RU2552332C1 - Method of treating of planocellular intraepithelial lesions of cervix of uterus - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: vaginal suppositories "Cervicon-DIM", containing diindolylmethane, are applied. The medication is applied for not less than 6 months in dosage 200 mg/day of diindolylmethane. The claimed method ensures durative anti-recurrence effect for not less than a year after the completion of the therapy, as well as stimulates the immune system, including general and local immunity of the vaginal mucosa.

EFFECT: invention contributes to the acceleration of epithelisation after carrying out destructive therapy concerning cervical intraepithelial neoplasia, makes it possible to reduce or eliminate the negative impact of side effects.

4 cl, 5 tbl, 5 ex, 2 dwg

Description

The claimed invention relates to medicine, namely to a method for the treatment of squamous intraepithelial lesions of the cervix.

Human papillomavirus infection (PVI) is considered the most common sexually transmitted infection (about 80% of the sexually active population is infected with HPV) (Tota J.E., 2011). At the same time, the effect of PVI on the development of benign and malignant neoplasms of the genital organs was confirmed. The most common clinical manifestation of HPV infection is genital warts. However, the etiological role of HPV in the development of cervical cancer as a result of infection of cervical tissues with the 16th and 18th (predominantly) HPV types of high oncogenic risk (Herrington KS Human papilloma viruses and cervical neoplasia. Interaction of HPV with other factors, 1995 , STD. No. 5. S. 3-10).

In infected cells in the initial stages, the viral genetic material persists in an episomal form, and the cell is capable of producing viral particles. At later stages, the viral genome is integrated into the cellular genome, as a result of which the ability to reproduce the virus is lost. Integration of viral DNA into the cellular DNA is often accompanied by the loss of viral material, but with the mandatory preservation of the E6 and E7 genes, instability of the cellular genome is induced. When the viral genome is integrated into the cellular genome, the transcription of viral genes, primarily E6 and E7, is preserved, and the transcription of some cellular genes is also activated. Proteins E6 and E7 can interact with tumor suppressor proteins, which play a key role in the regulation of cell division. So the product of the E6 gene is able to interact with the protein of the p53 gene, and E7 with pRb and inactivate their function. In addition, these genes affect cyclins, which are regulators of the cell cycle.

Until now, there is no clear certainty regarding the factors that determine the different rates of progression of human papillomavirus infection, while it is against the background of persistent PVI that intraepithelial neoplasia develops (Kiselev V.I., Ashrafyan L.A. et al., 2004; Bekkers R. , Meijer C, et al. Effects of HPV detection in population-based screening programmers for cervical cancer: a Dutch moment // Gynecol. Oncol. 2006; Mar: 100 (3): 451-4).

The role of the cellular component of immunity in the pathogenesis of HPV infection is evidenced by data on the complete remission of HPV-associated diseases after specific stimulation of the T-cell immune response to E6 and E7 (Kiselev V.I., Kiselev O.I. Human papilloma viruses in the development of cervical cancer uterus - St. Petersburg. - M., 2003). The various treatments for PVI currently offered, such as the use of interferons and immunostimulating active substances, are often not very effective and give a significant number of relapses of infection. Experimental studies have shown that interferons are not able to suppress HPV universally (Kim KY, Kim, L. Blatt, MW Taylor. The effects of interferon on the expression of human papillomavirus oncogenes J. Gen. Virol., 81 (2000), pp. 695 -700). The fact is that E7 oncoprotein is a powerful immunosuppressive agent, significantly reducing the effectiveness of immunocorrective treatment methods such as interferon therapy, interferon inducers (cycloferon) and immunomodulators. The use of drugs that have a cytostatic effect on virus-infected cells is also ineffective due to the peculiarities of the HPV life cycle (lack of cytopathic properties), which allow the pathogen to escape from the control of the immune system (Molochkov V.A., Kiselev V.I., Rudykh I.V. , Scherbo SN Papillomavirus infection: clinic, diagnostics, treatment. A manual for doctors. Moscow, 2005). HPV, on the other hand, includes mechanisms that disrupt the activation of the immune system.

The use of preventive HPV vaccines is still in the early stages of experimental testing, and published research data are often contradictory (Franco E., Harper D. Vaccination against human papillomavirus infection: a new paradigm in cervical cancer control, 2005, Vaccine. 23 (17-18 ): 2388-94).

