RU2479585C1 - Photosensitiser for photodynamic therapy - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: described is a novel meso-tetra[1-(4'-bromobutyl)-3-pyridyl]bacteriochlorin tetrabromide of formula

as a photosensitiser for photodynamic therapy (PDT).

EFFECT: low phototoxicity, high photo-induced anti-tumour activity for further use thereof in PDT.

1 cl, 6 ex, 3 dwg

Description

The present invention relates to medicine, namely to photosensitizers (PS) for photodynamic therapy (PDT) of malignant neoplasms and a number of other pathological conditions.

The PDT method is based on the use of natural or synthetic PSs that are capable of selective accumulation (tropism) in tumor tissue. When irradiated with light of a certain wavelength, the FS transforms into an activated state, which initiates the formation of cytotoxic agents - singlet oxygen and free radicals, which cause the destruction of the structural elements of the tumor tissue.

Prospective FS are phthalocyanine derivatives. Thus, the Photosens preparation based on sulfonated aluminum phthalocyanine has a long-wavelength absorption maximum at λ _max = 675 nm with a high molar extinction coefficient ε (over 100,000) and a high fluorescence quantum yield (RF patent No. 2220722, A61K 31/409, 2004), which allows achieve photoinduced necrosis to a depth of 1.0 cm. However, Photosens does not have a sufficiently high selectivity of accumulation in tumor cells, long-term preservation in tissues, which can lead to phototoxicity of the skin.

The successful application of the PDT method for the treatment of malignant neoplasms stimulates the search for new FS with improved properties. The most promising for PDT are FS with a maximum absorption in the red and near infrared ranges (700-800 nm), the so-called “therapeutic window”, where the intrinsic absorption of biological tissue is minimal, which allows deeper penetration of radiation into the tissue and, as a result, high efficacy of therapy (Bonnett RJ Heterocyclic Chem. 2002. V.39. P.455-470).

Bacteriochlorins (tetrahydroporphyrins) are promising PS for PDT absorbing in the near infrared range of the spectrum. Hydrogenation of another double bond in the chlorin molecule leads to a further bathochromic shift of the long-wavelength absorption band. So, tucad, a palladium derivative of bacteriochlorin (Tookad) with a maximum absorption at 760 nm, is allowed to treat the prostate.

The objective of the invention is the search for FS, characterized by absorption in the near infrared range of the spectrum, high tropism for tumor tissue, reduction of side effects due to the reduction of circulation time in the body and having high photoinduced activity.

To solve this problem, PS was synthesized, which is a positively charged bacteriochlorin - meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide (3-RuBC) of the following formula:

PSs of a number of cationic salts of meso-tetra (N-alkyl-3-pyridyl) bacteriochlorin are known (US Patent 6410568, 2002). The proposed PS is characterized by the presence in the alkyl (n-butyl) chain of the terminal bromine atom. The introduction of bromine atoms increases the likelihood of an intercombination transition to the triplet state and thereby increases the yield of photoinduced singlet oxygen and, more importantly, allows further quaternization with the participation of terminal bromine atoms.

3-RuBC is synthesized by alkylation of meso-tetra (3-pyridyl) bacteriochlorin with 1,4-dibromobutane. The starting meso-tetra (3-pyridyl) porphyrin was obtained by condensation of equimolar amounts of pyrrole with 3-pyridinaldehyde in boiling acetic acid in the presence of air. Porphyrin was isolated and purified by chromatography on silica gel with chloroform.

The recovery of meso-tetra (3-pyridyl) porphyrin was carried out with diimide generated under the reaction conditions from p-toluenesulfonylhydrazine in the presence of dry potash in dry pyridine. The cationic water-soluble quaternary salt of 3-RuBC was obtained by boiling meso-tetra (3-pyridyl) bacteriochlorin with an excess of 1,4-dibromobutane in nitromethane in an inert atmosphere.

In the ESP spectrum of the tetracationic 3-RuVS salt in methanol, the Q1 and Q2 bands are located at 761 and 515 nm. Figure 1 shows the 1 H NMR spectrum of 3-RuBC in CD3OD in the region of aromatic protons. A singlet of β-pyrrole protons is observed in the spectrum at 8.21 ppm. and four signals of protons of meso-pyridinium rings - at 8.51 ppm (H-5), 9.17 ppm. (H-6), 9.39 ppm. (H-4) and 9.81 ppm. (H-2).

Physico-chemical and photophysical properties of 3-RuVS

3-RuVS is readily soluble in water and water-salt solutions at room temperature.

