RU2699670C1 - Method for increasing the frequency of formation of double-strand breaks of dna in human cells under action of ionizing radiations under conditions of radio modifiers - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine. Disclosed is a method for increasing the frequency of double-strand DNA stripping in human cells under the action of ionizing radiations in conditions of radio modifiers. As inhibitors of DNA repair before irradiation, a combination of formal preparations 1-β-D-arabinofuranosylcytosine and hydroxyurea.

EFFECT: invention provides higher effectiveness of radiation exposure on tumor cells and reduced local radiation dose.

1 cl, 3 dwg

Description

The scope of the invention. The invention relates to studies relating to increasing the effectiveness of radiation therapy. The invention is applicable to methods of remote or contact (intracavitary) radiation therapy and radionuclide therapy of malignant neoplasms using various radiation such as X-rays, γ-radiation, neutron radiation, charged particle beams, radiation from decay of radionuclide sources.

The level of invention. Russia ranks fifth in the world in mortality of cancer patients and leads in the number of cancer patients per 100 thousand people. On average, in Russia, as well as around the world, there is an increase in the incidence of cancer by 1.5% per year. According to medical statistics, at present 500 thousand Russians annually fall ill with various forms of cancer, up to 300 thousand die a year. 900 billion rubles will be allocated from the federal budget until 2024 for the program of the fight against cancer.

In the treatment of cancer (malignant tumors), surgical methods, chemotherapy, immunological methods and ionizing radiation are used. As ionizing radiation in radiation therapy, both electromagnetic radiation (gamma rays and x-rays) and particle types of radiation (electrons, neutrons, protons, heavy ions) are used. The use of gamma rays and x-rays is the most affordable method of radiation therapy. A more promising method of hadron therapy for cancer is much more expensive, since it requires the creation of specialized accelerators of charged particles (this is especially true for carbon cancer therapy). Today in the Russian Federation only 60% of the need for radiotherapy is provided, so increasing its effectiveness is a priority. Long fractionated treatment using only radiation (currently in clinical practice the total radiation dose is from 40 to 60 Gy) is difficult due to various adverse reactions. To enhance the antitumor radiation effect, the combined use of chemotherapeutic drugs and radiation is used as one of the methods, which makes the results of this treatment more effective than the separate use of the radiation factor and chemotherapeutic drugs. In recent years, active searches have been carried out for various radiosensitizing agents: inhibitors that prevent cell division and their dissemination, sensitizers that increase cellular radiosensitivity and cause a decrease in the total radiation dose to the tumor.

With the passage of ionizing radiation through sensitive targets of the cells, a wide spectrum of deoxyribonucleic acid (DNA) structure disorders is formed. These damages are formed either due to the direct destruction of covalent bonds of the DNA molecule, or the indirect action of the formed free water radicals. The most serious violations leading to cell death are simultaneous violations of the integrity of two DNA chains — double-strand breaks (DR) [1, 2]. Double-strand breaks are formed either as a result of a direct break of two complementary sites — direct DRs (PDRs), due to the transfer of energy to a local DNA site, leading to a violation of its integrity, or are formed from other injuries as “repair costs” in the process of repair enzymes. This type of damage belongs to the category of enzymatic DR (EDR) [3, 4]. Under the action of rare-ionizing radiation, the output of single-strand breaks (OR) and DR of DNA is correlated as 10: 1. With an increase in linear energy transfer (LET), changes are observed in the spectrum of induced damage to the DNA of cells. At low LET values, damage to the bases and single-stranded DNA breaks are formed to a greater extent. OR DNA is efficiently repaired by cells. Double DNA breaks underlie the formation of chromosomal aberrations and cause cell death.

