RU2781892C1 - Method for photodynamic inactivation of gram-negative pathogenic microorganisms - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to microbiology and medicine and is intended for photodynamic inactivation of gram-negative pathogenic microorganisms in vivo, including antibiotic-resistant nosocomial microflora, using a photosensitizer, which is a mono-cationic derivative of chloride e₆, the formula indicated below, consisting in introduction into the focus of inflammation of a composition with a photosensitizer concentration of 0.0035 mol/kg containing, wt.%: 1 nonionic surfactant Twin 80 as a carrier, 0.1 disodium salt of ethylenediaminetetraacetic acid (Na₂H₂Edta), 10 glycerin, 2 dimethyl sulfoxide and 1 sodium alginate, and irradiation with red light with a wavelength of λ=660 nm and a light energy density of 80 J/cm².

EFFECT: destruction of pathogenic gram-negative microorganisms, including antibiotic-resistant nosocomial microflora in vivo, by carrying out their photodynamic inactivation.

1 cl, 1 tbl

Description

The invention relates to microbiology and pharmaceutical chemistry and is intended for photodynamic inactivation of Gram-negative pathogenic microorganisms, including antibiotic-resistant nosocomial microflora, using a photosensitizer.

Antimicrobial photodynamic therapy (APDT) is an alternative to the usual methods of combating pathogenic microflora, and can also be used as an effective addition to antibiotic therapy. APDT is a fundamentally new and different from antibiotic therapy strategy for inactivation of pathogenic microflora, based on the selective accumulation of colored substances - photosensitizers (PS) in microbial cells. These substances, when exposed to visible light of a certain wavelength and corresponding power, generate reactive oxygen species, which makes it possible to carry out photodynamic inactivation of microorganisms by triggering a cascade of photochemical reactions.

Neutral and anionic macroheterocyclic PSs based on porphyrins, chlorins, and phthalocyanines, introduced into clinical practice for the treatment of tumors, are effective in photodynamic inactivation of mainly gram-positive microorganisms; however, gram-negative bacteria with an outer lipopolysaccharide membrane usually turn out to be insensitive to APDT.

It is known to use the Photosens photosensitizer based on sulfonated hydroxyaluminum phthalocyanine for APDT of infected wounds and trophic ulcers with antibiotic-resistant microflora [Stranadko E.R., Tolstykh M.R., Riabov M.V., Krivikhin D.V. (2003) Photodynamic therapy of persistent purulent wounds and trophic ulcers of lower extremeties. DC World Congress of the International Photodynamic Association. abstracts. Miyazaki, P. 28].

However, the anionic nature of the proposed PS does not allow effective photodynamic inactivation of Gram-negative bacteria.

The use of neutral and cationic zinc complexes of phthalocyanines for photodynamic inactivation of Gram (-) microorgasms in vitro is described. It has been shown that the presence of cationic groups in the PS molecule makes it possible to reduce the number of colony-forming units of Escherichia coli (CFU) by several orders of magnitude [Scalise I, Durantini EN. Synthesis, properties, and photodynamic inactivation of escheric.iia coli using a cationic and a non-charged zn(ii) pyridyloxyphthalocyanine derivatives. Bioorg Med Chem 2005. 13. P. 3037-3045].

However, the PSs used do not allow complete inactivation of pathogenic microflora and, in addition, have a noticeable residual phototoxicity.

It is known to use low-toxic derivatives of chlorin e ₆ (neutral, anionic and cationic forms) against gram-negative and gram-positive bacteria in vitro [Drulis-Kawa Z, Bednarkiewicz A, Bugla G, Strek W, Doroszkiewicz W. // Adv Clin Exp Med 2006; 15(2). P. 279-83; Karygianni L, Ruf S, Folio M, Hellwig E, Bucher M, Anderson AC, Vach K, Al-Ahmad A. // Appl Environ Microbiol 2014; 80: 7324-7336; Mesquita MQ, Menezes JC, Neves MG, Tome AC, Cavaleiro JA, Cunha A, Almeida A, Hackbarth S, Roder B, Faustino MA. // Bioorg Med Chem Lett 2014; 24:808-812; Kustov AV, Belykh DV, Smimova NL, Venediktov EA, Kudayarova TV, Kruchin SO, Khudyaeva IS, Berezin DB. // Dyes & Pigments. 2018; 149: 553-559]. Despite the successful photoinactivation of Gram (+) pathogens, against Gram-negative microorganisms, photodynamic inactivation was not effective enough. In addition, all studies were carried out in vitro in relation to planktonic forms of microorganisms.

