RU2549953C2 - Photosensitiser for photodynamic therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: what is presented is using meso-tetra(3-pyridyl)bacteriochlorin of structural formula (I)

as a near-infrared photosensitiser for a photodynamic therapy. Doses 1.0-2.5 mg/kg of the declared photosensitiser have provided 70-100% tumour growth inhibition, 80-131% increase in life expectancy and 25-100% animals' recovery by selective tumour collection and fast clearance.

EFFECT: high photoinduced activity on human tumour cells of various epithelial origin and high dose-dependent anti-tumour effectiveness in the animal with tumours of various origins.

5 dwg, 8 ex

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 PSs, which are capable of selective accumulation (tropism) in tumor tissue and, when irradiated with light of a certain wavelength, go into an activated state, which initiates the formation of cytotoxic agents - singlet oxygen and free radicals, which destroy structural elements of tumor tissue.

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 FS with a maximum absorption in the far 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 effectiveness of therapy (Bonnett RJ Heterocyclic Chem. 2002. V.39. P.455-470).

Bacteriochlorins (tetrahydroporphyrins) (Mironov A.F., Green M.A., Tsiprovsky A.G. et al. // Bioorganic Chemistry. 2003. V.29. Promising PS for PDT that absorb in the near infrared range of the spectrum). .2. S.214-221). So, tucad, a palladium derivative of bacteriochlorin (Tookad), with a maximum absorption at 760 nm is allowed for the treatment of the prostate.

However, the predominant amount of bacteriochlorins is semi-synthetic preparations, for the preparation of which substances of natural origin are used as starting compounds. Well-known bacteriochlorins of a purely synthetic origin are very difficult to obtain — they are obtained by multi-stage syntheses, as a rule, are unstable during storage or photochemically unstable as a result of oxidative processes.

The objective of the invention is the search for FS, which would be characterized by absorption in the near infrared range of the spectrum, high photoinduced activity, high tropism for tumor tissue, reduction of side effects due to the reduction of circulation time in the body, which would significantly increase the effectiveness of treatment.

To solve this problem as a PS for PDT, it is proposed to use a very affordable purely synthetic bacteriochlorin - meso-tetra (3-pyridyl) bacteriochlorin (H ₂ Py ₄ BC) of the following formula:

This compound is described as an intermediate in the synthesis of meso-tetra [1- (4′-bromobutyl) -3-pyridyl] bacteriochlorin tetrabromide (RF Patent No. 2479585, C07D 487/22, 2013). H ₂ Py ₄ BC was obtained by two-stage synthesis starting from pyrrole and 3-pyridinaldehyde. For its solubilization in aqueous solutions, Cremophor EL non-ionic surfactant (4% solution), approved for medical use, was used.

The invention is illustrated by the following figures:

Figure 1 - Absorption spectrum of a solution of 0.41 mg / ml H ₂ Py ₄ BC in 4% Cremophor EL.

Figure 2 - Fluorescence spectra of a solution of H ₂ Py ₄ BC in a 0.9% solution of NaCl (A) and containing 10% ETS medium Needle MEM (B) when incubated in dark conditions (1-ex tempore, 2 - after a month).

Figure 3 - Fluorescence spectra of H ₂ Py ₄ BC in 0.9% NaCl solution (A) and containing 10% ETS medium Needle MEM (B) upon irradiation (1-ex tempore, 2-5 J / cm ² , 3- 10 J / cm ² ).

Figure 4 - Photo-induced activity of H ₂ Py ₄ BC against HEp2 cells (solid line) and EJ (dashed line).

Figure 5 - Photo-induced antitumor activity of H ₂ Py ₄ BC in mice with S37 tumor depending on the dose and the interval between administration and irradiation (1 - 2.5 mg / kg, 30 min; 2 - 2.5 mg / kg, 2 h; 3 - 1.0 mg / kg, 30 min; 4 - 1.0 mg / kg, 2 h; 5 - 0.5 mg / kg, 30 min).

