WO2021188013A1 - Method for treating endometriosis and concomitant formation of adhesions - Google Patents

Abstract

The invention relates to the field of medicine and may be used in treatment and prophylaxis of female reproductive system diseases. A method for treating endometriosis and concomitant formation of adhesions consists in an oral administration of a drug comprising a complex of 3,3'-diindolylmethane with β-cyclodextrin at a dosage of 400-600 mg of 3,3'-diindolylmethane per day. Said ingredients are present in the following ratio, wt %: 3,3'-diindolylmethane 10-20, β-cyclodextrin 80-90. A tablet dosage form is preferred for administration of the drug. The use of the active substance 3,3'-diindolylmethane complexed with β-cyclodextrin makes it possible to increase the bioavailability of the active substance, hence to increase the treatment efficacy.

Description

METHOD FOR TREATMENT OF ENDOMETRIOSIS AND ASSOCIATED ADHESION PROCESS

TECHNICAL FIELD The invention relates to the field of 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 are heard at the reception of gynecologists all the time; 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 one of the most common gynecological diseases today, affecting women of the most active age (usually in the range from 20 to 40 years, although there are cases of much earlier manifestation). It is known that endometriosis is found in half of the patients undergoing treatment for infertility, and in the majority of women (up to 80%) with chronic pelvic pain. The Caucasoid race is susceptible to this pathology to a greater extent than the other two [L. V. Adamyan and others. Endometriosis: A Guide for Physicians (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 abundance of menstruation), persistent nausea. As mentioned above, endometriosis can cause infertility and constant 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 regulating the vital activity of endometrial cells, in particular, suppressing 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 the administration of 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 lesions of endometriosis. 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 alter 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 aggravates. 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, intestinal obstruction, etc.

For this reason, fundamental studies of the etiology and pathogenesis of endometriosis remain 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.O pp. 1-12; M.-H. Wu et al. Hypoxia: The force of endometriosis (2019) J. Obstet. Gynaecol. Res. doi: 10.111 l / 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 the 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.O, No.O pp. 1-10].

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

3,3'-diindolylmethane (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 (13 C) 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, 13 C almost instantly turns into DIM [Ameson 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 studying the antitumor activity of 13 C are inclined to believe that most of the clinical effects observed when taking 13 C are in fact due to the dimeric form of indole-3-carbinol - DIM. It has been experimentally proven that DIM has a very wide spectrum of antiproliferative activity [Chang X, Toy 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- Spl 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 13 C- 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 most important molecular target, the blocking of the activity of which is directed by the action of modern targeted drugs (targeted drugs) being developed and introduced into clinical practice, is the nuclear transcription factor NF-kB. 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. Found that in vitro DIM [Rahman KM, AN 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-kB contributes to 3,3'-dHndolylmethane-induced apoptosis in breast cancer cells (2005) Cancer Res., 65, 364-371], as well as its metabolic precursor 13 C, effectively suppress nuclear translocation and the activity of NF-kB factor ... This means that in addition to the antiproliferative and antiangiogenic action, 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 PBK / Akt / mTOR / NF-kV signaling pathway, which is inhibited by DIM, which can 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-KB 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 migratory and invasive ability 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 an oral 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 patent 6086915 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.

The essence of the invention

The oral method of dosing drugs based on DIM is the most preferred, since it contains a number of advantages over other methods of dosing, such as the greatest comfort for the patient, the possibility of flexible treatment regimens, as well as the 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 increases the bioavailability by only 2 times.

Considering this, as well as the aforementioned molecular mechanisms of DIM action, 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 the oral administration of a drug containing 3,3'-diindolylmethane, which is characterized by the fact that a complex of 3,3'-diindolylmethane and b-cyclodextrin is used as a drug at a dosage of 400 -600 mg 3,3'-diindolylmethane per day.

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

3,3'-diindolylmethane 10-20 b-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 concomitant adhesive process.

Examples of implementation of the invention

Example 1.

A method of obtaining a dosage form of a complex of 3,3'-diindolylmethane and b-cyclodextrin (DIM + pCD).

The b-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 was added to the b-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 - b-cyclodextrin (IM + bOϋ) was 5350 g. The dry product was sieved through a 1 mm sieve.

The ratio of DIM and dry b-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.

A solution of the ϋIM + bOϋ complex was subjected to chromatographic analysis. The results of physicochemical analysis showed almost complete coincidence of the chromatograms of pure 3,3'-diindolimethane and the IM + bOϋ complex. The complex was also prepared with the following ratios of DIM and dry b-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 the other 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 the water-soluble DIM + PCD complex and crystalline 3,3'-diindolylmethane in equal dosages. In all studied groups of patients, there was a pronounced positive dynamics when taking the DIM + PCD 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.

Результаты испытаний показали, что устойчивые параметры биодоступности достигнуты при использовании водорастворимого комплекса DIM+PCD в дозировке 100 мг в день. При этом концентрация DIM в плазме при приеме 100 мг водорастворимых комплексов составила более 300 нг/мл. Из результатов испытаний также видно, что биодоступность при использовании комплекса DIM+PCD намного превышает биодоступность комплекса DIM+TPGS+PS по прототипу. Table 1

The test results showed that stable bioavailability parameters were achieved when using a water-soluble DIM + PCD 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 + PCD complex is much higher than the bioavailability of the DIM + TPGS + PS complex according to the prototype.

Example 3.

Composition of the preparation.

Oral tablets, composition:

DIM + PCD complex - 71%

Microcrystalline cellulose - 21%;

Sodium starch glycolate - 7%;

Magnesium stearate - 1%

Tablet options:

1) For resorption or chewable, uncoated

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

Example 4

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

Materials and methods: The experiment was carried out on sexually mature female rats (60 animals). The 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 DIM + PCD complex at a dosage of 5 mg / kg, in groups 3-5, modeling was carried out endometriosis, by autologous transplantation of endometrial tissue into the abdominal cavity. Animals in group 3 received a 1% starch solution, in group 4 they received an active drug at a dosage of 1 mg / kg, animals in 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) of endometriomas, M ± w.

table 2

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

Conclusions: The study showed that diindolylmethane 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 stage 2 endometrioid ovarian cyst using an oral dosage form based on the DIM + pCD complex. For a 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. Patients received oral tablet form DIM + pCD. 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.

Примечание: Интенсивность выраженных симптомов оценивалась какTable 3

Note: The intensity of severe symptoms was assessed as

++++ - pronounced +++ - moderately pronounced ++ - weakly expressed + - episodic manifestations

- no symptoms

Example 6

Preclinical study of the specific pharmacological activity of various doses of the drug DIM + pCD on the model of the adhesion 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. Table 4

The experimental results are shown in table 5.

Table 5

Note:

According to all evaluative + + + + + pronounced adhesion process criteria + + + + pronounced adhesion process

+ + + signs of inflammation + + lack of inflammation

Claims

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