3-RuVS is stable in aqueous multicomponent solutions for 6 months with varying concentrations from 2 to 40 μM under dark conditions (λmax = 761 nm in the absorption spectra λ _max = 751 nm in the fluorescence spectra). Figure 2 shows the absorption spectra of a solution of 3-RuBC (5 μM) in a 0.9% NaCl solution immediately after dissolution (solid line), one month later (dashed line), and after 6 months (dashed line) storage at + 4 ° C .

The 3-RuVS photosensitizer in a cell-free medium is prone to fading, which is accompanied by a decrease in fluorescence intensity without changing the shape of the spectrum.

Photobleaching of 3-RuVS was also observed in vivo during irradiation of mouse skin, which was carried out under the control of fluorescence. A decrease in the fluorescence intensity in the irradiated area to the background level (skin area without exposure) was shown, while the nature of the spectrum remained unchanged. This happened, apparently, both due to the cessation of blood flow as a result of vascular damage and / or blood clots, as well as intense dye burnout in the irradiation zone and was not associated with dye degradation during irradiation. After its completion, fluorescence in the irradiated area of the skin began to increase, indicating the entry of dye into the irradiated area from the bloodstream.

Photoinduced activity of 3-RuBC in the in vitro system

The dye possessed high photoinduced activity against human tumor cells of various epithelial origin: epidermoid carcinoma of the larynxopharynx (HEP2), lung adenocarcinoma (A549) and colon carcinoma (HT29) with varying dye solution concentrations from 0.15 to 70 μM, incubation time to light exposure to 0.5-6 hours and an energy density of 2-10 J / cm ² . The value of the IC ₅₀ estimated by the MTT test ranged from 0.4 to 5 μm.

Distribution and fluorescence contrast of 3-RuVS in animals with tumors of various origins

Assessment of the distribution of 3-RuVS in mouse tumors (Lewis lung carcinoma (LLC), sarcoma S37 (S37), melanoma B16 (B16), lymphocytic leukemia P388 (P388)) and surrounding tissue, performed by local fluorescence spectroscopy, showed that the photosensitizer accumulated as in the tumor and in the surrounding tissues.

In the tumor tissue, the normalized fluorescence (FN) of the dye reached a maximum value 30-60 minutes after administration, and then by 72 hours it decreased by 72% -85% of the maximum value depending on the histological type of the tumor. The highest levels of FN in the skin and muscle were also observed 30-60 minutes after administration of the substance. The maximum fluorescence contrast of the dye, both with respect to the skin and relative to the muscle, was recorded 5 seconds to 30 minutes after administration and amounted to 2.3-3.3 srvc. units and 1.6-2.3 conv. units respectively.

Skin phototoxicity 3-RuVS

The effectiveness and safety of the photosensitizer is determined, on the one hand, by the photo-destruction of the tumor tissue, on the other hand, by photo-damage to the surrounding tissue, especially the skin, which occurs both during the PDT session (a characteristic local skin reaction) and after its completion (general skin reaction if not observed light mode).

Assessment of skin phototoxicity in intact mice, carried out at various time intervals between the administration of 3-RuVS and irradiation (30 minutes to 28 days) showed that the photosensitizer has low skin phototoxicity, the minimum skin response at the control level was recorded in animals irradiated at 7-28 day after the introduction of the dye. No deep skin damage was detected. The presence of background fluorescence values of 3-RuVS in the skin did not significantly affect its cutaneous phototoxicity.

3-RuVS has lower skin phototoxicity compared to phthalocyanines [photosens, phthalosens and holosens (RF patents: 2183635, C07D 487/22, 2002; 2282646, С09В 47/32, 2006)].

Photo-induced antitumor activity of 3-RuBC in animals with tumors of various histogenesis

In the study of photo-induced antitumor activity of 3-RuBC in animals with Lewis lung carcinoma (LLC), S37 sarcoma (S37), B16 melanoma (B16), P388 lymphocytic leukemia (P388) depending on the dye dose (1.0-10.0 mg / kg), the interval between its administration and irradiation (5 minutes - 24 hours) and energy density (50-300 J / cm ² ) revealed a high dose-dependent antitumor efficacy: SRW was 63.6-100%, UPZ 26.2-48, 5%, the cure rate of animals reached 33.3% -100%, depending on the tumor model. The maximum antitumor effect was obtained in animals with an LLC tumor, the minimum - in mice with B16 melanoma and P388 lymphocytic leukemia.