At different times, many conservative and destructive methods of treating pathological conditions of the cervix have been proposed. Of conservative methods, chemical coagulants such as Solkovagin have found widespread use. The mechanism of action of the drug is due to the morphofunctional difference between the stratified squamous and ectopic cylindrical epithelium. The cylindrical epithelium is much larger than the squamous multilayer epithelium and is susceptible to a solution consisting of nitric and acetic acids, oxalic acid dihydrate and zinc nitrate hexahydrate. With this, a selective effect is achieved on the altered tissue of the cervix, without causing disturbances in the surrounding healthy stratified squamous epithelium. The depth of action of the drug on the tissue ensures the destruction of the pathologically changed area. The effectiveness of treatment with this drug in uncomplicated forms of pseudo-erosion in young nulliparous women is 74.3% (Kostava M.N. The effectiveness of treating background diseases of the cervix in young nulliparous women with Solkovagin and a low-intensity laser. - M., 1994. - 24 p. )

Diathermocoagulation, cryotherapy, laser therapy are also used, but now surgical methods (due to the high percentage of relapses after their use) have begun to play a secondary role. Etiopathogenetic therapy has come to the first place, having two main directions: 1) effect on the etiological factor - HPV; 2) blocking the basic mechanisms of carcinogenesis.

Epigen-intimate spray provides inhibition of DNA and RNA viruses, inactivation of viral particles, blocking the introduction of active viral particles through the membrane into the cell, impaired synthesis of new structural components of viruses. The main mechanism of the antiviral activity of glycyrrhizic acid is the inhibition of P kinase, which leads to inhibition of phosphorylation of cellular and virus encoded proteins in infected cells and in a free state. However, due to the low efficiency (30-40%), the duration of treatment, the development of undesirable side effects and complications, many doctors refused these methods (Prilepskaya V.N., Kondrikov N.I., Bebneva T.N. Papillomavirus infection and neck pathology uterus (literature review) // Gynecology. - M., - 2000. - T. 2. - N 3. - P. 80-82).

The closest analogue is the treatment of neoplastic diseases of the reproductive organs, known from patent RU 2318510 C1, 03/10/2008, based on the use of vaginal suppositories based on 3,3′-diindolylmethane. However, this source does not suggest the use of this drug for the treatment of intraepithelial lesions of the cervix that are not associated with HPV, and the most optimal and effective dosages are not disclosed.

Based on this, the object of the invention is to develop a method for the treatment of intraepithelial lesions of the cervix that is not associated with HPV, which has increased efficiency and reduced or eliminated the negative effects of side effects.

The objective of the invention is solved by the method of treatment of squamous intraepithelial lesions of the cervix, independent of the presence of their association with HPV. The method includes the use of vaginal suppositories "Cervicon-DIM" containing diindolylmethane. In this case, the drug is used for at least 6 months at a dosage of 200 mg / day of diindolylmethane.

The claimed method provides a long anti-relapse effect for at least a year after the end of therapy, and also stimulates the immune system, including general and local immunity of the vaginal mucosa. In addition, the claimed method helps to accelerate epithelialization after destructive therapy for cervical intraepithelial neoplasia.

Using the claimed method for the treatment of squamous intraepithelial lesions of the cervix also allows to increase the effectiveness of therapy and reduce or eliminate the negative impact of side effects.

Thus, there is a need to find new alternative methods of treatment of intraepithelial lesions of the cervix, as well as prophylactic agents, taking into account the genesis of development. The reason for the research was published experimental, clinical and epidemiological studies confirming the antitumor activity of I3C, the precursor of DIM, against a huge number of pre-tumor and tumor diseases of the breast, ovaries, cervix, endometrium, prostate, skin, gastrointestinal tract, lungs [Bradlow HL, Sepkovic DW, Telang NT, Osborne MP. Multifunctional aspects of the action of indole-3-carbinol as an antitumor agent. Ann. NY Acad. Sci., 1999, 889, 204-213; Telang NT, Katdare M, Bradlow HL et al. Inhibition of proliferation and modulation of estradiol metabolism: novel mechanisms for breast cancer prevention by the phytochemical indole-3-carbinol. Proc. Soc. Exp. Biol. Med., 1997, 216, 246-252; Chinni SR, Li Y, Upadhyay S et al. Indole-3-carbinol (I3C) induced cell growth inhibition, G1 cell cycle arrest and apoptosis in prostate cancer cells. Oncogene, 2001, 20, 2927-2936; Jin L, Qi M, Chen DZ et al. Indole-3-carbinol prevents cervical cancer in human papilloma virus type 16 (HPV16) transgenic mice. Cancer Res, 1999, 59, 3991-3997; Huh SW, Bae SM, Kim YW, Lee JM et al. Anticancer effects of (-) - epigallocatechin-3-gallate on ovarian carcinoma cell line. Gynecol. Oncol, 2004, 94 (3), 760-768].

