RU2774589C1 - Method for carrying out photodynamic therapy of solid ehrlich carcinoma in mice - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention relates to experimental medicine and can be used for photodynamic therapy of grafted Ehrlich solid carcinoma in mice. To do this, the photosensitizer Heliochlorin is administered intravenously at a dose of 5 mg/kg. Then the tumor is irradiated with laser radiation E=150 J/cm² with a wavelength of 662 nm, power density Ps=0.48 W/cm², drug-light interval is 45 minutes.

EFFECT: invention provides the maximum antitumor effect in this category of laboratory animals on the 90th day after the photodynamic therapy.

1 cl, 3 tbl

Description

The invention relates to experimental medicine, in particular to photodynamic therapy of grafted Ehrlich solid carcinoma in mice.

During PDT, a photosensitizer (PS) is introduced, which selectively accumulates in malignant neoplasms. Then the tumor is subjected to remote irradiation with laser light of a certain wavelength, in accordance with the excitation spectrum of the PS. As a result of irradiation, a photochemical reaction occurs, where the PS actually plays the role of a catalyst, with the participation of which the formation of reactive oxygen species (the main of which is singlet oxygen) and various active radicals, which are cytotoxic agents and cause direct destruction of tumor cells. Also, the PDT mechanisms include: destruction of the endothelium of blood vessels in the area of laser irradiation, which results in vascular thrombosis and hemodynamic disturbance of the tumor node, inflammatory reaction and immune response.

The effectiveness of photodynamic exposure depends on three components: PS, light and oxygen. The selectivity of tumor destruction is associated with the selectivity of PS accumulation in the tumor with respect to healthy tissue and with exposure to light of a certain wavelength. Tissues not affected by a tumor absorb PS to a lesser extent, but as a result of laser irradiation, undesirable partial destruction of healthy tissues may occur if the photosensitizer does not have sufficient selectivity.

A known method of PDT of malignant tumors (application for invention N 96115789/14, IPC A61N 5/06, publ. Bull. 1998 N 30). The photosensitizer is administered immediately before sessions of therapeutic laser light irradiation (at least before two), while the dose of the photosensitizer at the first injection is from 0.1 mg/kg to 0.5 mg/kg of body weight, with subsequent injections from 0.05 mg /kg to 0.25 mg/kg of body weight. As a result of fractional administration, the total dose of PS before laser irradiation remains equal to the generally accepted therapeutic dose. Under these conditions, intense laser exposure, carried out in a short period of time after PS administration, leads to a full therapeutic effect.

The disadvantage of this method are the undesirable consequences of therapy: deep necrosis of the tumor and parts of the surrounding normal tissues.

There is a known method of PDT of malignant tumors (RU 2169015 C1), in which PDT is carried out by introducing the FS "Photosens" at a dose of 0.3-0.8 mg/kg of the patient's body weight, followed by exposure to the tumor growth zone with remote laser radiation with a power of 100-500 mW / cm ² . Under these conditions, intense laser exposure, carried out in a short period of time after PS administration, leads to a complete therapeutic effect when exposed to superficial tumors and is accompanied by deep necrosis of the tumor and part of the surrounding normal tissues.

However, the second-generation photosensitizer Photosens causes long-term (up to 2 months) photosensitivity due to its slow removal from the skin. Increased skin photosensitivity requires compliance with a strict light regimen from patients, which creates a number of inconveniences.

The closest (prototype) is the method of PDT of malignant neoplasms (EN 2704202 C1). The photosensitizer "Photoran E6" is administered in doses of 5.0 mg/kg, which, when extrapolated to a human dose, is 0.85 mg/kg of the patient's body weight. 2.5 hours after the introduction of the photosensitizer, laser irradiation is carried out with a wavelength of 660-670 nm with a power density of 0.25 W/cm ² and an energy density of 300 J/cm ^{2 of} laser radiation, the irradiation time is 20 minutes.

However, the known method is aimed at the therapy of a connective tissue solid tumor - M-1 sarcoma and is not designed for use in PDT of other types of tumors, in particular, tumors such as Ehrlich solid carcinoma of mice.

The technical solution is to develop a regimen for the treatment of transplanted tumor Ehrlich carcinoma of mice by photodynamic therapy, leading to the maximum antitumor effect with 100% cure of animals on day 90 after the therapy session.

The technical solution is achieved by the fact that, as in the known method, a photosensitizer is introduced at a dose of 5 mg/kg, followed by irradiation of the tumor with laser radiation E = 150 J/cm ² with a wavelength of 662 nm.

A feature of the proposed method is that the photosensitizer Heliochlorin is administered intravenously, power density Ps=0.48 W/cm ² , drug-light interval is 45 minutes.

The invention is illustrated by a detailed description, tables and examples.

The method is carried out as follows.

White outbred laboratory mice were used in the in vivo experiment; Ehrlich's carcinoma was used as an experimental tumor model. This tumor has a tumor cell transplantation rate of 100%. According to the histological structure, it is an undifferentiated tumor that has lost its epithelial character. The work was carried out in compliance with international recommendations for research using laboratory animals set forth in the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" (Strasbourg, 1987), on mature female mice weighing 20-25 g with an implanted subcutaneously from the outside of the thigh with Ehrlich's carcinoma. Ascitic fluid, 0.05 ml, was injected subcutaneously into the thigh area to reproduce a solid tumor. The tumor strain was obtained from the bank of tumor materials of the Russian Cancer Research Center. N.N. Blokhin. On the 3rd day after inoculation, when the largest diameter of the tumor nodes was 0.4-0.6 cm, the animals were randomly divided into experimental and control groups.

Conducting PDT.

