RU2705098C2 - Pharmaceutical composition for treating mycotic lesions of mucous membranes - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and concerns a pharmaceutical composition for treating fungal infections of mucous membranes, containing as active substance peptide SETRPVLNRLFDKIRQVIRKFEKGIKEKSKRFF, additionally containing hydroxypropyl methylcellulose, polyethylene glycol-400 and water.

EFFECT: invention provides a peptide composition having optimum viscosity, stability and biological activity.

1 cl, 5 ex, 2 dwg, 1 tbl

Description

The technical field to which the invention relates.

The present invention relates to biochemistry, and in particular to a pharmaceutical composition based on the peptide SE33 (SETRPVLNRLFDKIRQVIRKFEKGIKEKSKRFF) for the treatment of fungal infections of the mucous membranes. More specifically, this invention relates to a gel composition for external use.

The prior art.

Fungal diseases in medicine are called mycoses, and are a fairly common reason for people to go to a dermatological clinic. Mycoses can occur regardless of age. Specialists have studied about 100 varieties of fungi - pathogens. The result of the parasitic activity of the fungus is continuous itching, redness, peeling of the skin.

The difficulty in treating fungal diseases lies in the increased resistance of fungi to the effects of drugs and disinfectants. Fungi can be in the wardrobe and household items of a person for more than 5 years. The source of infection is usually people infected with mycosis and their things. The disease itself is represented by various forms, the simplest of which is peeling of the skin. In addition to this main symptom, slight redness and slight itching may appear on the feet.

Peptide drugs are promising drug candidates for the treatment of fungal diseases. Currently, the biological activity and the mechanism of action of the peptide LL37 (human cathelicidin) are well studied and its high antimicrobial effect against gram-positive, gram-negative bacteria, fungi and enveloped viruses has been proved (WO 2009/001087, WO 2004/067025, WO 96/08508, RU 2346950).

Studies of peptides homologous to the LL37 peptide, and having the same antifungal and immunostimulating properties, should allow the development of a new antifungal drug similar in effect to LL37-based drugs.

Studies have shown that the analogue of cathelicidin with the amino acid sequence: SETRPVLNRLFDKIRQVIRKFEKGIKEKSKRFF (SE33) has a strong antimicrobial effect based on the physicochemical characteristics of the peptide sequence (RU 2434880).

, Vitaliy V. Khutoryanskiy, European Polymer Journal 65 (2015) 252-267)However, in order to use these peptides in practice for the treatment of bacterial and fungal infections of the skin and mucous membranes, it is necessary to create a suitable dosage form. To date, known compositions of ointments containing antimicrobial peptides (RU 2467753, RU 2012103653). However, these compositions contain an oil base, which complicates the diffusion of the peptide from the ointment into the infection site, since antimicrobial peptides in a non-aqueous environment form an amphipathic alpha-helix, which has a high affinity for saturated and unsaturated hydrocarbons. Therefore, the development of pharmaceutical compositions containing antimicrobial peptides is an urgent task. In the case of antimicrobial peptides, it is preferable to use water-soluble bases (

, Vitaliy V. Khutoryanskiy, European Polymer Journal 65 (2015) 252-267)

The closest analogue of the claimed invention is the composition of the hydrogel published in WO 2009/001087.

Disclosure of the invention.

The present invention solves the problem of expanding the arsenal of funds for the treatment of fungal lesions of the mucous membranes. Studies were conducted on compositions that contained polyethylene glycol-400 (PEG-400), hydroxypropyl methylcellulose (HPMC) and the peptide SE33, as an active substance. Moreover, the concentration of polyethylene glycol-400 was from 1 to 10% mass, HPMC from 1 to 8%, the concentration of the peptide was constant 1 mg / ml (0.001%). Several parameters were investigated: the viscosity of the composition, stability and its biological activity. The viscosity of the composition makes possible its physical use, since too fluid composition does not allow the gel to be fixed on the lesion, and too viscous (dense) composition makes it difficult for the peptide to exit the gel into the wound. Stability - an important characteristic for the consumer properties of the composition. The biological activity of the composition is a technical result based on its properties (ratio of components). As a result of research, the optimal ratio of components, masses was found. %:

PEG-400 5% GPMC 2-5% Peptide SE33 0.001% Water The rest is up to 100%

The invention is illustrated in FIG. 1 and 2:

FIG. 1. The killer effect of the peptide SE33 in relation to Candida albicans cells:

And the concentration of the peptide is 1 mg / ml,

B - peptide concentration of 3 mg / ml,

B - peptide concentration of 10 mg / ml.

