RU2767272C2 - Method for treatment of rat m-1 transferable connective tissue sarcoma under combined impact of photodynamic therapy and radiation therapy - Google Patents

Abstract

FIELD: experimental medicine.

SUBSTANCE: invention relates to experimental medicine, namely to oncology, and can be used for combined therapy of grafted superficial solid connective tissue tumor M-1 sarcoma in rats. The combined effect of radiation therapy and photodynamic therapy is carried out according to the scheme: RT + PDT, with a time interval of 48 hours; a PDT session is performed with the photosensitizer amidoamine chlorine e6 (AAC), which is administered intraperitoneally at a dose of 1.25 mg/kg of animal weight, which is extrapolated to the human dose of 0.21 mg/kg, the drug-light interval between drug administration and laser irradiation is 3.0 hours, the parameters of laser exposure are: energy density E = 300 J/cm², power density Ps = 0.48 W/cm², during radiation therapy, the dose of γ-radiation was 20 Gy.

EFFECT: method allows to determine the optimal conditions for conducting combination therapy in its various sequences, at different time intervals, with a low dose of a photosensitizer and low γ-radiation parameters to achieve a pronounced positive effect, consisting in the elimination of a neoplasm, due to a set of techniques of the claimed invention.

1 cl, 3 dwg, 2 tbl

Description

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

Over the years of the fight against cancer, there has been an improvement in already well-studied methods of treatment and the active development of new methods of antitumor therapy. All methods of treatment are aimed at the maximum destruction of tumor cells. Each method is characterized by different mechanisms of action on tumor cells, causing their death. The effectiveness of the therapeutic effect is determined by the duration of the delay in tumor growth from the onset of exposure.

PDT has advantages over radiation and chemotherapy. This is a local form of therapy, which has the absence of severe local and systemic complications. The main advantages of PDT over conventional methods of treating malignant tumors are the selectivity of lesions, the absence of the risk of surgical intervention and severe systemic complications, and the low cost of treatment. During PDT, a very complex set of changes occurs in cells. The targets of photochemical influences are many cellular structures: cell membranes, mitochondria, DNA and microtubules. As membrane damage progresses, other electrolyte disturbances may be observed. Sublethal damage to cells through the involvement of many signaling systems can induce apoptosis. However, indirect effects such as ischemic necrosis due to vascular injury may also be important in vivo. PDT, in addition, is a "trigger" mechanism in the activation of the antitumor immune response associated with an increase in the process of tumor cell death, as well as the development of an acute inflammatory process. Thus, the mechanism of action of PDT includes a whole complex of direct and indirect reactions of the interaction of various components, ultimately leading to cytotoxic effects. At the same time, PDT cannot be called a panacea for cancer; the method has its limits. This is, first of all, the limited depth of damage to the tumor tissue (up to 1.0 cm).

Today, scientists and clinical specialists in many countries of the world are conducting research aimed at creating new, more effective and safe drugs - photosensitizers (PS). Currently, derivatives of the chlorophyll series, one of which is the PS amidoaminechlorin ( Fig. 1 ), developed by Professor G.V. Ponomarev (Institute of Biomedical Chemistry, Russian Academy of Medical Sciences).

Photosensitizer - Amidoaminechlorin e6 (AAX e6) developed on the basis of the interaction of methylpheophorbidebutwith various primary amines and is a chemical modification of the peripheral substituents of chlorin e6. A characteristic feature of chlorin e6 is the fact that, depending on the solvent, its spectral characteristics vary greatly. Thus, it is known that in aqueous media its absorption maximum shifts to the short-wavelength region from 662 nm. For experimental studies, a solution in aqueous dimexide for intraperitoneal administration can be used. One of the absorption peaks is at 662 nm, which correlates with the high quantum yield of singlet oxygen.

Radiation therapy (RT) (radiotherapy) is a method of treating malignant neoplasms. It is the most affordable and one of the most common cancer treatments. Tumor cells turned out to be the most vulnerable, because intensive cell division makes them especially sensitive to the effects of radiation. During such therapy, a devastating effect on cancer cells is exerted, the process of their division and growth stops. The delay in cell division may be temporary, transient and does not affect the viability of the irradiated cell. Reactions of this type - reversible reactions - also include various metabolic disorders, incl. inhibition of nucleic acid metabolism and oxidative phosphorylation, adhesion of chromosomes, etc. The reversibility of this type of radiation reactions is explained by the fact that they are the result of damage to some of the multiple structures, the loss of which is very quickly replenished or simply goes unnoticed. Effects that lead an irradiated cell to death—lethal radiation reactions—have a substantially different nature. In radiobiology, cell death is understood as the loss of a cell's ability to divide. The relatively low rates of local treatment and adverse reactions after RT dictate the need to find new approaches to this treatment method.

