RU2775382C1 - Method for photo-activated treatment of tumour cells for producing cellular agents - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine, namely, to experimental oncology, and can be used in photo-activated treatment of cells for producing cellular agents. The method involves treating cells with a photosensitiser, followed by photo activation thereof. Transplanted cells of the Ehrlich tumour are injected subcutaneously into the body of the mouse, and after the tumour reaches the size of 10x10 mm, photosensitiser radachlorine at a dose of 10 mg/kg or fotoditazin at a dose of 5 mg/kg is injected into the body of the mouse. After removing the tumour from the body of the animal, before or after preparing suspended tumour cells from the tumour, the tumour cells are photo-activated by irradiation with a laser with a wavelength of 662 nm at a dose of 300 J/cm².

EFFECT: possibility of creating cellular agents from tumour cells, accounting for the pharmacokinetics of the photosensitiser in the body of the animal, conducted study of the dependence of the level of accumulation of the photosensitiser on the injected dose and accumulation time.

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Description

The invention relates to medicine, namely to experimental oncology and can be used in photodynamic processing of cells for the preparation of cell preparations.

A known method of photodynamic treatment of cells for the preparation of cell preparations, consisting of a two-stage photodynamic treatment of cells ex vivo (WO 2016/131960, publ. 25.08.2016).

The disadvantage of this method is the introduction of the photosensitizer directly into the cell culture medium, which does not take into account the pharmacokinetics of the photosensitizer in the animal body.

The technical result of the invention is the possibility of creating cell preparations from tumor cells, taking into account the pharmacokinetics of the photosensitizer in the animal body, the study of the dependence of the level of accumulation of the photosensitizer on the administered dose and accumulation time.

The specified technical result is achieved in a method for photodynamic treatment of tumor cells for the preparation of cell preparations, including treatment of cells with a photosensitizer and their subsequent photoactivation, characterized in that transplanted Ehrlich tumor cells are injected subcutaneously into the mouse body, after reaching a tumor size of 10x10 mm, the photosensitizer radachlorin is administered at a dose of 10 mg/kg or photoditazine at a dose of 5 mg/kg into the mouse body, after removing the tumor from the animal body, before or after preparing a suspension of tumor cells from the tumor, photoactivation of tumor cells is carried out by irradiation with a laser with a wavelength of 662 nm at a dose of 300 J/cm ² .

The method is confirmed by the following experimental examples.

Example 1. The method of photodynamic treatment of Ehrlich carcinoma cells ex vivo with the introduction of a photosensitizer (FS, Radachlorin, RADA-PHARMA LLC, Russia) to BALB/c female mice with subcutaneously grafted Ehrlich tumor.

Female mice of the BALB/c line underwent subcutaneous transplantation of Ehrlich's tumor in the thigh area in the amount of 5×10 ⁵ tumor cells. When the tumor size reached 10×10 mm, the animals received a single dose of a photosensitizer (Radachlorin, 10 mg/kg). PS accumulation is assessed using the Fluovizor non-invasive fluorescence imaging system (LLC Atkus, Russia) with an excitation wavelength of 660 nm and fluorescence registration in the infrared wavelength range. 6 hours after the accumulation of the photosensitizer in the tumor tissue with a high intensity of the fluorescence of the photosensitizer in the tumor (Table), euthanasia is performed, the tumor is isolated. A suspension of tumor cells is prepared from the isolated tumor. Tumor pieces are crushed with scissors, rubbed through a sterilized fine-meshed metal sieve. Sterile 0.9% sodium chloride solution is added to the obtained crushed tumor tissue. The cell suspension is collected in a sterile syringe and further crushed by passing it with a syringe through needles of smaller diameters. The cellularity of the resulting suspension is monitored (counting in the Goryaev chamber). The obtained suspension of cells is subjected to photoactivation by laser irradiation Alod (LLC "Alkom medica", Russia) with a wavelength of 662 nm. Laser radiation is delivered by a light guide with a lens for external irradiation (OOO Polironic, Russia), which forms a uniform spot in the impact zone with a diameter of 10 to 20 mm, depending on the size of the tumor. The radiation power was from 0.5 to 1.2 W, the time was from 8 to 13 minutes. The energy density or energy dose in all experiments was 300 J/cm ³ . Received the cell preparation for subsequent use.

The death of all or part of the cells is confirmed by the following experimental examples.