Radiosensitizing agents according to their mechanism of action are divided into 2 groups: a) enhancing the primary radiation damage to DNA; b) inhibitory processes of post-radiation recovery. For example, cisplatin is a group 1 radiosensitizer, causing the formation of DNA-protein, DNA-DNA crosslinks. An example of the use of a sensitizer of the 2nd group is the combination of radiation therapy with the preliminary administration of 5-fluorouracil. With radiotherapeutic use, this agent disrupts DNA synthesis and causes the formation of structurally imperfect RNA, inhibiting cell division. It inhibits the synthesis of ribonucleic acid by incorporating 5-fluoruridine triphosphate into its structure, instead of uridine triphosphate. This leads to disruption in the processing of ribonucleic acid and protein synthesis. The use of 5-fluorouracil in radiation therapy in the pre- and postoperative period is a common method [5-7], for example, it is a standard treatment for stage II-III rectal cancer. This method (a combination of radiotherapy with the introduction of the radiomodifier 5-fluorouracil) is a prototype of the present invention [8]. However, the effectiveness of the combined use of 5-fluorouracil and ionizing radiation is relatively low. For example, it is indicated [9] that "the combination of radiation therapy with chemotherapy ... in most cases, slightly improves the disease-free and overall survival in patients with common forms of cervical cancer." Therefore, the search for effective sensitizers is currently ongoing.

Disclosure of the invention. Thus, the present invention is the use of an enhancing agent for radiotherapeutic methods of treating tumor diseases, aimed at increasing the frequency of the formation of double-stranded DNA breaks in human cells. As a result, this will increase the efficiency of radiation exposure to tumor cells, reduce the local therapeutic dose of radiation, and reduce adverse side radiation reactions. The present invention provides, as a reinforcing agent for increasing the frequency of the formation of double-stranded DNA breaks in human cells, a 1-β-D-arabino-furanosylcytosine DNA repair inhibitor in combination with a hydroxyurea supplementant that is administered prior to irradiation.

1-β-D-arabinofuranosylcytosine (AraC) is an official drug (trade names cytosar, alexan, cytarabine, cytostadin, cytarabine-LENS, cytastadine, NovaMedica cytosar) and is used in the clinic for the treatment of acute and chronic leukemia. AraC (Russian name cytarabine) is a synthetic nucleoside containing arabinose in the carbohydrate part of the molecule. The chemical name is 4-amino-1-beta-B-arabinofuranosyl-2 (1H) -pyrimidinone. The structural formula of AraC (cytarabine):

The mechanism of action of AraC (cytarabine) is associated with the incorporation of its active metabolite, arabinosidcytosine triphosphate, into the end unit of the DNA molecule during its synthesis during replication, which leads to disruption of replicative processes. In this regard, cytarabine is active only in relation to cells in the DNA synthesis phase (S phase) of the cell cycle. AraC (cytarabine) also blocks the activity of DNA polymerase α, and to a lesser extent β, leading reparative DNA synthesis, as a result of which long-term fixation of single-strand DNA breaks resulting from irradiation occurs. Unrecoverable OR DNAs are sites of the formation of double-stranded DNA breaks as a result of attack of sections of strands opposite to unrepaired ORs by endonuclease type S ₁ . Thus, in the presence of AraC (cytarabine) under the action of ionizing radiation, unlike other radiosensitizers (for example, 5-fluorouracil), single-stranded DNA breaks transform into double-stranded breaks leading to cell death.

Hydroxyurea (GM) is an inhibitor of ribonucleotide reductase and affects the intracellular pool of nucleotides, in particular, cytosine and reduces it [10]. In combination with AraC (cytarabine) can be considered as a complementary agent that enhances the action of AraC (cytarabine). The structural formula of hydroxyurea:

Hydroxyurea is also an official drug and is used in the clinic for the treatment of head and neck tumors, skin melanoma, colon and rectal cancer, cervical cancer, kidney and prostate cancer. Thus, there are no obstacles to the practical use of AraC (cytarabine) and GM in combination with radiation therapy.

To confirm the possibility of using the invention, the modifying effect of AraC (cytarabine) was investigated. It was experimentally established that in cells irradiated with rare- and dense-ionizing radiation, under the influence of AraC (cytarabine) and hydroxyurea, the output of DR DNA in the post-radiation period is modified to varying degrees [11]. Thus, under γ-irradiation under the conditions of their influence, the yield of DR DNA increased significantly during the post-radiation incubation of human lymphocytes and cells in culture. At the same time, when the cells were irradiated with accelerated heavy ions, the modifying effect of the agent was much smaller or absent.

