assessed: 1) the presence and intensity of pain syndrome; 2) the frequency and intensity of menstrual bleeding. The research results are shown in Table 3.

Example 6

Conducting a preclinical study of the specific pharmacological activity of various doses of the drug DIM + βCD on the model of the adhesive process in rats

When setting up the experiment, the protocol described in the work of Yue Y, Yan S, Li H, Zong Y, Yue J, Zeng L. was taken as a basis. The role of oral fluvastatin on postoperative peritoneal adhesion formation in an experimental rat model. Acta Chir Belg. 2018 Dec; 118 (6): 372-379. doi: 10.1080 / 00015458.2018.1444549. Epub 2018 Feb 26.

The experiment is shown in Table 4.

Claims (3)

1. A method for the treatment of endometriosis and concomitant adhesions, which consists in the patient taking a drug containing 3,3'-diindolylmethane, characterized in that a complex of 3,3-diindolylmethane and β-cyclodextrin is used as a drug at a dosage of 400-600 mg 3,3'-diindolylmethane per day with the following ratio of components, wt%: 3,3'-diindolylmethane 10-20 β-cyclodextrin 80-90 2. The method according to claim 1, characterized in that the drug is used in tablet form.