RU2329790C1 - Treatment-and-preventive agent of antibacterial and spermicidal action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry, in particular to production of new treatment-and-preventive agent of antibacterial and spermicidal action which can be used in gynaecology. Offered treatment-and- preventive agent characterised by antibacterial and spermicidal action, is made as tablet for intravaginal introduction and contains chlorhexidine or its salt and base which as a main component contains lactose and other pharmaceutically acceptable adjuvant. Besides, specified agent can contain regenerant, in particular dexpanthenol or methyluracil or sea-buckthorn oil. Agent in the form of vaginal tablets is characterised by more effective antibacterial and spermicidal action and is more easy-to-use.

EFFECT: production of agent in the form of vaginal tablets characterised by antibacterial and spermicidal action.

40 ex, 10 tbl, 2 dwg, 2 cl

Description

The invention relates to medicine and the pharmaceutical industry, in particular to the creation of a new therapeutic and prophylactic agent with antibacterial and spermicidal action, which can be used in gynecology.

Currently, the spread of gynecological diseases and sexually transmitted diseases (STDs) has a tendency to increase and is gaining increasing social significance.

For the treatment and prevention of gynecological diseases, drugs are widely used that have antibacterial and antiseptic effects in the form of dosage forms containing chlorhexidine.

Chlorhexidine is one of the most active antibacterial and antiseptic agents. It has a quick and strong bactericidal effect on gram-positive and gram-negative bacteria. It is also known that chlorhexidine has spermicidal activity. Therefore, it can be used not only as an agent for the prevention of sexually transmitted diseases (STDs), but also as a spermicidal agent.

So, a female swab with a prolonged therapeutic and preventive effect, made of an absorbent material and having a treatment shell, which is a composition of biocompatible gelling polymers and antiseptic and antimicrobial drugs, in particular chlorhexidine (patent of the Russian Federation No. 2178312 for “Female swab with prolonged therapeutic and prophylactic effect ”).

This swab provides a prolonged therapeutic and / or prophylactic effect for the treatment of gynecological diseases and the prevention of STDs. However, the use of a tampon leads to a decrease in the supply of oxygen to the vaginal mucosa, which leads to a violation of the natural balance of the vaginal microflora and, as a result, the development of vaginosis, as well as when the content of chlorhexidine in the preparation is ~ 55%, increases the risk of side effects.

Known chemical composition for application to a tampon having spermicidal, bactericidal and virucidal activity, containing chlorhexidine and / or polyethylene oxide and nonoxynol-9 (PCT application No. 0003682 for “Chemical composition having spermicidal, bactericidal and virucidal activity, and a preparation for administration to vaginal canal ").

This composition has spermicidal, bactericidal and virucidal activity for intravaginal use as a contraceptive, as well as preventing the transmission of STDs. However, the use of antibacterial action in the composition of the two active substances leads to damage to the cells of the vaginal mucosa.

Known pharmacological composition of prolonged action for the treatment of various inflammatory diseases, in particular in dermatology, made in the form of a gel or film containing chlorhexidine, dimexide and a stabilizing agent (RF patent No. 2201219 "Pharmacological composition of prolonged action").

This pharmaceutical composition provides a prolonged effect of a medicinal substance - chlorhexidine - for the treatment of inflammatory diseases of various etiologies. However, the disadvantage of this composition, made in the form of a gel or film, is the inconvenience of use for the treatment of inflammatory diseases in dermatovenerology, as well as the use of dimexide leads to burns of the vaginal mucosa.

A vaginal agent in the form of a suppository is known containing an effective amount of at least one topical antimicrobial agent selected from the group consisting of, in particular, chlorhexidine gluconate and a viable colony of microencapsulated lactic acid bacteria (RF patent No. 2147431 for “Virocidic, bactericidal and spermatocidal suppository ").

This vaginal suppository is highly effective in preventing infection by almost all sexually transmitted diseases, and at the same time it is a contraceptive and also makes its users immune to yeast infections. However, it is known that the combined use of antibacterial agents and bacteria is undesirable, and the content of chlorhexidine in an amount of 10 to 2000 mg per suppository increases the risk of side effects.

The closest to the claimed tool, in terms of essential features, is the Hexicon therapeutic and prophylactic agent with antibacterial and antiseptic effects (RF patent No. 2166314 for the Hexicon therapeutic and prophylactic drug with antibacterial and antiseptic effects).

The known agent, made in the form of a suppository for intravaginal administration, containing chlorhexidine bigluconate and a hydrophilic base, is widely used to treat diseases such as syphilis, gonorrhea and trichomonasis.

However, the disadvantage of this suppository is that when using the suppository, the effect is not achieved in full due to a decrease in the bioavailability of chlorhexidine, due to the fact that vaginal infections are associated with abundant vaginal discharge, which lead to a decrease in the release of chlorhexidine from this base.

At the same time, the use of this drug leads to inhibition of the growth of normal microflora of the vagina, which increases the risk of developing vaginal dysbiosis. From practice, it is also known that when using this tool, a negative effect of the base on the vaginal mucosa is sometimes observed, which manifests itself in the form of a burn.

In addition, leakage of the suppository base creates a feeling of discomfort for consumers.