In order to enhance the antitumor effect, as well as to expand the scope of PDT as an effective antitumor, but sparing organ-preserving method of treatment, a combination therapy regimen combining PDT and RT for M-1 sarcoma was developed.

The use of PDT to improve the results of combined treatment poses the problem of finding its optimal place in therapeutic schemes. It is necessary to determine the practical possibility of combining methods, the best time sequence for their implementation, as well as the safety of their combined use. In order to determine the direct contribution of PDT to the results of combined treatment, the results of treatment of animals treated with PDT alone or only RT and PDT were compared with previous or subsequent RT.

Known way combined organ-preserving treatment of malignant tumors of the orbit Russian patent 2006 according to the IPC A61N5/67 A61F9/08 Description of the patent for the invention RU2286187C1. Irradiation of the orbit is carried out using remote gamma therapy. The goal of radiation therapy is post-radiation regression of the residual tumor (radical radiation program) or prevention of recurrence. At the same time, the total dose of remote gamma therapy depends on the stage of the neoplastic process and ranges from 40 to 60 Gy (1.5-1.8 Gy per fraction). The technical result is achieved through a combination of three methods: orbitotomy with excision of the tumor ad maximum, photodynamic therapy using a dose of photosensitizer Photosense 10 mg/kg and with a uniform distribution of light energy in the orbital cavity after excision of the tumor and with the selected mode of laser exposure, as well as chemotherapy. Irradiation with laser radiation with a wavelength of 670 nm, which is in the maximum absorption spectrum of the applied photosensitizer with a total power density of 120-800 mW/cm².

A known method of combined PDT and LT in the treatment of lung cancer (Ragulin Yu. A., Kaplan M. A., Medvedev V. N., Afanasova N. V., Kapinus V. N. Russian Biotherapeutic Journal. 2011. T. 10. No. 2. S. 33-36). The study included 57 patients with obstructive ventilation disorders caused by tumor stenosis of the main or lobar bronchus, who received radiation and chemoradiotherapy. All patients underwent radiation therapy according to the method of splitting the daily dose of 2.5 Gy into two fractions of 1 Gy and 1.5 Gy with an interval of 4–5 hours. therapy. 10 patients from each group received simultaneous chemotherapy according to the cisplatin + etoposide or carboplatin + paclitaxel scheme. Before radiation (or chemoradiation) therapy, 28 patients underwent PDT to recanalize the bronchus and restore pneumatization of the lung tissue, 29 patients made up the control group. The study showed the high clinical efficacy of PDT and the safety of its use. PDT before radiation therapy in patients with tumor stenosis of the large bronchi allows to achieve a pronounced positive effect, consisting in the elimination of atelectasis and the elimination of hypoventilation (in 85.7 patients versus 62.1% of the positive effect in patients receiving only radiation therapy). All patients underwent radiation therapy according to the method of splitting the daily dose of 2.5 Gy into two fractions of 1 Gy and 1.5 Gy with an interval of 4–5 hours. therapy. 10 patients from each group received simultaneous chemotherapy according to the cisplatin + etoposide or carboplatin + paclitaxel scheme.

The known methods used in the treatment of lung cancer methods have their advantages and disadvantages. Usually, a combination of two or three methods leads to an increase in the likelihood of tumor cure. At the same time, it is possible to potentiate the advantages of both methods and minimize their disadvantages. Thus, PDT has a highly effective local effect, but does not sufficiently affect micrometastases in normal tissue and regional collectors. Radiation therapy, on the contrary, effectively affects micrometastases, but at the same time, in some cases, does not achieve the full effect when exposed to the tumor itself. Considering all of the above, it seems possible and very promising to combine radiation therapy and PDT.