The closest in terms of essential features to the claimed invention is carrying out photodynamic inactivation of microorganisms in vivo with the photosensitizer "Photoditazin", which was used to treat long-term non-healing wounds containing a mixed microflora, represented by both gram-positive and gram-negative bacteria [Geynits A.V., Tolstykh P.I., Derbenev V.A. and others. Photodynamic therapy of purulent and long-term non-healing wounds. A guide for doctors. M., 2004. 15 p.]. It turned out that after irradiation, the level of contamination decreased by two orders of magnitude from 10 ⁷ to 10 ⁵ CFU, however, no photoinactivation of gram-negative microflora was noted.

The technical result of the invention is the destruction of gram-negative pathogenic microorganisms in vivo, including antibiotic-resistant nosocomial microflora, by carrying out their photodynamic inactivation.

This result is achieved by the fact that in the method of photodynamic inactivation of gram-negative pathogenic microorganisms in vivo, which consists in exposing them to a photosensitizer based on chlorin e ₆ , according to the invention, a drug based on a monocationic derivative of chlorin e ₆ is used as a photosensitizer:

by introducing into the inflammation focus its composition with a photosensitizer concentration of 0.0035 mol/kg, containing 1 wt. % nonionic surfactant Twin 80 as a carrier, 0.1 wt. % disodium salt of ethylenediaminetetraacetic acid (Na ₂ H ₂ Edta), 10 wt. % glycerol, 2 wt. % dimethyl sulfoxide and 1 wt. % sodium alginate, and irradiation with red light with a wavelength of λ=660 nm and a light energy density of 80 j/cm ² .

The technical result is achieved due to the fact that the photosensitizer used is capable of effective photodynamic inactivation of gram-negative microorganisms in vivo by realizing the following advantages:

1) the presence of one cationic group in the structure of the PS1 molecule makes it possible to provide the necessary solubility in aqueous solutions with a sufficiently high affinity for the lipophilic environment, including the outer membrane of gram-negative bacteria;

2) the presence of an iodine atom in the structure of the PS 1 molecule potentially increases phototoxicity with respect to pathogenic microflora through the production of reactive iodide radicals during photoinactivation;

3) the use of Tween 80 as a carrier and Na ₂ H ₂ Edta makes it possible to increase the aggregative stability of the photosensitizer in aqueous solutions, increase the photostability of PS molecules, and destabilize the outer lipopolysaccharide membrane of Gram-negative bacteria.

The present invention can be used to carry out photodynamic inactivation of gram-negative microorganisms in vivo, including antibiotic-resistant nosocomial pathogens.

To implement photodynamic inactivation with PS 1, the following substances and devices are used:

- photosensitizer;

- bidistilled water;

- Twin 80 (Panreac, >98%);

- glycerin (Panreac, >98%);

- Na ₂ H ₂ Edta 2 H ₂ O (Panreac, >99%);

- dimethyl sulfoxide (Panreac, >99%);

- sodium alginate (Aldrich, Pharm. sec. standard);

- diode laser or LED panel with a wavelength of 660 nm (Elomed LLC (Moscow), BMC CJSC (Minsk)).

Photoinactivation of pathogenic microorganisms is realized by introducing a composition with a photosensitizer concentration of 0.0035 mol/kg, containing 1 wt. % nonionic surfactant Twin 80 as a carrier, 0.1 wt. % disodium salt of ethylenediaminetetraacetic acid (Na ₂ H ₂ Edta), 10 wt. % glycerol, 2 wt. % dimethyl sulfoxide and 1 wt. % sodium alginate, and irradiation with red light with a wavelength of λ=660 nm and a light energy density of 80 j/cm ² .

Information confirming the possibility of reproducing the invention

The method was tested by photodynamic inactivation of gram-negative nosocomial antibiotic-resistant microorganisms using PS 1 in vivo.