The objective of the invention is to provide a therapeutic and prophylactic agent in solid dosage form — in the form of an intravaginal tablet containing chlorhexidine or its salt, which has a high therapeutic effect due to increased bioavailability, with good consumer properties and the ability to stimulate the growth of normal microflora, and in addition, an expanding arsenal of means with chlorhexidine for intravaginal administration, with antibacterial and spermicidal action tweed.

The problem is achieved by the proposed tool with antibacterial and spermicidal action, containing chlorhexidine or its salt and a base, made in a dosage form for intravaginal administration, according to the invention, the tool is made in the form of a tablet, the base of which contains lactose as the main component, as well as other pharmaceutically acceptable excipients, in the following ratio of components, wt.%:

Chlorhexidine or salt 1.0-4.0 The basis: Lactose 60.0-95.0 Other pharmaceutically acceptable up to 100 Excipients

It is preferred that bigluconate or acetate or dihydrochloride is used as the chlorhexidine salt.

Preferably, the agent may further comprise a regenerating agent selected from dexpanthenol, methyluracil, sea buckthorn oil in an amount of 0.5-10 wt.%.

The technical result of the proposed structure is to create a therapeutic agent in the form of tablets, allowing

- increase efficiency by increasing the bioavailability of chlorhexidine;

- stimulate the growth of normal microflora after application of the drug;

- improve consumer properties.

The fact that the proposed product with chlorhexidine has high therapeutic efficacy and good consumer properties, due to the fact that the tool is made in the form of a dosage form for intravaginal administration - in the form of a tablet containing lactose as the main component of the base, which contributes to the complete release of chlorhexidine from the base, which allows to increase its bioavailability and, as a result, to increase its antibacterial and spermicidal effect. The complete release of chlorhexidine from the base is due to the introduction of lactose as the main constituent component of the base (with its content of 60.0-95.0 wt.%) And allows you to evenly distribute chlorhexidine in the tablet mass (see table 1), which leads to a uniform distribution of chlorhexidine in the vaginal mucosa.

In addition, lactose stimulates the growth of normal microflora of the vagina, which reduces the risk of developing vaginal dysbiosis against the background of the use of the proposed tool.

The fact that the proposed tool is made in the form of a tablet leads to the absence of leakage of the base and, as a result, the absence of a feeling of discomfort.

An additional introduction to the composition of regenerating substances that can stimulate the restoration of tissues damaged as a result of infection leads to the rapid restoration of vaginal mucosa cells and reduces the risk of secondary infection, which eliminates the negative effect of chlorhexidine on the vaginal mucosa.

Table 1
Uniform distribution of chlorhexidine by tablet mass Series No. Chlorhexidine Content Average deviation from face value min max min max 490606 95.0 (1.9) 99.6 (2.7) 2.1 2.6 500606 93.6 (2.6) 98.9 (2.7) 510606 99.7 (2.4) 104.2 (2.0) 520606 95.3 (1.6) 99.7 (2.9)

Thus, the proposed tool is effective both in the treatment of bacterial inflammation, and when it is used as a spermicidal drug, with minimal risk of developing vaginal dysbiosis. In addition, the proposed tool has improved consumer properties.

The content of the components in the proposed tool is established experimentally.

The content of chlorhexidine or its salts of less than 1 wt.% Does not provide the required antibacterial and spermicidal action.

A content of chlorhexidine or its salt of more than 4 wt.% Increases the risk of side effects.

A lactose content of less than 60.0 wt.% Does not provide a uniform distribution of chlorhexidine, which means achieving the maximum bioavailability of chlorhexidine, and also does not provide sufficient stimulation of the growth of normal vaginal microflora.

The lactose content of more than 95.0 wt.% Does not allow you to get a tablet with the appropriate tablet requirements.

As other pharmaceutically acceptable excipients, an antifriction substance or a mixture of antifriction substances, excipients, binders, disintegrants, buffering agents to produce a tablet are used.

As an anti-friction substance, the proposed tool may contain, in particular, stearic acid or its salts, talc, aerosil.

As salts of stearic acid, the proposed tool may contain, in particular, magnesium and / or calcium salts.

As a filler, the proposed tool may contain, in particular starch, microcrystalline cellulose (MCC), magnesium carbonate.

As a binder, the proposed tool may contain, in particular, polyvinylpyrrolidone, hydroxypropyl methylcellulose, gelatin, sodium alginate.

As a disintegrant, the agent may contain, in particular, tween-80, crospovidone.

As a buffering agent, the agent may contain, in particular, adipic or lactic acid.

The method of obtaining the proposed tool.

The calculated amount of a solution of chlorhexidine or its salt, lactose and other pharmaceutically acceptable excipients are mixed. The resulting mass is dried, calibrated, and then dusted with an anti-friction substance or a mixture of anti-friction substances, or with a mixture of an anti-friction substance and a filler, then tableted.

The following are examples of the manufacture of the proposed funds in the form of vaginal tablets.

