RU2189232C2 - Antimutagenic agent - Google Patents

Abstract

FIELD: medicine, pharmacology. SUBSTANCE: invention relates to the use of iodoantipyrine as an agent decreasing the mutagenic effect of cyclophosphane. Using of said preparation ensures to provide an antimutagenic effect. EFFECT: valuable property of agent. 1 cl, 17 tbl

Description

 The invention relates to medicine, namely to pharmacology, and relates to agents with antimutagenic effect.

 Prevention of mutagenic effects is one of the most pressing problems of medicine. Its implementation provides for both measures aimed at limiting human contact with mutagens, and the development of means to protect heredity from mutagenic effects (1).

 Induction of mutations is a complex, multi-stage process, the components of which are the entry, distribution, bioconversion and excretion of a genetically active agent, its accumulation in the region of the cell and the target molecule, interaction with DNA and the effect on reparative and replication systems. The effect on any of these stages of the formation of mutations can modify the effect of the mutagen. On this basis, almost all the existing classifications of antimutagens have been created (2). Antimutagenic properties were found in many natural and synthetic compounds: vitamins, amino acids, natural metabolites, food additives, drugs, and other substances of an organic and inorganic nature.

 However, most of the work on the study of antimutagenesis was carried out in vitro, most often on microorganisms, which significantly reduces the prognostic value of this information, complicates the interpretation of experimental data and their extrapolation to humans.

 In the last decade, interest in the problem of antimutagenesis has rapidly increased and research in this area has intensified. In the performed studies, the dependence of the severity of the antimutagenic effect on the antimutagen use scheme, on the specificity of the existing mutagenic factor, was established.

 The search for antimutagens in each case is an independent pharmacological task.

 Closest to the proposed antimutagenic agent (prototype) is interferon. It was found that γ-interferon reduces by 1/3 the total level of chromosomal aberrations induced in the bone marrow cells of mice 24 hours after the administration of 20 mg / kg of cyclophosphamide (3).

 Attempts to find stronger antimutagenic agents under the influence of the cyclophosphamide preparation widely used in oncological practice have led to the discovery that antimutagenic properties were found in a preparation with the already known properties of iodantipyrine under the above conditions.

 One of the directions in the search for new antimutagenic agents is the study of drugs with already known effects as drugs with antimutagenic effects.

 The problem solved by the invention is to expand the arsenal of funds with antimutagenic action.

 This object is achieved in that iodantipyrine is used as an antimutagenic agent.

 The use of iodantipyrine as an antimutagenic agent was made possible thanks to experimental studies of the protective effect of iodantipyrine on the genome of bone marrow cells of CBA / Calac mice under the conditions of the introduction of the most common mutagenesis inducer, cyclophosphamide. Studies have revealed a new (antimutagenic) property of iodantipyrine.

 It is known that iodantipyrine (1-phenyl-2,3-dimethyl-4-iodopyrazolone) is an active inducer of late-type β-interferon, has anti-inflammatory, antiviral and immunomodulating properties along with the absence of embryotoxic, immunotoxic and allergenic effects. The greatest activity of the drug is manifested in relation to the non-polio enteroviruses Koksaki and Esno, viruses of vesicular stomatitis, tick-borne encephalitis, influenza and parainfluenza. It is recommended for the treatment and prophylaxis of tick-borne encephalitis, influenza, and in the treatment of urogenital chlamydia (4, 5).

 The experiments were performed on 230 male mice of the CBA / Calac line at the age of 2 months weighing 18-20 g (nursery "Dawn", Tomsk, certificate is available). Cyclophosphamide (CF) (Saransk plant "Biochemistry") was used as an inducer of mutagenesis, which was dissolved ex tempore in a sterile isotonic sodium chloride solution and was administered intraperitoneally once at doses of 10 mg / kg, 20 mg / kg, 40 mg / kg and 1 / 2 MTD (125 mg / kg) calculated by the method of graphic probit analysis (6) when observing animals for 30 days. Iodantipyrine (IA) (AOOT, Aspharma) at a dose of 150 mg / kg was dissolved in 1% starch gel and was administered intragastrically in a volume of 0.2 ml through a probe three times 2 days before the introduction of mutagen, combining the last injection of the modifier with CF injection (2) .

