RU2776449C1 - Method for photodynamic therapy of rat surface solid connective tissue sarcoma m-1 - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention relates to experimental medicine, namely to oncology, and can be used for carrying out photodynamic therapy (PDT) of transplanted superficial solid connective tissue tumor M-1 sarcoma in rats. To do this, the photosensitizer Heliochlorin is administered intravenously at a dose of 2.5 mg/kg. Then, after 30 minutes, the tumor is exposed to laser radiation with an energy density of E = 150 J/cm², a wavelength of 662 nm and a power density of Ps = 0.48 W/cm².

EFFECT: method provides maximum antitumor effect, 100% cure of animals on the 90th day after PDT session with no neoplasm recurrence, while reducing the amount of photosensitizer administered and the dose of laser exposure to animals.

1 cl, 1 dwg, 3 tbl

Description

The invention relates to experimental medicine, specifically to oncology, in particular to photodynamic therapy (PDT) of a transplanted superficial solid connective tissue tumor M-1 sarcoma in rats.

For this, for the first time in experimental studies, PDT was carried out with a new domestic photosensitizer Heliochlorin. Heliochlorin contains trismeglumine salt of chlorin e6, and meglumine as a cryostabilizer in the ratio 1:0.1-0.2 wt. h., has the ability to accumulate in the tumor and, under local exposure to laser radiation with a wavelength in the range of 660–670 nm, provides a photosensitizing effect. The photosensitizer was administered intravenously to rats at a dose of 2.5 mg/kg. Observing the optimal time between drug administration and irradiation, the so-called drug-light interval (DLI) from 15 to 90 minutes, and specifically 30 minutes after administration, and using laser radiation parameters (laser radiation power density of 0.48 W / cm ² and energy density (E) - 150 J/cm ² ), using the Atkus-2 semiconductor device (CJSC Semiconductor Devices, St. Petersburg) with a wavelength of 662 nm, we obtained complete regression of tumors up to 21 days.

During PDT, a patient is injected with a photosensitizer (PS), which selectively accumulates in malignant neoplasms. Then the tumor is irradiated with laser light of a certain wavelength, in accordance with the PS excitation spectrum. As a result of irradiation, a photochemical reaction occurs, where the PS actually plays the role of a catalyst, and the formation of reactive oxygen species (the main of which is singlet oxygen) and various radicals, which are cytotoxic agents and cause destruction of tumor cells, occurs. The second mechanism of PDT is the destruction of the endothelium of blood vessels in the zone of laser irradiation, which results in vascular thrombosis and malnutrition in the tumor.

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 occurs.

The known method of quantum PDT (RU 2448745). The method includes the introduction of the drug Photoditazin at a dose of 5 mg per 20 ml of saline. 1-1.5 hours after the injection, laser irradiation of the neoplasm is carried out using an emitter with an optical element output power of 100 mW, light energy of 60-80 J/cm2 and a wavelength of 402 nm, which corresponds to the maximum absorption peak of Photoditazin administered intravesically and the formation of free radicals or atomic oxygen, which irreversibly damage the cellular structures of the tumor.

The used PS Photoditazine is not formed in the body of animals and, when it is administered, accumulates mainly in the pathologically altered tissue. At the same time, its tropism is still not high enough, and with a realistically possible time of exposure to radiation on the tumor, complete regression of tumor nodes is not achieved.

There is also known a method for PDT of malignant neoplasms (application for invention N 96107054/14, IPC A 61 N 5/00, publ. Bull. 1998 N 21), including, as well as the claimed invention, the introduction of a photosensitizer into the patient's body and laser irradiation of the tumor growth zone with a light dose of 300-500 J/cm ² .

The proposed method is aimed at increasing the concentration of the photosensitizer in the tumor, however, the laser irradiation parameters themselves remain high, which leads to the development of local complications during and after PDT.

There is also known a method of PDT of malignant tumors (RU 2146159), including systemic administration of PS and laser remote irradiation of the tumor growth zone with a light dose of 300-500 J/cm ² .

However, laser irradiation with the parameters used leads to the development of hemorrhagic necrosis in healthy tissues during and after PDT.

