Antifungal agent for elimination of candida fungi

Abstract

FIELD: microbiology; gynecology.SUBSTANCE: used to treat infections caused by fungi of the Candida genus. An antifungal agent for the elimination of Candida fungi is proposed, containing a chitosan solution at a concentration of 3 to 12 mg / ml with a chitosan molecular weight of 30 kDa and a deacetylation degree of 80%, and cell-free metabolites of lactobacilli obtained from Lactobacillus bacteria, pre-grown to a concentration of 107–1011 CFU / ml, in a 1:1 ratio.EFFECT: invention ensures effective elimination of Candida fungi.1 cl, 3 dwg, 6 ex

Description

[0001] The present invention relates to the fields of microbiology and medicine, particularly gynecology. The invention consists of an antifungal agent containing chitosan and acellular lactobacilli metabolites for the elimination of Candida fungi. This agent is proposed for use in medical practice for the treatment of infections caused by Candida fungi.

[0002] The relevance of the developed antifungal agent is determined by the widespread prevalence of candidiasis of the human mucosa. Candidiasis is an infectious and inflammatory process caused by yeast-like fungi of the genus Candida. The most common forms of candidiasis are oral candidiasis and genital candidiasis, including vulvovaginal candidiasis in women.

[0003] Treatment of candidiasis is complicated by a number of interrelated factors. Firstly, the development of resistance in Candida fungi due to improper use of antifungal agents, which reduces the effectiveness of standard medications. Secondly, the ineffectiveness of candidiasis therapy and, consequently, the increased risk of relapse can be attributed to the patient's compromised immune status. Thirdly, existing standard antifungal drugs have limitations related to their inability to be used in certain patient groups, such as pregnant women, children, and patients with comorbidities. Some antifungal drugs are toxic to mammals and humans, limiting the dosage and duration of treatment.

[0004] Taken together, these factors create significant challenges for the successful and safe treatment of candidiasis. If treatment of mucosal candidiasis is unsuccessful, the inflammatory process can spread to other organs and lead to dangerous complications for the patient.

[0005] Thus, candidiasis can cause socioeconomic and demographic losses, deteriorating quality of life and public health. All this demonstrates the need to develop new, more effective and safer agents with antifungal activity.

[0006] Candida fungi are eliminated using the drug Fluconazole®. According to the official instructions for use, published on the website of the State Register of Medicines (https: / / grls.rosminzdrav.ru / grls.aspx), its primary mechanism of action is the inhibition of the fungal cytochrome P-450-mediated demethylation reaction of 14-alpha-lanosterol, a key step in the biosynthesis of ergosterol in fungi. Fluconazole® has been shown to be more selective for fungal cytochrome P-450 enzymes than for various mammalian cytochrome P-450 enzymes. A disadvantage of this drug is the presence of resistance to it in some strains of Candida fungi, as well as side effects such as allergic reactions, nausea, vomiting, and headaches.

[0007] Amphotericin B® is a well-known drug used to eliminate fungi, including Candida species. According to the official instructions for use published on the website of the State Register of Medicines (https: / / grls.rosminzdrav.ru / grls.aspx), Amphotericin B® has a fungicidal or fungistatic effect depending on its concentration in biological fluids and the sensitivity of the pathogen. It binds to sterols (ergosterols) found in the cell membrane of the fungus sensitive to the drug. This disrupts membrane permeability, allowing intracellular components to leak into the extracellular space, leading to fungal lysis. A drawback of the drug is its toxic effect on the human body. Mammalian cell membranes also contain sterols, so it is believed that the same mechanism underlies the action of Amphotericin B® on human and fungal cells.

[0008] The drug Nystatin® is a polyene antifungal antibiotic effective against Candida fungi. According to the official instructions for use on the website of the State Register of Medicines (https: / / grls.rosminzdrav.ru / grls.aspx), Nystatin® binds to sterols in the fungal cell membrane, disrupting its permeability, leading to the release of key cellular components. A drawback of the drug is the presence of resistance in some Candida strains.

