RU2438652C1 - Drug for human papilloma virus associated diseases of uterine neck, presented in form of solution for vaginal irrigations, kit and method for preparing - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry, namely to gynaecology and can find application for treating the pre-cancer diseases of uterine neck. A drug presented in the form of a solution for vaginal irrigations, characterised by the fact that it contains a recombinant composition of proteins E7 of HPV type 16 and 18 types linked to an amino acid sequence of heat-shock proteins of m. w. 70 kDa from M. tuberculosis in the form of a lyophilised powder, chitosan of a degree of acetylation 86%, a preserving agent, carboxymethyl cellulose sodium salt of molecular weight 250000, a foaming agent selected from glycyrrhizic acid and its pharmaceutically acceptable salts, purified water. Also, there is offered a kit and method for preparing it.

EFFECT: invention provides stability of recombinant proteins in its composition throughout a long period of time.

7 cl, 1 tbl, 5 ex

Description

The invention relates to medicine, in particular to gynecology, and may find application for the treatment of cervical diseases associated with human papillomavirus. Currently, the role of HPV in the development of genital warts and cervical cancer can be considered proven. It turned out that in most cases all over the world (65-77%), human papillomaviruses are detected - HPV types 16/18 related to high oncogenic risk viruses.

The spread of infection occurs mainly through sexual contact, reaching a maximum of infection up to 30 years. Over 50% of the sexually active population of the world is infected with the human papillomavirus during their lifetime, and this is the “primary” event in the pathogenesis of cervical cancer. Most infections result in spontaneous recovery. However, in some cases, a persistent infection develops that can trigger the mechanisms of cellular transformation of epithelial cells. So with CIN 1, active replication of the virus and its asymptomatic isolation is observed. The conversion of CIN 1 to invasive cancer occurs with a high frequency and, as a rule, is accompanied by the integration of viral DNA into the genome of the host cell [V. Kiselev. (2004) Human papillomaviruses in the development of cervical cancer. M .: Dimitrade Graph Group® Company].

HPV of a high degree of oncogenic risk (mainly of types 16 and 18) is found in 50-80% of samples of moderate and severe cervical squamous dysplasia and in 90% of invasive cancer. An important fact is that the clinical manifestations of HPV infection are not detected for a sufficiently long time, but the consequences of the persistence of the virus from this do not become easier. The development of mild and moderate dysplasia in severe occurs, respectively, in 10 and 20% of cases.

Due to the simplicity of organizing the viral genome, it turned out to be rather difficult to develop a drug that selectively inhibits the reproduction of the virus. For this reason, the main efforts are aimed at creating vaccines, since the role of immunity in spontaneous regression of the clinical manifestations of a viral infection is well substantiated.

In the patent of the Russian Federation No. 2229307 describes the development of a vaccine based on the hybrid proteins E7 and Hsp 70 M. tuberculosis. The authors describe an injectable dosage form with which it is possible to achieve the regression of tumors induced by the virus in experimental models with transplantable tumors. The demonstrated vaccination efficacy in these models confirms the correct choice of the E7 protein as the molecular target against which immune responses are induced.

In the application of the Russian Federation 2008139869 describes the possibility of introducing various active substances using a bioadhesive carrier with a slow release for mucous membranes containing a wetting agent comprising an active component, from 1 to 75 wt.% Diluent and from 1 to 10 wt.% Alkali metal sulfate, from 0.5 to 5% by weight of a binder and from 5 to 80% by weight of at least one bioadhesive polymer selected from the group consisting of natural polymers, said natural polymers being polysaccharides or natural proteins of animal origin, such as proteins of milk, or of vegetable origin, such as soy protein, or synthetic polymers, as well as mixtures of these substances, and from 5 to 80 wt.%, at least one polymer, providing a slow release of the active component. This carrier does not provide the necessary degree of adhesion and bioavailability of protein preparations.

RF patent 2377305 describes topical mucosal vaccines for diseases associated with human papillomavirus, including cervical cancer. It is noted that the vaccine in combination with chitosan can be used as a spray or suppository. However, this source does not disclose the composition in the form of a vaginal solution for irrigation.

This source can be indicated as the closest analogue prototype.

The objective of the invention is to provide a tool that is effective in use, eliminating the injection route of administration as traumatic and unsafe, as well as ensuring the stability of the recombinant proteins included in its composition for a long time.