Example 1

To a solution containing 10.0 g of chlorhexidine (in terms of dry matter), 30.0 g of methylcellulose is added. 950.0 g of lactose are added to the resulting mixture, the mass is stirred, dried, calibrated, dusted with 10.0 g of aerosil and tableted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 1.0

Lactose - 95.0

Aerosil - 1.0

Cellulose 3.0

Example 2

To a solution containing 40.0 g of chlorhexidine (in terms of dry matter), 9.0 g of lactic acid is added. 950.0 g of lactose are added to the resulting mixture, the mass is stirred, dried, calibrated, 1.0 g of stearic acid is dusted and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 4.0

Lactic acid - 0.9

Lactose - 95.0

Stearic acid - 0.1

Example 3

To a solution containing 10.0 g of chlorhexidine (in terms of dry matter), 50.0 g of polyvinylpyrrolidone, 30.0 g of tween-80, 20.0 g of lactic acid are added. 600.0 g of lactose and 289.1 g of starch are added to the resulting mixture, the mass is mixed, dried, calibrated, 0.9 g of calcium stearate is dusted and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 1.0

Lactose - 60.0

Calcium Stearate - 0.09

Polyvinylpyrrolidone - 5.0

Twin 80 - 3.0

Lactic acid - 2.0

Starch - up to 100

Example 4

In a solution containing 40.0 g of chlorhexidine (in terms of dry matter), 20.0 g of gelatin is added. To the resulting mixture was added 20.0 g of crospovidone, 309.1 g of MCC and 600.0 g of lactose, the mass was stirred, dried, calibrated, dusted with 0.9 g of talc and 10.0 g of adipic acid, tableted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 4.0

Lactose - 60.0

Talc - 0.09

Gelatin - 2.0

Crospovidone - 2.0

Adipic acid - 1.0

MCC - up to 100

Examples 5-8.

Carried out analogously to examples No. 1-4, but as the active substance, chlorhexidine bigluconate is used.

Examples 9-12.

Carried out analogously to examples No. 1-4, but as the active substance, chlorhexidine acetate is used.

Examples 13-16.

Carried out analogously to examples No. 1-4, but as the active substance, chlorhexidine dihydrochloride is used.

Example 17

5.0 g of dexpanthenol, 10.0 g of sodium alginate, 10.0 g of tween-80 are added to a solution containing 10.0 g of chlorhexidine (in terms of dry matter). 14.1 g of starch and 950.0 g of lactose are added to the resulting mixture, the mass is mixed, dried, calibrated, 0.9 g of magnesium stearate is dusted and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 1.0

Dexpanthenol - 0.5

Lactose - 95.0

Magnesium Stearate - 0.09

Sodium Alginate - 1.0

Twin 80 - 1.0

Starch - up to 100

Example 18

5.0 g of dexpanthenol are introduced into a solution containing 40.0 g of chlorhexidine (in terms of dry matter). 4.1 g of MCC and 950.0 g of lactose are added to the resulting mixture, the mass is stirred, dried, calibrated, dusted with 0.9 g of stearic acid and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 4.0

Dexpanthenol - 0.5

Lactose - 95.0

Stearic acid - 0.09

MCC - up to 100

Example 19

10.0 g of gelatin and 5.0 g of dexpanthenol are introduced into a solution containing 10.0 g of chlorhexidine (in terms of dry matter). 5.0 g of crospovidone, 349.0 g of MCC and 600.0 g of lactose are added to the resulting mixture, the mass is mixed, dried, calibrated, dusted with 20.0 g of adipic acid and 1.0 g of a mixture of magnesium stearate and calcium stearate (in the ratio 1: 1) and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 1.0

Dexpanthenol - 0.5

Lactose - 60.0

Magnesium Stearate / Calcium Stearate - 0.1

Gelatin - 1.0

Crospovidone - 0.5

Adipic acid - 2.0

MCC - up to 100

Example 20

To a solution containing 40.0 g of chlorhexidine (in terms of dry matter), 50.0 g of polyvinylpyrrolidone, 15.0 g of tween-80, 5.0 g of dexpanthenol are introduced. 600.0 g of lactose and 265.0 g of magnesium carbonate are added to the resulting mixture, the mass is stirred, dried, calibrated, dusted with 10.0 g of Aerosil and 15.0 g of adipic acid, and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 4.0

Dexpanthenol - 0.5

Lactose - 60.0

Aerosil - 1.0

Polyvinylpyrrolidone - 5.0

Twin-80 - 1.5

Adipic acid - 1.5

Magnesium carbonate - up to 100

Example 21

To a solution containing 10.0 g of chlorhexidine (in terms of dry matter), 100.0 g of dexpanthenol is introduced. 880.0 g of lactose are added to the resulting mixture, the mass is stirred, dried, calibrated, 10.0 g of talcum powder is dusted and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 1.0

Dexpanthenol - 10.0

Lactose - 88.0

Talc - 1.0

Example 22

To a solution containing 40.0 g of chlorhexidine (in terms of dry matter), 100.0 g of dexpanthenol is introduced. 850.0 g of lactose are added to the resulting mixture, the mass is stirred, dried, calibrated, 10.0 g of calcium stearate is dusted and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 4.0

Dexpanthenol - 10.0

Lactose - 85.0

Calcium Stearate - 1.0

Examples 23

To a solution containing 10.0 g of chlorhexidine (in terms of dry matter), 100.0 g of dexpanthenol, 10.0 g of polyvinylpyrrolidone, 5.0 g of tween-80, 10.0 g of lactic acid are added. 600.0 g of lactose and 264.0 g of starch are added to the resulting mixture, the mass is stirred, dried, calibrated, 1.0 g of magnesium stearate is dusted and tabletted.

