RU2670201C1 - Photosensitizers for photodynamic inactivation of bacteria, including in biofilms - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to microbiology, pharmaceutics and medicine, namely to photosensitizers for photodynamic inactivation of bacteria. Synthetic cationic bacteriochlorins of general formula:

, where R=CH₂CH₂Br, or C₇H₁₅, or CH₂CH₂N⁺C₅H₅Br^-, as photosensitizers for photodynamic inactivation of bacteria, including in biofilms.

EFFECT: proposed photosensitizers have a high efficiency of photodynamic inactivation of bacteria.

1 cl, 3 dwg, 2 tbl, 5 ex

Description

The present invention relates to microbiology, pharmaceuticals and medicine, and more particularly to photosensitizers (PS) for the photodynamic inactivation of bacteria, including in biofilms. The invention can be used to inactivate local foci of chronic infection.

The fight against infectious diseases associated with the increasing resistance of pathogenic bacteria causing chronic diseases to broad-spectrum antibiotics has become one of the main problems of modern medicine. It has been established that organized communities of pathogenic bacteria (the so-called bacterial biofilms) having a complex structure are practically not amenable to antibiotic therapy and cause not only chronic infectious diseases (purulent wounds, trophic ulcers), but also serious complications arising in cardiac surgery, urology, and other areas of medicine in which medical devices (catheters, stents, artificial valves and joints) are introduced into the patient’s body. The complex infrastructure and hierarchy of bacteria in the bacterial biofilm, the formation of special means of life support and protection in the form of a matrix, lead to the fact that for the treatment of such infectious lesions, the doses of antibiotics must be increased hundreds of times, and such doses go beyond the real capabilities of the body.

It was found that photodynamic therapy can be used to inactivate biofilms, and bacteria do not form resistance to this treatment method. To increase the efficiency of the photodynamic inactivation of bacteria and biofilms, it is most appropriate to use cationic photosensitizers that can effectively act on both gram-positive and gram-negative bacteria, since infectious foci in most cases have a multivariate nature).

Known cationic photosensitizers based on synthetic bacteriochlorin of the general formula 3-Py ₄ BC (BuR) ₄ Br _n , where:

R = Br, n = 4 [meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide with a charge of molecule +4, RF patent No. 2479585]

or

R = Py, n = 8 [meso-tetra [1- (4'-pyridiniobutyl) -3-pyridyl] bacteriochlorin octabromide with +8 molecule charge, RF patent No. 2476218].

A significant positive charge of the molecules of these photosensitizers allows them to interact with both gram-positive and gram-negative bacteria, and when exposed to radiation with a wavelength in the absorption band of molecules of 760 ± 20 nm, exert a photodynamic effect on them.

These photosensitizers are selected as the closest analogues.

A disadvantage of the known photosensitizers is associated with the insufficiently high efficiency of the photodynamic inactivation of pathogenic bacteria in the free state and biofilms.

The invention solves the problem of creating a photosensitizer with enhanced photodynamic inactivation of pathogenic bacteria in a free state and biofilms.

To solve this problem, synthetic cationic bacteriochlorins of the general formula are proposed:

where R = CH ₂ CH ₂ Br, or C ₇ H ₁₅ , or CH ₂ CH ₂ N ⁺ C ₅ H ₅ Br ^- ,

as photosensitizers for the photodynamic inactivation of gram-positive and gram-negative bacteria, including biofilms,