Example 2. The method of photodynamic treatment of Ehrlich carcinoma cells ex vivo with the introduction of a photosensitizer (Photoditazine) to obtain a cell preparation in the form of a suspension after photoactivation by laser irradiation and subsequent implantation in mice with an assessment of survival.

In the first stage, nine mice were subcutaneously implanted with 10 ⁶ Ehrlich tumor cells. 7-10 days after inoculation, when the tumor size was 10 × 10 mm, mice were injected with Photoditazine (5 mg/kg, Veta Grand, Russia) intraperitoneally once 4 hours before cell isolation and their photoactivation (the time was determined as optimal in additional experiments on FS accumulation). 4 hours after PS administration, the tumor was isolated from the mouse body. Next, a preparation was prepared from the tumor tissue without macroscopic signs of necrosis in the form of a suspension of tumor cells (as in example 1), and the count was performed in the Goryaev chamber. Then, 4.5 million cells from each tumor were placed in 2 wells of a 6-well flat-bottomed culture plate with low adhesion. In one well, tumor cells were subjected to laser photoactivation, and the second served as a control. PS photoactivation was carried out using an Alod laser device (OOO Alkom Medica, Russia), the laser wavelength was 662 nm. A light guide with a lens for external irradiation (OOO Polironic, Russia) formed a uniform spot in the impact zone with a diameter of 11 to 25 mm, depending on the size of the irradiated object, the radiation power was from 0.6 to 1.5 W, the energy density, or the radiation dose in all experiments was 300 J/cm ² .

At the second stage, 1.5 million tumor cells from each well were transplanted subcutaneously into three healthy mice. Mice were monitored with registration of tumor size for 32 days. Survival of the tumor suspension in this modification of the method was 100% in all groups. Upon administration of the cell preparation to recipient mice without photoditazine photoactivation, the tumor volume by the 32nd day was 3.14±1.47 cm ³ , and upon its photoactivation it decreased to 1.67±0.26 cm ³ . In the control group with laser exposure, by day 32, the tumor volume reached 2.74±0.29 cm ³ . Thus, inhibition of tumor growth of 39% was obtained compared to the control with photoactivation, indicating the death of a large part of the cells in the resulting preparation and/or a cytostatic effect on tumor cells in the obtained preparation.

Example 3. The method of photodynamic treatment of Ehrlich carcinoma cells ex vivo with the introduction of a photosensitizer (Photoditazine) with the isolation of a piece of tumor tissue from the mouse body, photoactivation by laser irradiation and preparation of a cell preparation in the form of a suspension, followed by their implantation in mice with an assessment of survival.

In the first stage, nine mice were subcutaneously implanted with 10 ⁶ Ehrlich tumor cells. 7–10 days after inoculation, when the tumor size was 10 × 10 mm, the mice were injected with Photoditazine (5 mg/kg, LLC Veta Grand, Russia) intraperitoneally once 4 hours before the isolation of a tumor piece and its photoactivation (time is defined as optimal in additional experiments on PS accumulation). 4 hours after PS administration, the tumor was isolated from the mouse body, and a piece was obtained without macroscopic signs of necrosis.

After photoactivation, a preparation was prepared from a piece of tumor in the form of a suspension of tumor cells (as in example 1), cellularity was counted in a Goryaev chamber.

At the second stage, 1.5 million tumor cells from each well were transplanted subcutaneously into three healthy mice. Mice were monitored with registration of tumor size for 32 days. The survival rate of the tumor suspension of the second modification in the group with photoactivation was 33.3%, in the group without laser exposure - 83.3%, which indicates a cytotoxic effect on tumor cells in the resulting preparation.

The method provides the possibility of creating cell preparations from tumor cells, taking into account the pharmacokinetics of the photosensitizer in the animal body, studying the dependence of the photosensitizer accumulation level on the administered dose and accumulation time.

Claims (1)

A method for photodynamic treatment of tumor cells for the preparation of cell preparations, including treatment of cells with a photosensitizer and their subsequent photoactivation, characterized in that transplanted Ehrlich tumor cells are injected subcutaneously into the mouse body, after reaching a tumor size of 10x10 mm, the photosensitizer radachlorin is injected at a dose of 10 mg/kg or photoditazine at a dose of 5 mg/kg into the mouse body, after removing the tumor from the animal body, before or after preparing a suspension of tumor cells from the tumor, photoactivation of tumor cells is carried out by irradiation with a laser with a wavelength of 662 nm at a dose of 300 J/cm ² .