RU2230110C2 - Method for inactivation of pathogenic fungi - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: invention can be used in medicine and veterinary science for inactivation of pathogenic fungi. Method involves treatment of fungi with inactivating agent, photosensitizing agent of chlorin nature, taken in the concentration 10-20 mcM. Treatment of fungi is carried out for 5 min followed by irradiation by electromagnetic radiation in the range 400-700 nm for 10 min, not less. Method provides high effectiveness and selectivity of inactivation in a single using and in small nontoxic concentrations of preparations and irradiation doses. EFFECT: improved inactivation method. 5 tbl, 5 ex

Description

The present invention relates to microbiology and can be used in medicine and veterinary medicine to inactivate pathogenic fungi, for example, the genus Candida.

A known means of destroying pathogenic fungi is the antibiotic levorin. The disadvantage of this drug is the need for multiple treatments to achieve reliable antifungal action.

Many other chemical preparations are also known - antimycotics (lamisil, nizoral, batrafen, etc. [1]), effective to a greater or lesser extent.

The disadvantages of these drugs are the need for repeated use, the rapid development of fungal resistance to them and the toxic effect on the gastrointestinal tract, blood, human brain, as well as causing allergic reactions.

Also known antifungal drug [2] in the form of an aqueous solution of trace elements.

The disadvantage of this drug is the lack of effectiveness, to increase which it is used in combination with other drugs.

There is also a method of destroying infectious microorganisms by processing them with electromagnetic radiation from the microwave range [3].

The disadvantage of this method is the inability to use in medicine and veterinary medicine for the treatment of fungal diseases due to the need to use powerful microwave generators that have a harmful effect on human and animal organisms.

There is also known a method of using oxidizing agents as antimycotics [4].

The disadvantage of this method is its lack of effectiveness due to the low concentration of peroxides in the preparations and the complicated application for a long time.

Also known is the method chosen by the authors for the prototype for treating patients with cutaneous candidiasis using photochemotherapy based on the combined action of amifurin (0.3% solution) and ultraviolet radiation (UV) [5]. The method consists of photosensitized amifurin containing a mixture of furocoumarins, and UV inactivation of Candida pathogenic fungal cells. The mechanism of furocoumarin photosensitized inactivation of cells of various organisms has not been fully studied, it is assumed that it is mainly based on the ability of these compounds to bind to DNA and in the photoexcited state induce the formation of lethal crosslinks in it. A photodynamic pathway for cell inactivation involving furocoumarins is also suggested, but the quantum yield of singlet oxygen generation by these compounds is very low (0.002-0.05).

The disadvantages of this method are: the lack of selectivity between sensitivity to photosensitized furocoumarins inactivation of cells of fungi and animals and humans (used, in particular, for PUVA therapy of psoriasis); the use of a high (0.3%) toxic concentration of the drug ammifurin; photosensitizer absorption of optical radiation only in the ultraviolet (320-390 nm) range with low transmission in the biological medium, which leads to insufficient treatment method, especially in patients with recurrent candidiasis with dense clusters of pathogenic fungal cells and deep lesions; the need for this in connection with conducting multiple (on average 12) sessions of ultraviolet radiation and repeated courses of photochemotherapy; complications of the combined effect of artificial and natural ultraviolet radiation in the summer months.

Using the present invention, a technical result is achieved, consisting in the manifestation of high efficiency and selectivity of inactivation of pathogenic fungi with a single application and the use of small non-toxic drug concentrations and radiation doses.

In accordance with the invention, the technical result is achieved by the fact that as a photosensitizer a chemical substance of chlorine nature is selected in a concentration of 10-20 μM (0.001-0.002%), having a high quantum yield of generation of singlet oxygen (0.5-0.7), processing fungi which are kept for 5 minutes, after which they are irradiated for at least 10 minutes with electromagnetic radiation in the range 400-700 nm. In the process of irradiation, chlorine-type photosensitizer molecules in solution under the influence of irradiation repeatedly go into an excited state and produce oxidizing inactivating cells of microorganisms - active oxygen species (ROS), mainly singlet oxygen. ROS induce lethal oxidative destructive processes in subcellular structures.

The method is as follows.

In a medium containing cultures of yeast fungi of the genus Candida (C. albicans or C. guilliermondii), a chlorine-type photosensitizer is added at a concentration of 10 to 20 μM, kept for 5 minutes, and then treated for at least 10 minutes with visible electromagnetic radiation ( 400-600 nm) range with an intensity of 5 mW / cm ² or monochromatic laser radiation of the far red range with an intensity of 5-20 mW / cm ² .

Evaluation of the inactivation results is carried out to determine the colony forming ability (survival) of yeast fungi subjected to the action of a photosensitizer and radiation, in comparison with control untreated cultures or cultures treated only with a photosensitizer or only with radiation.

Example 1. In three-day-old, pure cultures of C. guilliermondii VSB-656 suspended in saline, at a concentration of 10 ⁶ fungal cells per milliliter, a photosensitizer photoditazine (glucosamine salt of chlorin e6) at a concentration of 10-20 μM is added or cells are resuspended in 0.3% ammifurin solution and incubated for 30 minutes in the dark. The repetition of experiments three times. When exposed to 10-20 μM of the photosensitizer photoditazine in the absence of illumination, 98-100% of fungal cells in C. guilliermondii cultures retained the ability to reproduce, and when exposed to 0.3% ammifurin, 100% of fungal cells lost it. The experimental results are presented in table 1.

