RU2282646C1 - Photosensitizing agents for photodynamic therapy - Google Patents

Abstract

FIELD: medicine, in particular photosensitizing agents for photodynamic therapy.

SUBSTANCE: invention relates to quaternary phthalocyanines of general formula MPc(CH₂X)_nCl_n, wherein Pc is phthalocyanine rest C₃₂H₁₆N₈; M is Zn, AlY; n = 6-8; X is

Y is Cl, OH, OSO₃H, useful as photosensitizing agents in photodynamic therapy having high photoinduced activity in vivo and in vitro.

EFFECT: new class of effective photosensitizing agents useful in treatment of various tumors by photodynamic therapy.

1 dwg, 8 ex

Description

The present invention relates to medicine, namely to photosensitizers 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 photosensitizers (PS), which have a tropism for tumor tissue. When irradiated with light of a certain wavelength, the PS transforms into an activated state, which initiates the formation of cytotoxic agents, in particular free radicals and singlet oxygen, which cause destruction of the tumor tissue.

Currently, in clinical practice, FSs belonging to the class of hematoporphyrins, such as photofrin-2 and photogem, are used to treat tumors of various superficial localizations (Van Lier J. Phthalocyanines as sensitizers for PDT of cancer // Photodynamic Therapy of Neoplastic Disease, CRS Press, Boca Raton, FL, 1990, vol. 1, pp. 279-291). The disadvantages of FS of this class are: absorption at a wavelength of 620-640 nm, which allows you to hit a tumor with a depth of up to 0.5 cm; low absorption intensity at maximum (extinction ε = 3000), low selectivity of accumulation in tumor tissue; slow elimination from the body and, as a result, increased skin photosensitivity.

Chlorin-based PSs, such as radochlorin and photoditazine, have advantages in some respects. A drug based on chlorin e ₆ photoditazine with a maximum absorption at 662 nm has a higher selectivity of accumulation in the tumor and penetration into the tissue (Romanko Yu.S., Tsyb AF, Kaplan MA et al. Effect of photodynamic therapy with photoditazine on the morphological and functional characteristics of M1 sarcoma // Bulletin of Experimental Biology and Medicine. 2004. T.138. No. 12. P.658-665).

Promising PSs are phthalocyanine derivatives, for example photosens. Photosens preparation based on sulfonated derivatives of aluminum phthalocyanine has a maximum absorption in the longer wavelength region of the spectrum (λ _max = 675 nm), a high molar extinction coefficient ε (over 100,000), and a high fluorescence quantum yield (RF Patent N 2220722. A 61 K 31/409 , 2004). This allows achieving photoinduced necrosis to a depth of 1.0 cm, as well as conducting multiple PDT courses using one injection of photosens (Vakulovskaya E.G., Shental V.V. Photodynamic therapy and fluorescence diagnostics in patients with head and neck cancer // Materials 6 Annual Russian Oncology Conference, Moscow, 2002. P.44-45).

However, in the course of a clinical study, photosensitivity deficiencies were revealed: the selectivity of accumulation in tumor cells was not high enough, long-term preservation in the tissues, and, as a result, an increase in skin phototoxicity.

The objective of the invention is to increase the effectiveness of the treatment of malignant neoplasms using PDT based on FS, characterized by high tropism to the tumor tissue, absorption in the longer wavelength region of the spectrum (675-700 nm), reduction of side effects due to the reduction of circulation time in the body and having high ph -induced activity.

To solve this problem, the use of positively charged phthalocyanines, cationic quaternized chloromethyl substituted phthalocyanines with various side substituents and central metal atoms of the following general formula, is proposed as a PS for PDT:

where Pc = phthalocyanine residue C ₃₂ H ₁₆ N ₈ ,

M = Zn, AlY,

n = 6 ÷ 8,

Y = Cl, OH, OSO ₃ H, for example:

ZnPcChol ₈ - octakis (2-hydroxyethyl dimethylammoniomethyl) phthalocyanine zinc octachloride;

AlPcChol ₈ - octakis (2-hydroxyethyl dimethylammoniomethyl) phthalocyanine chloroaluminum octachloride;

ZnPcPym ₈ - octakis (pyridiniomethyl) zinc phthalocyanine octachloride;

AlPcPym ₈ - octakis (pyridiniomethyl) phthalocyanine chloraluminium octachloride;

ZnPcTmed ₈ - octakis (N- (2- (dimethylamino) ethyl) -N, N-dimethylammoniomethyl) phthalocyanine zinc octachloride;

ZnPcThea ₈ - octakis (N, N, N-tri (2-hydroxyethyl) ammoniomethyl) phthalocyanine zinc octachloride;

ZnPcPmed ₈ - octakis (N, N-dimethyl-N- (2-trimethylammonioethyl) ammoniomethyl) phthalocyanine zinc octaiodide octachloride.