The proposed tetracationic bacteriochlorins (meso-tetra [1- (2'-bromoethyl) -3-pyridyl] bacteriochlorin tetrabromide (3-Py ₄ BC (EtBr) ₄ Br ₄ ) at R = (CH ₂ ) ₂ Br and meso tetra 1-heptyl-3-pyridyl) bacteriochlorin tetrabromide (3-Py ₄ BCH _p4 Br ₄ ) at R = C ₇ H _{15 were} synthesized by alkylation of meso-tetra (3-pyridyl) bacteriochlorin with 1,2-dibromo-ethane or heptyl bromide, respectively in nitromethane in an inert atmosphere for 2 hours with high yields of 83.9% and 88.4%, respectively. Octacationic meso-tetra [1- (2'-pyridinioethyl) -3-pyridyl] bacteriochlorin octabromide (3-Py ₄ BC (EtPy) ₄ Br ₈ ) (at R = (CH ₂ ) ₂ NC ₅ H ₅ Br ₄ ) was synthesized by boiling a tetracationic (3-Py ₄ BC (EtBr) ₄ Br ₄ ) with an excess of dry pyridine in methanol in an inert atmosphere for 3 hours with a yield of 91.9%.

The present invention is characterized by the following examples.

Example 1. Obtaining the meso-tetra [1- (2'-bromoethyl) -3-pyridyl] bacteriochlorin tetrabromide (3-Py ₄ BC (EtBr) ₄ Br ₄ ).

Meso-Tetra (3-pyridyl) bacteriochlorin (0.20 g, 0.32 mmol) [RF Patent No. 2549953] is dissolved in 10 ml of 1,2-dibromoethane and 10 ml of nitromethane and 2 ml of methanol are added. The reaction mass is stirred while boiling for 2 hours in an inert atmosphere. After cooling to room temperature, the precipitate formed is filtered off, washed with benzene and dried in air in the dark. Then the residue is dissolved in 10 ml of methanol, filtered through a membrane filter (Millipore) with a pore size of 0.22 μm, the filtrate is evaporated to dryness in vacuo. 0.37 g (83.9%) of 3-Py ₄ BC (EtBr) ₄ Br _{4 is} obtained. Electronic absorption spectrum, water, λ _max , nm (ε): 349 (86590), 372 (78340), 490 (5980), 519 (39570), 701 (4420), 762 (96510) (Fig. 1). Found,%: C, 41.96, 41.74; H, 3.56, 3.75; N, 7.41, 7.53. C ₄₈ H ₄₆ Br ₈ N ₈ . Calculated,%: C, 41.95; H, 3.37; N, 8.15%.

Example 2. Obtaining the mesotetra [1- (2'-pyridinioethyl) -3-pyridyl] bacteriochlorin octabromide (3-Py ₄ BC (EtPy) ₄ Br ₈ ).

Meso-Tetra [1- (2'-bromoethyl) -3-pyridyl] bacteriochlorin tetrabromide (3-Py ₄ BC (EtBr) ₄ Br ₄ ) (0.15 g; 0.11 mmol) was dissolved in 7 ml of methanol and added 2 ml dry pyridine. The reaction mass is stirred while boiling for 3 hours in an inert atmosphere. After cooling to room temperature, the reaction mixture was evaporated to dryness in vacuo, the residue was washed with benzene to a colorless filtrate. The residue is then dissolved in 10 ml of methanol, filtered through a membrane filter (Millipore) (0.22 μm), the filtrate is evaporated to dryness in vacuo. 0.17 g (91.9%) of 3-Py ₄ BC (EtPy) ₄ Br _{8 is} obtained. Molecular Weight of 1690.56. Electronic absorption spectrum, water, λ _max , nm (ε): 348 (90750), 370 (78840), 485 (5520), 519 (38050), 699 (4160), 764 (95720) (Fig. 2). Found,%: C, 48.21, 48.54; H, 4.49, 4.58; N, 9.25, 9.25. C ₆₈ H ₆₆ Br ₈ N ₁₂ . Calculated,%: C, 48.31; H, 3.94; N, 9.94.

Example 3. Obtaining meso-tetra (1-heptyl-3-pyridyl) bacteriochlorin tetrabromide (3-Py ₄ BCH _p4 Br ₄ ).