Fig 2. The dependence of the seeding of the vagina of mice with yeast Candida albicans on the dose of the drug in groups No. 3-7 in the dynamics of treatment. The first seeding was carried out before the start of treatment, the second and third seeding - one week and two weeks after the start of treatment, respectively. Wilcoxon Signed Rank Test and ANOVA on Ranks with Tukey's and Dunnet's post hoc tests, p <0.01-p <0.1.

Examples of carrying out the invention.

Example 1. The preparation of a gel based on hydroxypropyl methylcellulose (HPMC)

To prepare the gel, a 2.5% HPMC solution is first prepared. The solution is prepared with stirring on a magnetic stirrer until completely dissolved at room temperature a day before the preparation of the doses. For further use, the prepared HPMC solution is stored in a refrigerator of 5 ± 3 ° C for 1-7 days. On the day of preparation of the doses, the necessary aliquot of the HPMC solution is selected. An aliquot is placed on a heated magnetic stir bar or water bath (60-70 ° C). After about 5 minutes, PEG-400 is added to the heated HPMC solution, bringing the solution to 5% with PEG-400. Then, after 2-5 minutes, the calculated amount of the peptide is added to the prepared solution (the minimum dose concentration is 1.0 mg / ml; the maximum dose concentration is 50 mg / ml). The solution was stirred on a magnetic stirrer until the peptide was completely dissolved in about 60 seconds. Next, the prepared solution is placed in a cold water bath (temperature of cold running water) and after about 5 minutes it is transferred for use.

Example 2. The study of chronic toxicity of the gel containing the peptide SE33.

The chronic toxicity of the gel containing the SE33 peptide was studied by repeated cutaneous application to male and female SD rats for 28 days followed by a 14-day withdrawal period.

During the study, the death of animals or clinical signs of intoxication of animals was not. In animals of all groups, positive dynamics of body weight was observed during the study. In this case, there were no statistically significant differences between groups of animals of both males and females with the application of the test substance to the skin relative to the corresponding control groups. Cutaneous application of the test gel did not lead to a statistically significant change in animal feed intake. The study of the effects of local irritating effects using the Draise scale revealed insignificant reddening of the skin, which was equally characteristic for control animals and rats of the experimental groups. The revealed effects were probably the result of minor skin damage when cutting hair. The rest of the studied parameters (hemogram, biochemical parameters of blood serum, mass of internal organs) also did not statistically significantly differ for the experimental and control groups of animals.

During planned necropsy of animals during a visual examination of the external condition of the body, internal surfaces and passages, the cavity of the skull, chest, abdominal and pelvic cavities with organs and tissues located in them, the neck with organs and tissues, the skeleton and the musculoskeletal system, the skin - places application of the drug, morphological signs of the toxic effect of the drug were not detected. A microscopic analysis of the following organs was also performed: liver, kidneys, adrenal glands, heart, lungs, ovaries / testes, spleen, thymus, stomach, submandibular and mesenteric lymph nodes, brain and skin from the area of application. In these organs, microscopic lesions and deviations from the norm associated with the action of the test drug were not detected.

As a result of the study of chronic toxicity, it was shown that the gel under study does not cause toxic effects both when using a dose of 6 mg / kg, which corresponds to a therapeutic dose taking into account conversion to this animal species, and in a dose of 75 mg / kg, which exceeds the equivalent dose therapeutic 12.5 times.

Example 3. Determination of the minimum inhibitory concentration (MIC).

The MIC was determined by the method of microdilutions in a modified synthetic nutrient medium RPMI-1640 containing salts, amino acids, trace elements, vitamins, antibiotic and glucose [Yarrow D., 1998]. The effect of the peptide SE-33 was studied in the concentration range of 0.5-1024 μg / ml. The experiments were carried out in 96-well round-bottom plates. Horizontally, the drug was applied at a final concentration of 1024 μg / ml and lower with a double dilution step (20 μl), after which a suspension of yeast cells was added to a final concentration of 103 CFU / ml (180 μl). The tablets were placed in a thermostat at a temperature of 320C and incubated for 48 hours, after which the result of a change in the color of the indicator was taken into account. Such experiments were performed twice with an interval of 1 week.

In two independent experiments, the minimum concentration inhibiting the growth of fungi for 2 days was 1024 μg / ml. Based on these data, the lowest concentration of SE33 was chosen for in vivo experiments.

Example 4. The antifungal effect of the peptide SE33 in blitz experiments.

