RU2724480C2 - Method of combined radiotherapy and photodynamic therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to oncology, and can be used for photodynamic therapy. Single intravenous introduction of a photosensitizer Chlorine E6 in dose of 10 mg/kg of body weight is performed. Spectrometry is performed 3 hours after the preparation administration. Method includes a session of remote therapy by electrons in single basic dose of 32 Gy at dose rate 3.3 Gy/min, energy 10 MeV. That is followed by repeated spectrometry, after which the fluorescence values are compared before and after the exposure, and the photo burnout photosensitizer is evaluated to confirm the photodynamic component of the therapy arising from activation of the photosensitizer induced by Cerenkov radiation.

EFFECT: method provides higher anticancer action, reduced total time of therapeutic procedure, increased depth of penetration, without increasing damage of surrounding healthy tissues due to use of induced radiation occurring directly in tumour when exposed to electron beam with specified parameters.

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Description

The invention relates to medicine, namely to experimental research in oncology and can be used to increase the effectiveness of radiation therapy of malignant neoplasms.

Radiation therapy along with surgical treatment and chemotherapy plays an important role in the treatment of malignant neoplasms. (Helen H.W. Chen, Macus Tien Kuo. Improving radiotherapy in cancer treatment: Promises and challenges. Oncotarget. 2017; Sep 5; 8 (37): 62742-62758. Doi: 10.18632 / oncotarget. 18409). Despite the discovery and introduction into practice of types of radiation with high linear energy transfer (proton, ion radiation), significant costs for the construction and operation of accelerator plants limit access to their widespread use. Photon and electron radiation remain one of the most frequently used radiation sources in modern radiation therapy (Kaprin A.D., Mardinsky Yu.S., Smirnov V.P., Ivanov S.A., Kostin A.A., Polikhov S. A., Reshetov I.V., Fatyanova A.S., Denisenko M.V., Epatova T.V., Korenev SV., Tereshchenko A.V., Filonenko E.V., Gafarov M.M., Romanko Yu.S. On the history of the development of radiation therapy (Part I). Biomedical Photonics. 2019; 8 (1): 52-62. Https://doi.org/10.24931/2413-9432-2019-8-1-52- 62 Kaprin AD, Mardinskiy YS, Smirnov VP, Ivanov SA, Kostin AA, Polikhov SA, Reshetov IV, Fatianova AS, Denisenko MV, Epatova TV, Korenev SV, Tereshchenko AV, Filonenko EV, Gafarov MM, Romanko YS The history of radiation therapy (part I). Biomedical Photonics. 2019; 8 (1): 52-62. (In Russ. https://doi.org/10.24931/2413-9432-2019-8-1-52-62).

The main mechanisms of antitumor action for all types of radiation therapy are direct cytotoxic effects due to damage to the genetic apparatus of the cell, namely the induction of double-stranded DNA breaks. It should not be forgotten that similar changes also occur in surrounding normal tissues that have fallen into the zone of influence. (Maria Rita Fabbrizi, Kacie E. Warshowsky, Cheri L. Zobel, Dennis E. Hallahan, Girdhar G. Molecular and epigenetic regulatory mechanisms of normal stem cell radiosensitivity. SharmaCell Death Discov. 2018; 4: 117.doi: 10.1038 / s41420- 018-0132-8)

There is a direct correlation between the effectiveness of radiation therapy and the degree of tissue oxygenation. At the same time, hypoxia due to the uneven distribution of newly formed vessels, as well as their functional immaturity, is a common feature of solid tumors. This fact determines one of the most important reasons for the failure of radiation therapy of the primary tumor and causes the occurrence of radioresistant relapses (Kuwahara Y, Tomita K, Urushihara Y, Sato T, Kurimasa A, Fukumoto M. Association between radiation-induced cell death and clinically relevant radioresistance. Histochem Cell Biol. 2018 Dec; 150 (6): 649-659. Doi: 10.1007 / s00418-018-1728-z.)