The present invention is illustrated by the following examples.

Example 1

To 2.5 g (2.58 mmol) octakis (chloromethyl) zinc phthalocyanine (found,%: Cl 29.05; calculated,%: Cl 29.36) obtained as described in patent No. 84,4338, 1953, 5 ml of dimethylformamide and 5 ml of N, N- dimethylaminoethanol. The mixture was stirred for 2 hours at 100 ° C, the precipitate was filtered off, washed with acetone, dried and 3.4 g (78.3%) octakis (N- (2-hydroxyethyl) -N, N-dimethylammoniomethyl) phthalocyanine zinc octachloride (ZnPcChol ₈ ) were obtained. Electronic absorption spectrum, λ _max , nm (H ₂ O): 679-680. Found,%: C 51.41; H 6.84; N 13.07; Cl 15.88. Calculated for C ₇₂ H ₁₁₂ AlCl ₈ N ₁₆ O ₈ Zn,%: C 51.50; H 6.72; N 13.35; Cl 16.89.

Example 2

Analogously to example 1, from hexachloromethyl substituted zinc phthalocyanine (found,%: Cl 24.98; calculated for ZnPc (CH ₂ Cl) ₆ ,%: Cl 24.48), hexakis (N- (2-hydroxyethyl) -N, N-dimethylammoniomethyl) zinc phthalocyanine hexachloride is obtained (ZnPcChol ₆ ). Electronic absorption spectrum, λ _max , nm (H ₂ O): 677-679.

Example 3

A mixture of 0.150 mg octakis (chloromethyl) zinc phthalocyanine and 2 ml of pyridine is heated under reflux for 1 h, then the mixture is filtered, washed with acetone, dried, and 0.228 g (87.6%) octakis (pyridiniomethyl) phthalocyanine zinc octachloride (ZnPcPym ₈ ) is obtained. Electronic absorption spectrum, λ _max , nm (H ₂ O): 679-680.

Example 4

Analogously to example 3, octakis (pyridiniomethyl) phthalocyanine chloroaluminum octachloride (AlPcPym ₈ ) is obtained from octakis (chloromethyl) phthalocyanine of chloraluminium. Electronic absorption spectrum, λ _max , nm (H ₂ O): 680. Found,%: C 59.09; H 4.36; N, 13.49; Cl 18.41. Calculated for C ₈₀ H ₆₄ AlCl ₉ N ₁₆ ,%: C 60.22; H 4.04; N, 14.04; Cl 19.99.

Example 5

To 0.50 g (0.51 mmol) of octakis (chloromethyl) zinc phthalocyanine, 2 ml of dimethylformamide and 3 ml of N, N, N ', N'-tetramethylethylenediamine are added. After the initial phthalocyanine was dissolved, the mixture was stirred for 1 h at 100 ° C, the precipitate was separated, washed with dimethylformamide, acetone, dried and 0.71 g (73%) octakis (N- (2- (dimethylamino) ethyl) -N, N-dimethylammoniomethyl) was obtained zinc phthalocyanine octachloride (ZnPcTmed ₈ ). Electronic absorption spectrum, λ _max , nm (H ₂ O): 679. Found,%: C 55.45; H, 7.68; N 17.09; Cl 15.05. Calculated for C ₈₈ H ₁₅₂ Cl ₈ N ₂₄ Zn,%: C 55.70; H 8.08; N, 17.70; Cl 14.92.

Example 6

A mixture of 0.1 g of the complex obtained in Example 5, 5 ml of methanol and 3 ml of methyl iodide was stirred at 40 ° C for 3 hours. The precipitate was separated, washed with methanol, dried and 0.12 g (75%) octakis (N, N-dimethyl- N- (2- (trimethylammonio) ethyl) ammoniomethyl) phthalocyanine zinc octaiodide octachloride (ZnPcPmed ₈ ). Electronic absorption spectrum, λ _max , nm (H ₂ O): 680. Found,%: C 38.20; H 5.65; N, 10.75; Cl 9.05. Calculated for C ₉₆ H ₁₇₆ Cl ₈ I ₈ N ₂₄ Zn,%: C 38.04; H 5.85; N 11.08; Cl 9.35.

Example 7

Analogously to example 3, from hexachloromethyl-substituted aluminum phthalocyanine with a degree of substitution of approximately six (found,%: Cl 23.27; calculated for HO ₃ SOAlPc (CH ₂ Cl) ₆ ,%: Cl 22.93), hexakis (pyridiniomethyl) -phthalocyanine hydrosulfatoaluminium (hexahloroaluminium (hexahloroaluminium) AlPcPym ₆ ). Electronic absorption spectrum, λ _max , nm (H ₂ O): 680-682.