A known method of x-ray induced photodynamic therapy: a combination of radiation therapy and photodynamic therapy (X-Ray Induced Photodynamic Therapy: A Combination of Radiotherapy and Photodynamic Therapy. PNAS. August 20, 2019. vol. 116. no. 34. 16823-16828). This method uses X-rays as an energy source to activate the PDT process. In addition to breaking the dogma of small tissue penetration, studies have shown that X-PDT (100 J/cm ² , power density ~ 0.2 W/cm ² ) is more effective in killing tumor cells than radiotherapy (RT) alone. The studied PS was Metvix cream (Galderma, France), since its lipophilicity allows relatively deep penetration into tumors. This second generation PS contains methylaminolevulinate (MAL) as hydrochloride (C6H11NO3 • HCl) (160 mg/g) and, among others, cetostearyl alcohol (40 mg/g), methyl parahydroxybenzoate (2 mg/g), propyl parahydroxybenzoate (1 mg/g). d) and peanut butter (30 mg/g). Metvix represents excitation maxima at 405 and 635 nm. Contained methylaminolevulinate is metabolized to intracellular porphyrins, including protoporphyrin IX (PpIX), which is highly photosensitive and mainly accumulates in the tumor rather than in healthy cells. However, the mechanisms of cytotoxicity have not been elucidated. In the present, the authors are studying the mechanisms of action of X-PDT on cancer cells. The results show that X-PDT is more than just a derivative of PDT, but is essentially a combination of PDT and RT. These two modalities target different cellular components (cell membrane and DNA, respectively), resulting in enhanced therapeutic effects. As a result, X-PDT not only reduces the short-term viability of cancer cells, but also their long-term clonogenicity. From this point of view, X-PDT can also be considered as a unique method of radiosensitivity, and as such it offers clear advantages over RT in tumor therapy, especially for radioresistant cells. This has been demonstrated not only in vitro but also in vivo with H1299 tumors that are either subcutaneously inoculated or implanted in the lungs of mice. These results and achievements are of great importance for the development of X-PDT as a new method of treatment against cancer.

However, in this study, the photosensitizer cream was applied to the tumor rather than administered intravenously, and the number of animals was only 4 animals, with one of the animals being the control, which is insufficient to reliably establish antitumor efficacy. In addition, antitumor efficacy was determined only by the weight of the tumor on day 12 after exposure.

A comparative analysis of the radiological results of self-radiation therapy and radiation therapy with previous photodynamic therapy is known (Ragulin Yu.A., Kaplan M.A., Medvedev V.N., Afanasova N.V., Kudryavtsev D.V., Kapinus. Radiation and risk / Bulletin of the National Radiation and Epidemiological Register, 2015, vol. 24, no. 4, pp. 53-61). Radiation therapy was carried out according to the method of splitting the daily dose of 2.5 Gy into two fractions of 1 Gy and 1.5 Gy with an interval of 4-5 hours. 24 patients underwent endoscopic PDT 2-4 weeks before the start of the course of radiation therapy. The control X-ray study was performed when SOD-45-50 Gy was reached. The groups of patients were comparable in terms of gender, age, and morphological structure of the tumors. Positive radiological dynamics was achieved in 17 (65.4%) patients in the RT group and in 20 (83.3%) patients in the RT+PDT group. Resolution of atelectasis with complete recovery of pneumatization was observed in 4 patients out of 10 (40%) of the PDT+LT group, while in the control group this event was observed only in 2 cases out of 10 (20%). The lack of effect and negative dynamics were recorded in 9 (34.6%) patients of group No. 1, in group No. 2 there was no increase in ventilation disorders, the absence of dynamics was noted in 4 (16.7%). Complications (pneumonia) were observed in 3 (15%) patients in the RT+PDT group and in 2 (7.7%) patients in the RT group.

However, when analyzing the frequency of complications in the RT + PDT group, it was noted that in 14 patients who received antibiotic therapy immediately after PDT, pneumonia developed only in one case (7.1%). The use of PDT before radiation therapy in patients with tumor stenoses of the large bronchi, causing ventilation disorders, allows to achieve better immediate results of treatment, consisting in the elimination of atelectasis and hypoventilation.