Given the fact that recurrent tumors are less susceptible to treatment, many strategies have been developed to overcome hypoxia-induced radioresistance. The most common of them are the addition of radiosensitizers, saturation of the tumor with hyperbaric oxygen, the use of cytotoxins selective for hypoxia (Hui Wang, Heng Jiang, Melissa Van De Gucht, Mark De Ridder. Hypoxic Radioresistance: Can ROS Be the Key to Overcome It? Cancers (Basel) . 2019 Jan; 11 (1): 112. doi: 10.3390 / cancersl 1010112) (Higgins GS, O'Cathail SM, Muschel RJ, McKenna WG. Drug radiotherapy combinations: review of previous failures and reasons for future optimism. Cancer Treat Rev . 2015 Feb; 41 (2): 105-13. Doi: 10.1016 / j.ctrv.2014.12.01.01).

In recent years, hypoxia has also been identified as the main factor in suppressing immunity in the tumor microenvironment. In this connection, the combination of radiation therapy and immuno-targeted agents is of particular interest. (Eckert F, Zwirner K, Boeke S, Thorwarth D, Zips D, Huber SM. Rationale for Combining Radiotherapy and Immune Checkpoint Inhibition for Patients With Hypoxic Tumors. Front Immunol. 2019 Mar 12; 10: 407. Doi: 10.3389 / fimmu. 2019.00407).

However, adequate clinical application of the above methods is limited by their lack of effectiveness, side effects, and often high cost.

Photodynamic therapy is a non-invasive, effective treatment method that has proven itself in various areas of medicine, including the treatment of malignant neoplasms (Benov L. Photodynamic Therapy: Current Status and Future Directions. Med Princ Pract. 2015; 24 (suppl 1): 14-28.doi.org/10.1159/000362416).

Ferreira dos Santos, Daria Raquel Queiroz de Almeida, Leticia Ferreira Terra,

Baptista, Leticia Labriola. Photodynamic therapy in cancer treatment - an update review. J Cancer Metastasis Treat. 2019;5:25.10.20517/2394-4722.2018.83). Благодаря способности фотосенсибилизатора поглощать и испускать свет на определенных длинах волн возможен мониторинг фотодинамической терапии методом спектрометрии, условно позволяющей оценить разницу концентраций фотосенсибилизатора в опухоли до и после сеанса терапии и косвенно оценить эффективность проведенного лечения (Atif, М., Zellweger, М., Wagnieres, G.. Review of the role played by the photosensitizer's photobleaching during photodynamic therapy. Journal Of Optoelectronics And Advanced Materials. 2016; 18, 3-4,338-350).The effect is achieved due to the simultaneous presence of three components: a photosensitizer, molecular oxygen and laser light of a certain wavelength, none of which is toxic or by itself damages cells or tissues. One of the significant advantages of the method is its high selectivity due to the targeted accumulation of the photosensitizer in target cells. This allows the exposure to only the desired area to avoid generalized toxicity. When the laser light is absorbed by the drug, a whole cascade of events is triggered, leading ultimately to the formation of free radicals that kill target cells, damage the vasculature associated with the tumor, and activate the immune response (

Ferreira dos Santos, Daria Raquel Queiroz de Almeida, Leticia Ferreira Terra,

Baptista, Leticia Labriola. Photodynamic therapy in cancer treatment - an update review. J Cancer Metastasis Treat. 2019; 5: 25.10.20517 / 2394-4722.2018.83). Due to the ability of the photosensitizer to absorb and emit light at specific wavelengths, it is possible to monitor photodynamic therapy by spectrometry, which conditionally allows you to evaluate the difference in the concentration of photosensitizer in the tumor before and after the treatment session and indirectly evaluate the effectiveness of the treatment (Atif, M., Zellweger, M., Wagnieres, G .. Review of the role played by the photosensitizer's photobleaching during photodynamic therapy. Journal Of Optoelectronics And Advanced Materials. 2016; 18, 3-4, 3338-350).