Daily cultures of test strains of microorganisms on beveled beef agar were washed off with saline and adjusted to a concentration of 10 ⁹ CFU per 1 ml. A seed dose of 10 ⁸ cells per 1 ml was prepared from the original standard suspension by dilution. In an in vivo inactivation model, 0.1 ml of a microorganism test culture solution (10 ⁸ cfu/ml) was applied to the burn area obtained on the back of Wistar female laboratory rats or by applying a suspension of microorganisms to the forearm of volunteers. All work was carried out in a specialized laboratory of the Ivanovo State Medical Academy and was approved by the ethics committee of IvGMA (EC 2017.25.10).

Immediately prior to testing, laboratory animals were put into deep sleep by injection of a mixture of Zoletil 100 (Virbac Sante Animale) and Rometar (Bioveta). After that, the surface of the back of the animals was subjected to epilation, and then with a special device, heated to a temperature of 220-230 ° C, several circular burns with a radius of 1 cm were carried out, each of which 0.1 ml of a suspension with a culture of microorganisms was applied after 5 minutes. After 20 minutes, a composition was applied to the area of each burn, with a PS concentration of 0.0035 mol/kg, and after another 20-25 minutes, an antimicrobial PDT session was performed by irradiating the surface with red light. At the end of the session, the biomaterial from each burn was collected with a sterile swab, starting from the center to the edge of the wound surface, and sown on the appropriate nutrient media. After the end of the experiment, the sleeping animals were injected with a lethal dose of the above drugs, after which they were disposed of in the prescribed manner.

When carrying out photoinactivation of gram-negative microorganisms on the surface of the skin of volunteers, 0.1 ml of a solution with a test culture of microorganisms (10 ⁸ CFU/ml) was applied to the area of the forearm with an area of ~4 cm ² . After 20 minutes, the FS composition was applied to it, and after another 20-25 minutes, an antimicrobial PDT session was performed by irradiating the surface with red light with a wavelength of 660 nm. At the end of the session, the biomaterial from each area of application was collected with a sterile swab, starting from the center to the edge of the wound surface, and sown on the appropriate nutrient media. The skin surface was then washed off and treated with an antimicrobial agent.

The results of in vivo tests with the claimed PS during photoinactivation of nosocomial antibiotic-resistant Gram-negative bacteria Pseudomonas aeroginosa and E. coli in laboratory animals in a model of a burn wound and in volunteers when applied to the skin are shown in the table.

As can be seen from the table, irradiation of the burn surface with a dose of light energy of 80 J/cm2 without PS ¹ (control) reduces the number of microorganisms on the wound surface by more than one and a half orders of magnitude, indicating the presence of a residual bactericidal effect of the skin in laboratory animals. The use of PS 1 makes it possible to completely inactivate the pathogenic microorganism Pseudomonas aeruginosa and reduce the number of CFUs in E. coli by almost two orders of magnitude in a single PDT session.

Studies of photoinactivation of microorganisms from the surface of the skin of volunteers showed that surface irradiation with a dose of light energy of 80 J/cm2 without PS ¹ (control) reduces the number of microorganisms on the wound surface by more than three orders of magnitude, indicating a significant bactericidal effect of the skin. The use of PS 1 makes it possible to completely inactivate both pathogenic microorganisms. From the data in the table, we can conclude that the inactivation of Gram (-) microorganisms in vivo is highly effective.

Claims (3)

A method for photodynamic inactivation of gram-negative pathogenic microorganisms in vivo, which consists in exposing them to a photosensitizer based on chlorin e ₆ , characterized in that for photodynamic inactivation of gram-negative pathogenic microorganisms, including antibiotic-resistant nosocomial microflora, a drug based on a monocationic derivative of chlorin e is used as a photosensitizer ₆ , formulas:

by introducing into the inflammation focus its composition with a photosensitizer concentration of 0.0035 mol/kg, containing 1 wt. % nonionic surfactant Twin 80 as a carrier, 0.1 wt. % disodium salt of ethylenediaminetetraacetic acid (Na ₂ H ₂ Edta), 10 wt. % glycerol, 2 wt. % dimethyl sulfoxide and 1 wt. % sodium alginate, and irradiation with red light with a wavelength of λ=660 nm and a light energy density of 80 j/cm ² .