A comparative analysis of the effectiveness of radiation photodynamic treatment of an experimental tumor is known (N.M. Rostovtsev, N.A. Kotlyarov. Pediatric Bulletin of the Southern Urals. 2015. No. 1. P. 29-32). For experiments, 258 outbred mice and lines CBA, C57B1, C3NA, hybrids (CBA* C57B1) were used. The average body weight of the experimental animal was about 25 ± 2.5 g. Tumor-bearing animals were withdrawn from the experiment on the 7th, 14th, 21st, and 28th days from the moment of inoculation of Ehrlich's carcinoma obtained from donor mice. We used a 2nd generation photosensitizer, a chlorin E 6 derivative, Radachlorin (manufactured in Russia), which was administered intraperitoneally. For close-focus radiation therapy of animals with a single X-ray irradiation at a dose of 5 Gy, an X-ray unit was used. Tumor resection was also included. Animals were treated on the 7th day after tumor inoculation. All experimental animals were divided into 6 groups, including the control group. In groups 1-III, tumor-carrying animals underwent PDT using a laser irradiation dose of 100 J/cm ² , 200 J/cm ² , 400 J/cm ² , respectively. An X-ray unit was used for close-focus radiation therapy in the IV group of animals with a single X-ray irradiation at a dose of 5 Gy. The effectiveness of the treatment was determined by the inhibition of tumor growth (TRO%) and partial tumor regression (PR%) within the established time limits. Compared with these groups, a significantly smaller tumor volume at all periods of the experiment was recorded after X-ray therapy (P < 0.05). The most pronounced suppression of tumor growth rate, which persisted throughout the entire observation period, was observed with PDT at a dose of 400 J/cm ² . Conclusion. As a result of experimental studies on transplanted Ehrlich ascitic carcinoma, it was found that the optimal dose of laser irradiation corresponds to 400 J/cm ² . Complete tumor regression was achieved with a combination of tumor resection and intraoperative PDT at a given dose on the residual tumor.

However, in this publication, in the description of the method, the power density of laser radiation is not indicated, which does not allow comparison with our study, in addition, a large dose of PS - 5.0 mg/kg and a high energy density - up to 400 J/cm ^{2 were} used.

The technical solution is to determine the optimal conditions for conducting combination therapy in a different sequence of its implementation (PDT + LT or LT + PDT), at different time intervals, with a low dose of the photosensitizer and low γ-radiation parameters to achieve a pronounced positive effect, which consists in the elimination of the neoplasm.

The technical result is achieved by the fact that, just as in the known method, photodynamic therapy (PDT) is used to treat an experimental tumor.

A feature of the proposed method is that a combined effect of radiation therapy and photodynamic therapy is carried out according to the scheme: RT + PDT, with a time interval of 48 hours, a PDT session is performed with a photosensitizer amidoaminechlorin e6 (AAC), which is administered intraperitoneally at a dose of 1.25 mg/kg animal weight, which, when extrapolated to a human dose, is 0.21 mg/kg, the drug-light interval between drug administration and laser irradiation is 3.0 hours, laser exposure parameters: energy density E = 300 J/cm ² , power density Ps = 0.48 W/cm ² , during radiation therapy, the dose of γ-radiation was 20 Gy.

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

Fig.1 - The formula of the photosensitizer amidoaminechlorin.

Figure 2 - Dynamics of accumulation of AAX e6 in the tumor and healthy tissue of the thigh of rats after intraperitoneal injection at a dose of 2.5 mg/kg. The abscissa shows the period after intraperitoneal administration of PS; along the y-axis: on the left, the fluorescence intensity in arb. units, on the right is the contrast index (red lines on the graphs).

Fig. 3 - The percentage of animals with complete regression of M-1 sarcoma on day 21 after combination therapy in various sequences of its implementation (PDT + RT or RT + PDT), at different time intervals (24 h or 48 h) and laser irradiation parameters (PDT - E \u003d 150 or 300 J / cm ² , Ps \u003d 0.34 or 0.48 W / cm ² ); FS amidoaminchlorin at a dose of 1.25 mg/kg; LT - dose of γ-radiation 20 Gy:

- I series of experiments (PDT + LT) - four groups of animals with different parameters of laser irradiation and with different time intervals;

- II series of experiments (LT + PDT) - four groups of animals with different time intervals and with different parameters of laser irradiation;

- Monotherapy - three groups of animals. Two groups of animals with PDT with different parameters of laser irradiation and one group with RT.

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 source of laser radiation 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).

Source γ - radiation was the installation "Luch - 1" (gamma-therapeutic installation with a source of Co⁶⁰). RT was performed remotely (ESWL), distance on a scale of 65 cm, locally once with a low radiation dose of 20 Gy.

Sexually mature female rats weighing 160–195 g on days 7–9 after inoculation, when the largest diameter of tumor nodes is 0.8–1.0 cm

Experimental part, proving the effectiveness of the proposed method.

