RU2337693C1 - Agent limiting lymphotoxin effect of methotrexate - Google Patents

Abstract

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to application of miliacin as agent limiting lymphotoxin effect of methotrexate.

EFFECT: extended range of agents limiting lymphotoxin effect of methotrexate.

3 tbl, 8 dwg, 1 ex

Description

The invention relates to medicine, namely to oncology and immunology, and may find application in immunorehabilitation after the use of immunosuppressants.

The folic acid antagonist methotrexate (MT) used in clinical oncology is a classic immunosuppressant (Yushkov V.V., Yushkova T.A., Kazyanin A.V., 2002). This side effect limits the use of MT, reduces the duration of treatment, and makes it difficult to prescribe repeated courses. It seems promising the use for this purpose of biologically active substances of plant origin with immunomodulatory effects and minimal side effects. Among the latter, triterpenoids as substances with pronounced immunotropic activity deserve attention. In particular, the stimulating effect of soap tree triterpenoids on the production of antibodies to envelope antigens of the influenza virus (S. Behboudi et al., 1996), to the Epstein-Barr virus in high- (CBA; H-2K) and low-response (Balb / c; H-2D) lines of mice (E. Dotsika et al., 1997), which is associated with increased activity of antigen-presenting cells and T _x1 lymphocytes (S. Behboudi et al., 1997; B. Morein et al., 1998). The data obtained in recent years indicate the manifestation of an immunostimulating effect in other triterpenoid saponins (S. Loganbal et al., 2000), as well as in the triterpenes themselves - glycerrhizic and urosolic acids and their derivatives (T.N. Il'icheva et al. , 2001). Despite the extensive biological properties of triterpenoids, there are no published data on the limitation of the lymphotoxic effect of methotrexate.

In this regard, the search for funds aimed at limiting the lymphotoxic effect of MT and increasing the effectiveness of immunorehabilitation after its use is relevant.

Among the triterpenoids that have found practical application in medicine, is miliacin, which is part of millet oil (Olifson L.E., Osadchaya N.D., Nuzov B.G. et al., 1991), used to treat trophic ulcers, infected wounds and some other diseases (B.G. Nuzov et al., 2001). It has been experimentally shown that, having a wide spectrum of biological activity, miliacin stimulates nonspecific protection factors and prevents their severe depression in conditions of toxic damage (M.M. Pavlova, 1984), causes hypercellular lymphoid organs (Kirillova A.V., Korneev G.I. ., 2003), stimulates the immune response (Kirillova A.V., Skachkov M.V., Panfilova T.V. et al., 2003), has antioxidant activity (Panfilova T.V., Shtil A.A., Frolov B.A., 2006) and limits glucocorticoid-induced lymphocyte apoptosis (Panf TV fishing, Calm AA Polosuhina ER et al., 2003).

The novelty of the invention is the property of plant triterpenoid miliacin to weaken the lymphotoxic effect of methotrexate and to provide faster restoration of morphological parameters in the central (thymus, bone marrow) and peripheral (spleen) organs of the immunogenesis system.

A significant difference of the invention is that miliacin is used as a means of limiting the lymphotoxic effect of methotrexate.

The proposed tool 3-β-methoxy-Δ18-oleanen (mylacin) belongs to the group of natural cyclic triterpenoids, is contained in millet oil and is a white substance with a melting point of 285-286 ° C. It is optically active, insoluble in water, slightly soluble in ethyl alcohol, diethyl ether, acetone, soluble in chloroform. Milyacin has good tolerance in the range of doses from 2 to 1000 mg / kg. The LD _{50 of} this compound is more than 1000 mg / kg (Olifson L.E. et al., 1991), which indicates the absence of toxic properties.