The problem is solved by creating a solution for irrigation, which contains a recombinant composition of the E7 proteins of human papillomavirus types 16 and 18, connected to the amino acid sequence of the heat shock protein mm 70 kDa from M. tuberculosis in the form of a lyophilized powder, chitosan with a degree of deacetylation of 86%, preservative, sodium salt of carboxymethyl cellulose with a molecular weight of 250,000, a foaming agent selected from glycerrhizic acid and its pharmaceutically acceptable salts, water purified in the following combination of components per dose for intravaginal application, parts by weight:

Recombinant composition of E7 proteins of human papillomavirus types 16 and 18, coupled to the amino acid sequence of heat shock protein m.m. 70 kDa from M. tuberculosis 0.1-1.0 Chitosan with a degree of deacetylation of 86% 0.055-0.18 Preservative 1.0-2.5 Sodium salt of carboxymethyl cellulose with a molecular weight of 250,000 0.5-1.5 Blowing agent 0.1-0.25 Purified water Up to 100

Chitosan with a degree of deacetylation of 86% is able to enhance the absorption of hydrophilic compounds, for example, protein and peptide drugs, on the epithelium. It increases the permeability of apical cell membranes and redistributes cytoskeleton proteins due to its cationic nature. Its interaction with the cell membrane leads to a structural reorganization of proteins associated with plasma membranes, which, in turn, increases the permeability of drugs.

In addition, the introduction of chitosan with a degree of deacetylation of 86% in the composition of drugs with protein compounds provides an increase in their stability in the dosage form due to the formation of a complex between protein and chitosan.

The sodium salt of carboxymethyl cellulose with a molecular weight of 250,000 has gelling properties. It is the claimed ratio of the sodium salt of carboxymethyl cellulose with a molecular weight of 250,000 that is able to form a gel of the necessary consistency, which allows controlling the release process of the recombinant composition of the E7 proteins of human papillomavirus type 16 and 18, coupled to the amino acid sequence of the heat shock protein m.m. 70 kDa from M. tuberculosis, from the dosage form onto the vaginal mucosa. An increase in concentration leads to the formation of a gel, which condenses at storage temperature, which stops the release process, and a decrease in concentration leads to a decrease in viscosity and the spread of the recombinant composition of E7 proteins of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m.m. 70 kDa from M. tuberculosis, along the vagina, which complicates the application of proteins at the injection site.

A foaming agent selected from glycerrhizic acid and its pharmaceutically acceptable salts, such as potassium, sodium, ammonium salts, also has antiviral properties that increase the effectiveness of treatment of the pathological process. In the stated ratio, it provides the necessary level of pricing for additional application of the recombinant composition of the E7 proteins of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m.m. 70 kDa from M. tuberculosis, on the vaginal mucosa. A preferred foaming agent is the mono-ammonium salt of glycerrhizic acid.

As a preservative, any physiologically acceptable preservatives exhibiting antifungal and antibacterial properties are used. Preferably, nipagin, nipazole, benzalkonium chloride and the like can be used as preservative.

The irrigation solution can be placed in a syringe for intravaginal application.

Also, the problem is solved by creating a kit for obtaining a solution for irrigation.

The kit for irrigation includes two bottles. One bottle contains a gel containing a preservative, sodium salt of carboxymethyl cellulose with a molecular weight of 250,000, a foaming agent selected from glycerrhizic acid and its pharmaceutically acceptable salts, chitosan with a deacetylation degree of 86% and purified water.

In another vial, there is a lyophilized-dried protein E7 of human papillomavirus 16 connected to the amino acid sequence of the heat shock protein m. 70 kDa from M. tuberculosis, and lyophilized-dried human papillomavirus E7 protein 18, coupled to the amino acid sequence of heat shock protein m. 70 kDa from M. tuberculosis (0.1-1.0 parts by weight based on the total weight of the resulting solution).

An irrigation kit may also include an extra syringe for intravaginal application.

The preparation procedure is as follows: the contents of the bottle with the gel are poured into the bottle with lyophilized-dried proteins, screwed on with a lid. The vial is shaken for 1-2 minutes, after which, if necessary, the contents are taken with a syringe for intravaginal use and an application is made in the cervical region.

The proposed dosage form and the ratio of active and auxiliary substances provides the necessary quality and effective therapeutic effect, as well as ease of use.

The proposed products meet all the requirements for dosage forms: high bioavailability and stability of the active ingredients, thermal and colloidal stability, ease of use.

Example 1

The composition of the solution for irrigation in hours

Recombinant composition of E7 proteins of human papillomavirus types 16 and 18, coupled to the amino acid sequence of heat shock protein m.m. 70 kDa from M. tuberculosis one Chitosan with a degree of deacetylation of 86% 0.165 Nipagin 1,5 Sodium salt of carboxymethyl cellulose with a molecular weight of 250,000 one Monoammonium salt of glycerrhizic acid 0.2 Purified water Up to 100

Example 2

The composition of the solution for irrigation in hours

Recombinant composition of E7 proteins of human papillomavirus types 16 and 18, coupled to the amino acid sequence of heat shock protein m.m. 70 kDa from M. tuberculosis one Chitosan with a degree of deacetylation of 86% 0,055 Nipagin 2.5 Sodium salt of carboxymethyl cellulose with a molecular weight of 250,000 1,5 Monoammonium salt of glycerrhizic acid 0.1 Purified water Up to 100

Example 3

Set for obtaining a solution for irrigation (components, including on the final solution) with a syringe for intravaginal application.