Meso-Tetra (3-pyridyl) bacteriochlorin (0.10 g, 0.16 mmol) is dissolved in 5 ml of chloroform and 5 ml of nitromethane and 1 ml of methanol are added. Then, 2 ml of heptyl bromide is added to the reaction mass and stirred under reflux for 2 hours in an inert atmosphere. After cooling to room temperature, the reaction mass was diluted with 10 ml of benzene and cooled with ice. The precipitate formed is filtered off, washed with benzene and air dried in the dark. Then the residue is dissolved in 10 ml of methanol, filtered through a membrane filter (Millipore) with a pore size of 0.22 μm, the filtrate is evaporated to dryness in vacuo. 0.19 g (88.4%) of 3-Py ₄ BCH _p4 Br _{4 is} obtained. Electronic absorption spectrum, methanol, λ _max , nm (ε): 350 (90920), 374 (88400), 488 (11150), 517 (51590), 693 (10120), 760 (101550) (Fig. 3). Found,%: C, 60.62; 60.69; H, 6.62; 6.70; N, 7.90; 7.89. C ₆₈ H ₉₀ Br ₄ N ₈ . Calculated,%: C, 60.99; H, 6.77; N, 8.37.

Example 4. Preparation of a dispersion (3-Py ₄ BCH _p4 Br ₄ ) in 4% Kolliphor ELP with a concentration of 1 mm.

A portion of (3-Py ₄ BCH _p4 Br ₄ ) (13.4 mg) was dissolved in 10 ml of methanol and mixed with a solution of 0.4 g of Kolliphor ELP in 10 ml of chloroform. Mix the solution in a 1 L round-bottom flask using a magnetic stirrer, heating to 40-50 ° C. Then the solvents are evaporated on a rotary evaporator under vacuum at a temperature of a water bath of 40-50 ° C. The resulting film is dried under vacuum until the chloroform odor disappears completely. The resulting film is then hydrated in 10 ml of distilled water until the Kolliphor ELP is completely dissolved with solubilized (3-Py ₄ BCH _p4 Br ₄ ). The solution is filtered through a membrane filter (Millipore) with a pore size of 0.22 μm.

Example 5. Comparative studies of the photodynamic inactivation of pathogenic bacteria S.aureus 15 and P. aeruginosa32 using known photosensitizers (3-Py ₄ BC (BuBr) ₄ Br ₄ ) and (3-Py ₄ BC (PyBu) ₄ Br ₈ ) in water solutions of the proposed photosensitizers (3-Py ₄ BC (EtBr) ₄ Br ₄ ) and (3-Py ₄ BC (PyEt) ₄ Br ₈ ) in aqueous solutions, and (3-Py4BCH _p4 Br ₄ ) 4% Kolliphor ELP

In vitro experiments evaluated MBC (minimum bactericidal concentration) in relation to plankton bacteria of antibiotic-resistant clinical isolates of S.aureus15 and P.aeruginosa32 of known photosensitizers (3-Ru ₄ BC (BuBr) ₄ Br ₄ ) and (3-Ru4BC (PyBu) ₄ Br ₈ ) in aqueous solutions of the proposed photosensitizers (3-Py ₄ BC (EtBr) ₄ Br ₄ ) and (3-Py4BC (PyEt) ₄ Br ₈ ) in aqueous solutions, and (3-Py ₄ BCH _p4 Br ₄ ) 4 % Kolliphor ELP under standard conditions: incubation of bacteria with PS for 30 min, radiation dose density of 30 J / cm ² . The initial bacterial titer is 1 × 10 ⁸ CFU / ml (colony forming units in ml). Twice dilutions of PS were used, starting with 100 μM. After incubation, the bacterial suspension was centrifuged, the FS was removed, the bacteria were resuspended in physiological saline, and 100 μl from each concentration and control without PS was poured into the wells of 96-well flat-bottomed irradiation plates. After irradiation, 50 μl from each well was plated on Petri dishes with LB agar, incubated in the dark at 37 ° C for 20 h. The lowest concentration of PS was noted, from which it did not grow. This concentration was taken as the minimum bactericidal concentration (MBC).

