RU2438702C1 - Medicine for treatment of uterus neck diseases, associated with human papilloma virus, in form of vaginal capsules - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, in particular, to gynecology and can be applied for treatment of precancerous diseases of uterus neck. Medication is made in form of vaginal capsules, characterised by containing recombinant composition of proteins E7 of human papilloma virus of 16 and 18 types, bound with amino acid sequence of heat sock protein m.m. 70 kDa from M. tuberculosis, gelatin, glycerin, distillated monoglycerides, purified water, chitosan with degree of deacetylation 86%, lactose, dextran 40000, preservative, natural oil.

EFFECT: medication is more efficient and stable in storing.

2 cl, 1 tbl, 4 ex

Description

The invention relates to medicine, in particular to gynecology, and may find application for the treatment of precancerous and cancer diseases of the cervix. 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 the 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 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.

The patent application No. 2008146809 of 11/27/08 describes 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 m.m. 70 kDa from M. tuberculosis in the form of vaginal suppositories. The disadvantages of this composition is the use of cocoa butter and type A solid fat as the basis for suppositories, since these bases are not stable at high temperatures, give metastable modifications and cannot be reused at the stages of the process, which significantly increases the cost of production. Also, this application does not address the stability issues 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 in suppositories.

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 in the form of suppositories composition:

- a solution of recombinant proteins, E716-Hsp 70, E718-Hsp 70;

- Chitosan with a degree of deacetylation of 86%;

- PEG 1500;

- solid fat type A;

- vitepsol HW;

- TRIS from MR Biomedicals Inc. cat. 195605.

This source does not specifically disclose the capsule form, but is similar in effect and composition, therefore, it can be indicated as the closest analogue prototype.

Gelatin capsules for vaginal use are more promising compared to suppositories from a technological, biopharmaceutical and economic point of view. The gelatin shell protects the drug from the effects of environmental factors. In particular, they withstand temperatures of more than 40 degrees, unlike candles.

The release of the drug substance occurs faster and easier than in suppositories, since under the influence of a weakly alkaline medium (pH 7.3-7.6) of the mucous membranes, the gelatinous membranes swell, and in this state, even a weak effect on them is enough to break it and release the contents . The amount of drug substance in the capsule, which has the necessary therapeutic effect, can be significantly higher than in suppositories.

Thus, the production of vaginal preparations based on proteins in a gelatin shell allows significantly expanding the range of products for the treatment of cervical diseases associated with human papillomavirus.

The objective of this 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 in various climatic regions.

The problem is solved by a vaginal soft gelatin capsule, which 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, gelatin, glycerin, distilled monoglycerides, purified water, chitosan with a deacetylation degree of 86%, lactose, dextran 40,000, preservative, vegetable oil, with the following combination of components per 1 capsule, parts by weight:

- Recombinant composition of proteins E7 of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m. 70 kDa from:

M. tuberculosis 3.0-6.0 - Gelatin 10.0-12.5 - Glycerin 20.0-30.0 - Distilled monoglycerides 0.35-0.65 - Purified water 30.6-36.0 - Chitosan with a degree of deacetylation of 86% 18.0 - Lactose 50, -100.0 - Dextran 40,000 150.0-200.0 - Preservative 5.0-10.0 - Vegetable oil up to 1000

Distilled gelatin, glycerin and monoglycerides and purified water are used to form capsule shells. In the stated ratio, they provide the necessary structural integrity and elasticity of the shell. When the ratio is changed, the shell either does not form, or its disintegration time exceeds 30 minutes.

Chitosan with a deacetylation degree of 86%, preservative, lactose, dextran 40,000 and vegetable oil are used to form the internal contents of the capsule. In the stated ratio, they form a suspension with a recombinant composition of the E7 proteins of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein mm 70 kDa from M. tuberculosis, which has the necessary consistency to delay the recombinant composition of proteins on the cells of the vaginal mucosa for the necessary time for the manifestation of a therapeutic effect.

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.

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.