The effectiveness of the modifying effect of AraC (cytarabine) + GM was established by studying the frequency of formation of DR of DNA in various human cells irradiated with a therapeutic proton beam at the Bragg peak without the introduction of modifying agents and with their preliminary introduction before irradiation. Experimental studies were performed on proton beam No. 1 of the Medical and Technical Complex at the phasotron of the Laboratory of Nuclear Problems of the Joint Institute for Nuclear Research, Dubna. The therapeutic proton beam is formed by decelerating the extracted beam of a phasotron with an energy of 660 MeV to an energy of 150-200 MeV using a carbon degrader with subsequent collimation of the beam and magnetic analysis. During the experiments, a proton beam with an energy of 170 MeV created a uniform irradiation field of 10 × 10 cm in size. Then, using a absorber and a comb filter in the irradiation zone, a modified Bragg peak up to 2 cm wide was formed (in water and biological tissue) at a level of 90% maximum dose. In the LET spectrum, the contribution to the absorbed dose of protons with low LET (2-25 keV / μm) was ~ 67%, with LET 25-50 keV / μm ~ 23% and with high LET (50-100 keV / μm) ~ 10% . The dose rate during irradiation was about 1.5 Gy / min. Thus, the experiment completely simulated the irradiation of deep-seated local neoplasms in a patient.

Normal human skin fibroblasts (NHDF 22873, Lonza, CC-2509) were cultured in IMDM medium (Sigma-Aldrich, Germany) with the addition of 10% fetal calf serum (Sigma-Aldrich, Germany) and 1% gentamicin-glutamine solution (Sigma-Aldrich, Germany) in plastic bottles with a growth surface area of 25 cm ² (Corning, USA) at 37 ° С and a 5% CO ₂ content in the atmosphere. For all experiments, cells were used at passage 9-12. 16-18 hours before irradiation, 300 μl of the cell suspension was applied to coverslips with a diameter of 14 mm and a thickness of 0.17 mm placed in plastic Petri dishes with a diameter of 35 mm (MatTek Corporation, P35G-0.170-14-C, USA). After 2 hours of incubation, 3.5 ml of culture medium was added to the attached cells. 1 h before irradiation, AraC (Sigma-Aldrich, Germany) and GM (Sigma-Aldrich, Germany) were added to the fibroblast culture medium at a final concentration of 20 μM and 2 mM, respectively, after which the cells were incubated in an incubator at 37 ° C for 1 hour

In experiments on a medical proton beam, the proton beam before crossing the cell monolayer passed through the glass bottom of a Petri dish (MatTekCorporation, P35G0.170-14-C, USA) 170 μm thick at an angle of 10 °, i.e. through 980 μm glass with ρ = 2.58 g / cm ³ . The energy spectrum of slow protons at the Bragg peak in this case extended from 0 to ~ 44 MeV.

To quantify the frequency of formation of DR DNA in individual cell nuclei and their visualization under the action of radiation under ordinary conditions and in the presence of AraC (cytarabine) + GM, an immunocytochemical method was used to determine γH2AX / 53 BP1 foci. The DNA focus method is based on the use of individual enzymes labeled with fluorescent dyes involved in the repair of DR DNA. When using a confocal microscope in the nuclei of cells, it is possible to register fluorescent DNA foci that reflect the sites of formation of DNA DR. This method allows us to study not only the quantitative aspects of the formation and elimination of radiation-induced foci (RIF), since the focus output is proportional to the number of DRs, but also to analyze the structure of complex DNA damage. As a result of studies, it was shown that the introduction of AraC (cytarabine) + GM before irradiation with protons leads to a sharp increase in the frequency of formation of DR in the post-radiation period.

Irradiation of human cells (skin fibroblasts and glioblastoma cells) in the absence and presence of AraC (cytarabine) + GM was also carried out on a beam of boron-11 nuclei at the U400M cyclotron of the JINR Laboratory of Nuclear Reactions. Boron nuclei (B-11) with an energy of 8 MeV / nucleon (LET = 139 keV / μm) at a dose rate of ~ 2 Gy / min were irradiated with monolayers of cells in Petri dishes with a diameter of 14 mm. Irradiation of samples was carried out on a Genom-M unit at a dose of 1 Gy. This experiment actually reproduced the irradiation conditions of human cells at the Bragg peak during carbon therapy due to the proximity of the physical characteristics of B-11 and C-12.