The closest prototype is a method of photodynamic therapy of malignant tumors ( RU 2704202 C1) and is intended for the treatment of malignant neoplasm Sarcoma M-1 rats. To do this, the photosensitizer "Photoran E6" is administered to the animals at a dose of 5.0 mg/kg, and after 2.5 hours, laser irradiation is performed with a wavelength of 660-670 nm with an energy density of 300 J/cm ² , a power density of 250 mW/cm ² . The exposure time is 20 minutes. This makes it possible to achieve complete eradication of tumor nodes up to the 90th day after the PDT session.

However, this method involves the introduction of a large dose of photosensitizer and the use of laser exposure for a long time and with a high energy density.

In order to reduce the amount of the photosensitizer used and reduce the dose of laser exposure to obtain a sparing organ-preserving method of treatment, as well as to reduce the exposure time, a therapy regimen was developed that leads to the maximum antitumor effect (100% cure of animals on day 90 after therapy) with improved parameters.

The technical result of the invention is the development of a method for achieving complete eradication of neoplasms.

The technical result is achieved by the fact that, as in the known method ( RU 2704202 ) , a photosensitizer is introduced once and the tumor is exposed to laser radiation with a wavelength of 662 nm.

A feature of the proposed method is that the photosensitizer Heliochlorin is administered intravenously to animals at a dose of 2.5 mg/kg, and after a drug-light interval of 30 minutes, exposure to light with a power density Ps = 0.48 W/cm ² is applied.

The invention is illustrated by a detailed description, tables and illustrations, which show:

Fig. 1 - Dynamics of accumulation of heliochlorin in the tumor and surrounding tissue of the thigh of rats after intravenous administration at a dose of 2.5 mg/kg; on the abscissa axis - the period after intravenous administration of PS; along the y-axis: fluorescence intensity on the left, contrast index on the right.

The method is carried out as follows.

In the in vivo experiment, we used white outbred laboratory rats; M-1 sarcoma was used as an experimental tumor model. The work was carried out in compliance with international recommendations for conducting 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).

The work was performed on 48 mature female rats weighing 165–190 g with M-1 sarcoma implanted subcutaneously on the outer side of the thigh (pieces of a donor tumor, 1*1*2 mm ³ in size). Upon reaching the largest diameter of the tumor nodes of 0.8-1.0 cm, the animals were randomly divided into experimental and control groups.

To determine the time of laser irradiation after PS administration, the drug light interval (DLI) was determined. The photodynamic activity of photosensitizers and the safety of surrounding tissues are realized due to the selective accumulation of photosensitizers in the tumor tissue. Therefore, their registration in order to determine the concentration and dynamics of the content in tissues in vivo is necessary to determine the optimal time from the moment of PS administration to laser exposure - LSVI. The accumulation of PS in the tumor and healthy thigh tissue was studied by laser spectrometry using a LESA-01-Biospec, Russia. The level of PS concentration was estimated from the intensity of fluorescence in arb. units, selectivity was determined by the contrast index (tumor/surrounding tissue). PS was administered to rats intravenously at a dose of 2.5 mg/kg. The first measurement was carried out before the administration of the drug (0 hours), and then 15, 30, 45, 60, 75, 90, 105, 120 minutes.

Conducting PDT.

control tumor-bearing rats served without any exposure (Table 1).

Table 1

Experiment scheme

Tumor Test system A drug Dose of PS
mg/kg Method
Introductions Irradiation parameters E
J / cm ² PS
W / cm ² Sarcoma
M-1 rats Heliochlorin 2.5 Intravenously 150 0.48 Control rats -

The source of laser radiation during PDT was an Atkus-2 semiconductor laser device manufactured by CJSC Semiconductor Devices (St. Petersburg) with a radiation wavelength of 662 ± 1 nm. The light spot diameter was 1.5 cm. Animals during irradiation, they were under general thiopental anesthesia (intraperitoneally 2.5% solution in a volume of 0.2 ml/100 g of animal weight). PDT was performed locally once, PS was administered intravenously.