The work was performed on 119 mature female rats weighing 165–190 g with M-1 sarcoma implanted subcutaneously on the outer side of the thigh. Upon reaching the largest diameter of the tumor nodes of 0.8-1.0 cm, the animals were divided into experimental and control groups by randomization.

Experimental animals are subjected to a combined effect of photodynamic therapy and radiation therapy according to the schemes: PDT + RT or RT + PDT, with a time interval of 24 or 48 hours. During PDT, PS amidoaminchlorin e6 (AAC) was administered at a dose of 1.25 mg/kg of animal weight (which, when extrapolated to a human dose, is 0.21 mg/kg), the drug-light interval was 3.0 hours, with laser radiation parameters energy density E \u003d 150 J / cm ² , power density Ps \u003d 0.34 W / cm ² or E \u003d 300 J / cm ² , Ps \u003d 0.48 W / cm ^{2 of} laser radiation and during radiation therapy (dose γ- radiation 20 Gy).

The dose of PS 1.25 mg/kg and the dose of gamma radiation 20 Gy were the same in all studies.

Control tumor-carrying animals without any treatment.

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 dynamics of PS accumulation in the tumor and healthy thigh tissue was studied by laser spectrometry using a LESA-01-Biospec setup. The level of PS concentration was assessed by the fluorescence intensity, selectivity was determined by the contrast index (tumor/healthy tissue). PS AAX was administered to rats intraperitoneally at a dose of 1.25 mg/kg. The first measurement was carried out before the administration of the drug (0 hours), and then after 20 minutes; 1.0; 1.5; 3.0 hours and 4.0 and 5.5 hours.

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.

Carrying out combination therapy:

The first series of experiments (PDT + LT) - four groups of animals. PDT session with a dose of photosensitizer AAX e6 1.25 mg/kg, with different parameters of laser radiation and at intervals (24 or 48 hours) RT (radiation exposure - γ-radiation - 20 Gy):

1. group. PDT (E = 150 J/cm ² , Ps = 0.34 W/cm ² ) and after 24 hours RT;

2. group. PDT (E = 300 J/cm ² , Ps = 0.48 W/cm ² ) and after 24 hours RT;

3. group. PDT (E = 150 J/cm ² , Ps = 0.34 W/cm ² ) and after 48 hours RT;

4. group. PDT (E = 300 J/cm ² , Ps = 0.48 W/cm ² ) and after 48 hours RT.

The second series of experiments (LT + PDT) - four groups of animals. Carrying out RT (radiation exposure - γ-radiation - 20 Gy) and at intervals (24 or 48 hours) a PDT session with a dose of AAX xe6 photosensitizer 1.25 mg / kg, with different parameters of laser radiation:

1. group. RT and after 24 hours PDT (E = 150 J/cm ² , Ps = 0.34 W/cm ² );

2. group. RT and after 24 hours PDT (E = 300 J/cm ² , Ps = 0.48 W/cm ² );

3. group. RT and after 48 hours PDT (E = 150 J/cm ² , Ps = 0.34 W/cm ² );

4. group. RT and after 48 hours PDT (E = 300 J/cm ² , Ps = 0.48 W/cm ² ).

Monotherapy - three groups of animals. Two groups of animals undergoing PDT with a photosensitizer dose of 1.25 mg/kg and different laser parameters and one group with LT (radiation exposure - γ-radiation - 20 Gy):

1. group. PDT (1.25 mg/kg, E = 150 J/cm ² , Ps = 0.34 W/cm ² );

2. group. PDT (1.25 mg/kg, E = 300 J/cm ² , Ps = 0.48 W/cm ² );

3. group. RT once at a dose of gamma radiation of 20 Gy.

Controls tumor-bearing rats served without any exposure.

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. Percentage of partial tumor regression (PR %). PR was taken as a decrease in tumor growth from the initial volume (-1.00 < K <0). Significant PD ≥50%.

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

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 tumor and healthy tissue.

The level and selectivity of PS accumulation in the tumor, as well as the rate of its excretion from normal tissue, are the most important characteristics for PDT. They affect both the effectiveness of the method and the likelihood of reducing side effects (Fig. 2).

From the data presented in Fig. 2 shows that within 5.5 hours after intraperitoneal administration of AAX e6 at a dose of 2.5 mg/kg, there is a gradual increase in the level of its accumulation, both in the tumor and in healthy thigh tissue.