The invention is illustrated by the following examples, but is not limited to.

Example 1. Meso-Tetra (3-pyridyl) bacteriochlorin was synthesized according to the previously described method (RF Patent No. 2479585, C07D 487/22, 2013) by reduction of the meso-tetra (3-pyridyl) porphyrin with diimide generated under the reaction conditions from n-toluenesulfonyl hydrazide , in the presence of dry potash in dry pyridine. λ _max , nm (logε), chloroform: 747 (5.05), 683 (3.78), 521 (4.71), 491 (3.78), 380 (5.08), 367 (4.95) ), 357 (5.00).

The starting meso-tetra (3-pyridyl) porphyrin was obtained by condensation of equimolar amounts of pyrrole with 3-pyridinaldehyde in boiling propionic acid in the presence of air.

Example 2. Preparation of a solution of H ₂ Py ₄ BC in 4% Cremophor EL

Samples of H ₂ Py ₄ BC (4.1 mg) and non-ionic surfactant Cremophor EL (0.4 g) were dissolved in 20 ml of chloroform. The solution was stirred in a 1 L round-bottom flask with a magnetic stirrer, heating to 40-55 ° C to intensify the dissolution process. Then the solvent was evaporated on a rotary evaporator in vacuum at a temperature of a water bath of 30-40 ° C. The resulting film was dried in vacuo and then hydrated by adding 10 ml of phosphate buffered saline (PBS) with pH = 7.34. Stirring was carried out until the film was completely dissolved; the resulting solution was filtered through a membrane filter (Millipore, Type GS) with a pore size of 0.22 μm. All stages of the preparation of the solution were carried out with minimal exposure to external lighting.

The solutions were stored at a temperature of 6-10 ° C in a dark place. The absorption spectrum of a solution of 0.41 mg / ml H ₂ Py ₄ BC in 4% Cremophor EL is shown in Figure 1 (recorded relative to a 4% solution of Cremophor EL in a cuvette l = 0.02 cm).

Example 3. Evaluation of the stability of H ₂ Py ₄ BC.

The stability of the FS was evaluated using a fluorescent analysis method. Research solutions were prepared ex tempore, reaching a selected concentration (5 μg / ml) by successive dilutions of the stock solution. The concentration of the stock solution was 0.41 mg / ml. As a solvent, Eagle’s medium containing 10% ETS and a 0.9% NaCl solution were used. The fluorescence of solutions was recorded in dynamics by the contact method on a laser spectral analyzer for fluorescence diagnosis of tumors "LESA-6" (BioSpek LLP, Russia). Fluorescence was excited by a He-Ne laser at a wavelength of 632.8 nm, the spectral range from 600 to 950 nm.

Figure 2 presents the fluorescence spectra of a solution of H ₂ Py ₄ BC in 0.9% NaCl solution (A) and containing 10% ETS medium Needle MEM (B) during incubation in dark conditions. The absence of changes in the spectrum profile and fluorescence intensity indicates its stability in the selected time range.

Example 4. Assessment of the photostability of H ₂ Py ₄ BC in a cell-free medium.

Photo fading was evaluated in a 0.9% sodium chloride solution and in Needle MEM medium containing 10% ETS under irradiation with polychromatic light. A 500 W halogen lamp with a KS-13 broadband filter (λ≥680 nm) and a 5 cm thick water filter were used as a light source. The light dose was 5 and 10 J / cm ² with a power density of 32.0 ± 1.0 mW / cm ² . Fluorescence measurements were carried out by the contact method on a LESA-06 laser spectral analyzer in the spectral range 600–950 nm. Fluorescence spectra were recorded immediately after preparation of the solution and after exposure to light.

When PS was irradiated (Fig. 3) in a 0.9% NaCl solution (A) and in the Needle MEM (B) medium, only a slight decrease (not more than 18-20%) of the fluorescence intensity (λ _max = 745 ± 2 nm) was observed without changes in the profile of the spectrum, which indicated its photostability.