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 1.0

Dexpanthenol - 10.0

Lactose - 60.0

Magnesium Stearate - 0.1

Polyvinylpyrrolidone - 1.0

Twin-80 - 0.5

Lactic acid - 1.0

Starch - up to 100

Example 24

100.0 g of dexpanthenol, 40.0 g of hydroxypropyl methylcellulose and 30.0 g of tween-80 are added to a solution containing 40.0 g of chlorhexidine (in terms of dry matter). 600.0 g of lactose and 184.0 g of MCC are added to the resulting mixture, the mass is stirred, dried, calibrated, dusted with 5.0 g of adipic acid and 1.0 g of a mixture of calcium stearate and magnesium stearate (in a 1: 1 ratio), tableted .

Get a tablet mass having the following composition, wt.%:

Chlorhexidine 4.0

Dexpanthenol - 10.0

Lactose - 60.0

Calcium Stearate / Magnesium Stearate - 0.1

Oxypropyl methylcellulose 4.0

Twin 80 - 3.0

Adipic acid - 0.5

MCC - up to 100

Examples 25-28.

Conducted similarly to examples No. 17-20, but as a regenerating substance, methyluracil is used.

Examples 29-32.

Conducted similarly to examples No. 17-20, but as a regenerating substance, sea buckthorn oil is used.

Example 33-36.

Conducted similarly to examples No. 21-24, but as a regenerating substance, methyluracil is used.

Examples 37-40.

Conducted similarly to examples No. 21-24, but as a regenerating substance, sea buckthorn oil is used.

The quantitative determination of chlorhexidine or its salt in tablets is carried out by high performance liquid chromatography (HPLC) on a Waters liquid chromatograph (or the like) with a spectrophotometric detector.

The test for microbiological purity of the tablets is carried out in accordance with the requirements of the Global Fund XI, issue 2, p.193, and Changes No. 3 (category 2). The results obtained comply with regulatory requirements.

The resulting tablets in appearance comply with the requirements of the Global Fund XI, issue 2, p. 154.

The following are the results of preclinical studies of the proposed tool, consisting of two parts.

In order to determine the general toxic effect and evidence of the effectiveness of the proposed drug and its spermicidal activity, its preclinical trials were conducted at the central research laboratory of the Nizhny Novgorod State Medical Academy.

The experimental results were processed on an IBM PC / AT using the Statistica 5.5 application software packages. For all quantitative data, the arithmetic group mean (M) and standard deviation (SD) were calculated, paired intragroup and intergroup mean comparisons were determined according to Wilcoxon criteria. Differences were considered significant at a significance level of p <0.05.

1. This part of the report presents the results of a preclinical study of the proposed chlorhexidine-based agent, vaginal tablets developed by Nizhpharm OJSC (Russia), according to the parameters of bactericidal action, histology of the vaginal mucosa, and pH of vaginal mucus when modeling bacterial vaginitis in rats. The contents of the components are presented in table 2.

table 2
The composition of the proposed product (content per tablet) Components Formulation Options (wt.%) one 2 3 Chlorhexidine 2.9 2.9 0 Twin 80 0.50 0.50 0.50 Dexpanthenol 0 10 0 PVP 2 2 2 MCC 5 5 5 Calcium stearate one one one Lactic acid 0 one 0 Lactose up to 100 up to 100 up to 100

Toxicological studies were carried out in accordance with the guidelines “Guidelines for the experimental (preclinical) study of new pharmacological substances”, 2000.

In the experiments, 70 sexually mature rats of Wistar females weighing 220 ± 3.2 g were used.

Animals were randomly assigned to groups using body weight as a criterion so that the individual weight of the animals did not differ by more than 20% from the average weight of animals of the same sex.

Each animal was assigned an individual number, according to which the animal was labeled with either a puncture of the auricle or coloring with dyes (eosin and methylene blue) of the tail surface according to the developed schemes. On the label of a cell of a certain color, the group, animal number, label, study code, leader were indicated.

The basic rules for keeping and caring were in accordance with the guidelines given in the Guide for care and use of laboratory animals. ILAR publication, 1996, National Academy Press. All procedures for routine animal care were performed in accordance with the SOP laboratory.

Animals were kept in polycarbonate cages, 5-7 animals per cage on the bedding. The cages are equipped with steel grid covers with a feed recess.

Sawdust of non-coniferous trees was used as litter.

Complete feed for laboratory animals was given ad libitum in the feed recess of the cell lid.

Filtered tap water was supplied in standard autoclaved drinking bottles.

There was no contamination of bedding, feed and water that could affect the results of the study. The laboratory conducts periodic analysis of water, litter and feed for microbiological contamination.

To evaluate the effectiveness, the effect of the proposed agent on intravaginal use was studied.

The calculation of doses was based on taking into account the mass and surface of the animal’s body, customer recommendations on the use of the drug.

The calculated daily dose of chlorhexidine for humans was 0.46 mg / kg, using a coefficient of 2.6 mg / kg for rat body surface area. On a rat weighing 200 g - 0.54 mg / day, given a 2-time use (see table 3).

The proposed tool was used 2 times a day for 10 days. Used tablets weighing 0.1 g, the content of chlorhexidine in which was 2.6 mg. For a single administration, the tablet was divided into parts and a portion of the tablet was intravaginally administered.

For humans: max TD = 0.46 mg / kg (two tablets) / day. For rats, when recalculating doses based on body surface: max TD = 0.46 mg / kg × 39/7 = 2.6 mg / kg.