[0009] An antifungal agent is known that suppresses the growth of yeast fungi using biologically active compounds—substituted indolin-3-ones, namely (Z)-2-(2-oxopropylidene)indolin-3-one (patent RU (11) 2 803 747 (13) C1, published September 19, 2023). A disadvantage of this agent is its low efficacy against Candida fungi.

[0010] No analogues of the invention were found in patent and scientific literature.

[0011] The technical result of the claimed invention is to expand the arsenal of known antifungal agents by creating an antifungal agent, the effect of which is achieved due to the identified effect of potentiating the antifungal properties of chitosan by cell-free metabolites of lactobacilli. The technical result is achieved in that an antifungal agent is proposed containing from 3 to 12 mg / ml of chitosan. with a molecular weight of 30 kDa and a degree of deacetylation of 80%, and cell-free metabolites of lactobacilli obtained from bacteria of the genus Lactobacillus, pre-grown to a concentration of 10 7 - 10 11 CFU / ml, in a 1:1 ratio.

[0012] The aim of this invention is to create an antifungal agent that is effective against Candida fungi, including strains that are resistant to antifungal drugs.

[0013] The essence and implementation of the invention

[0014] The goal is achieved by creating a product with antifungal action against Candida fungi, containing from 3 to 12 mg / ml of chitosan with a molecular weight of 30 kDa and a deacetylation degree of 80%, and cell-free metabolites of lactobacilli obtained from bacteria of the genus Lactobacillus, pre-grown to a concentration of 10 7 - 10 11 CFU / ml, in a 1:1 ratio.

[0015] To achieve this goal, a study was conducted using 20 strains of Candida fungi. To obtain extracellular lactobacilli metabolites, 10 lactobacilli strains isolated from cervicovaginal fluid samples and two strains isolated from probiotic medicinal products (Lactonorm® and Lactozhinal®) were used.

[0016] To obtain cell-free metabolites of lactobacilli, a suspension was prepared from each lactobacilli strain in a liquid nutrient medium MRS containing 10 lactobacilli 3 - 10 5 CFU / ml. Lactobacilli were then cultured to 10 7 - 10 11 CFU / ml under microaerophilic conditions at 37°C. The culture medium was then centrifuged at 3500 g for 10 minutes, and the resulting supernatant, which consisted of extracellular metabolites, was filtered through a 0.22 µm membrane filter and used to create an antifungal agent.

[0017] To prepare the antifungal agent, lactobacillus metabolites were mixed in a 1:1 ratio with a chitosan solution with a molecular weight of 30 kDa and a deacetylation degree of 80% at concentrations of 3 mg / ml, 6 mg / ml, or 12 mg / ml. Chitosan is insoluble in water but soluble in an acidic medium, so physiological saline was used as a solvent for the chitosan, to which lactic acid was added to create an acidic pH. Its concentration in the solution was 0.5%. The resulting mixture was filtered through a membrane filter with a pore size of 0.22 μm.

[0018] Next, the antifungal activity of chitosan, lactobacillus metabolites, and the prepared antifungal agent was assessed using the well diffusion method. For this purpose, a pure culture of Candida fungi was inoculated as a microbial suspension in saline, containing approximately 10 fungi. 6CFU / ml onto a solid Sabouraud nutrient medium containing chloramphenicol, followed by spreading the suspension over the surface with a spatula. Wells were then formed in the agar using a punch, and the following solutions were added dropwise in 50 µl volumes:

[0019] 1) manufactured antifungal agent,

[0020] 2) chitosan solution,

[0021] 3) lactobacilli metabolites,

[0022] 4) solvent for chitosan as a control

[0023] 5) MRS liquid medium without lactobacilli metabolites as a control.

[0024] The scheme of the experiment is shown in Figure 1.

[0025] The antifungal efficacy was assessed by the presence of growth inhibition zones around the wells 24 hours after culturing under microaerophilic conditions at 37°C. The antifungal effect was assessed by the diameter of the growth inhibition zones. An example of the experimental results is shown in Figure 2.