Example 5. Photo-induced activity and dark cytotoxicity of H ₂ Py ₄ BC in relation to human cell culture HEp2 and EJ.

Studies were performed on human tumor cells. Cell cultures of epidermoid carcinoma of the larynxopharynx (HEp2) and bladder carcinoma (EJ) were obtained from the Institute of Virology. DI. Ivanovo RAMS, which were cultivated at 37 ° C in a humidified atmosphere containing 5% CO ₂ (standard conditions).

Cells were scattered into the wells of a flat-bottomed 96-well microplate. Test compounds were added to the wells 24 hours after plating, varying the concentration from 13 to 3200 nM. To assess phototoxicity after 0.5, 2, 4, and 6 hours of incubation with a photosensitizer, the cells were irradiated with a halogen lamp through a KS-13 broadband filter (λ≥680 nm). The power density was 32.0 ± 1.0 mW / cm ² , the calculated light dose was 10 J / cm ² . Studies were also carried out both with the removal of PS from the medium before irradiation, and without removal.

After irradiation, the cells were incubated for 24 hours under standard conditions. To assess the cytotoxic activity of the plate was placed in darkened conditions for 24 hours. Survival score was determined visually and colorimetrically using the MTT test. Inhibition of cell growth in culture by more than 50% was considered a biologically significant effect. This value was calculated as the average according to the results of three independent tests.

It was found that PS showed maximum photoinduced activity against HEp2 and EJ culture cells during 4-hour incubation, IC ₅₀ was 32 ± 2 nM and 35 ± 3 nM, respectively, with an increase in incubation time up to 6 hours, the IC ₅₀ value did not change (Figure 4 ) Incubation of cells with dye at concentrations up to 3200 nM in the absence of light exposure for 24 hours did not affect the growth of cell cultures.

Removal of PS from the culture medium before exposure to light slightly decreased the efficiency of the photodynamic effect (by 15–20%), which indicates the realization of photo-induced activity mainly due to activation of intracellular PS.

Thus, in vitro results showed that H ₂ Py ₄ BC accumulates in the cells and has a high photoinduced activity.

Example 6. The distribution of H ₂ Py ₄ BC in the tumor S37 and fluorescence contrast relative to the surrounding tissue.

The distribution of FS in the tumor and surrounding tissues was evaluated in mice with S37 sarcoma in the range from 5 minutes to 48 hours using local fluorescence spectroscopy (LFS). A photosensitizer was administered intravenously at a dose of 2.5 mg / kg. Fluorescence was recorded by the contact method on a laser spectral analyzer for fluorescence diagnosis of tumors and monitoring PDT "LESA-06".

In the tumor tissue, the normalized fluorescence of PS reached its maximum value (2.1 ± 0.7 conv. Units) after 5 minutes and remained at a high level for 1 hour after administration, and then decreased by 95% of the maximum value by 48 hours. The highest levels of normalized fluorescence in the skin (1.8 ± 0.6 srvc. Units) were observed 4 hours after the introduction of FS, in the muscle (3.3 ± 0.3 srvc. Units) - after 5 minutes - 2 hours . The maximum fluorescence contrast of PS relative to the surrounding normal skin tissues was recorded 5 minutes after administration and amounted to 2.3 ± 0.4 srvc. units, and relative to the muscle - 0.9 ± 0.1 srvc. units 5 minutes and 24 hours after administration.

Example 7. Photo-induced antitumor activity of H ₂ Py ₄ BC in animals with S37 sarcoma grafted subcutaneously from the outside of the right thigh to BDF ₁ mice.

A study of the effectiveness of photodynamic therapy was performed on the 7th day after tumor inoculation. PS was administered to animals once intravenously at doses of 0.5, 1.0 and 2.5 mg / kg. Irradiation was carried out after 0.5 and 2 hours after the introduction of the dye. For irradiation, we used an LED source (FSUE “SSC“ NIOPIK ”) with a wavelength of 754 ± 14 nm and a power density of 150 mW / cm ² (light dose of 150 J / cm ² ). The control group of animals without exposure.