The maximum level of PS accumulation in the tumor was observed 3.0–5.5 hours after PS administration. As for the contrast index, the highest (1.8) was determined after 3 hours.

Thus, the optimal time for laser irradiation after PS administration occurs after 3 hours, 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 antitumor efficacy

The first series of PDT + LT experiments (Table 1, Fig. 3)

Group 1 . During combined therapy (PDT - E =150 J/cm ² , Ps = 0.34 W/cm ² and after 24 hours of RT), a slight inhibitory effect was obtained on day 21 after treatment. PR was observed in 30% of the animals. PR was observed in 10% of animals, the percentage of regression from V ₀ did not exceed 32%, and tumor growth was observed in these individuals on day 21. The coefficient of absolute tumor growth on the 21st day in animals with continued growth is reduced compared to the control, but is not significantly significant.

Group 2. (PDT - E=150 J/cm ² , Ps=0.34 W/cm ² and after 48 hours of RT) a more significant inhibitory effect was obtained on the 21st day of the study compared to group 1 with the same parameters of laser radiation. Complete regression of the tumor was observed in 50% of the animals. PR was observed in a significant number of animals, and the percentage of regression was more than 50% of V ₀ . The coefficient of absolute growth in animals with tumor growth is reduced and significant in comparison with the control (p < 0.050).

Group 3. With an increase in the energy density and power density of laser radiation (PDT - E = 300 J / cm ² ; Ps = 0.48 W / cm ² and after 24 hours of RT ) , an increase in the inhibitory effect was obtained compared with groups 1 and 2. Tumor PR on the 21st day after therapy already in 66.7% of animals. CR did not exceed 39% of V ₀ and on day 21 they had tumor growth. The coefficient of absolute growth in animals with tumor growth was significantly lower than in the control (p < 0.050).

Group 4. (PDT - E = 300 J/cm ² ; Ps = 0.48 W/cm ² and after 48 hours RT ) - received an increase in the inhibitory effect. The PR of the tumor was 64.5% on the 21st day after the combined exposure. PR was observed and amounted to 54% of V ₀ and further there was a complete regression of the tumor. The growth rate in animals with continued tumor growth is already significantly lower than in controls (p < 0.010).

Thus, on the 21st day after the combination therapy in the sequence of PDT + RT , the antitumor effect depended primarily on the parameters of laser radiation. At a light dose E = 300 J/cm ² ; Ps = 0.48 W/ cm ² the antitumor effect was more significant 66.7% and 64.5% compared with E=150 J/cm ² , Ps=0.34 W/cm ² - only in 30% and 50 % of animals showed complete regression of the tumor.

With laser irradiation parameters E=150 J/cm ² , Ps=0.34 W/cm ² a more pronounced effect was noted after 48 hours (50%) versus (30%) after 24 hours.

With laser irradiation parameters E=300 J/cm ² , Ps=0.48 W/cm ² , the antitumor efficacy was practically the same both after 24 hours (66.7%) and after 48 hours (64.5%).

The second series of experiments LT + PDT (Table 1, Fig. 3)

Group 1. During combination therapy(LT and after 24 hours PDT - E=150 J/cm ² , Ps=0.34 W/cm ² ) on the 21st day of the study, 50% of the animals had tumor PR, which was higher than in the first series of experiments with the same parameters. PR was observed in a significant number of animals and ranged from 40% to 54% of V_0.Subsequently, tumor PR was observed in these animals. The growth rate in animals with continued tumor growth was significantly lower than in controls (p < 0.050).

Group 2. (LT Andafter 48 hours PDT with the same parameters of laser radiation as in group 1(E = 150 J/cm ² ; Ps = 0.34 W/cm ²) on the 21st day of the study, a significant inhibitory effect was observed. At this time, after the combined exposure to PR, tumors were already observed in 77.8% of the animals. This is more than in the I series with these parameters. The decrease in the coefficient of absolute growth in animals with tumor growth is significant compared to the control (p < 0.050).

Group 3. ( RT and PDT 24 hours later , but with an increase in laser radiation parameters (E = 300 J/cm2 ; Ps ⁼ 0.48 W/cm2 ) – tumor PR ^on day 21 in 75% of animals. PR was observed in a significant number of animals, it was noted up to 80% of V _0. Continued tumor growth was observed only in one animal.