Similar experiments were carried out when cells were irradiated with Co-60 gamma-quanta on the Rocus-M medical irradiation facility. An increase in the number of DNA DRs in the presence of AraC (cytarabine) and GM upon γ-irradiation, as was shown by us earlier [12], is accompanied by a more than twofold increase in the radiosensitivity of Chinese hamster cells in culture, evaluated by their survival.

The results obtained are well demonstrated by figures 1-3.

Description of figures:

The figure 1 presents the image of individual γH2AX / 53 BP1 foci in the nuclei of human cells at different times after irradiation with protons and accelerated boron ions in doses of 0.6 Gy and 1 Gy, respectively, without modifiers and in the presence of AraC (cytarabine) + GM. DNA damage is visible as bright dots. The arrows in the images show complex lesions, including multiple DNA DRs, formed as “tracks” along the charged particle. It is clearly seen that 24 hours after irradiation, the amount of DNA damage during proton irradiation with the preliminary introduction of AraC (cytarabine) + GM is much larger than the number of damages formed as a result of irradiation with protons and nuclei without the introduction of radio modifiers.

Figure 2 shows the kinetics of the formation and elimination of γH2AX / 53 BP1 foci in the nuclei of human cells upon irradiation with protons at the Bragg peak in the presence and absence of AraC (cytarabine) + GM radio modifiers. The ordinate shows the number of tricks per cell. The abscissa is the time after irradiation. On the left are the results for a dose of 0.6 Gy, on the right for a dose of 1.25 Gy.

From figure 2 it can be seen that without AraC (cytarabine) + GM, the largest number of foci (25 and 35 per cell, respectively) is formed 1 hour after irradiation. After 24 hours, their number decreases to a minimum (~ 5 per cell). A completely different type of kinetics of the formation of γH2AX / 53 BP1 foci is observed upon irradiation of cells with protons in the presence of AraC (cytarabine) + GM. Within 24 hours of post-radiation incubation of the cells, there is not a decrease in the number of RIFs, but their sharp increase to 65 per cell.

Figure 3 shows a comparison of the kinetics of the formation and elimination of γH2AX / 53 BP1 foci in the nuclei of human cells upon irradiation with protons and boron-11 nuclei at the Bragg peak in the presence and absence of AraC (cytarabine) + GM radio modifiers. The ordinate shows the number of tricks per cell. The abscissa is the time after irradiation. The yield of γH2AX / 53 BP1 foci in the absence of AraC (cytarabine) + GM radio modifiers when irradiated with boron-11 nuclei in the Bragg peak is higher than expected when compared with protons irradiated with Bragg peak at comparable doses (45 and 35 per cell, respectively, after 1 h after irradiation and ~ 20 and 5 foci per cell, respectively, 24 hours after irradiation). However, the introduction of AraC + Gm makes proton irradiation significantly more effective than nuclear irradiation in the post-radiation period (up to 65 per cell 24 hours after irradiation).

Thus, the use of AraC (cytarabine) + GM before irradiation can lead to an increase in the number of lethal DNA damage in tumor cells in the post-radiation period, i.e. to increase the effectiveness of radiation therapy. This effect is manifested not only during radiation therapy, but will also occur during radionuclide therapy, when (orally, intravenously, intracavitary or interstitial tissue) are introduced radiopharmaceuticals that act directly on pathological foci, since the mechanism of the damaging effect of radiation on DNA chains in all cases is the same . Summing up, it can be assumed that the combined use of used official drugs with radiation of rare-ionizing radiation is promising for use in the clinic.

Literature

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Claims (1)

A method of increasing the frequency of the formation of double-stranded DNA breaks in human cells under the action of ionizing radiation under the influence of radio modifiers, characterized in that a combination of official preparations of 1-β-D-arabinofuranosylcytosine and hydroxyurea is used as inhibitors of DNA repair before irradiation.

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