RU2196568C1 - Pharmaceutical composition for prophylaxis and treatment of uterus cervix dysplasia and cancer and larynx papillomatosis and method of prophylaxis and treatment of said sicknesses based on thereof - Google Patents

Abstract

FIELD: medicine, pharmacology, pharmacy. SUBSTANCE: invention relates to pharmaceutical composition used for prophylaxis and treatment of tumors associated with human papilloma viruses, namely, uterus cervix dysplasia and cancer and larynx papillomatosis. Invention proposes composition based on indole-3-carbinol as active component and methods of prophylaxis and treatment based on thereof. Invention provides another mechanism of inhibition of tumor growth with indole-3-carbinol being applicable for uterus cervix and larynx tumor cells. EFFECT: improved method of prophylaxis and treatment, valuable antitumor properties. 5 cl, 2 dwg, 6 ex

Description

 The invention relates to the field of medicine and pharmacology, specifically to a composition for the treatment and prevention of cervical dysplasia and laryngeal papillomatosis, a method for the prevention and / or treatment of these diseases using the proposed composition.

 Approximately 1 million women in the world are annually diagnosed with cervical cancer, which ranks second as the cause of death from cancer among women. Despite screening programs in the United States that detect cervical cancer at an early stage, the incidence of invasive cervical cancer is not decreasing.

 The level of estrogen synthesis is one of the risk factors in the process of cancerous degeneration of epithelial cells in estrogen-dependent zones, which primarily include the female reproductive organs: uterine cavity, ovaries, mammary glands and including the cervix (Seprowich et al. "Estrogen metabolitic retions and rick assessment of hormone related cancer, Assay validation and prediction of cervical cancer rick ", Annals of NY of Science, v. 768, pp. 312-316, 1995).

 Currently, however, in medical practice, endocrine therapy, in particular antiestrogen therapy, is used to prevent and treat mainly breast cancer, where antihormonal drugs are first-line drugs. Breast tumors can have estrogen, progesterone, and androgen receptors. In this case, they are significantly more sensitive to hormonal influences. The selection of the most effective method is based on the analysis of tumor cell receptors.

 Cervical cancer refers to tumors in which chemotherapy is often ineffective (group B, while breast carcinoma belongs to group B, in which chemotherapy is indicated), while cytotoxic drugs such as mitomycin are classified as first and second row drugs, methotrexate, cyclophosphamide, bleomecin (D.R. Lurie. Clinical Pharmacology, v. 1, p. 553-573). Treatment measures for dysplasia and cervical cancer are reduced mainly to methods of surgical removal, cryo- or laser destruction of the lesions (Big Medical Encyclopedia, t. 15, p. 409/2, t. 28, p. 355/3, Pat Russia 931035244. A method for the treatment of precancerous changes in the cervix with laser destruction). Laryngeal papillomamatosis is treated exclusively by surgery, most recently - by the method of laser tumor destruction (BME, t. 6, laryngeal papillomatosis).

 In medical practice (for the treatment and prevention of breast cancer), estrogen antagonists such as raloxifene and tamoxifen are most widely used (see structural formulas). Therapy with these antiestrogens leads to side effects, such as an increased risk of ovarian cancer and osteoporosis. Some of these side effects are due to the fact that in some tissues they act as estrogen agonists.

Одним из перспективных направлений поиска антагонистов эстрогена является скрининг веществ-аналогов природных индолов, содержащихся в капустных культурах и показавших способность ингибировать канцерогенез грудных желез в ряде модельных экспериментов, а именно индолкарбинола или его димера (Kojima J. et al. Cancer Re. 54,1446-1449,1984; Crubbs C.J. et al. Anticancer Re. 15, 709-716,1995).

One of the promising directions in the search for estrogen antagonists is the screening of analogous substances of natural indoles contained in cabbage cultures and showing the ability to inhibit breast carcinogenesis in a number of model experiments, namely indolcarbinol or its dimer (Kojima J. et al. Cancer Re. 54.1446 -1449.1984; Crubbs CJ et al. Anticancer Re. 15, 709-716.1995).

где хотя бы один из заместителей не водород, применяемые для обработки эстрогензависимых раковых опухолей груди и эндометрия (пат. РСТ 98/50357).So known substituted indole-3-carbinol, diindolylmethane General formula

where at least one of the substituents is not hydrogen, used to treat estrogen-dependent breast and endometrial cancers (Pat. PCT 98/50357).

(пат.США 5877202)

(пат. США 6159959), композиции на их основе для лечения неопластических заболеваний, в частности шейки матки.Indole derivatives of the general formula are also known.

(US Pat. 5877202)

(US Pat. US 6159959), compositions based on them for the treatment of neoplastic diseases, in particular the cervix.

 The disadvantage of the above substances is their greater toxicity compared to natural analogues. In addition, these substances and compositions based on them are suitable for estrogen receptor-positive dysplasias and cervical cancers. The last of these two analogues can be taken as a prototype.