Group 4 ( RT and after 48 hours PDT with the same parameters of laser radiation as in group 3 (E = 300 J/cm ² ; Ps = 0.48 W/cm ² ) received an increase in the inhibitory effect. PR was 21 days after treatment already in 90% of animals.Compared to series I, this is a significant increase in the antitumor effect.Continued tumor growth was also observed in only one animal.The coefficient of absolute growth is almost zero (K = 0.09).

Thus, on the 21st day after combined therapy in the RT + PDT sequence, the antitumor effect was significantly higher than in the first series of experiments (PDT + RT) and depended both on the parameters of laser radiation and on the time interval.

With a time interval of 48 hours, the antitumor effect was more significant 90% ( E = 300 J/cm ² ; Ps = 0.48 W/ cm ² ) and 77.8% ( E=150 J/cm ² , Ps=0.34 W/cm ² ) compared with the time interval after 24 hours - only 75% and 50% of the animals showed complete regression of the tumor with the same parameters of laser exposure.

The use of RT before PDT (with a time interval of 48 hours) in animals with M-1 sarcoma makes it possible to achieve a pronounced positive effect, consisting in complete regression of malignant tumors in 90% of animals.

Table 1.

Dynamics of M-1 sarcoma in rats after combination therapy in various sequences of its implementation PDT and RT or RT and PDT,

with different time intervals, with a dose of photosensitizer e6 1.25 mg/kg,

PDT (E = 150 J/cm ² , Ps = 0.34 W/cm ² or E = 300 J/cm ² , Ps = 0.48 W/cm ² ); LT (20 Gr)

No. Parameters of laser and gamma radiation Time interval Absolute tumor growth rate (K) (M±m) in rats with tumor growth Complete tumor regression (CR%) Partial tumor regression (PR %) 3 days 7 days 10 days 14 day 21 days PDT + LT one. E \u003d 150J / cm ² ,
P _S \u003d 0.34 W / cm ²
20 Gr 24 hours 1.82 ± 1.02
20.0%
10.0% 1.34±0.39*
20.0%
10.0% 4.47 ± 1.66*
20.0%
10.0% 4.90±1.86*
30.0%
0% 12.39, ± 2.72
30.0%
0% 2. E \u003d 150J / cm ² ,
P _S \u003d 0.34 W / cm ²
20 Gr 48 hours 0.29 ± 0.11*
11.1%
33.3% 0.12*
44.4%
44.4% 0.78 ± 0.00*
55.6%
22.2% 0.82 ± 0.24*
50.0%
0% 3.09±0.83*
50.0%
0% 3. E \u003d 300J / cm ² ,
P _S \u003d 0.48 W / cm ²
20 Gr 24 hours 0.86±0.42*
66.7%
0% 1.29*
88.7%
0% 0.47*
77.8%
11.1% 1.14 ± 1.00*
77.8%
0% 3.27 ± 1.94*
66.7%
0% 4. E \u003d 300J / cm ² ,
P _S \u003d 0.48 W / cm ²
20 Gr 48 hours 1.31 ± 1.17
75.0%
12.5% 1.88
87.5%
0% 0.50
87.5%
0% 0.50
87.5%
0% 0.90 ± 0.36*
64.5%
0% LT + PDT one. 20 Gy E \u003d 150J / cm ² ,
P _S \u003d 0.34 W / cm ² 24 hours 0.70 ± 0.37*
12.5%
37.5% 1.07 ± 0.96*
50.0%
25.0% 1.99 ± 0.13*
50.0%
25.0% 1.64 ± 1.09*
50.0%
0% 5.36 ± 2.66*
50.0%
0% 2. 20 Gy E \u003d 150J / cm ² ,
P _S \u003d 0.34 W / cm ² 48 hours 6.43
88.9%
0% 3.99*
77.8%
11.1% 4.7*
77.8%
11.1% 4.66 ± 3.03*
77.8%
0% 7.43 ± 6.72*
77.8%
0% 3. 20 Gy E \u003d 300J / cm ² ,
P _S \u003d 0.48 W / cm ² 24 hours 1.16±0.33
11.1%
44.4% 0.76 ± 0.10*
44.4%
33.3% 0.64 ± 0.04*
44.4%
33.3% 0.59*
66.7%
33.3% 0.57*
75.0%
12.5% 4. 20 Gy E \u003d 300J / cm ² ,
P _S \u003d 0.48 W / cm ² 48 hours 1.97±0.18
0%
0% 0.25±0.17*
50.0%
0% 0.15*
80.0%
10.0% 0.12*
90.0%
0% 0.09*
90.0%
0% Control (K) 2.11±0.25 8.08±0.95 17.49±2.54 43.78±10.23 81.94±22.56

Note: level of significance compared to control – * p < 0.050

Monotherapy (Table 2, Fig. 3)

Group 1. When conducting PDT - E=150 J/cm ² , Ps=0.34 W/cm ² on the 21st day after treatment, only 30% of the animals showed complete regression of the tumor. In animals with continued tumor growth, although a decrease in the absolute tumor growth rate was observed, this decrease is not significant compared to the control.