1 bottle contains a recombinant composition of proteins E7 of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m.m. 70 kDa from M. tuberculosis one 2 bottles contain a gel consisting of: Chitosan with a degree of deacetylation of 86% 0.165 Nipazole 1,5 Sodium salt of carboxymethyl cellulose with a molecular weight of 250,000 one Potassium salt of glycerrhizic acid 0.25 Purified water Up to 100

Example 4

A comparative study of the stability of recombinant proteins E7-16-Hsp and E7-18-Hsp

Suppositories containing recombinant proteins E7-16-Hsp and E7-18-Hsp at a concentration of 250 μg each are made in accordance with the recipe described in patent application No. 2377305.

Determination of the content of recombinant proteins was carried out by enzyme-linked immunosorbent assay described in the article "Development of a diagnostic system for the determination of oncoproteins E7 of human papillomavirus type 16 and type 18 by enzyme-linked immunosorbent assay" Kiselev VI, Solopova ON, Sveshnikov P .G and others. Molecular Medicine, 2010, No. 2, pp. 52-57.

The stability of proteins in suppositories was compared with the stability of proteins in a set of vaginal solution for irrigation, where the proteins are in lyophilized form.

Table 1 Stability of recombinant proteins in various dosage forms. Storage at room temperature. Suppositories Set Dates of measurement (days) E7-16 (mcg) E7-18 (mcg) E7-16 (mcg) E7-18 (mcg) 250 250 250 250 one 154 120 250 250 fourteen 110 70 250 250 thirty 60 40 250 250 60 fifty 35 250 250 90

Example 5

Results of pharmacokinetic studies

The studies were conducted on experimental animals (rabbits) in accordance with Karkishchenko N.N., Khoronko V.V., Sergeeva S.A. "Pharmacokinetics", Rostov, 2001.

It was found that the maximum concentration level of the recombinant protein E7-16-Hsp 70 (within 180-250 ng / ml) in the blood of experimental animals with intravaginal administration is achieved 1.5 hours after intravaginal administration in a dose corresponding to the composition of the solution according to example 1 , while with the injection route of administration, this indicator is 0.5 hours at the same concentration.

The duration of the drug is about 13.5 hours. Data from pharmacological tests confirm the preservation of the pharmacological properties of the active principle. The composition can be effectively used to treat various cervical diseases associated with human papillomavirus, such as dysplasia, cervical cancer, and candidiasis.

Claims (7)

1. An agent for treating cervical diseases associated with human papillomavirus in the form of a vaginal solution for irrigation, characterized in that it contains a recombinant composition of proteins E7 of human papillomavirus 16 and 18 types, coupled with the amino acid sequence of the heat shock protein mm 70 kDa from M. tuberculosis in the form of a lyophilized powder, chitosan with a degree of deacetylation of 86%, preservative, sodium salt of carboxymethyl cellulose with a molecular weight of 250,000, a foaming agent selected from glycerrhizic acid and its pharmaceutically acceptable salts, water purified at the following content of components per 1 dose for intravaginal application, parts by weight:
Recombinant composition of the E7 virus proteins human papillomas of the 16th and 18th types connected with the amino acid sequence of a protein heat shock m.m. 70 kDa from M. tuberculosis 0.1-1.0 Chitosan with a degree of deacetylation of 86% 0.055-0.18 Preservative 1.0-2.5 Carboxymethyl Cellulose Sodium with a molecular weight of 250,000 0.5-1.5 Blowing agent 0.1-0.25 Purified water up to 100 2. The tool according to claim 1, characterized in that as a foaming substance contains monoammonium salt of glycerrhizic acid. 3. The tool according to claim 1, characterized in that the solution is placed in a syringe for intravaginal application. 4. A kit for the preparation of a medicament for treating cervical diseases associated with human papillomavirus in the form of a vaginal solution for irrigation according to any one of claims 1 and 2, characterized in that it consists of two bottles, the first of which contains a lyophilized dried recombinant protein composition E7 human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m.m. 70 kDa from M. tuberculosis, and the second contains a gel of chitosan with a degree of deacetylation of 86%, a preservative, sodium salt of carboxymethyl cellulose with a molecular weight of 250,000, a foaming agent selected from glycerrhizic acid and its pharmaceutically acceptable salts, purified water. 5. The kit according to claim 4, characterized in that it further comprises a syringe for intravaginal application. 6. A method of obtaining a means for treating cervical diseases associated with human papillomavirus in the form of a vaginal solution for irrigation according to any one of claims 1 and 2, characterized in that the contents of the second vial of the kit according to claim 4, with a chitosan gel with a degree deacetylation of 86%, preservative, sodium salt of carboxymethyl cellulose with a molecular weight of 250,000, a foaming agent selected from glycerrhizic acid and its pharmaceutically acceptable salts, purified water is poured into the first bottle of the kit according to claim 4 with lyophil composition but dried recombinant protein E7 of human papillomavirus types 16 and 18 connected to heat shock of the amino acid sequence of the protein M.W. 70 kDa from M. tuberculosis, and shakes for 1, 2 minutes. 7. The method of obtaining funds according to claim 6, characterized in that the resulting solution is placed in a syringe for intravaginal use.