Table 3
Dose conversion factors (in mg / kg per mg / m ² ) for rats and humans depending on body weight (Freireich EJ, 1966; Ulanova I.P. et al., 1968) Kind of animal Body weight (kg) Conversion factor Rat 0,200 6.5 Person 70 39

Placebo animals were intravaginally administered similarly sized tablet particles consisting of a pill base.

The specific pharmacological effect of the drug was studied in modeling bacterial vaginitis.

To test the proposed tool used typical strains of Staphylococcus aureus strain 7M (NNIEM, N. Novgorod), and clinical isolate Esherichia coli strain 782-2. For the cultivation of staphylococci and E. coli meat-peptone agar (MPA).

A suspension of microorganisms containing 10 ⁷ microbial bodies in 1 ml (initial concentration) was prepared by washing with buffered saline (pH 7.2-7.4) 18-hour agar culture. The microbial suspension should be standardized according to the turbidity standard of the State Control Institute of Medical Biological Equipment named after L.A. Tarasovich for bacteria and spectrophotometrically for candid.

Rats were infected intravaginally with the administration of 0.025 ml of S. aureus and 0.025 ml of E. coli. A day after infection, the introduction of the proposed funds within 10 days.

10 days after the application of the proposed tool, seeding was made from the vaginal cavity to detect Staphylococcus aureus, Esherichia coli. For this, material was taken from the vaginal cavity with a sterile swab, placed in 1 ml of sterile physiological saline and carefully resuspended, diluted 1 to 10 in sterile physiological saline. The resulting suspension (0.1 ml) was placed on selective solid media to detect the corresponding microorganisms. For the cultivation of staphylococci, yolk-salt agar (LCA) was used; for the cultivation of Escherichia coli, Endo medium was used. The material on Petri dishes was triturated with a spatula, incubated in a thermostat (37 ° C, 24-48 h).

The number of colony forming units (CFU) characteristic of these microorganisms was calculated on Petri dishes with selective media. All crops were carried out in 2 repetitions.

15 minutes after application of the proposed drug, as well as 6 and 12 hours, a pH study of intravaginal mucus was performed. A similar measurement mode is caused by a 2-fold introduction of the proposed funds per day.

Before the experiment, female rats were selected with the same estrous cycle according to the “vaginal smear cytology” test so that the rats were at the stage of proestrus before infection and during the collection of histological analyzes. During the experiment, a vaginal smear was taken daily for cytological studies. On the 10th day of the experiment, samples of the vaginal mucosa were taken for histological examination. The experimental material was fixed in 10% neutral formalin, dehydrated in alcohols of increasing concentration and embedded in paraffin. Sections of 5-7 μm thick were obtained on a Leica SM 2000R microtome, stained with hematoxylin and eosin and examined using a Leica DMLS microscope. Video images were obtained on a video system using a CCD camera.

Bactericidal activity of the proposed tool. During the entire period of observation, septic phenomena were not detected, the mass of animals did not significantly decrease. In a number of rats of different groups, vaginal discharge was observed. Over the entire observation period, the animals remained mobile, looked healthy outwardly. Clinical examination data indicate the absence of a generalized systemic reaction of the body with intravaginal infection.

After treatment, a decrease in the number of CFU was observed in some groups (see tables 4, 5).

Table 4
A comparative amount of S. aureus strain 7M in the vaginal cavity of rats after intravaginal application of various compositional options of the proposed drug (M ± SD) Animal groups (series of preparations) The number of S. aureus strain 7M (KOE, M ± SD) Native material Breeding 1:50 The proposed tool, option No. 1 69.7 ± 21.9 * 5.8 ± 2.4 * The proposed tool, option No. 2 16.5 ± 7.8 * 0.2 ± 0.1 * The proposed tool, option No. 3 (placebo) 302.5 ± 85.6 75.3 ± 14.2 Control (vaginitis without treatment) 314.2 ± 94.3 73.8 ± 12.4 * Significant decrease relative to control (placebo) (p <0.05) Table 5
The comparative amount of E. coli strain 782-2 (CFU) in the vaginal cavity of rats after intravaginal use of various options for the composition of the proposed funds (M ± SD) Groups of animals (series of drugs) The number of E. coli strain 782-2 Native material Breeding 1:50 The proposed tool, option No. 1 8.3 ± 4.5 * 1.2 ± 0.9 * The proposed tool, option No. 2 3.5 ± 1.2 * 0.2 ± 0.1 * The proposed tool, option No. 3 (placebo) 333.3 ± 94.1 70.2 ± 34.9 Control (vaginitis without treatment) 345.6 ± 83.5 95.2 ± 28.5 * Significant decrease relative to control (placebo) (p <0.05)

A decrease in the number of CFU of both S. aureus and E. coli was observed in the group composition No. 1 and No. 2 compared with the placebo group. The most pronounced decrease in microbial contamination was noted in the group using series No. 2 (Tables 4, 5).

Thus, there was a significant decrease in the number of Staphylococcus aureus and Esherichia coli observed only in groups using the proposed tool option No. 1 and option No. 2. In this case, the most pronounced bactericidal effect is characteristic of the group "option No. 2".

The effect of the proposed tool on the reaction of the vaginal mucus. Data on measuring the pH of vaginal mucus are presented in table 6.

