Radiodynamic and contact radiation therapy of inoculated tumor in an in vivo experiment

• 1    - STATE INSTITUTION “N. N. ALEXANDROV NATIONAL CANCER CENTER OF BELARUS”, Minsk, Belarus

• 2    - MINSK REGIONAL CLINICAL HOSPITAL, Minsk, Belarus

Introduction. In recent years, interest in the use of such physical factors as magnetic fields, ultrasound, and hyperthermia to activate Photosensibilizers (PS) has increased in experimental oncology. One of the promising scientific areas is radiodynamic therapy (RDT), a new method of therapy based on the combined use of PS and ionizing radiation [1]. The first PS with the radiosensitizing properties proven in the in vitro/in vivo experiments were 5-aminolevulinic acid, Hematoporphyrin, and Photofrin II [2, 3]. The main mechanisms of the implementation of the antitumor response of RDT remain poorly studied. According to Shaffer M. et al., on the one hand, PS (for example, "Photofrin II"), under the influence of ionizing radiation, can enhance the radiolytic effect due to reactive oxygen species (hydroxyl radical, superoxide anion and singlet oxygen) formed in the tumor cell upon exposure to the proper radiation [2]. On the other hand, the exposure to ionizing radiation leads to sublethal and lethal damage to tumor cells. Further, sublethal changes are, as a rule, reversible, associated with the implementation of the mechanisms responsible for restoring the functions of the tumor cell. In case of activation of the PS "Photofrin II" by ionizing radiation, the oligomeric components of this PS, interacting with intermediate free radicals (hydroxyl radicals) formed in the tumor cell under radiation, prevent the development of these processes, and, therefore, this combination leads to the production of antitumor effects. The result of such interactions is the initiation of the processes of apoptosis and autophagy, leading to the death of tumor cells [1-4].

The results of experimental studies on the radiosensitizing effect of PS, published in 2019–2021, are presented in a number of scientific reports [1-3]. In the framework of some pilot projects, testing of the RDT method has begun in patients with various malignant neoplasms of the cervix and bladder, pelvic sarcomas, melanoma, gliomas, including inoperable cases. The obtained preliminary data show good tolerability of the above method (no serious adverse reactions found), acceptable antitumor efficacy (an increase in the rate of objective responses and an increase in the % reduction in tumor volume, which made it possible to transfer them to a resectable state) [5-8]. Taking into account all of the above, the study of the radiosensitizing properties of PS of the chlorin series seems to be a topical promising issue. This research work is a continuation of the studies, the results of which were published earlier [9], and it is aimed at optimizing the modes and parameters of the combined use of ionizing radiation in contact radiation therapy (CRT) and PS PHOTOLON.

The aim of the study was to investigate the antitumor efficacy of the RDT method with chlorin PS in combination with CRT in an experiment on laboratory animals with transplanted tumors.

Materials and methods . The study was performed in 35 white outbred rats of both sexes (hereinafter referred to as the rats) (animal breeding facility at the N.N. Alexandrov National Cancer Center), weighing from 170 to 300 g, aged 2.5–3 months. The duration of quarantine before inclusion in the experiment was 14 days. The rats were kept under standard conditions of food and water ration ad libitum, with 12-hour illuminance, at a temperature of 19–22°C and a humidity of 55–60%, in individual cages, 5 individuals in each. The studies were carried out in accordance with international legislation and the regulatory legal acts in force in the Republic of Belarus applicable to conducting experimental studies with laboratory animals, namely, the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Strasbourg, France, dd. March 18, 1986) as amended in accordance with the provisions of the Protocol (E.T.S. No. 170 of December 2, 2005) and TPC 125-2008 (02040) “Good Laboratory Practice” (Resolution No.56 of March, 28, 2008 issued by the Ministry of Health of the Republic of Belarus). The study was approved by the Ethics Committee at the N.N. Alexandrov National Cancer Center (Extract from the Protocol No. 180 dated February 25, 2022). Before the experiments, the rats were anesthetized (neuroleptanalgesia: 0.005% fentanyl solution + 0.25% droperidol solution, in a ratio of 2:1, 0.2 ml per 100 g of body weight, intramuscularly). Pliss lymphosarcoma (PLS) (Russian Collection of Cell Cultures, Institute of Cytology RAS, St. 7 Cardiometry, Issue 24, November 2022