[0026] According to the data shown in Figure 2, the diameter of the growth inhibition zones around the well (1) containing the prepared antifungal agent is larger than the diameter of the growth inhibition zones around the wells containing each of the components of the agent separately (2, 3). There was no growth inhibition zone around the control wells (4, 5).

[0027] The results of measuring the diameter of growth inhibition zones around the wells are shown in Figure 3.

[0028] Thus, the authors in an experimental study for the first time found that an antifungal agent containing chitosan and extracellular metabolites of lactobacilli, prepared in the above manner, has a more pronounced antifungal effect than each of the components separately.

[0029] Examples of specific use of the invention

[0030] Example #1

[0031] A suspension containing a strain of Candida albicans was seeded onto a solid Sabouraud nutrient medium, followed by trituration of the suspension over the surface with a spatula. Wells were then formed in the nutrient medium, into one of which an antifungal agent prepared according to the invention formula was added. The agent contained chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 80% at a concentration of 3 mg / ml and cell-free metabolites of lactobacilli at a concentration of 10 7 CFU / ml at a 1:1 ratio. Metabolites were obtained from a Lactobacillus gasseri strain isolated from cervicovaginal fluid samples. Each component of the product was added separately to two other wells. The fourth well served as a control, containing a solvent for chitosan.

[0032] The fifth well also served as a control, with liquid MRS culture medium without lactobacilli metabolites added to it.

[0033] After 24 hours of culturing, the diameter of the growth inhibition zone around the well containing the indicated antifungal agent was 13 mm. The diameter of the growth inhibition zone around the well containing only the chitosan solution was 8 mm. The diameter of the growth inhibition zone around the well containing only lactobacillus metabolites was 8 mm. There was no growth inhibition zone around the control wells, indicating that the claimed antifungal agent has a pronounced antifungal effect.

[0034] Example #2

[0035] A suspension containing a Candida glabrata strain was seeded onto a solid Sabouraud nutrient medium, followed by trituration of the suspension over the surface with a spatula. Wells were then formed in the nutrient medium, into one of which an antifungal agent prepared according to the invention formula was added. The agent contained chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 80% at a concentration of 6 mg / ml and cell-free lactobacilli metabolites at a concentration of 10 7 CFU / ml at a 1:1 ratio. Metabolites were obtained from a Lactobacillus acidophilus strain isolated from the medicinal product Laktonorm®. Each component of the product was added separately to two other wells. The fourth well served as a control; it contained a solvent for chitosan. The fifth well also served as a control; it contained MRS liquid nutrient medium without lactobacillus metabolites.

[0036] After 24 hours of culturing, the diameter of the growth inhibition zone around the well containing the antifungal agent was 16 mm. The diameter of the growth inhibition zone around the well containing only the chitosan solution was 10 mm. The diameter of the growth inhibition zone around the well containing only lactobacillus metabolites was 9 mm. There was no growth inhibition zone around the control wells, indicating that the claimed antifungal agent has a pronounced antifungal effect.

[0037] Example #3

[0038] A suspension containing a strain of Candida tropicalis was seeded on a solid Sabouraud nutrient medium, followed by trituration of the suspension over the surface with a spatula. Wells were then formed in the nutrient medium, into one of which an antifungal agent prepared according to the invention formula was added. The agent contained chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 80% at a concentration of 12 mg / ml and cell-free metabolites of lactobacilli at a concentration of 10 7 CFU / ml at a 1:1 ratio. Metabolites were obtained from the Lactobacillus casei rhamnosus Doderleini strain isolated from the medicinal product Lactozhinal®. Each component of the product was added separately to two other wells. The fourth well served as a control; it contained a solvent for chitosan. The fifth well also served as a control; it contained liquid MRS nutrient medium without lactobacillus metabolites.

[0039] After 24 hours of culturing, the diameter of the growth inhibition zone around the well containing the indicated antifungal agent was 20 mm. The diameter of the growth inhibition zone around the well containing only the chitosan solution was 14 mm. The diameter of the growth inhibition zone around the well containing only cell-free lactobacillus metabolites was 9 mm. There was no growth inhibition zone around the control wells, indicating that the claimed antifungal agent has a pronounced antifungal effect.