Group 2. During PDT-E=300 J/cm ² ,Ps = 0.48 W/cm ² on the 21st day after treatment, already in 40% of the animals there was a complete regression of the tumor and the coefficient of absolute tumor growth (p < 0.050) compared with the control.

Group 3. When conducting RT with a dose of γ-radiation - 20 Gy on the 21st day after treatment, only 30% of the animals showed complete regression of the tumor. In animals with continued tumor growth, but absolute tumor growth rate (p < 0.050) compared to control.

Table 2.

Dynamics of M-1 sarcoma in rats after monotherapy:

PDT (E = 150 J/cm ² , Ps = 0.34 W/cm ² or E = 300 J/cm ² , Ps = 0.48 W/cm ² ); LT (20 Gr)

No. Monotherapy Parameters of laser-
and gamma radiation Absolute tumor growth rate (K) (M±m) in rats with tumor growth Complete tumor regression (CR%) Partial tumor regression (PR %) 3 days 7 days 10 days 14 day 21 days one. PDT E \u003d 150J / cm ² ,
P _S \u003d 0.34 W / cm ² -0.85 ± 0.10*
50.0%
0% -0.14±0.52*
40.0%
0% 0.70 ± 1.17*
30.0%
0% 5.52 ± 4.32*
30.0%
0% 28.11 ± 14.34
30.0%
0% 2. PDT E \u003d 300J / cm ² ,
P _S \u003d 0.48 W / cm ² -0.90 ± 0.15*
70.0%
0% -0.10 ± 0.64*
70.0%
0% 0.34 ± 1.90*
50.0%
0% 3.28 ± 3.02*
50.0%
0% 7.34 ± 2.93*
40.0%
0% 3. LT 20 Gr 0.53 ± 0.12*
0%
10 % 0.81 ± 0.34*
0%
40% 1.76 ± 0.47*
15.0%
15.0% 3.20 ± 0.83*
25.0%
10.0% 8.81 ± 2.02*
30.0%
0% Control (K) 2.97±0.50 14.01 ± 2.05 20.73 ± 2.04 36.00 ± 3.85 65.31 ± 7.70

Note: level of significance compared to control – * p < 0.050

The effectiveness of combination therapy exceeds the therapeutic effect of monotherapy carried out under the same conditions, which was expressed in an increase in the percentage of animals with partial and complete tumor regression and a significant decrease in the absolute growth rate in animals with continued tumor growth under all experimental parameters.

In the control group, progressive tumor growth was observed throughout the study period.

The conducted studies have established the advantage of the second treatment regimen (RT + PDT). Experiments with the use of RT and after 48 hours PDT (E = 300 J/cm ² , Ps = 0.48 W/cm ² ) of animals with M-1 sarcoma turned out to be the most effective, which makes it possible to achieve a pronounced positive effect, which consists in the elimination of the neoplasm in 90% versus 30% positive effect in rats receiving only radiation therapy and 40% - only PDT .

The proposed method of treatment allows to achieve a pronounced positive effect and eliminate neoplasms, which is of great importance for the development of a combined effect as a new method of cancer treatment.

Claims (1)

A method for treating grafted connective tissue sarcoma M-1 in rats under the combined effect of photodynamic therapy and radiation therapy, characterized in that the combined effect of radiation therapy and photodynamic therapy is carried out according to the scheme: RT + PDT, with a time interval of 48 hours, the PDT session is carried out with the photosensitizer amidoaminechlorin e6 (AAX), which is administered intraperitoneally at a dose of 1.25 mg/kg of animal weight, which, when extrapolated to a human dose, is 0.21 mg/kg, the drug-light interval between drug administration and laser irradiation is 3.0 hours, the parameters of laser exposure: the energy density E is 300 J/cm ² and the power density Ps is 0.48 W/cm ² during radiation therapy, the dose of γ-radiation was 20 Gy.

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