 Animals were sacrificed under ether anesthesia with the dislocation of the cervical vertebrae. Material for the study was taken after 6 and 24 hours, as well as on the 2nd, 3rd, 4th, 5th, 7th, 10th day after the combined administration of drugs. Comparison of experimental animals was carried out with the corresponding values in intact mice (background), as well as in animals that received only CF (positive control).

 Bone marrow cell metaphase chromosome preparations were prepared by the conventional dry air method, as described previously (7), using colchicine at a rate of 2.5 mg / kg in order to accumulate metaphases. Cytogenetic analysis (10 × 10, Biolam I microscope) took into account cells with structural chromosome abnormalities (single and paired fragments, chromosome exchanges, cells with multiple chromosome damage — more than 5 chromosomal aberrations in the cell), metaphases with a change in the number of chromosomes were recorded. Genes were recorded, but not taken into account as aberrations. For each animal, 50 metaphase plates with a good spread of chromosomes were analyzed, 5-6 animals were examined in each group, the background group was 10 individuals.

 The total number of cells (BCC) of the bone marrow was determined, myelogram counts (8), and the number of red blood cells with micronuclei in the peripheral blood (9) were estimated by conventional methods. The data obtained were processed by the method of variation statistics using the Statgrafics static software package.

 It was found that IA has a pronounced antimutagenic effect at all used doses of CF. The maximum decrease (by 55.2%) in the number of cells with cytogenetic abnormalities after 24 hours of experiment was observed with the introduction of CF at a dose of 10 μ / kg (Table 3). This indicator decreased by 41.3% with a CF dose of 20 mg / kg and by 38.75% with a CF dose of 40 mg / kg (Tables 4 and 5, respectively). It should be emphasized that the introduction of IA led to a drop in the level of cytogenetically defective cells (to background values) by the 48th hour of the experiment. At the same time, when using one CF, the number of cells with cytogenetic disorders remained significantly higher than the background (Tables 3, 4, 5).

 For a preliminary assessment of the proportion of “yield” of aberrations in the cells of various bone marrow growths, myelogram analysis was performed. So, with the introduction of CF at a dose of 10 mg / kg, inhibition of bone marrow hematopoiesis was not observed, OCC was within the background during the entire observation period (Table 2). In this case, a decrease in the absolute content of erythroid cells in the bone marrow was compensated by irritation of the granulocytic germ of hematopoiesis. It can be assumed that the registered aberrations in bone marrow cells in this case are the result of mitotic activity of the elements of the myeloid series (Table 10). An indirect confirmation of what has been said can be the absence of an increased “exit” of red blood cells with micronuclei to peripheral blood (Table 1).

 Administration of CF to mice at a dose of 20 mg / kg led to a decrease in OCC after 24 and 48 hours of experiment by 26.6 and 23.9%, respectively (Table 2). The use of IA in this case allows preventing the drop in OCC due to an increase (in comparison with one CF) of mitotic activity and the number of erythro- and granulocytopoiesis elements (Tables 8, 9).

 Administration of CF at a dose of 40 mg / kg led to increasing inhibition of bone marrow hematopoiesis (after 24 h, OCC decreased by 23% and after 48 h - by 48% of the initial level) - (Table 2). The introduction of IA in this case did not change the direction of the process, but had an antimutagenic protective effect on bone marrow cells. After 48 hours of the experiment, the CCC was significantly (p <0.01) 21% higher than that with the administration of one CF, mainly as a result of a higher level of lymphoid elements (Tables 2, 6, 7).