[0040] Example #4

[0041] A suspension containing a Candida glabrata strain resistant to Fluconazole® was inoculated onto a solid Sabouraud nutrient medium, followed by trituration of the suspension over the surface with a spatula. Wells were then formed in the nutrient medium, into one of which an antifungal agent prepared according to the invention formula was added. The agent contained chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 80% at a concentration of 6 mg / ml and cell-free lactobacilli metabolites at a concentration of 10 9CFU / ml at a 1:1 ratio. Metabolites were obtained from a Lactobacillus acidophilus strain isolated from the medicinal product Laktonorm®. Each component of the product was added separately to two other wells. The fourth well served as a control; chitosan solvent was added to it. The fifth well also served as a control; liquid MRS nutrient medium without lactobacillus metabolites was added to it. A sixth well was prepared and supplemented with Fluconazole® at a concentration of 6 mg / ml.

[0042] After 24 hours of culturing, the diameter of the growth inhibition zone around the well containing the antifungal agent was 16 mm. The diameter of the growth inhibition zone around the well containing only the chitosan solution was 10 mm. The diameter of the growth inhibition zone around the well containing only lactobacillus metabolites was 11 mm. There was no growth inhibition zone around the control wells or the well containing Fluconazole®, indicating that the claimed antifungal agent has a pronounced antifungal effect, including against strains resistant to Fluconazole®.

[0043] Example #5

[0044] A suspension containing a strain of Candida tropicalis was seeded on a solid Sabouraud nutrient medium, followed by trituration of the suspension over the surface with a spatula. Wells were then formed in the nutrient medium, into one of which an antifungal agent prepared according to the invention formula was added. The agent contained chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 80% at a concentration of 12 mg / ml and cell-free metabolites of lactobacilli at a concentration of 10 11 CFU / ml at a 1:1 ratio. Metabolites were obtained from the Lactobacillus casei rhamnosus Doderleini strain isolated from the medicinal product Lactozhinal®. Each component of the product was added separately to the other two wells. The fourth well served as a control; it contained a solvent for chitosan. The fifth well also served as a control; it contained MRS liquid nutrient medium without lactobacillus metabolites.

[0045] After 24 hours of culturing, the diameter of the growth inhibition zone around the well containing the antifungal agent was 18 mm. The diameter of the growth inhibition zone around the well containing only the chitosan solution was 14 mm. The diameter of the growth inhibition zone around the well containing only cell-free lactobacillus metabolites was 11 mm. There was no growth inhibition zone around the control wells, indicating that the claimed antifungal agent has a pronounced antifungal effect.

[0046] Example #6

[0047] A suspension containing a strain of Candida albicans resistant to Fluconazole® was inoculated onto a solid Sabouraud nutrient medium, followed by trituration of the suspension over the surface with a spatula. Wells were then formed in the nutrient medium, into one of which an antifungal agent prepared according to the invention formula was added. The agent contained chitosan with a molecular weight of 30 kDa and a degree of deacetylation of 80% at a concentration of 3 mg / ml and cell-free metabolites of lactobacilli at a concentration of 10 9CFU / ml at a 1:1 ratio. Metabolites were obtained from a Lactobacillus iners strain isolated from cervicovaginal fluid samples. Each component of the product was added separately to two other wells. The fourth well served as a control; chitosan solvent was added to it. The fifth well also served as a control; it contained MRS liquid culture medium without lactobacillus metabolites. A sixth well was prepared and added with Fluconazole® at a concentration of 6 mg / ml.

[0048] After 24 hours of culturing, the diameter of the growth inhibition zone around the well containing the antifungal agent was 14 mm. The diameter of the growth inhibition zone around the well containing only the chitosan solution was 8 mm. The diameter of the growth inhibition zone around the well containing only cell-free lactobacillus metabolites was 9 mm. There was no growth inhibition zone around the control wells or the well containing Fluconazole®, indicating that the claimed antifungal agent has a pronounced antifungal effect, including against strains resistant to Fluconazole®.

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