The results of studies of the specific effectiveness of I3C and its metabolic derivative of 3,3′-diindolylmethane (DIM) for cervical cancer, as well as for cell cultures of HPV-induced dysplasia and cervical carcinoma, were analyzed. It turned out that DIM selectively inhibits the synthesis of E7 in epithelial cells infected with HPV [Schwartz S.M, Daling J.R, Shera K.S. et al. Human Papillomavirus and Prognosis of Inva-sive Cervical Cancer: A Population-Based Study. J. Clin. Oncol. 2001, 19: 1906-1915].

Currently, the participation of estrogen metabolites in the development of neoplastic processes in the tissues of the cervix has been confirmed. The metabolite 16-OHE1 in estrogen-dependent tissues of the organs of the female reproductive system greatly enhances the expression of estrogen-dependent genes. At the same time, 2-OHE1 has antiproliferative properties and induces apoptosis. Numerous experimental and clinical studies have proven the need to maintain a balance between the metabolites of 2-OHE1 / 16b-OHE1 in a ratio of 2: 1. It was established that with persistence of HPV infection, the formation of an aggressive metabolite of estrogen 16b-OHE1 occurs 200 times more often than 2-OHE1. In a study (Gynecol. Oncol., 78 (2), 123-129), the ratio of 2-OHE1 and 16-OHE1 metabolites was measured in the urine of patients with grade II and III cervical intraepithelial neoplasia (CIN II-III) daily taking 200- 400 mg I3C. It turned out that in women with a pronounced regression of dysplasia, there was a significant change in the concentrations of estradiol metabolites towards estrogen - 2-OHE1 (Appendix, Fig. 1).

In the K14-HPV16 transgenic mouse model, it was shown that I3C added to experimental animals feed increases the expression of tumor suppressor phosphatase PTEN in cervical epithelium, inhibiting manifestations of genetic instability on the part of the viral oncogenes E6 and E7 and inhibiting the development of cervical cancer (Mol. Med ., 11, 59-63)

It was found that DIM, as well as its metabolic precursor, I3C, induced apoptosis of human C33A line cervical tumor cells. Moreover, DIM showed several times more pronounced proapoptotic activity compared to I3C (Nutr., 131 (12), 3294-3302).

Search results in this direction allowed us to apply and evaluate the clinical effectiveness of the long-term use of vaginal suppositories containing diindolylmethane as the active substance (Cervicon-DIM preparation) (RF patent No. 325310). The drug is a vaginal suppository containing 3,3′-diindolylmethane (DIM), a lipophilic base containing solid confectionery fat, polyvinylpyrrolidone and butylhydroxyanisole or butylhydroxytoluene in the following content, wt. % (see table 1):

Table 1. The composition of the vaginal suppositories "Cervicon-DIM". Diindolylmethane 2.0-6.0 Polyvinylpyrrolidone 0.5-1.2 Butylhydroxyanisole or butylhydroxytoluene 0.3-0.5 Lipophilic base rest Diindolylmethane is contained in an amount of 0.045-0.110 g per dose.

The effectiveness of the use of the drug Cervicon-DIM in in vivo experiments was determined.

Example 1. Modeling erosion (dysplasia) of the cervix.

In vivo experiments were performed on white outbred female rats (4-6 years old) obtained from the Rappolovo nursery, St. Petersburg.

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, temperature 20-25 ° C, relative humidity 50-70% and free access to feed and water.