The present invention is characterized by the following examples.

Example 1

Meso-Tetra (3-pyridyl) bacteriochlorin. A mixture of meso-tetra (3-pyridyl) porphin (0.2 g, 0.32 mmol), p-toluenesulfonyl hydrazide (0.12 g, 0.65 mmol) and dry potash (0.44 g, 3.2 mmol) in 12 ml of dry pyridine is heated with stirring to 108-110 ° C in argon atmosphere. Stirring is continued at the same temperature for 12 hours, adding a new portion of p-toluenesulfonyl hydrazide (0.12 g, 0.65 mmol) to the reaction mass every 1.5 hours. Next, the reaction mass is cooled, the precipitate is filtered off, the filtrate is evaporated to dryness, washed with water and dried in air. The technical product is dissolved in chloroform and chromatographed on neutral alumina, eluting with chloroform. The first pink fraction was collected and, after removal of the solvent, 0.08 g (40%) of meso-tetra (3-pyridyl) bacteriochlorin was obtained. Mass spectrum (MALDI-TOF), m / z: 622.025 [M] ⁺ . ESP, λ _max , nm (log ε), chloroform: 747 (5.05), 683 (3.78), 521 (4.71), 491 (3.78), 380 (5.08), 367 ( 4.95), 357 (5.00).

Meso-Tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide. Meso-Tetra (3-pyridyl) bacteriochlorin (0.15 g, 0.24 mmol) is dissolved in 5 ml of nitromethane and 1.5 ml of 1,4-dibromobutane is added. The reaction mass is stirred while boiling for 1 h in an inert atmosphere. After cooling to room temperature, the reaction mass was diluted with 5 ml of benzene and cooled with ice. The precipitate formed is filtered off, washed successively with a mixture of nitromethane: benzene (1: 1), then with benzene and hexane. 0.24 g (66.2%) of meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide is obtained. Electronic absorption spectrum, water, λ _max , nm (log ε): 763 (5.02), 702 (4.24), 518 (4.72), 374 (4.95), 351 (4.98). Electronic absorption spectrum, methanol, λ _max , nm (log ε): 761 (5.04), 700 (3.76), 516 (4.71), 488 (3.89), 374 (4.93), 349 (4.96). Spectrum ¹ H NMR (CD ₃ OD), δ, ppm: 2.07-2.14 (m, 8H, CH ₂ ), 2.38-2.45 (m, 8H, CH ₂ ), 3 61 (t, 8H, CH ₂ ), 4.13-4.28 (m, 8H, β-H), 4.94-4.98 (m, 8H, CH ₂ ), 8.21 (s, 4H, β-H), 8.49-8.52 (m, 4H, H-5 (En)), 9.17 (w, 4H, H-6 (En)), 9.39 (d, 4H H-4 (En), 9.78-9.83 (m, 4H, H-2 (En)).

Found,%: C 43.39, 43.20; H 4.65, 4.67; N, 7.31; 7.13. C ₅₆ H ₆₂ Br ₈ N ₈ × 3H ₂ O. Calculated,%: C 43.66; H 4.45; N, 7.27.

Example 2

Photo-fading of 3-RuVS in a cell-free environment. Evaluation of the photo-fading of fluorochrome was carried out in Eagle's medium containing 7% ETS. The concentration of the working solution was 13 μM. The fluorescence spectra were recorded immediately after the preparation of the solutions and after light exposure. The light dose was 5 and 10 J / cm ² , the power density of 16.0 mW / cm ² . A 500 W halogen lamp with a KS-13 broadband filter (λ≥640 nm) and a water filter with a thickness of 5 cm was used as an optical radiation source. Fluorescence measurements were performed by the contact method on a LESA-06 laser spectral analyzer (CJSC BIOSPEK, Russia) in the spectral range of 649-850 nm. A significant decrease in the fluorescence intensity was revealed at a maximum at 751 nm without changing the profile of the spectrum, which testified to the photobleaching of 3-RuVS in solutions when exposed to light. Figure 3 shows the fluorescence spectra of 3-RuVS before exposure to light (1), when irradiated with 5 J / cm ² (2) and 10 J / cm ² (3) in a culture medium.