Example 2. In three-day old, suspended in physiological saline, pure cultures of C. albicans (reference strain ATCC 24433 from the American Type Culture Collection) or C. guilliermondii VSB-656 at a concentration of 10 ⁶ fungal cells per milliliter, a photosensitizer photoditazine (glucosamine salt of chlorine e6 ) in a concentration of 10 to 20 μM, they are incubated for 5 minutes and irradiated for 5-20 minutes with electromagnetic radiation of the visible (400-600 nm) range with an intensity of 5 mW / cm ² obtained from a DRSh-1000 mercury lamp in combination with ZhS-10 filters and SZS-21. The repetition of experiments three times. With a single combined exposure to the photosensitizer photoditazine and radiation of 400-600 nm, up to 97% of fungal cells in cultures of C. albicans and up to 100% - C. guilliermondii lost their ability to reproduce. The experimental results are presented in table 2.

Example 3. In three-day old, suspended in physiological saline, pure cultures of C. albicans, freshly isolated from clinical material (strains 55 from feces, 109 from the pharynx, 575 from sputum, 623 from urine), at a concentration of 10 ⁶ mushroom cells in a milliliter add a photosensitizer photoditazine (glucosamine salt of chlorin e6) at a concentration of 10 μM, incubated for 5 minutes and irradiated for up to 20 minutes with electromagnetic radiation of the visible (400-600 nm) range with an intensity of 5 mW / cm ² received from a DRSh mercury lamp -1000 combined of filters ZS-10 and SES-21. The repetition of experiments three times. Photosensitivity (1 / D ₁₀ , i.e., the dose of light necessary to achieve a 10% survival rate of fungal cells in cultures or a 90% inactivation level, respectively) of strains freshly isolated from the clinical material to the proposed inactivation method is close to that for the reference C. albicans strain ATCC 24433. The results are presented in table 3.

Example 4. In three-day old, suspended in physiological saline, pure cultures of C. albicans (reference strain ATCC 24433 from the American Type Culture Collection) or C. guilliermondii VSB-656 at a concentration of 10 ⁶ fungal cells per milliliter add a 3-formyl-3- photosensitizer devinylchlorin p6 at a concentration of 10 to 20 μM, incubated for 5 minutes and irradiated for up to 20 minutes with monochromatic laser radiation of 690 nm with an intensity of 20 mW / cm ² obtained from a semiconductor laser based on an IDL-50-M-690 laser diode. The repetition of experiments three times. With a single combined exposure to the photosensitizer 3-formyl-3-devinylchlorin p6 and radiation of 690 nm, up to 81% of fungal cells in cultures of C. albicans and up to 88% - C. guilliermondii lost their ability to reproduce. The results are presented in table 4,

Example 5. In a three-day, suspended in physiological saline solution, pure culture of C. guilliermondii VSB-656 at a concentration of 0.5 × 10 ⁶ to 500 × 10 ⁶ fungal cells in a milliliter, a 3-formyl-3-devinylchlorin p6 photosensitizer at a concentration of 10 μM, incubated for 5 minutes and irradiated for 20 minutes with monochromatic laser radiation of 690 nm with an intensity of 20 mW / cm ² obtained from a semiconductor laser based on an IDL-50-M-690 laser diode, or electromagnetic radiation of the visible (400-600 nm) range with an intensity of 5 mW / cm ² obtained from a mercury lamp DRSh-1000 in combination with filters 10 and ZS-21-ESS. The photosensitivity of yeast at a concentration of 0.5 × 10 ⁶ cells per milliliter is taken as 100%. The repetition of experiments three times. When using laser radiation of 690 nm, a more effective inactivation of fungal cultures with high cell concentrations is observed. The results are presented in table 5.

The results of the studies allow us to conclude that a single combined effect of dark non-toxic micromolar (up to 20 μM) concentrations of chlorine-type photosensitizers and non-inactivating cells in the absence of photosensitizers of low-intensity (5-20 mW / cm ² ) doses of visible electromagnetic radiation (400-600 nm) or far red (690 nm) ranges allows to achieve high (up to 97-100%) levels of inactivation of cultures of yeast of the genus Candida. Photosensitized inactivation of animal cells is achieved at significantly higher intensities and radiation doses [6], which ensures the selectivity of the proposed method.

Using the proposed method allows reliable inactivation of various strains of pathogenic fungi C. albicans, including freshly isolated from clinical material.

The introduction of a method of photosensitized chlorins inactivation of pathogenic fungi in medicine will help reduce the ever-increasing prevalence of fungal diseases.

It is currently difficult to quantify the economic effect, but the proposed method involves the use of low concentrations of domestically produced chlorin-based photosensitizers obtained from cheap plant materials and cheap sources of low-intensity electromagnetic radiation.

Sources of information

1. RF patent No. 2116066, IPC A 61 K 7/32.

2. RF patent No. 2153876, IPC A 61 K 31/7135.

3. RF patent No. 2098134, IPC A 61 L 2/12.

4. RF patent No. 2008898, IPC A 61 K 9/06.

5. Bikbulatova N.N. Development of a pathogenetically substantiated method for the treatment of patients with cutaneous candidiasis using photochemotherapy. Abstract of dissertation. M. 1995 - prototype.

6. Zeina B., Greenman J., Corry D., Purcell W.M. Cytotoxic effects of antimicrobial photodynamic therapy on keratinocytes in vitro. Br. J. Dermatol., 2002; V. 146, N 4, P. 568-573.

Claims (1)

A method of inactivating pathogenic fungi, including treating the fungi with an inactivating substance, characterized in that a chlorine-type photosensitizer at a concentration of 10-20 μM is selected as the inactivating substance, the mushrooms are treated for 5 minutes, after which they are irradiated for at least 10 minutes with electromagnetic radiation of the range 400-700 nm.

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