Table 6
The change in pH after intravaginal use of the proposed tool (M ± SD) Groups of animals (series of drugs) pH Before After 15 min In 6 hours After 12 hours The proposed tool, option No. 1 7.00 ± 0.2 5.5 ± 0.22 * 6.00 ± 0.30 7.0 ± 0.28 The proposed tool, option No. 2 7.20 ± 0.22 8.5 ± 0.22 * 8.2 ± 0.20 7.5 ± 0.26 The proposed tool, option No. 3 (placebo) 7.20 ± 0.20 6.0 ± 0.30 * 7.0 ± 0.24 7.5 ± 0.28 Control (vaginitis without treatment) 7.00 ± 0.20 7.0 ± 0.28 7.2 ± 0.20 7.0 ± 0.28 * Significant decrease relative to control (placebo) (p <0.05)

It should be noted that the normal pH of vaginal mucus in rats ranges from 7.0-7.2. During the entire treatment period and in the control, no significant differences were revealed.

The effect of drugs on the structure of the vaginal mucosa. In the control series of experiments and placebo on the 10th day there was a pronounced inflammatory infiltrate, which is diffusely distributed in its own plate of the mucous membrane, as illustrated in figure 1. Accumulations of leukocytes were observed in the thickness of the epithelial layer.

Figure 1 shows the structure of the vagina in the stage of proestrus on the 11th day of modeling of bacterial vaginitis in the control series without the use of the proposed tool (staining with hematoxylin and eosin; magnification × 200).

When treating animals with the proposed agent, option No. 1 and option No. 2, there were no pathological changes in the structure of the vagina: the mucous membrane is covered with a stratified squamous epithelium of the skin type, in which there are three layers - basal, intermediate and superficial. The basis of its own plate of the mucous membrane is a loose unformed connective tissue. The submucosa in the vagina is not expressed, and the plate of the mucosa directly passes into the muscularis, which mainly consists of longitudinally extending bundles of smooth muscle cells, as illustrated in FIG. Blood vessels with an open lumen are determined.

Figure 2 shows the structure of the vagina in the stage of proestrus in the treatment of animals with the proposed tool: a - option No. 1 - the absence of structural disorders in the mucous membrane; b - option No. 2 - the absence of inflammatory manifestations in the structure of the mucous membrane (staining with hematoxylin and eosin; magnification × 200).

Thus, a histological examination of the vagina revealed normalization of the structure in the treatment of animals with the proposed tool, options No. 1 and No. 2.

Conclusion According to the degree of severity of the bactericidal action, the variants of the compositions of the proposed product in comparison with the control were located in the following order: No. 2-1-3.

For preclinical studies, the developer provided various options for the proposed chlorhexidine-based agent in the form of vaginal tablets developed by Nizhpharm OJSC, Russia.

This study was conducted to study the specific pharmacological action of drugs with intravaginal use.

The test substance (as a medicine) was used in an equitherapeutic dose. As a control substance, a tablet base was used.

The effectiveness of the preparations was evaluated by the parameters of bactericidal action, histology of the vaginal mucosa, and pH of vaginal mucus when modeling bacterial vaginitis in rats.

It was shown that a significant decrease in the number of Staphylococcus aureus and Esherichia coli was observed in groups with the proposed tool, options No. 1 and No. 2. In this case, the most pronounced bactericidal effect is characteristic for option No. 2.

Histological examination of the vagina revealed normalization of the structure in the treatment of animals with the proposed tool, options No. 1 and No. 2.

According to the degree of severity of the bactericidal action of the proposed funds compared with the control options were located in the following order: No. 2-1-3.

This part of the report presents data on the preclinical study of vaginal tablets developed by Nizhpharm OJSC (Russia), according to the parameters of the spermicidal activity of the proposed drug. The contents of the components are presented in table 2.

The study used ejaculate healthy men aged 18-20 years.

The procedure for introducing the substance. To evaluate the effectiveness, the effect of the test drug in vitro in the ejaculate was studied.

The dose of the proposed drug in the test system was 1 tablet per serving of donor ejaculate (3.5 ml).

In vitro study of spermicidal action. The method of obtaining and evaluating ejaculate. Ejaculate for analysis was obtained after 4 days of abstinence, smoking cessation and alcohol from young men aged 19-20 years. Before taking the ejaculate, each subject filled out an informed consent form.

The ejaculate was collected in a clean glass graduated container with a wide neck in a special laboratory room immediately before analysis. On the container indicate the name of the donor, date and time of receipt of the ejaculate. The ejaculate is a suspension of sperm in the secret of the testicles and their appendages, which at the time of ejaculation is mixed with the secretion of the prostate gland, seminal vesicles and onion-urethral glands. In general, ejaculate is a viscous liquid.

When working with ejaculate in the laboratory, the safety rules generally accepted in laboratory practice were observed (WHO, 1983).

Initial macroscopic evaluation of the ejaculate. Ejaculate was evaluated immediately after liquefaction. Normally, at room temperature, the ejaculate sample liquefies within 15-30 minutes.

The sample was moved in the same container in which it was received. Since sperm motility was evaluated at a temperature of 37 ° C, the sample was gradually brought to this temperature during liquefaction and stirring.

The volume of ejaculate was measured using a graduated conical tube.

Preparation of the drug for a routine spermogram. The volume of ejaculate applied to the slide and the size of the coverslip were standard. A fixed volume of ejaculate was applied to a clean slide and covered with a coverslip (22 × 22 mm), making sure that there were no air bubbles between the glasses.