Example 8

Analogously to example 1, from hexachloromethyl-substituted aluminum phthalocyanine (found,%: Cl 23.27; calculated for HO ₃ SOAlPc (CH ₂ Cl) ₆ ,%: Cl 22.93), hexakis (N- (2-hydroxyethyl) -N, N-dimethylammoniomethyl) phthalocyanine is obtained hydro-sulfatoaluminum hexachloride (AlPcChol ₆ ). Electronic absorption spectrum, λ _max , nm (H ₂ O): 680-682.

The study of photoinduced activity in vitro was carried out on 2 cell lines: epidermoid carcinoma of the human larynxopharynx (Hep2) and human lung adenocarcinoma (A 549). The cells were cultured using Igla-MEM medium (Hep2, A 549) with the addition of 2 mM L-glutamine and 8-10% fetal calf serum. Cultivation was carried out at 37 ° C in a humid atmosphere with 5% CO ₂ content. The cells were scattered into the wells of a flat-bottomed 96-well microplate (Costar, USA) with 100 μl of a suspension of cells at a concentration of 0.5 × 10 ⁵ cells / ml. After 24 hours, 50 μl of the test compounds were introduced into the well in serial dilutions and incubated for 2 hours under standard conditions and then irradiated with a 500 W halogen lamp through a 5 cm thick water filter and a KS-13 broadband filter (λ≥640 nm). The power density was 14 ± 2 mW / cm ² , the irradiation time was 13-15 minutes, and the calculated light dose was 10 J / cm ² .

After irradiation, cells were incubated under standard conditions for 24 hours. To assess the cytotoxic activity, the cells were placed in darkened conditions for 24 hours. Cell survival was determined visually, assessing morphological changes in cells using light microscopy, and colorimetric using the MTT test.

The level of inhibition of cell growth was calculated by the formula (1):

where IR is the level of inhibition of cell growth in culture,

OD _{o the} optical density in the experiment,

_To OD - optical density in control.

Inhibition of cell growth in culture by more than 50% was considered a biologically significant effect. The values of IR ₅₀ and IR ₉₀ , corresponding to the concentration of the test compound, at which 50 and 90% inhibition of culture growth is observed, was calculated as the average value according to the results of three independent tests.

The substance ZnPcChol ₈ has photoactivity in relation to the two studied cell cultures, and the values of IR ₅₀ and IR ₉₀ are close in their values. When irradiated in the presence of the test compound in the incubation medium (incubation time 2 hours), the concentration of IC ₅₀ is 0.24 ± 0.08 μM (culture HEP 2) and 0.29 ± 0.09 μM (culture A 549), and IR ₉₀ is 1.31 ± 0.08 μM and 1.30 ± 0.08 μM, respectively.

ZnPcTmed ₈ , ZnPcThea _8, and ZnPcPmed ₈ zinc phthalocyanine derivatives have high photoinduced activity with respect to Hep2 cell culture: the IR ₅₀ values of these compounds were 0.3 ± 0.1 μM, 0.69 ± 0.4 μM, and 0.18 ± 0.2 μM, respectively. The dark toxicity of PS on cell culture in the studied concentration range was not detected.

The specific in vivo activity of positively charged PS was studied in animals with transplantable tumors. We studied the fluorescence properties of the ability of PS to accumulate in the tumor, evaluating the level of normalized fluorescence (PF) in mouse tissues, as well as the antitumor photoinduced activity of the dyes. All PSs accumulated in the tumor tissue and destroyed it upon irradiation.

Normalized fluorescence was determined in the tissues of mice grafted with subcutaneous thigh lymphocytic leukemia R-388. The content of ZnPcChol ₈ was evaluated by a fluorescence method on a LESA laser diagnostic setup (Biospek LLP, Moscow) based on a He-Ne laser with a wavelength of 633 nm using an ex vivo contact method for various observation periods from 5 seconds to 72 hours. The integrated fluorescence intensity in the range of 641-724 nm was normalized to the integrated intensity of the back diffuse scattering signal of the exciting laser radiation, thus normalized fluorescence (PF) in the tissue was determined.

ZnPcChol ₈ was administered intravenously at a dose of 1.0 mg / kg. In the course of the study, the dye PF was evaluated and the ratio of PF in the tumor to PF in the skin and muscle was calculated, thus determining the selectivity index (Ks) of PS accumulation in the tumor compared to surrounding tissues.

Studies have shown that ZnPcChol ₈ has a maximum fluorescence in mouse tissues at 696 nm. In the muscle, the drug accumulates over 5 minutes, and in the P-388 tumor tissue and skin, the maximum value of Fn is reached after 30 minutes. In the tissues of the body, the drug is delayed for 1 hour, and then quickly excreted (see drawing).

Drawing. Normalized fluorescence (Fn) of ZnPcChol ₈ in the tissues of mice with tumor P-388.