Example 3

The photoinduced activity of 3-RuBC against human epidermoid carcinoma cells of the larynxopharynx (HEp-2 cell culture). The photosensitizer was introduced into the culture medium at a concentration of from 0.4 to 15 μM. The incubation time ranged from 30 minutes to 4 hours. Irradiation was carried out with a 500 W halogen lamp using a KS-13 broadband filter (λ≥640 nm) and a 5 cm thick water filter. The level of growth inhibition of the culture was calculated by the formula:

IR (%) = [(P _to -P _o ) / P _to ] × 100%

where IR - inhibition of culture growth, in percent;

P _about and P _to - the number of viable cells, expressed in units of optical density, respectively, in the experimental (with dye) and control (without dye) samples. Inhibition of culture growth by 50% (IC ₅₀ ) was considered a biologically significant effect.

It was shown that the 3-RuBC photosensitizer was highly toxic to HEp2 culture cells. The maximum photoinduced activity was observed at 2 hours of incubation (IC ₅₀ was 0.40 ± 0.05 μM), with an increase in the time interval, the IC ₅₀ value changed slightly. 3-RuVS did not exhibit dark toxicity during the day of observation (IR ₅₀ >> 20 μg / ml).

Example 4

Distribution of 3-RuBC in an LLC tumor and fluorescence contrast relative to surrounding tissue. The distribution of 3-RuVS in the tumor and surrounding tissue was evaluated in mice with LLC carcinoma in the range from 5 seconds to 72 hours using local fluorescence spectroscopy. The photosensitizer was administered intravenously in an effective (therapeutic) dose of 5.0 mg / kg. Fluorescence was recorded by the contact method on a laser spectral analyzer for fluorescence diagnosis of tumors and monitoring PDT "LESA-06" (CJSC "BIOSPEC", Russia). Fluorescence was excited by radiation from a He-Ne laser (lasing wavelength 632.8 nm).

In LLC tumor tissue, the normalized dye fluorescence reached a maximum value 30 minutes after administration, and then decreased by 85% from the maximum value by 48 hours. The highest levels of normalized fluorescence in the skin and muscle were observed 30 minutes after administration of the substance. The maximum fluorescence contrast of the dye with respect to both the skin and the muscle was also recorded 30 minutes after administration and amounted to 3.3 ± 0.7 and 2.3 ± 0.4 srvc. units respectively.

Example 5

Cutaneous phototoxicity of 3-RuVS was evaluated in intact mice 30 minutes later (maximum dye accumulation in the skin), as well as 1, 7, and 28 days after administration of the photosensitizer.

3-RuVS was administered intravenously in one of the effective doses for mice - 5.0 mg / kg. For irradiation of the depilated skin area, the standard modes were chosen, the same as those for PDT of tumors (power density - 100 mW / cm ² , energy density - 150 J / cm ² ). An LED red light source was used, the wavelength of which corresponded to the maximum absorption of the dye - 759 ± 18 nm.

After irradiation, all animals developed edema during the first day, the intensity of which depended on the time interval between administration of 3-RuVS and irradiation. The most pronounced edema was recorded in animals one day after the irradiation session. The maximum edema was recorded in mice irradiated 30 minutes after dye administration; upon irradiation after 24 hours, the edema size sharply decreased and amounted to only 27% of the maximum, after 7 and 28 days - 10% and 4%, respectively, and was comparable with edema in control animals (3% of the maximum recorded). No deep skin damage was detected.

Example 6

Photo-induced antitumor activity of 3-RuVS

The study was conducted in animals with Lewis lung carcinoma (LLC), depending on the dose of the dye (2.5-5.0 mg / kg). Irradiation was carried out by an LED source (759 ± 18 nm) with a power density of 100 mW / cm ² (energy density of 150 J / cm ² ) 30 minutes after the introduction of the photosensitizer. Antitumor efficacy was assessed by inhibition of tumor growth (SRW), an increase in life expectancy (VL) and the criterion of cure (KI).

During the day after irradiation, the animals formed intense edema in the irradiation zone, which lasted up to 4-5 days. After irradiation, no animal deaths were noted. When using a tetrabromide derivative of bacteriochlorin at a dose of 2.5 mg / kg, the SRW was 70.9-100%, VL did not reach a biologically significant level, and no cured animals were found. With an increase in dose to 3.75 mg / kg, an increase in efficacy was observed. SRW was already 94.4-100%, UPZH - 48.5%, CI - 33.3%. For a dose of 5.0 mg / kg, an even higher efficiency was revealed: 100% inhibition of tumor growth and 100% cure of animals (within 120 days of observation).

Thus, the proposed FS has all the necessary properties for its further use for PDT.

Claims (1)

Meso-tetra [1- (4'-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide as a photosensitizer for photodynamic therapy

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