The value of the fluorescence parameters and their influence on the effectiveness of photodynamic therapy is confirmed in clinical works (Fluorescence imaging for photodynamic therapy of non-melanoma skin malignancies-a retrospective clinical study Gamayunov S., Turchin I., Fiks I., Kleshnin M., Shakhova N. , Korchagina K. Photonics and Lasers in Medicine. 2016. T. 5. No. 2. C. 101-111).

The contribution of certain mechanisms to the antitumor effect depends on the chemical structure of the photosensitizer used and the related bio-distribution of the drug. Chlorin E6 derivatives most commonly used in clinical practice are hydrophilic, which allows them to accumulate in the vascular bed of the tumor, causing damage to the basement membrane and endothelium, release of vasoactive molecules, adhesion of leukocytes, increased vascular permeability and narrowing of their lumen. This leads to the destruction of the tumor due to vascular collapse, thrombosis and hemorrhage in the tissue (Krammer B. Vascular effects of photodynamic therapy. Anticancer Res. 2001 Nov-Dec; 21 (6B): 4271-7).

Despite the rather successful application of the method, one of the main disadvantages of photodynamic therapy is the limited depth of laser light penetration, which makes this method possible for treating only superficially located formations (Zheng Huang, Heping Xu, Arlen D. Meyers, Ali I. Musani, Luowei Wang, Randall Tagg, Al B. Barqawi, Yang K. Chen. Photodynamic therapy for treatment of solid tumors - potential and technical challenges. Technol Cancer Res Treat. 2008 Aug; 7 (4): 309-320.doi: 10.1177 / 153303460800700405).

A theoretical justification for the proposed version of the combined method of radiation and photodynamic therapy is a series of articles.

It is a known fact that when charged particles pass through a medium with a uniform speed exceeding the phase velocity of light in the same medium, radiation arises called “Cherenkovsky” (Xiaowei Ma, Jing Wang, Zhen Cheng. Cerenkov radiation: a multi-functional approach for biological sciences. Front. Phys., 2014 (2), 1-14. doi.org/10.3389/fphy.2014.00004).

Recently, the use of Cherenkov radiation in biological research has been of great interest. For example, much attention is paid to the use of the Cherenkov effect in dosimetry - to control the depth of penetration of the proton beam (Andy Lau, Salahuddin Ahmad and Yong Chen. A simulation study investigating a Cherenkov material for use with the prompt gamma range verification in proton therapy. Journal of X -Ray Science and Technology. 24 (2016), 565-582 DOI 10.3233 / XST-160575) or surrogate absorbed dose rate ((Kyoung Won Jang, Sang Hun Shin, Seon Geun Kim, Jae Seok Kim, Wook Jae Yoo, Young Hoon Ji, Bongsoo Lee. Measurement of Cerenkov Radiation Induced by the Gamma-Rays of Co-60 Therapy Units Using Wavelength Shifting Fiber. Sensors (Basel). 2014 Apr; 14 (4): 7013-7025. Doi: 10.3390 / s140407013.

These works are based on the idea that the plots of depth distribution of gamma and electron radiation and Cherenkov emission coincide (Adam K Glaser1, Rongxiao Zhang2, David J Gladstone3 and Brian W Pogue. Optical dosimetry of radiotherapy beams using Cherenkov radiation: the relationship between light emission and dose. Phys. Med. Biol. 59 (2014) 3789-3811 doi: 10.1088 / 0031-9155 / 59/14/3789).

Cherenkov radiation has unique spectral and spatial properties. Its emission spectrum is continuous from infrared to ultraviolet and is proportional to 1 / λ ² . In this case, the maximum value falls on the region of 200-400 nm (Xu Cao, Shudong Jiang, Mengyu Jeremy Jia, Jason R. Gunn, Tianshun Miao, Scott C. Davis, Petr Bruza, Brian W. Pogue. Cherenkov excited short-wavelength infrared fluorescence imaging in vivo with external beam radiation. J. of Biomedical Optics. 2018; 24 (5). https://doi.Org/10.1117/1.JBO.24.5.051405).