 The objective of the invention is to expand the arsenal of funds for this purpose with less toxicity, suitable for both receptor-positive and receptor-negative cancers, cervical dysplasia, to develop a new approach to the treatment of laryngeal papillomatosis, to increase the effectiveness of treatment and prevention of these diseases based on the mechanism of action of the active the beginning of the proposed pharmaceutical composition. (The high effectiveness of the proposed pharmaceutical composition in the treatment and prevention of cervical cancer and laryngeal papillomatosis is explained by the fact that the activity of human pipalloma virus (HPV) genes is the basis of tumor degeneration of epithelial cells of the cervix and larynx. The authors found that indole-3-carbinol inhibits expression of HPV oncogenes, thus stopping the carcinogenesis process).

 The problem is solved in that a pharmaceutical composition is proposed for the prevention and treatment of cancer and cervical dysplasia and laryngeal papillomatosis, containing as an active principle indole-3-carbinol in an effective amount and a suitable pharmaceutical carrier.

 The composition may additionally contain no more than 100 nM actinomycin D, when used in the form of vaginal suppositories.

 The preferred dosage form for this composition are capsules or suppositories necessary for the proposed methods of their use.

 The problem is also solved by the fact that a method for the prevention and treatment of dysplasia and cervical cancer is proposed, which consists in analyzing a tumor or cervical dysplasia for the presence of infection of epithelial cells with human papillomavirus type 16, 18, 31, 45 and, in the case of a positive result, a course of indole-3-carbinol or its mixture with actinomycin-D in the form of vaginal suppositories is prescribed as the main or auxiliary effect.

 The problem is also solved by the fact that a method for the treatment of laryngeal papillomatosis by laser tumor destruction is proposed, characterized in that in the postoperative period, long-term (throughout the year) indole-3-carbinol therapy is prescribed in the form of capsules for oral administration.

 The content of active principle in the composition is from 0.1 to 99%. The carrier may be a solid, semi-solid or liquid material and acting as a carrier, excipient or medium for the active component. Examples of suitable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, phosphate, calcium silicate, microcrystalline cellulose, vegetable oils, talc, magnesium stearate, water and mineral oils.

 Compositions may also include wetting agents, lubricants, preservatives, perfumes and stabilizers.

 The compositions are preferably used in the form of capsules (for oral administration) or vaginal suppositories in the form of a standard dose: for capsules - 200-400 mg of active principle, for suppositories - 10-100 mg of active principle, suppositories may contain additional 1-100 nM actinomycin D.

 Indole-3-carbinol used in the framework of the present invention is effective in wide dosage ranges. Daily doses are in the range of 1-50 mg / kg body weight, preferably 5-10 mg / kg body weight in a single or divided dose. However, it is understood that the amount of the compound actually administered is determined by the physician depending on the circumstances, including the condition being treated, the weight and reaction of the individual patient, the severity of the symptoms and the chosen treatment path.

 The analysis of the cervical epithelium for infection with the human papillomavirus is carried out by the polymerase chain reaction method according to the known method (Polymerase chain reaction in the diagnosis of urogenital infections. - Kiselev V.I.-M., 2000. A manual for doctors).

 As already mentioned, indole-3-carbinol is able to slow the growth of breast tumor cells in model experiments. An anticarcinogenic effect on liver cell models and a reduction in dysplasia of the upper stomach caused by benzopyrene have also been described (Shertrer Y. G. Chomico-Biol. Interections, 48, 81-90, 1996).

 The antitumor effect of indole-3-carbinol was associated with its antiestrogenic activity, but later it was found that he himself was not able to bind to estrogen receptors, but only his dimer or trimer, into which he converted at a low pH of the stomach.

 For the first time in US Pat. US 6001868, the mechanism of the antitumor action of indole-3-carbinol on breast cells has been disclosed. It shows that it does not depend on the estrogenic mechanism of action, but is the result of a suspension of the G-cell cycle, which correlates with a decrease in CDK6 protein. Indole-3-carbinol and its acid-resistant counterparts can inhibit the growth of estrogen-dependent and estrogen-independent breast cancer cells and other types of cancer cells capable of inducing CDK6.

 The basis of the invention is another mechanism of inhibition of tumor growth with indole-3-carbinol, applicable only to tumor cells of the cervix and larynx. Indole-3-carbinol, as established by the applicant, inhibits the expression of oncogenes E6 and E7 of human papillomavirus (types 16, 18, 31, 45), which infects epithelial cells of the cervix and larynx, the products of which inactivate two key suppressor proteins that control cell proliferation, namely, p53 and pRB proteins.

 It should be clarified that over the past 15 years, epidemiological and virological studies unequivocally confirm the connection of cervical cancer, cervical dysplasia with human papillomavirus (HPV). Although over 100 types of HPV are currently described, about 80% of cases of cervical cancer are associated with four types of HPV (16, 18, 31, 45). The most widespread type 16 virus, which occurs in 50% of cases.

 Actinomycin D, a well-known anti-cancer antibiotic, as established by the applicant, is also able to influence the development of neoplastic processes caused by infection of human cervical epithelial cells with human papillomavirus. It was shown that its introduction into cell cultures containing the integrated HPV-16 gene leads to a multiple increase in the content of p53 protein, a tumor suppressor. The mutual presence of indole-3-carbinol and actinomycin-D leads to a synergistic effect.