ZnPcChol ₈ has a tropism for tumor tissue P-388. The maximum value of the selectivity indicators, which is recorded 30 minutes after drug administration, is 3.3 ± 0.6 (fn tumor / fn skin) and 3.9 ± 0.7 (fn tumor / fn muscle).

The study of the specific antitumor activity of substances in vivo was carried out on mouse hybrids BDF ₁ females with lymphocytic leukemia P-388 on the 6th-7th day of growth using various doses of dye and irradiation modes. The P-388 tumor was inoculated subcutaneously on the hip at 0.7 × 10 ⁶ cells / mouse. PS was administered intravenously at a dose of 1 mg / kg, the interval between the introduction of PS and radiation was 5, 15, 30 and 60 minutes. An ATO-1 apparatus based on a 150 W halogen lamp with a fiber optic bundle and KS-10 and SZS-26 filters (wavelength - 600-800 nm) was used as a radiation source. The power density (100-300 mW / cm ² ) was controlled using a power meter IMPO (NPO Plus), the light dose of 90-270 J / cm ² . Before irradiation, the hair over the tumor was removed. A 5% ketamini solution at a dose of 2.5 mg / mouse was administered intraperitoneally as anesthesia. The duration of observation of the animals continued until their death. Tumor-bearing mice that were not exposed to any effect were used as control.

The efficacy of PDT with ZnPcChol ₈ was evaluated by inhibition of tumor growth (TPO) and increase in life expectancy (VL), which were calculated according to formulas (2) and (3):

where V _op is the volume of the tumor, equal to d ₁ × d ₂ × d ₃ , where d ₁ , d ₂ and d ₃ are three mutually perpendicular diameters of the tumor, LNG is the average life expectancy in the experimental and control group.

PDT with ZnPcChol ₈ led to the development of edema, followed by the formation of a necrotic scab in the irradiation zone, which disappeared 8-10 days after irradiation. The effectiveness of PDT is influenced by the light dose. Thus, an increase in the dose of light from 90 to 270 J / cm ² leads to an increase in the values of SRW with ZnPcChol ₈ from 47.1 ÷ 75.2% to 93.6 ÷ 97.1%.

An increase in the dose of ZnPcChol ₈ from 0.5 to 1.0 mg / kg also leads to an increase in SRW from 55.9–68.6% to 93.6–97.1% for all observation periods (15 days). With a further increase in the dose to 2 mg / kg, SRW remains at a high level (85.3-91.2%). The life expectancy of animals increases by 38.8% at a dose of 1 mg / kg and 23.1% at a dose of 2 mg / kg.

When irradiated at various time intervals Δt 5, 15, 30 and 60 minutes after dye administration at a dose of 1 mg / kg and a light dose of 270 J / cm ² , the therapeutic effect was higher at Δt 15 and 30 minutes: TPO is 87.8- 100% and 95.2-100%, and UPZ - 31.4% and 38.8%, respectively. An increase in the Δt interval to 60 minutes leads to a certain decrease in SRW - 73.7-91.2%.

The study of photoinduced activity in vivo in mice with Colo 26 tumor was performed on Balb / c female mice on the 6th-7th day of tumor growth. Colo 26 mouse colon adenocarcinoma was inoculated subcutaneously on the thigh at 0.05 × 10 ⁶ cells / mouse. The substance ZnPcChol ₈ was administered intravenously at a dose of 1 mg / kg. The interval between intravenous administration of FS and irradiation was 30 minutes. As a radiation source, we used an LED red light source with a maximum absorption at 685 nm with a regulator of light power density (25-100 mW / cm ² ); light dose of 90 J / cm ² . Before irradiation, the hair over the tumor was removed. For anesthesia, a 5% ketamini solution was used at a dose of 2.5 mg / mouse intraperitoneally. As a control, tumor-bearing mice that were not exposed to any effect were used. The effectiveness of PDT with ZnPcChol ₈ was evaluated by the criterion of cure (Ki), which was calculated by the formula (4).

where N _and is the number of cured animals,

N _o - the total number of animals in the experimental group.

PDT with ZnPcChol ₈ (dose 1.0 mg / kg) leads to complete resorption of the Colo 26 tumor (K _and = 100%) regardless of the irradiation conditions - 100, 50 and 25 mW / cm ² , 90 J / cm ² , Δt 30 minutes.

The compounds of the present invention, positively charged phthalocyanines with various side substituents and central metal atoms, are highly tumorous, have high photoinduced activity in vitro and in vivo, and represent a new class of effective photosensitizers that can be used to treat tumors of various localization by PDT .

Claims (1)

Quaternized phthalocyanines of the general formula MRc (CH ₂ X) _n Cl _n , where Pc is the rest of the phthalocyanine C ₃₂ H ₁₆ N ₈ , M - Zn, AlY, n = 6 ÷ 8,

Y is Cl, OH, OSO ₃ H, as photosensitizers for photodynamic therapy.