 In a special series of experiments, a study was performed on CBA / Calac mice of the effect of IA on the bone marrow cell genome under conditions of cytostatic myelodepression caused by the introduction of CF in 1/2 MPD (125 mg / kg).

 The study showed that the introduction of CF in 1/2 MTD (125 mg / kg) led to deep inhibition of bone marrow hematopoiesis (table. 13). After 24 hours of the experiment, the total number of myelocaryocytes was only 30% of the initial values. During this period, the maximum number of cells with cytogenetic disorders in the bone marrow was detected (45.00 ± 4.08%; p <0.001). Cells with changes in the structure of chromosomes were 38.80 ± 4.27% (p <0.001), of which 21.20 ± 3.44% were metaphases with multiple chromosome breaks; the number of cells with a change in the number of chromosomes (metaphases with 38, 39, 41, 42 chromosomes, polyploids) reached 6.80 ± 1.50% (p <0.001) - (Table 17). The data obtained are consistent with the results obtained previously (7, 10).

The maximum decrease in the total number of myelokaryocytes was observed on the 3rd day of the study (on average by 88% of the initial level) - (Table 13). The number of cells with cytogenetic disorders in the bone marrow in this case was practically impossible to calculate due to significant bone marrow hypoplasia. From the 4th day of the study, the restoration of the total number of myelokaryocytes was noted, which was accompanied by a sharp (more than 10-fold) increase in the number of mitoses of cells of the myeloid and erythroid rows of the bone marrow (0.31 ± 0.07 • 10 ⁶ / hip and 0.33 ± 0.06 • 10 ⁶ / hip, respectively) - (tab. 14). By the 7th day of the experiment, the total number of myelokaryocytes was 74% of the initial values (Table 13), the content of immature and mature neutrophilic granulocytes was completely normalized, but pronounced inhibition of the erythroid hematopoietic stem remained (Table 14). Along with this, a repeated peak in the increase in cytogenetically defective cells was observed in the bone marrow - up to 8.80 ± 2.40% (p <0.001) (Table 17). The yield of aberrant cells in this case was apparently associated with the activation of proliferation of the original hematopoietic cells in the post-cytostatic period (11).

Due to the fact that on the 7th day of the experiment the cells of the myeloid series made up the overwhelming majority of bone marrow nuclei and the number of their mitoses (0.10 ± 0.01 • 10 ⁶ / thigh) was 3 times higher than the number of erythrokaryocyte mitoses (0.03 ± 0.001 • 10 ⁶ / thigh), it is logical to say that the registered aberrations at the indicated time of the study were the result of mitotic activity of the elements of the myeloid and not the erythroid series (Table 14). An indirect confirmation of the aforesaid can be the absence of an increased "exit" of red blood cells with micronuclei to the peripheral blood after the second peak of an increase in the number of cells with cytogenetic disorders in the bone marrow under the influence of CF in 1/2 MPD (Table 12).

 With the combined introduction of CF and YA in the bone marrow of mice, there was a noticeable change in the dynamics of the number of cells with cytogenetic disorders. With a slight difference in the number of aberrant metaphases, a statistically significant decrease in this indicator was observed after 24 hours of the experiment, after 6 hours on the 4th day of the study, by almost 70 and 74%, respectively (Table 17). At the same time, 24 hours after the joint use of YA and CF, a decrease in the frequency of chromosome damage per cell was recorded. Thus, the number of cells with multiple ruptures was 13.20 ± 3.87%, which is 37.7% lower than the corresponding value with the introduction of a single mutagen. By the 5th day of the experiment, the number of cells with cytogenetic disorders in animals of this group did not differ from the initial level (Table 17).