Rats were anesthetized with ketamine at a dose of 30 mg / kg intramuscularly before being burned. A glass funnel was inserted into the vagina, a copper rod heated to 100 ° C was inserted into the inside of the vagina, and a burn of the cervix was caused through an opening in the cylindrical part of the funnel (2-3 seconds to avoid loss of temperature of the rod). After a burn, 2 drops of a 5% aqueous solution of caustic soda were injected into the same hole to change the acidic environment of the vagina. Treatment began in a day; at the same time, the preparations were introduced into the vagina: Solkovagin - in the amount of 3 drops, and Cervicon-DIM (0.1 g) - in the form of a strip at a dose of 100 mg / kg for 7 days.

The dynamics of regeneration and the histological picture were noted. In addition, indicators of the severity of inflammatory processes (the number of leukocytes, the content of lymphocytes and neutrophils, ESR, CRP and fibrinogen) were determined in the peripheral blood by conventional methods (see table 2).

table 2 The severity of inflammatory processes in the control and experimental groups. Indicators Control I (intact animals) n = 10 Control II (cervical erosion) n = 10 Experienced I (treatment with Cervicon-DIM) n = 10 Experienced II (treatment with Solkovagin) n = 10 White blood cells, 10 ⁹ / L 12.50.5 16.30.2 * 11.80.6 11.90.8 Lymphocytes,% 728 8611 705 696 Neutrophils,% 122 141 102 102 ESR, mm / hour 41 101 * 51 61 CRP, mm precipitation 0 41 * eleven eleven Fibrinogen, g / l 2.50.5 9.20.3 * 3.10.1 3.00.2

For histological examination, rats were euthanized by decapitation in dynamics and after the end of the experiment. Control rats were euthanized in the same manner. The cervix was fixed in 10% formalin and, after alcohol wiring, was embedded in paraffin. Sections were stained with hematoxylin-eosin. Sections were studied under a biological microscope MBI-6.

In a histological examination of the cervix in rats after application of Cervicon-DIM, complete epithelization of the erosion surface was observed. The epithelium covering the mucous membrane of the neck was stratified squamous. Inflammatory infiltration in the submucosal layer was absent. Collagen connective tissue fibers and single polymorphonuclear leukocytes and capillaries were visible in the submucosal layer. While monitoring the course of the erosive-inflammatory process in animals without treatment showed that there is necrobiosis of the epithelium, edema and infiltration of polymorphic nuclear leukocytes of the submucosal layer. The cell boundaries of the epithelium of the vaginal part of the uterus were indistinguishable, the nuclei were pyknotic and deformed. In places, the epithelium was absent, its dysplasia was observed (application, Fig. 2).

The long-term use of Cervicon-DIM in patients with genital warts was also analyzed. The study confirmed the ability of the drug to eliminate non-oncogenic types of the virus.

Example 2. The study of the effects on genital warts.

Studies have also been conducted on patients with a diagnosis of genital genital warts. Diagnosis was based on cytological, histological examination of biopsy samples, determination of antibodies to HPV and detection of HPV DNA and E7 oncoprotein. Of the 25 patients in the group, genital warts were associated with other urogenital infections in approximately 90% of cases. About 10% were previously treated by electrocoagulation. 15 patients were given 2 times a day suppositories according to example 2 and 10 patients received suppositories according to example 1 for 10 weeks - 12 weeks before genital warts resolved. At the follow-up examination after a year and a half, there was no relapse of genital warts in the main group, 3 patients had relapse. An additional treatment was prescribed with an increase in dose.

Example 3. Conducting a placebo-controlled multicenter clinical trial of the effectiveness and safety of the drug Cervicon-DIM (vaginal suppositories) in the treatment of cervical intraepithelial neoplasia.

The relapse rate was studied during long-term therapy with Cervicon-DIM, namely, the proportion of patients with no relapse within 3, 6 and 18 months was determined (table 3), as well as the proportion of patients who eliminated cells containing viral DNA (PCR test ) at the end of the treatment period (6 months). The patients in the control group received placebo therapy (Table 4).