Freshly prepared preparation was left for approximately one minute to stabilize. Sperm motility was evaluated at a temperature of 37 ° C on a heated stage, since temperature strongly affects sperm motility and speed.

The drug was examined under a microscope at first at a 100-fold cumulative increase, while obtaining a general idea of the formation of mucus filaments, aggregation and uniform distribution of sperm on the glass. Then the drug was investigated at a 400-fold cumulative increase.

Assessment of sperm concentration was carried out using a hemocytometer when diluting the ejaculate 1:20. Sperm concentration was expressed as 1 · 10 ⁶ / ml.

Sperm motility was evaluated after sequential viewing of 200 sperm in at least 5 fields of view of the microscope. The motility of each sperm is classified into categories (a), (b) and (c) using the following criteria:

a) rapid translational movement (i.e. ≥25 μm / s at 37 ° C, 25 μm approximately corresponds to the length of 5 heads or half the length of the tail of the sperm);

b) oscillatory (slow and sluggish movement) and inaccessible movement;

c) immobile sperm.

Secondary macroscopic evaluation of the ejaculate. Staining the drug to assess sperm viability. Viability meant the percentage (in percent) of “live” spermatozoa, which were determined either by exclusion of the dye or by hypoosmotic swelling. The proportion of living and dead spermatozoa was determined using staining methods based on the principle of uptake of dead cells with damaged plasma membranes by dead cells. Using a light microscope, 200 spermatozoa were counted, differentiating live (unpainted) and dead (stained) spermatozoa.

Assessment of sperm morphology. Cooking smear. At least two smears were prepared from a sample of fresh ejaculate for a double assessment and in case of problems with staining. A small drop of ejaculate was applied to the surface of a clean fat-free dry glass. At a sperm concentration of more than 20 × 10 ⁶ ml, 5 μm ejaculate is sufficient. To prepare smears for undiluted ejaculate, the glass smearing method was used.

Staining method. The preparations were stained according to Papanicolaou. The sperm head in the acrosomal region was stained pale blue, and in the postacrosomal region, it was dark blue. The middle part of the sperm stained red. The tail was also painted blue or reddish.

Morphological classification of sperm. The sizes of sperm heads in stained preparations are slightly smaller than the sizes of live sperm heads in ejaculate, but their shape does not change significantly. Strict criteria were used to define the concept of the morphological "norm" of the sperm.

The sperm was considered normal if its head, neck, middle part and tail were normal, i.e. morphological description met the following criteria:

1. The sperm head is oval, the length of the sperm head is 4.0-5.5 μm, the width is 2.5-3.5 μm, the ratio of length to width is from 1.50 to 1.75. Sperm length and width were measured using an eyepiece micrometer.

2. A well-defined acrosomal region, comprising 40-70% of the head area.

3. The middle part (neck) is thin, less than 1 μm wide, is 1.5 times the length of the head and is attached to the head along its axis.

4. The tail of the sperm is straight, of equal thickness throughout, smaller in width of the middle part, not twisted, about 45 μm long.

The sperm was considered as pathological if its head, neck, middle part and tail had violations:

1. Head defects, namely large, small, conical, pear-shaped, round and amorphous heads, heads with vacuoles (> 20% of the head area is filled with unpainted vacuoles), heads with a small acrosome region (<40% of the head area) and with double heads or any combination thereof.

2. Defects of the neck and middle part, namely the “inclined” neck (the neck and tail form an angle of more than 90 ° to the long axis of the head), asymmetric attachment of the middle part to the head, thickened or uneven middle part, pathologically thin middle part, or any combination of them . If the heads were absorbed, only individual tails or the so-called “pin heads” were found in the ejaculate. “Pinheads” were not taken into account as a defect in the head, but as a defect in the neck and middle part.

3. Tail defects: short, multiple, in the form of hairpins, broken, inclined (angle greater than 90 °), tails of uneven thickness, twisted or any combination thereof.

The procedure for counting spermatozoa in assessing their morphology. In the study of colored preparations under an immersion microscope, a lens with a magnification of × 100 with a brightly illuminated field of view and an eyepiece with an increase of at least × 10 were used.

Morphological evaluation was carried out in several successively selected sections of the smear. As the drug was systematically scanned from one field of view to another, all normal spermatozoa were evaluated and counted, and all their defects were noted. Spermatozoa that lay on top of each other and with heads lying on the rib were not taken into account. They could be evaluated by changing the focus of the microscope. Standards are presented in table 7.

Table 7
Standard values for ejaculate Ejaculate indicators Normative values Volume ≥2.0 ml Sperm concentration ≥20 · 10 ⁶ / ml Mobility ≥50% motile or ≥25% with translational motion within 60 min after ejaculation Morphology <15% morphologically normal sperm is associated with a decrease in the frequency of in vitro fertilization Viability ≥50% live, i.e. not stained

Sample Ejaculate Analysis Form Date of receipt of ejaculate Source After 30 min After 60 min one 2 3 four Duration of Abstinence The interval between ejaculation and the beginning of the analysis (min) Volume (ml) Mobility a) rapid forward movement; b) oscillatory (slow and sluggish movement) and inaccessible movement; c) motionless sperm Viability (% live) Concentration (10 ⁶ / ml) Morphology - normal - head defect - defect of the neck or middle part - tail defect

After determining the parameters of the ejaculate donors in the same test tubes with ejaculate at a temperature of 37 ° C was added the required amount of the proposed funds, equal to a single dose for intravaginal use. For sequential observation, left in the thermostat for 30 and 60 min at 37 ° C.