 Analysis of myelograms showed that an increase in OCC in mice after combined administration of CF and YA was due to the activation of almost all hematopoietic sprouts (Table 16). Moreover, the number of mitoses in the cells of the myeloid and erythroid rows was significantly (p <0.05; <0.001) higher than similar values under the action of a single cytostatic agent. The established fact of the absence of a repeated peak in the increase in the number of cells with cytogenetic abnormalities in the bone marrow in animals treated with IA against the background of the action of cytostatic should be recognized as important. The level of aberrant cells on the 7th day of the study in animals of this group did not differ from the initial one (Table 17). It should also be noted that the content of red blood cells with micronuclei in the peripheral blood of mice treated with CF exceeded their average blood level from 2 to 5 days inclusive, varying between 3.45-3.90% (Table 12 ) The use of IA in this case led to a significant decrease in this indicator. It must be emphasized that the introduction of IA into intact animals did not reveal its effect on spontaneous mutagenesis (Table 17). However, in the first 24 hours, the cellularity of almost all bone marrow growths decreased (Table 15).

 Thus, it was found that IA reduces the mutagenic effect of CF on hematopoietic cells of the bone marrow during the post-cytostatic regeneration of hematopoietic tissue. This, in turn, leads to an acceleration in the rate of restoration of bone marrow cellularity. Based on the kinetics of the restoration of hematopoiesis after exposure to cytostatics (11), it can be assumed that the effect of the drug is realized at least at the level of hematopoietic precursors. In this case, the mechanism of the antimutagenic effect is apparently associated with the induction of interferon, which has an inhibitory effect on the proliferation of hematopoietic progenitor cells (12) and a stimulating effect on excision DNA repair (13), which may have a protective effect under the conditions of CF application at high doses .

 Thus, the data presented convincingly indicate the possibility of using iodantipyrine as an antimutagen and expanding the scope of this drug.

LITERATURE
1. Durnev A. D., Seredenin S. B. Modern principles of protection of human heredity. - Ross. Nawion. congress "Man and medicine". - Moscow, April 21-25. 1998.-S.331.

 2. Seredenin S. B., Durnev A. D. Pharmacological protection of the genome. - M.: VINITI.-1992.-160 p.

 3. Ingel F.I., Sakharova T.A., Khripach L.V., Revazova Yu.A. // Bulletin of Ross. Acad. Honey. Sciences.-1993.- 3.-S.20-23.

 4. Yavorovskaya V.E., Saratikov A.S., Fedorov Yu.V., Evstropov A.N., Solyanik R.G., Anosova G.V., Prishchep TP, Gritsenko L.N. / / Q. Virology.-1994 .- 3.-C.136-138.

 5. Yavorovskaya V.E., Gritsenko L.N., Petrov S.A., Evstropov A.N. // Vopr. Virology.-1994 .- 4.-C.184-187.

 6. Litchfield G., Wilcokon F.G. // Pharma col. Expte. Jherap.-1949.-T.96. -S.99-113.

 7. Khlusova M.Yu., Novitsky V.V., Goldberg E.D. // Experiment. and clinic. Pharmacology.-1992.-T.55.- 2.-s. 49-52.

 8. Goldberg E. D., Dygay A. M., Shakhov V. P. Methods of tissue culture in hematology. Tomsk, 1992.

 9. Schlegel R., Mac Gregor G.T.//Mutat Res.-1983.-113.- 6.-P.481-487.

 10. Goldberg E.D., Dygay A.M., Zhdanov V.V. The role of the hematopoiesis-inducing microenvironment in the regulation of hematopoiesis in cytostatic myelosuppression.-Tomsk, 1999.

 11. Kinetic aspects of hematopoiesis / Ed. G.I.Kozintsa, E.D. Goldberg. Tomsk, 1982.

 12. Goldberg E.D., Dygay A.M., Udut V.V. and other Regularities of the structural organization of life support systems in the norm and in the development of the pathological process.-Tomsk, 1992.

 13. Yakubovskaya E.L., Makedonov G.L., Zasukhina G.D.// Dokl. USSR Academy of Sciences. 1987. -T.294.- 5.-S.1240-1242.

Claims (1)

 The use of iodantipyrine as a means of reducing the mutagenic effect of cyclophosphamide.

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