Experimental studies were performed on two groups of animals. control served as tumor-bearing mice without any exposure (Table 1)

Table 1.
Experiment scheme Tumor Test system A drug Dose of PS
mg/kg Method
Introductions Irradiation parameters Irradiation time E
J / cm ² PS
W / cm ² Ehrlich carcinoma mice Heliochlorin 2.5 Intravenously 150 0.48 5 minutes 30 seconds Ehrlich carcinoma mice Heliochlorin 5 Intravenously 150 0.48 5 minutes 30 seconds Control mice -

Laser irradiation was carried out during the period of maximum tumor/healthy tissue contrast index after PS administration (the optimal drug-light interval was 45 minutes). For the impact of laser radiation on mouse tumors, an Atkus-2 semiconductor device (CJSC Semiconductor Devices, St. Petersburg) was used. The radiation wavelength was 662 nm, the laser radiation energy density (E) was 150 J/cm2, ^the light spot diameter was ₁ cm. day (V _t ) after PDT.

Tumor volume was measured: before treatment (V ₀ ), and on days 3, 7, 10, 14, and 21 after therapy. The end date of the study was chosen based on the need to compare the dynamics of tumor growth in rats with continued growth after therapy with control animals, since the death of control animals already begins at this time of the study.

As criteria for determining antitumor efficacy, the following were used:

1. Coefficient of absolute tumor growth (K). To do this, the volumes of tumors were first calculated using the formula:

where: d _1, d ₂ , d _3, - three mutually perpendicular diameters of the tumor,

V is the volume of the tumor in cm ³ .

The coefficient of absolute tumor growth (K) is calculated by the formula:

where: V ₀ is the volume of the tumor before exposure,

V _t - tumor volume for a certain period of observation;

2. Inhibition of tumor growth (TPO%) according to the formula:

3. Percentage of animals in the group with complete regression (CR) of the tumor (K = -1.00). Complete tumor regression was defined as the absence of a visible and palpable tumor.

4. The average life span of animals (LES, days) of animals and the increase in life expectancy (LEL%) compared with the control. LLS ≥ 50% is considered significant.

5. The criterion of cure of animals is the absence of tumor recurrence within 90 days after PDT

Statistical processing of results for independent groups was performed using Statistica 6.0 software. Descriptive statistics are presented as arithmetic mean and standard error of the mean (M±m). To assess the level of significance of intergroup differences, the Mann-Whitney U-test was used at p<0.05.

Study of the effectiveness of PDT with Heliochlorin.

On the 3rd day after PDT, erosions covered with thin scabs appeared on the irradiated skin above the tumors. On days 7-10, dense scabs were formed with a demarcation line between the surrounding skin and the necrotic tissue of the neoplasms.

In the experimental groups in animals up to 21 days after the PDT session, a complete regression of the tumor was noted. In the control groups, progressive tumor growth was observed (Table 1)

In group I with a PS dose of 2.5 mg/kg, on the 58th day after PDT, the growth of Ehrlich's carcinoma was observed to resume, mainly along the periphery of the laser exposure zones. At a dose of FS 5.0 mg/kg on day 96, there were no recurrences of neoplasms.

Increasing the dose of Photoran to 5.0 mg/kg led to complete regression of Ehrlich's carcinoma in the absence of a visible and palpable tumor in 100% of the animals throughout the entire observation period (Table 2).

The results of a quantitative analysis of the studied indicators of the effectiveness of PDT with Heliochlorin on Ehrlich's carcinoma are given in Table 2.

table 2
Indicators of antitumor efficacy of PDT with intravenous administration of Heliochlorin in different doses at parameters of exposure to laser radiation E = 150 J/cm ² ; Ps ⁼ 0.48 W/cm2 for mouse Ehrlich carcinoma No.
gr. Dose
FS
(mg/kg) Observation period (days) 7 fourteen 21 90 one 2.5 (1) 100 (2) 66.6 2 5.0 (1) 100 (2) 100 Control V 0.09±0.02 0.94±0.16 4.05 ± 2.43 K 10.7 ± 3.2 49.2 ± 30.7 Note:
(1) percentage of animals with complete tumor regression (CR);
(2) percentage of cured animals;
* p < 0.05 compared to control.

The indicators of the increase in life expectancy and cure rate of animals on the 90th day after PDT are presented in Table 3.

Table 3
Increased lifespan of mice after PDT with intravenous administration of Heliochlorin Laser radiation parameters: E \u003d 150 J / cm ² , Ps \u003d 0.48 W / cm ² PS dose (mg/kg) life expectancy UPZH 2.5 >86 ±4.0 * >45 5 >90* >100* Control 59.3 ± 3.0 - Note: ALE - average life expectancy, days; UPZh - increase in life expectancy,%; * Significant increase in life expectancy compared to control (LLE ≥ 50%).

According to a comparative analysis, after PDT, carried out at the optimal time after PS administration and selection of therapy parameters, a significant increase in the lifespan of mice was obtained at a PS dose of 5.0 mg/kg, laser irradiation parameters E-150 J/cm ² , Ps-0 ,48 W/cm ² and achieved 100% cure for 90 days.

The proposed method allows you to determine the parameters of photodynamic therapy in the treatment of transplanted Ehrlich carcinoma of mice and increase the lifespan of animals.

Claims (1)

A method for carrying out photodynamic therapy of solid Ehrlich carcinoma in mice, including the administration of a photosensitizer at a dose of 5 mg/kg and subsequent irradiation of the tumor with laser radiation E = 150 J/cm ² with a wavelength of 662 nm, characterized in that the photosensitizer Heliochlorin with power density Ps = 0 is intravenously administered ,48 W/cm ² drug-light interval is 45 minutes.