This interval overlaps with a unique absorption peak of 402 nm common for all types of photosensitizers (Soret peak), and the quantum yield of singlet oxygen upon excitation of PS at this wavelength is maximum (Heidi Abrahamse, Michael R. Hamblin. New photosensitizers for photodynamic therapy. Biochem J. 2016 Feb 15; 473 (4): 347-364. Doi: 10.1042 / BJ20150942).

These theoretical models and their analytical interpretation suggest that, under appropriate conditions, activation of the photosensitizer accumulated in the tumor is possible under the action of a radiation agent capable of generating Cherenkov radiation.

The closest is a method of treating patients with nodular and radioresistant malignant tumors (RU 2570033 C1), including 8-10 sessions of combined remote gamma therapy and photodynamic therapy with an interval of 24 hours.

However, this method has the following disadvantages: the long half-life of Photosens increases the time for observing the light regime. The consistent use of photodynamic and radiation therapy methods lengthens the duration of the procedure. The use of a remote laser light source to excite a photosensitizer does not allow the treatment of tumors located at a depth of a laser more penetrating (4-8 mm).

The technical result of the claimed invention is the possibility of conducting combination therapy that allows you to simultaneously combine the effects of radiation and photodynamic effects, which would lead to an increase in antitumor activity and, as a result, to reduce the number of recurrent tumors, reduce the total time of the therapeutic procedure and would allow to combine radiation and photodynamic therapy of deeply located tumors due to leveling of restrictions on the penetrating power of laser light, without increasing damage to surrounding healthy tissues.

The technical result is achieved by the fact that, as in the known method, a combination of remote radiation therapy and photodynamic therapy is carried out.

A feature of the proposed method lies in the fact that Chlorin E6 photosensitizer is administered once intravenously at a dose of 10 mg / kg body weight and 3 hours after the end of drug administration, spectrometry is performed to determine the accumulation of the drug in the tumor relative to healthy tissues, then a distance therapy with electrons on the tumor is performed in a single focal dose of 32 Gy, with a dose rate of 3.3 Gy / min, an energy of 10 MeV and spectral measurements are repeated at the same points, after which the fluorescence values are compared before and after the exposure to the drug and the photosensitizer photoabsorption is evaluated to confirm the photodynamic component therapy arising from the activation of the photosensitizer induced by Cherenkov radiation.

The invention is illustrated by a detailed description, laboratory tests and illustrations, which depict:

FIG. 1 - Diagram and graphical representation of the fluorescence spectra at the points: a - normal skin before radiation therapy (6.4 p.u.), b - skin above the tumor before radiation therapy (13.6 p.u.), c - skin above tumor after radiation therapy (10.2 pu), d - normal skin after radiation therapy (6.1 pu).

FIG. 2 - Growth curves of a tumor of M1 Sarcoma in the control groups, independent radiation exposure and radiation exposure with the administration of Photoditazine (day 0 - day of radiation exposure, 2, 4, 6, 8, 11, 14, 17, 21 - day after exposure) . On a scale of U - tumor volume in cm ³ .

The method is as follows.

Chlorin E6 photosensitizer (for example, Photoditazine, Radachlorin or an analogue) is once administered intravenously to the animal at a dose of 10 mg / kg, spectrometry is performed 3 hours after drug administration to determine the accumulation of the drug in the tumor relative to healthy tissues. Next, a session of remote therapy with electrons on the tumor is carried out in a single focal dose of 32 Gy, with a dose rate of 3.3 Gy / min, an energy of 10 MeV. Immediately after irradiation, spectral measurements at the same points are repeated. Comparison of the fluorescence values before and after exposure allows us to judge the “burnout” of the drug in the process of photodynamic reaction.

The proposed combination therapy option was performed in 8 rats with an implanted subcutaneously from the outside of the left thigh with M-1 sarcoma.