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:

(1)

(one)

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:

(2)

(2)

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

V _t is the volume of the tumor for a certain period of observation;

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

(3)

(3)

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.

(4)

(four)

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). The level of significance of intergroup differences was assessed using the Mann–Whitney U-test at p<0.05.

I. Study of the dynamics of PS accumulation in the tumor and surrounding tissue.

The study of the accumulation of various photoactive substances in cells is an important aspect of screening studies, which makes it possible to optimize the conditions for photodynamic exposure (determination of the drug-light interval (DLI) - the time from the moment of PS administration to laser irradiation, which allows treatment at a high level of PS accumulation in the tumor with minimal its concentration in normal tissue - at the maximum contrast index.

From the data presented in the diagram (Fig. 1) it can be seen that within 90 minutes after intravenous administration of heliochlorin at a dose of 2.5 mg/kg, there is a gradual increase in the level of accumulation, both in the tumor and in the surrounding tissue of the thigh.

The maximum level of PS accumulation in the tumor was observed in the range of 15–90 min after PS administration. As for the contrast index, the highest (1.5) was determined after 15 minutes.

Thus, the optimal time for laser irradiation after PS administration occurs in 15–90 min, i.e. at this time, we have a high accumulation of PS in the tumor tissue and its minimum content in healthy tissues.

II. Study of the effectiveness of PDT with Heliochlorin.

Erosions covered with thin scabs appeared on the irradiated skin over the tumors on the 3rd day after PDT. 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 group, rats showed complete regression of the tumor up to 21 days after PDT (Table 2). Animals were left for further observation to determine life expectancy (Table 3) and the absence of tumor recurrence up to 90 days after PDT (Table 2).

According to a comparative analysis, after PDT, carried out at the optimal time after PS administration and selection of laser radiation parameters, a significant increase in the lifespan of rats was registered in the experimental group (Table 3).

table 2

Indicators of antitumor efficacy of PDT of M-1 sarcoma in rats with intravenous administration of Heliochlorin at a dose of 2.5 mg/kg and with the following parameters: energy density of laser radiation (E) - 150 J/cm ² ;. power density (Ps) - 0.48 W / cm ²

No.
gr. Dose
FS
(mg/kg) Observation period (days) 3 7 ten fourteen 21 90 one 2.5 1) 100 5) 100 Comte
role V 1.2±0.2 2.7±0.5 7.7±1.4 10.2±2.1 17.1±4.2 To 3.0±0.4 9.1±1.4 20.1±2.8 42.1±7.4 83.8±24.9

Note: ( ¹ ) tumor volume (V, cm3); (2) tumor growth rate in animals with continued growth (K); (3) inhibition of tumor growth (TPO,%); (4) percentage of animals with complete tumor regression (CR); (5) - percentage of cured animals on day 90

* p < 0.05 compared to control

Table 3

Increased lifespan of rats 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 >90* >100* Control 37.3 ± 2.2 -

Note: ALE – average life expectancy, days; LLS - increase in life expectancy,%; * Significant increase in life expectancy compared to control (LLE ≥ 50%).

Thus, the conducted in vivo study established the high antitumor efficacy of the studied drug on the transplanted malignant tumor M-1 sarcoma of rats up to 90 days after treatment with intravenous administration of Heliochlorin at a dose of 2.5 mg/kg (when extrapolated to the human dose is 0.42 mg /kg) and laser irradiation parameters E–150 J/ ^cm2 , Ps–0.48 W/cm2 ^.

The method made it possible to increase the life span of rats after PDT of a transplanted malignant tumor M-1 sarcoma with no recurrence of neoplasms up to 90 days after PDT.

Claims (1)

A method for photodynamic therapy of grafted superficial solid connective tissue sarcoma M-1 in rats, including the administration of a photosensitizer and subsequent exposure of the tumor to laser radiation with an energy density of E = 150 J/cm², a wavelength of 662 nm, characterized in that the photosensitizer Heliochlorin is administered intravenously at a dose of 2.5 mg/kg and light exposure with a power density of Ps = 0.48 W/cm is applied after a drug-light interval of 30 minutes².

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