Petersburg, Russian Federation) was used as a tumor strain. A tumor model in rats was established by subcutaneous passivation in vivo: injection of 0.5 ml of a suspension of tumor cells in a Hank's solution under the skin of the left inguinal region. After inoculation, the rats were randomly assigned to study groups, 5 animals in each group. The rats with PLS were involved into the experiment on the 6th day after the average diameter of transplanted tumors had reached a size from 3 to 5 mm. As a radiosensitizing agent, we used the PS agent of the chlorine series PHOTOLON (Belmedpreparaty, Minsk, Republic of Belarus), which represents a trisodium salt of chlorin e6 with povidone with a K-value of 17 (Registration number 16/11/886 dated 08.11.2016) (Pharmacotherapeutic group: drugs used in photodynamic and radiation therapy; ATC code: L01XD). The injection of the above PS agent was carried out once by intravenous infusion using a special catheter into the tail vein in a darkened room at a dose of 2.5 mg/kg of body weight. Radiation therapy was carried out by the contact method (CRT) with microSelectron-HDR V3 Digital equipment (Elekta, Sweden) using γ-radiation (192 Ir). For superficial CRT on the surface area of the transplanted tumor, the Leipzig applicator was used, fixed on the tumor surface with soft rubber holders. The study groups were composed as follows: group 1 as intact reference (IR) to cover rats with transplanted tumors, which were not injected with PS and did not undergo radiation (n=5); group 2 with 1 session of CRT at a single focal dose (SFD) of 2 Gy (n=5); group 3 with 2 sessions of CRT on the 1st and 2nd days of the experiment at a SFD of 2 Gy (n=5); group 4 with 3 sessions of CRT on the 1st, 2nd and 3rd days of the experiment at a SFD of 2 Gy (n=5); group 5 with PS 2.5 mg/kg + 1 RDT session 2.5-3 hours after the end of the PS infusion at a SFD of 2 Gy (n=5); group 6 with PS 2.5 mg/kg + 1 RDT session 2.5-3 hours after the end of the PS infusion at a SFD of 2 Gy + 1 CRT session on the 2nd day of the experiment at a SFD of 2 Gy (n=5); group 7 with PS 2.5 mg/kg + 1 RDT session 2.5-3 hours after the end of the PS infusion at a SFD of 2 Gy + 2 CRT sessions on the 2nd and 3rd days of the experiment at a SFD of 2 Gy (n=5 ). The total focal dose (TFD) in groups 2 and 5 was 2 Gy; in groups 3 and 6 the TFD values was 4 Gy, and in groups 4 and 7 the TFD value was 6 Gy.

The effectiveness of therapeutic actions and effects was estimated according to the generally accepted criteria applied to experimental oncology, which characterize the dynamics of changes in the volumes of the tumors: the average volume of tumors (Vav., in cm3) and the coefficient of tumor growth inhibition (TGI, in %).

The minimum biologically significant TGI was 50%. The rate of complete regressions (CR) of tumors was assessed 60 days after the beginning of therapeutic interventions in the absence of visual and palpatory signs of the tumor growth. After the end of the observation period, the rats were sacrificed using the euthanasia method (aether pro narcosi) in compliance with humane methods of manipulations with laboratory animals. Statistical data processing was carried out using 8 Cardiometry, Issue 24, November 2022

the Origin Pro (version 7.0) and Statistica (version 10.0) application softwares. Data are presented as M referred to as mean value and m (SEM) referred to as error of the mean. The significance of differences was assessed using the Mann–Whitney U test. Differences were considered as statistically significant at p<0.05.

Results . The efficiency of the inoculation of the tumor strain was 100%. No complications that led to the death of rats with the introduction of the PS agent and radiation of tumors were recorded. The obtained experimental data demonstrating immediate and long-term results are presented below. Vav. in the IR group on the 14th day of the experiment was 44.42±5.88 cm³. Irradiation of inoculated tumors (CRT) in groups 2-4 with CRT without prior administration of the PS agent allowed obtaining a moderately pronounced inhibition of the growth of inoculated tumors: 38.88±5.65; 30.25±7.24 and 3.41±6.29 cm³ (p=0.51; p=0.16 and p=0.033 in relation to the IR group values). The TGI indicators in the groups amounted to 12.47%; 31.90% and 47.30%, respectively. The rate of CR of the tumors was recorded to be 20%, 20% and 20%, respectively. Irradiation of inoculated tumors in groups 5-7 with CRT after preliminary administration of the PS agent led to a more pronounced inhibition of the growth of the inoculated tumors: 29.38±2.24; 24.45±4.52 and 13.55±4.55 cm³ (p=0.036; p=0.021 and p=0.0016 in relation to the IR group values). The TGI indicators in those groups amounted to 33.86%; 44.96% and 69.50%, respectively. The rate of the complete regression (CR) of the tumors was reported to be 0%, 20% and 40%, respectively.

Conclusions. The limited global experience in the combined use of PS and ionizing radiation and our own evidence data obtained testify to the fact that RDT is a new promising area in the scientific research in experimental oncology, and that the PS agent PHOTOLON has radiosensitizing properties, when using certain irradiation parameters, that is confirmed by the data on the dynamics of the growth of the inoculated tumors and the rate of the objective responses to the therapy. And the combined use of the two methods (RDT and CRT) allows summing up their antitumor effects, thereby optimizing the therapeutic effect produced on the inoculated tumors in the in vivo experiment.