Another 8 animals with a similar tumor model were included in the radiation therapy group in an independent mode. As control animals, there were 8 individuals.

The 1st group of rats "Photoditazine" was injected intravenously into the tail vein at a dose of 10 mg / kg of body weight. Spectroscopy was performed at 3 random points of normal skin (region of the anterior abdominal wall) and 3 points of skin above the tumor with preliminary removal of the coat. The data obtained were summed up with the calculation of the average value in each of the groups. When analyzing the data of normalized fluorescence, FN revealed that tumors accumulate the drug in significantly higher concentrations - 14 rel. units than healthy tissue - 6 rel. units (Fig. 1a, b). Further, the animals of both groups (with and without Photoditazine) were irradiated with electrons on the tumor in a single focal dose of 32 Gy, with a dose rate of 3.3 Gy / min, and an energy of 10 MeV. After that, rats of the 1st group were selected for repeated spectrometry at the points "norm / tumor". When analyzing the obtained values, it was shown that the drug “burned out” after the radiation therapy session, while the fluorescence indices in normal tissue remained at the same level (Fig. 1a, d), which indicates FS activation and its expenditure on the implementation of the photodynamic reaction (a decrease in fluorescence is recorded over the tumor).

Further, animals of all groups were observed in order to study the growth dynamics of Sarcoma M-1. Measurements were taken 1 time in 3 days with registration of 3 tumor sizes. The obtained graphs of the growth curves (Fig. 2) reflect the greater antitumor efficacy of the proposed combined method in comparison with electron irradiation in an independent mode, since radiation therapy affects tumor cells, exerting direct cytotoxic effects on them in the form of damage to the DNA molecule. At the same time, Cherenkov radiation resulting from electron irradiation activates the photosensitizer introduced and selectively accumulated in the pathological focus. This in turn leads to the realization of the effects of photodynamic therapy and, in the first place, damage to the microvasculature of the tumor by free radicals formed at the output of the photo-dynamic reaction. Due to the fact that a period of 2-3 days elapses from the moment of exposure to the damaging factor to the realization of vascular effects, the proportion of more radioresistant hypoxic cells does not increase directly at the time of irradiation due to the effects of stasis and thrombosis. At the same time, an increase in hypoxia in the long term reduces the ability of the tumor to repair DNA strand breaks. In addition, a significant advantage of this method is the possibility of photodynamic effects on deeply located tumors, given that the exciting radiation is formed directly in the epicenter of the tumor in the area of the beam. The exposure time is also reduced due to the simultaneous implementation of both therapeutic options.

When carrying out this method, a double effect on the tumor is realized due to the energy of electrons and the photodynamic reaction resulting from the excitation of the photosensitizer by the induced “Cherenkov” radiation.

The proposed method is fundamentally different from the combination of classical photodynamic therapy with radiation therapy by the absence of the need to deliver light energy to the pathological focus through the use of induced radiation that occurs directly in the tumor when it is irradiated with an electron beam with the specified parameters.

Using the developed method in the clinic leads to an increase in the antitumor effect and, as a result, a reduction in the number of recurrent tumors, a decrease in the total time of the therapeutic procedure, and an increase in the penetration depth due to the leveling of restrictions on the penetrating power of laser light without increasing damage to surrounding healthy tissues. The invention is industrially applicable, since when performing this method, standard medical equipment used in the treatment of cancer patients in specialized medical institutions is used.

Claims (1)

The method of photodynamic therapy, including a single intravenous administration of the Chlorin E6 photosensitizer at a dose of 10 mg / kg body weight, spectrometry 3 hours after the end of the drug administration, a distance electron therapy session in a single focal dose of 32 Gy at a dose rate of 3.3 Gy / min, energy of 10 MeV, repeated spectrometry, after which the fluorescence values are compared before and after exposure to the drug and the “photo-absorption” of the photosensitizer is evaluated to confirm the photodynamic component of therapy resulting from the activation of the photosensitizer by Cherenkov-induced radiation.

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