 The invention is illustrated by the following examples.

Example 1. The composition used in examples 3, 4, 5, consisted of the following components:
1. Composition for gelatin capsules:
Indole-3-carbinol - 200 mg
Microcrystalline Cellulose - 200 mg
Starch - 50 mg
2. Composition for vaginal suppositories:
Indole-3-carbinol - 200 mg
Actinomycin D - 50 nm
Vitepsol - up to 2 gr
Example 2

a) Suppression of the expression of genes E6 and E7 in the presence of indole-3-carbinol and
b) stimulation of expression of a gene encoding p53 in cervical carcinoma cells with actinomycin D.

 a) Studies were conducted on the CaSki uterine carcinoma cell line containing the integrated HPV-16 genome. Using the Northern hybridization method, the expression level of the HPV-16 E6 and E7 genes was measured. It was established that the addition of estradiol to the culture medium causes a multiple increase in transcription of the E6 and E7 genes. In the presence of indole-3-carbinol, this effect was not observed.

 b) The experiments were performed on CaSki and SiHa cell lines containing the integrated HPV-16 genome. The content of p53 protein in the nuclei of cells was determined by indirect immunofluorescence using monoclonal antibodies specific for p53. It was found that introducing actinomycin D into the culture medium at a concentration of 5 nm significantly increases the p53 content in the nuclei of the cell lines SiHa and CaSki.

 Example 3. The preventive effect of IR in relation to cervical cancer.

 The experiments were performed on transgenic mice containing the HPV-16 gene, in which cervical cancer develops with long-term administration of 17β-estradiol. The experiment involved two groups of mice that received 17β-estradiol in a total dose of 0.125 mg for 60 days. One group of animals received a standard diet, the other a diet containing 0.2% indole-3-carbinol.

 After 60 days, the animals were killed and the cervix was examined histologically and the degree of cell dysplasia was evaluated. The results are presented in figure 1.

 Example 4. The antitumor effect of a pharmaceutical composition containing indole-3-carbinol and actinomycin D.

 The experimental design is described in Example 3. Instead of a diet containing indole-3-carbinol, the experimental group of mice received vaginal suppositories containing 0.2 IR and 50 nM actinomycin D once every 2 days. The experimental results are shown in FIG. 2.

Example 5. The treatment of cervical dysplasia
To assess the clinical effect of indole-carbinol, 27 patients were selected with a diagnosis of cervical dysplasia of degrees 2 and 3 (CIN II-III), based on studies of biopsy material. None of the 10 patients receiving placebo showed positive dynamics. In 17 patients, an analysis was made for infection of epithelial cells with human papilloma viruses of types 16, 18, 31, 45. Moreover, in 14 patients the analysis was positive. Of the 17 patients who received 200 mg of IR in the form of hard gelatin capsules daily for 12 weeks, 9 of these 14 showed complete regression of dysplasia. The data obtained indicate a statistically significant clinical effect of the use of IR in comparison with placebo in the treatment of neoplastic cervical processes.

 Example 6. Treatment of recurrent laryngeal papillomatosis with indole-3-carbinol.

 To assess the effectiveness of treatment of recurrent laryngeal papillomatosis, two patients aged 3 and 4 years were selected. Symptoms of the disease consisted of difficulty breathing and systematic severe prolonged coughing. When examining both patients, it was found that the lumen of the larynx is closed by papillomatous outgrowths, which were subsequently removed by laser surgery.

 In the next 6 months, the symptoms of the disease resumed three times due to the intensive growth of tissues, which forced them to resort to surgical intervention. Laboratory study of biopsy material by polymerase chain reaction showed the presence of human papillomavirus types 6 and 11. In order to prevent relapse, patients were prescribed 200 mg of indole-3-carbinol daily for 6 months. Observation of patients for 1 year showed that there are no symptoms of the disease, and with laryngoscopy, papillomatous formations were not detected.

Claims (5)

 1. Composition for the prevention and treatment of dysplasia and cancer of the cervix uteri and laryngeal papillomatosis, comprising indole-3-carbinol in an effective amount and a suitable pharmaceutical carrier.  2. The composition according to p. 1, characterized in that it further comprises actinomycin D at a concentration of not more than 100 nM.  3. The composition according to p. 1, characterized in that it is a capsule or vaginal suppository.  4. A method for the prevention and treatment of cervical dysplasia and cancer, including as a main or auxiliary effect, a course of chemotherapy, characterized in that the analysis of a tumor or cervical dysplasia for the presence of infection of epithelial cells with human papilloma virus type 16, 18, 31, 45 and in case of a positive result, indole-3-carbinol is prescribed in the form of capsules for oral administration or vaginal suppositories or its mixture with actinomycin D in the form of vaginal suppositories.  5. A method of treating laryngeal papillomatosis by laser tumor destruction, characterized in that in the postoperative period, long-term therapy with indole-3-carbinol is prescribed in the form of capsules for oral administration.

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