The essence of the blitz experiments was as follows: 0.5 ml of physiological saline containing 0 mg / ml 1 mg / ml, 3 mg / ml and 10 mg / ml SE33 were added to test tubes of the Eppendorf type. 30 μl of Candida albicans exponential culture No. 927 (final cell content of 106 CFU / ml) was also added there. The mixture was incubated at 320 ° C in an incubator for 2 hours, after which the cells were precipitated by centrifugation, the supernatant was discarded, and the cells were stained by adding a 2 mM solution of bromocresol purple for 1 hour. After precipitation, the cells were microscopic, photographed, the data was transferred to a computer and the percentage of stained (i.e. dead) cells was counted. Blitz experiments showed that the effect of SE33 on Candida albicans No. 927 cells has a dose-dependent effect: for example, the proportion of dead (stained) cells (minus control) when exposed to peptide fungi at a concentration of 1 mg / ml was 40.4%, a concentration of 3 mg / ml - 76.9%, at a concentration of 10 mg / ml - 92.0% (Fig. 1). Thus, of the studied concentrations of SE33, the most effective was 10 mg / ml.

Example 5. The antifungal effect of the peptide SE33 in vivo.

To create an experimental vulvovaginal candidiasis, mice were estrogenized three times during the experiment (see chart) by rubbing 200 mg of estrogen (approximately 0.125 μg of estrogen) into the abdomen of the animal. The animals were infected by a single intravaginal injection of a suspension of C. albicans yeast cells in saline, the inoculum volume was 20 μl, and the cell concentration was 106 CFU / ml. "Treatment" of animals with drugs was carried out by daily intravaginal administration of gels (in the case of pimafucin - cream) for 5 days, the sample volume of 20 μl. Seeding was carried out three times by intravaginal administration of a sterile disposable 1 mm microbiological loop, twisting it twice and suspending the contents of the loop in 200 μl of sterile saline in an Eppen-Dorf tube. After transferring the tubes from the vivarium to the laminar, 10 μl were seeded from each tube into Petri dishes with GPA medium. The plates were incubated for 1 day at 320 ° C., after which the grown yeast colonies were taken into account.

When observing the behavior of mice, signs of pathological side effects were not recorded. All mice, regardless of belonging to a particular group, survived in this experiment. In the control groups (groups 1 and 2) not a single mouse was ill with IHC throughout the experiment, while in the experimental groups treated with estrogen (No. 3-7), all animals became carriers of Candida albicans.

The therapeutic effect of drugs.

The therapeutic effect of the drugs was evaluated according to two indicators: the dynamics of the reduction of vaginal contamination during treatment of animals and the proportion of “cured” (ie, with zero seeding) animals, calculated on the 28th day of the experiment. These indicators are reflected in FIG. 2 and in table 1.

From the data in Figure 2, it can be seen that the vaginal contamination before treatment (results of the first seeding) was comparable for all groups of animals infected with Candida albicans (groups 3-7). The drug SE33 in all studied doses (1 mg / ml, 10 mg / ml and 50 mg / ml - groups 3, 4 and 5, respectively) is statistically significant (p <0.1 for groups 3 and 4, p <0.01 for group 5) reduced vaginal contamination compared with the same indicator in the placebo group (group 6) two weeks after the start of treatment (third seeding). The effectiveness of treatment with SE33 in all doses was comparable to that for the reference drug - pimafucin at a dose of 20 mg / ml (group 7, third seeding, p <0.1). At the highest dose used (50 mg / ml - group 5), SE33 reduced vaginal contamination with a very high degree of confidence (p <0.01, third sowing). It is worth noting that an increase in the dose of SE33 from 1 mg / ml (group 3) to 10 mg / ml (group 4) and 50 mg / ml (group 5) led to a statistically significant (p <0.1) difference in vaginal colonization by compared with control group No. 6 a week after the start of treatment (second seeding), which was not observed for the comparison drug pimafucin (20 mg / ml, group 7). For groups 3-5 (SE33 1 mg / ml, 10 mg / ml and 50 mg / ml, respectively), there was a tendency to a decrease in vaginal dissemination in the dynamics of treatment (Table 1)

The most pronounced activity of SE33 in this study was observed when administered at a dose of 50 mg / ml. The introduction of SE33 at doses of 1 and 10 mg / ml led to a decrease in the median of vaginal seeding compared to placebo - by 1.12 times and an increase in the proportion of fully cured animals by 1.5-1.75 times compared with placebo. Administration of SE33 at a dose of 50 mg / ml resulted in a 1.15-fold decrease in the median vaginal contamination of the vagina compared to placebo and a 2.5-fold increase in the proportion of fully cured animals compared to placebo. The same indicators for pimafucin were 1.14 times and 2.25 times, respectively.

Claims (2)

A pharmaceutical composition for treating fungal infections of the mucous membranes, comprising as an active ingredient the peptide SEQ ID: 1, characterized in that it further comprises hydroxypropyl methylcellulose, polyethylene glycol-400 and water in the following ratio, wt.%: Peptide 0.001% Hydroxypropyl methylcellulose 2-5% Polyethylene glycol-400 5% Water The rest is up to 100%

Images