PDT efficacy was evaluated using criteria generally accepted in experimental oncology:

- inhibition of tumor growth TRO = [(V _to -V _op ) / V _to ] · 100%, where V _op and V _to are the tumor volume in the experimental and control groups, respectively;

- an increase in life expectancy of life expectancy = [(SPG _op- SPG _k ) / SPG _k ] · 100%, where SPG _op and SPG _k are the average life expectancy in the experimental and control groups, respectively;

- the criterion of healing, CI = [Ni / No] · 100%, where Ni and No are the number of cured animals and the total number of animals in the experimental group, respectively.

The tumor volume was calculated by the formula: V = d ₁ · d ₂ · d ₃ , where d ₁ , d ₂ and d ₃ are three mutually perpendicular diameters of the tumor.

Tumor volume was measured within 21 days after irradiation with the electronic digital caliper STORMtm 3C301 Central. The animals were observed for 120 days.

In the experimental groups, during the day after irradiation, the animals formed intense edema in the exposure zone, which lasted up to 5-15 days. A biologically significant antitumor effect was obtained when using FS in doses of 1.0 and 2.5 mg / kg when irradiated 30 minutes and 2 hours after administration and reached 70-100% SRW (Figure 5), 25-100% of the cure of animals ; the life expectancy of animals increased by 80-131%. The highest antitumor effect (100% TPO and 100% CI) with minimal damaging effect was obtained using PS at a dose of 2.5 mg / kg and irradiation 2 hours after administration.

Example 8. Pharmacokinetics of H ₂ Py ₄ BC in intact mice.

The pharmacokinetics of H ₂ Py ₄ BC was studied by LPS in the organs and tissues of intact mice at a dose of 2.5 mg / kg according to normalized fluorescence (FN).

The maximum of the PS fluorescence spectrum in animal tissues was recorded at 744 ± 2 nm. The fluorescent form of the dye quickly (within 5-30 minutes) entered the internal organs and tissues of the body, mainly the liver, then the FN decreased at different rates. The maximum fluorescence in the blood was determined immediately after intravenous administration and within 4 hours decreased by 95% of the maximum value and was not registered in a day.

In internal organs, after 24 hours, the normalized fluorescence level decreased in the liver by 87%, the kidneys - by 90%, the spleen - by 92% of the maximum value. A fluorescent form of PS at a dose of 2.5 mg / kg was determined in the kidneys and spleen for up to 2 days, and in the liver, the residual amount was determined up to 7 days.

In the skin, the maximum fluorescence value was recorded 4 hours after the administration of PS, then its normalized fluorescence decreased and after 24 hours amounted to 50% of the maximum value and was not determined after 5 days. This indicates the rapid elimination of FS from the skin. In muscle after 24 hours, the level of normalized fluorescence also decreased by 82%, and in adipose tissue - by 20%. The fluorescent form of PS was determined in the muscle up to 5 days, and in the adipose tissue a residual amount (26%) was recorded for more than 7 days.

The data obtained indicate the rapid circulation of H ₂ Py ₄ BC in the mammalian organism and its excretion mainly through the liver with bile.

Thus, tetra- (3-pyridyl) bacteriochlorin is a highly active new-generation PS that absorbs in the near IR region of the spectrum (λ _max = 745 ± 2 nm); it is stable during storage and has high photostability, exhibits high photoinduced antitumor activity in in vitro systems (at 4-hour incubation of HEp2 and EJ IC ₅₀ cultures it was 32 ± 2 nM and 35 ± 3 nM, respectively) and in vivo (100% TPO and 100% CI when using FS in a dose of 2.5 mg / kg and irradiation after 2 hours). FS has a rapid excretion of animals from the body (after 24 hours, the content in the internal organs decreases by 87-92% of the maximum value, the residual amount is observed up to 7 days).

Claims (1)

The use of meso-tetra (3-pyridyl) bacteriochlorin structural formula

as a near-infrared photosensitizer for photodynamic therapy.

Images