The results of comparative studies of MBC known photosensitizers (3-Py ₄ BC (BuBr) ₄ Br ₄ ) and (3-Py ₄ BC (PyBu) 4Br ₈ ) in aqueous solution, and the proposed photosensitizers (Py ₄ BC (EtBr) ₄ Br ₄ ) and (3-Py ₄ BC (PyEt) ₄ Br ₈ ) in aqueous solution and (3-Py ₄ BCH _p4 Br ₄ in 4% Kolliphor ELP on plankton cells of St.aureus strain are shown in Table 1.

The results of comparative studies of MBC known photosensitizers (3-Py ₄ BC (BuBr) ₄ Br ₄ ) and (3-Py ₄ BC (PyBu) ₄ Br ₈ ) in aqueous solution and in aqueous solution and (3-Py ₄ BCH _p4 Br ₄ ) in 4% Kolliphor ELP on plankton cells of P.aeruginosa32 MBK strain of the known photosensitizer (3-Py ₄ BC (BuBr) ₄ Br ₄ ) in an aqueous solution (prototype) and the proposed photosensitizers (3-Py ₄ BC (EtBr) ₄ Br ₄ ) in aqueous solution and 3-Py ₄ BCH _p4 Br ₄ ) (dispersion in 4% Kolliphor ELP) are shown in Table 2.

Example 5. Photoinactivation of bacteria in biofilms.

St.aureus bacterial biofilms were grown for 18 hours on 7 × 12 mm glass plates in a Petri dish in which LB broth with a seeded bacterial culture was poured. Then the glasses were transferred to a 24-well plate, washed with distilled water, poured with FS solutions of various concentrations, incubated for 1 hour in a thermostat, PS was removed and the glasses were irradiated in physiological saline with a light dose of 70 J / cm ² . Then the glasses and the liquid from the well were transferred to an Eppendorf tube, the biofilm was removed with a brush probe and treated with a brush for 10 minutes on a Vortex. 10-fold dilutions of the bacterial suspension were plated on LB agar plates to count the colonies and determine the CFU titer.

When using the well-known photosensitizer 3-Py ₄ BC (PyBu) ₄ Br ₈ , the CFU / ml values were reduced by 3 orders of magnitude, the well-known photosensitizer (3-Py ₄ BC (BuBr) ₄ Br ₄ ) - by 3.4 orders of magnitude.

When using the proposed 3-Py ₄ BC (PyEt) ₄ Br ₈ , (3-Py ₄ BC (EtBr) ₄ Br ₄ ) or (3-Py ₄ BCH _p4 Br ₄ ) photosensitizers, the CFU / ml values decreased by more than 6 orders of magnitude .

Thus, the proposed photosensitizers in comparison with the known photosensitizers have a significantly higher efficiency of the photodynamic inactivation of planktonic bacteria, including in biofilms.

For the photodynamic inactivation of local foci of chronic infection, they are sensitized by systemic (e.g., intravenous) administration of an aqueous solution (3-Py ₄ BC (EtBr) ₄ Br ₄ ) or (3-Py ₄ BC (PyEt) ₄ Br ₈ ), or cremophore dispersion ( 3-Py ₄ BCH _p4 Br ₄ ), or application compositions based on ((3-Py ₄ BC (EtBr) ₄ Br ₄ ) or (3-Py ₄ BC (PyEt) ₄ Br ₈ ), or cremophore dispersion (3- Py ₄ BCH _p4 Br _4), followed by irradiation of light emission in the spectral range 750-770 nm. Our studies have shown that the sensitization is advantageously carried out for 60-180 minutes. Optimally spends sensitization under the control of the fluorescence intensity of the photosensitizer in the spectral range 760-800 nm, and the irradiation is carried out after reaching the maximum fluorescence intensity.

Claims (4)

Synthetic cationic bacteriochlorins of the general formula:

where R = CH ₂ CH ₂ Br, or C ₇ H ₁₅ , or CH ₂ CH ₂ N ⁺ C ₅ H ₅ Br ^- , as photosensitizers for the photodynamic inactivation of bacteria, including in biofilms.

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