As vegetable oil can be used olive, sunflower, soybean and other oils. Soybean oil is preferably used. Capsules are prepared by methods generally accepted in pharmacy. For example, as follows.

The gelatin in the reactor is poured with water (temperature 15-18 ° C) for 1.5-2 hours, then it is melted at t = 45-75 ° C with stirring for 1 hour, glycerin is added, distilled monoglycerides and water, while stirring still 30 minutes. Then turn off the heating and the mixer, leave the mass in the reactor for 1.5-2 hours with the connection of a vacuum to remove air bubbles from the mass. The prepared mass is transferred to a thermostat and kept at t = 60 ° C to stabilize 2.5-3 hours. A suspension is prepared with a 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. 70 kDa from M. Tuberculosis, chitosan with a degree of deacetylation of 86%, preservative, lactose, dextran 40,000 and vegetable oil. Fill the capsules with a dispenser. The dispenser is a piston pump with a volume control of the working cylinder and an electronic pause controller. During the suction stroke of the metering piston, fluid enters the syringe. During the reverse stroke, it is pushed into the rubber tube, which ends with a pipette, which is introduced into the empty shells. After filling, the gelatin capsules immediately enter the sealing machine. Check the quality of the seal. Capsules are dried in a cabinet with forced air circulation at t = + 23 ... + 26 ° С for 20 hours. Grind the capsules.

The proposed dosage form and the ratio of active and auxiliary substances provide 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

Soft capsule composition

- Recombinant composition of proteins E7 of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m. 70 kDa from:

M. tuberculosis 4.0 - Gelatin 10.0 - Glycerin 30,0 - Distilled monoglycerides 0.65 - Purified water 30.6 - Chitosan with a degree of deacetylation of 86% 18.0 - Lactose 70.0 - Dextran 40,000 180.0 - Nipagin 7.0 - Soybean oil up to 1000

Example 2

Soft capsule composition

- Recombinant composition of proteins E7 of human papillomavirus types 16 and 18, coupled to the amino acid sequence of the heat shock protein m. 70 kDa from:

M.tuberculosis 6.0 - Gelatin 12.5 - Glycerin 25.0 - Distilled monoglycerides 0.55 - Purified water 36.0 - Chitosan with a degree of deacetylation of 86% 18.0 - Lactose 100.0 - Dextran 40,000 150.0 - Lemon acid 5,0 - Olive oil up to 1000

Example 3

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

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.

Stability of recombinant proteins in various dosage forms. Storage at room temperature Suppositories Capsules of Example 1 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 70 210 200 thirty 60 40 180 160 60 fifty 35 one hundred 90 90

Example 4

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 one hour after intravaginal administration in a dose corresponding to the capsule composition of example 1, while time as with the injection route of administration, this indicator is 0.5 hours at the same concentration value.

The duration of action of the drug in capsules is about 10.5 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 (2)

1. An agent for the treatment of cervical diseases associated with human papillomavirus in the form of vaginal capsules, characterized in that it contains a recombinant composition of the E7 proteins of human papillomavirus 16 and 18 types, coupled to the amino acid sequence of the heat shock protein m.m. 70 kDa from M. tuberculosis, gelatin, glycerin, distilled monoglycerides, purified water, chitosan with a deacetylation degree of 86%, lactose, dextran 40,000, preservative, vegetable oil in the following combination of components per 1 capsule, parts by weight:
Recombinant E7 Protein Composition human papillomavirus 16 and 18 types connected with amino acid thermal protein sequence shock m.m. 70 kDa from M. tuberculosis 3.0-6.0 Gelatin 10.0-12.5 Glycerol 20.0-30.0 Distilled monoglycerides 0.35-0.65 Purified water 30.6-36.0 Chitosan with a degree of deacetylation of 86% 18.0 Lactose 50, -100.0 Dextran 40,000 150.0-200.0 Preservative 5.0-10.0 Vegetable oil Up to 1000 2. The tool according to claim 1, characterized in that it contains soybean oil as vegetable oil.