Experimental groups (in each group n = 10):

1. Placebo (pill base).

2. The proposed tool option No. 1.

3. The proposed tool option No. 2.

The influence of the proposed funds on the viability of sperm in the ejaculate of healthy men in vitro. Macroscopic analysis of the ejaculate of healthy young men showed the following results (see table 8).

Table 8
Data of macroscopic evaluation of ejaculate in healthy young male donors, n = 30 (M ± SD) Volume (ml) Concentration (10 ⁶ / ml) Mobility (%) Viability (% live) but b from 3.82 ± 0.53 352 ± 149 71.5 ± 8.6 11.2 ± 4.04 16.3 ± 8.1 85 ± 7.1

When comparing with standards (see table 7), we can conclude that the studied ejaculates correspond to the values of normal indicators in a macroscopic description.

After adding the proposed tool, both motility and sperm viability significantly decreased, unlike placebo groups (see table 9), after 30 minutes.

Table 9
The effect of the proposed tool on the motility and viability of sperm in the ejaculate of healthy young male donors (M ± SD) Group Mobility (30 min) (%) Viability (% live) but b from 30 minutes 60 min Placebo pills 45.8 ± 8.2 15.0 ± 6.7 39.0 ± 22.4 74.25 ± 7.88 72.5 ± 5.31 The proposed tool, option No. 1 - - one hundred 2.0 ± 1.90 0 The proposed tool, option No. 2 - - one hundred 1.25 ± 0.35 0

Thus, a macroscopic analysis of the ejaculate after application of the proposed agent, in the form of vaginal tablets, revealed 1-2% of viable sperm after 30 minutes, although after 60 minutes the results were comparable with the action of other drugs - immobilization and death of sperm.

The results of the morphological analysis of smears, allowing to identify structural changes in sperm, after applying the proposed funds for 30 and 60 minutes are presented in table 10.

Adding the proposed funds option No. 1 and option No. 2 to a portion of the ejaculate in vitro has a damaging effect on the structure of sperm. The main point of application was the tail of the sperm, the morphology of which is most damaged 30 and 60 minutes after exposure.

Table 10
The effect of the proposed tool on the morphology of sperm in the ejaculate of healthy young male donors 30 and 60 minutes after exposure (M ± SD) Group Norm Pathology heads necks tail Source data (n = 30) 94.3 ± 2.9 2.2 ± 1.9 0.9 ± 0.8 2.7 ± 1.5 Placebo Pills (n = 10) 30 minutes 66.25 ± 20.2 2.5 ± 1.29 9.5 ± 4.35 27.75 ± 17.9 60 min 62.25 ± 23.0 2.5 ± 1.29 10.0 ± 9.52 26.25 ± 20.53 The proposed tool option No. 1 (n = 10) 30 minutes 63.5 ± 12.8 2.5 ± 1.7 13.0 ± 7.7 23.0 ± 9.8 60 min 54.3 ± 8.0 2.0 ± 1.0 10.3 ± 4.04 34.3 ± 10.5 The proposed tool option No. 2 (n = 10) 30 minutes 68 ± 12.8 2.3 ± 0.57 6.6 ± 4.04 24.7 ± 8.71 60 min 52.3 ± 5.03 2.6 ± 0.57 8.6 ± 3.7 38.3 ± 2.08

When comparing the results of microscopic analysis with the results of macroscopic analysis, it can be assumed that under the influence of drugs with chlorhexidine, immobilization and death of spermatozoa is due to a violation of the membrane permeability and cell metabolism, but not a violation of its structural integrity by rupture of the membrane.

Conclusion The study examined the effectiveness of the spermicidal action of the proposed agent in the form of tablets for intravaginal administration, developed by OJSC Nizhpharm, Russia, in vitro using semen from male donors.

The proposed tool was used in a dose of 1 tablet once per serving of human sperm (3.5 ml). As a control substance, a tablet base was used.

It was shown that in vitro when using various options No. 1 and No. 2 of the proposed agent, 1-2% of viable spermatozoa were detected after 30 minutes, although after 60 minutes complete immobilization and death of sperm cells was observed (100%).

When assessing the morphology of spermatozoa in an in vitro test, the main mechanism for the violation of their motility and death under the action of the proposed chlorhexidine-based agent was a violation of membrane permeability and sperm metabolism, but not structural integrity.

As studies have shown, the proposed tool in the form of vaginal tablets is an effective antibacterial and spermicidal agent and convenient to use.

Claims (3)

1. A therapeutic and prophylactic agent with antibacterial and spermicidal action, in a dosage form for intravaginal administration, containing the active substance - chlorhexidine or its salt and a base, characterized in that it is made in the form of a tablet, the base of which contains lactose as the main component, as well as other pharmaceutically acceptable excipients, in the following ratio, wt.%: Chlorhexidine or salt 1.0-4.0 The basis: Lactose 60.0-95.0 Other pharmaceutically acceptable Excipients up to 100 2. The tool according to claim 1, characterized in that as the salt of chlorhexidine it contains bigluconate, or acetate, or dihydrochloride. 3. The tool according to claim 1, characterized in that it may further comprise a regenerating substance, in particular dexpanthenol, or methyluracil, or sea buckthorn oil, in an amount of 0.5-10 wt.%.

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