Table 3. Assessment of relapse detection rates within visit groups. Study day Relapse rate Group Cervicon-dim Placebo Frequencies % in Group Frequencies % in Group 0 CIN is 31 100.0 31 100.0 0 CIN no 0 0 0 0 90 ± 5 CIN is 2 6.5 10 32,3 CIN no 29th 93.5 21 67.7 180 ± 5 CIN is / relapse 0 0 8 25.8 CIN no 31 100.0 23 74,2 540 ± 15 relapse 0 0 fourteen 45,2 CIN no 31 100.0 17 54.8

Table 4. Evaluation of indicators for detecting HPV by PCR within visit groups (HPV types 16 and 18). Study day HPV 16/18 Group Cervicon-dim Placebo Frequencies % in Group Frequencies % in Group 0 HPV 5 17.85714 2 6.45161 HPV + 23 82.14286 29th 93,54839 elimination 90 ± 5 reinfection 0 0 four 12.90323 no effect 19 70,37037 27 87,09677 elimination 8 29,62963 0 0 180 ± 5 reinfection 0 0 one 3,44828 no effect twenty 71,42857 27 93,10345 elimination 8 28.57143 one 3,44828

According to the PCR diagnosis of HPV infection, the overall effectiveness of the antiviral therapy in the group of patients taking Cervicon-DIM was about 30%. There is also a higher efficiency of Cervicon-DIM therapy to prevent recurrence of cervical intraepithelial neoplasia compared with the placebo group: 93.5% by the end of the 3rd month and 100% by the end of the 6th month. Remote observation showed that the achieved therapeutic effect persists throughout the year: no relapses were recorded in the group receiving Cervicon-DIM, despite the fact that their number in the placebo group reached 45.2%.

Thus, it was confirmed that long-term therapy with Cervicon-DIM promotes the elimination of human papillomavirus infection in patients with cervical intraepithelial neoplasia, preventing the recurrence of the disease and its possible progression to cervical cancer.

Example 4. Chronic viral infections are usually associated with impaired functioning of the immune system. The results of the study of the effect of the drug Cervicon-DIM on the severity of the immunodeficiency state induced by emotional stress were studied on the model of stressing mice by placing them in crowded conditions (Table 5).

Table 5. Effect on the immune system of stressed mice Immunoreactivity indicators, units Intact animals Stressed animals The control P ₁ < Experience (Cervicon-DIM) P ₂ < The number of AOK per 10 ⁶ cells per spleen (Mm) 4.9 (5.0-4.2) 2.3 (2.6-1.8) 0.01 5.9 (6.3-5.7) 0.01 HA titer on day 14 (Mm) 5.4 ± 0.11 4.4 ± 0.24 0.001 4.2 ± 0.32 0.01 Index of completion of phagocytosis,% (Mm) 36.2 ± 7.3 16.6 ± 3.3 0.05 33.4 ± 5.0 0.01 Mascara clearance, index (Mm) 4.41 ± 0.13 3.08 ± 0.21 0.01 3.60 ± 0.1 0.01 P1 - significance of differences with indicators of intact animals P2 - significance of differences with indicators of stressed animals

As can be seen from table 5, the crowding of animals for 10 days leads to the suppression of the humoral immune response (the number of AOKs and the titer of HA), the suppression of completeness of phagocytosis and the rate of blood purification from inert carcass particles. All of these disorders are induced by stress. A 14-day intravaginal administration of the drug at a dose of 1000 mg / kg / day prevents these disorders.

Example 5. In the patients of example 3, the level of secretory immunoglobulin class A (sIgA) in the vaginal discharge was studied. SIgA level was determined by enzyme-linked immunosorbent assay using test systems manufactured by Vector-Best CJSC.

The sIgA content was calculated per 1 g of total protein determined by the biuret reaction. The initial sIgA level in the placebo group was 210 ± 43 μg / g, and in the group of patients receiving Cervicon-DIM at a dose of 200 mg / day, 223 ± 37 μg / g (M ± m). After 6 months of therapy, the sIgA level in the placebo group was 239 ± 68 μg / g, and in the group of patients receiving Cervicon-DIM at a dose of 200 mg / day, 326 ± 55 μg / g (significant differences, p <0.001 according to the Student's test )

Claims (4)

1. A method of treating squamous intraepithelial lesions of the cervix, independent of their association with HPV, including the use of vaginal suppositories “Cervicon-DIM” containing diindolylmethane, characterized in that the drug is used for at least 6 months at a dosage of 200 mg / day of diindolylmethane . 2. The method according to p. 1, providing a long anti-relapse effect for at least a year after the end of therapy. 3. The method according to p. 1, providing stimulation of the immune system, including general and local immunity of the vaginal mucosa. 4. The method according to p. 1, providing accelerated epithelization after destructive therapy for CIN.

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