The chemical structure of the pentacyclic triterpenoid - miliacin (3-β-methoxy-Δ18-oleanene)

Experiment description

The experiments were performed on 219 male mice (CBAxC ₅₇ Bl ₆ ) F ₁ , divided into 4 groups: 1) intact mice (14 animals); 2) mice treated with MT (74 animals); 3) mice treated with MT followed by administration of the solvent of miliacin - Tween-21 in physiological saline (1.6 × 10 ^-7 mol / kg; 71 animals), and 4) mice treated with MT followed by the administration of miliacin (60 animals). MT was administered once intraperitoneally at a dose of 10 mg / kg. Miliacin or solvent was administered intraperitoneally in a single dose of 2 mg / kg 3 times for 3 days: 1 hour after MT injection and the next 2 days. Animals were taken out of the experiment on days 4, 7, 10, 14, and 21 after the introduction of MT by dislocation of the cervical vertebrae in compliance with the "Rules for the work using experimental animals." The mass of lymphoid organs (thymus, spleen) was determined, then the organs were fixed in 10% neutral formalin, histological preparations were prepared according to the standard method and stained with hematoxylin and eosin. The areas of the cortical and medulla of the thymus, the T- and B-dependent zones of the spleen, as well as the cell density in these areas were determined using a morphometric grid (G. Avtandilov 1973). From each organ, 1-5 preparations (sections) were prepared, 10 visual fields (70 × 70 μm) were analyzed in each preparation. The number of myelocaryocytes (from the femur) was determined and the relative content of cells of various hematopoietic growths in smears of bone marrow stained by Pappenheim was evaluated. At least 500 karyocytes were counted in each smear. The data were processed by standard methods of variation statistics from the Microsoft Excel application package using the Student t-test.

The results are presented in tables 1, 2 and 3 and graphs for them, reflecting the effect of miliacin on the morphological features of the thymus, spleen and red bone marrow of mice exposed to methotrexate.

In the early period (4th day) after MT administration, a sharp thymus hypoplasia occurs, accompanied by a decrease in organ mass (by 72.6 ± 2.3% of the values in the control group), and its cerebral area (by 57.7 ± 2.6 %) and cortical (by 73.7 ± 2.1%) zones with a decrease in the density of cell infiltrate in them by 41.0 ± 2.5% and 36.4 ± 1.5%, respectively (Table 1 and graphs 1a, 1b, 1c in Fig. 1, 2, 3). In these zones, cells of various sizes appeared with the formation of disoriented cords. The most pronounced changes were recorded in the brain zone, where fragmentation of nuclei, the formation of cell conglomerates (from 5-6 cells), forming 3-4 such foci, were noted in individual cells. On the 7th day of observation, there was a slight restoration of cell density in the brain and cortical areas with a continuing picture of degenerative changes. By 10 days, the growth of the studied parameters was more pronounced, and by 14 days the cell density in the area of the medulla reached the initial level. However, the normalization of the density of cellular infiltrate in the cortical zone was detected only on 21 days. A complete restoration of the morphological picture of the thymus was not noted even by this time, since the indicators of the areas of the studied zones and, accordingly, the mass of the organ remained reduced.

The introduction of a solvent (tween-21) against the background of MT did not affect the severity of the cellular emptying of the thymus and its weight reduction, nor the subsequent dynamics of these indicators.

On the 4th day, in the thymus of mice receiving mylacin during MT administration, a significantly smaller decrease in the area of the brain (by 47.9 ± 4.8%) and cortical (by 51.5 ± 2.4%) substances was recorded (Figure 16) ), a less pronounced decrease in cell density in the brain (by 16.1 ± 3.2%) and cortical (by 19.6 ± 2.0%) zones (graph 1c) and, accordingly, a significantly smaller decrease in its mass (by 56.0 ± 1.5%) compared with animals that received only MT or MT with a solvent (graph 1a). Cytological abnormalities in the form of nuclear degeneration, the formation of cell strands and cell conglomerates were recorded much less frequently. On the 7th day of observation, the area of cortical substance in mice treated with miliacin was 2 times higher than in animals exposed only to MT or MT with a solvent. A similar shift was noted for the thymus medulla, whose area on day 7 in animals treated with MT + miliacin was 33.3% higher than that in mice of groups 2 and 3. In group 4, the cells of these zones had clear boundaries and almost the same sizes without signs of degenerative changes in the nuclei. A similar pattern was revealed over the next observation period. As a result, the density of cell infiltrate was restored by 14 days not only in the brain, but also in the cortical zone of the organ; by 21 days the sizes of the areas of these zones and the mass of the thymus returned to normal.

On the 4th day after MT administration, a decrease in spleen mass was recorded (by 36.6 ± 4.0% compared with intact animals) and a decrease in the areas of B-dependent (by 45.2 ± 4.4%) and T-dependent (by 77 9 ± 3.8%) of the organ zones (Table 2 and graphs 2a, 2b, 2c in Figs. 4, 5, 6). This effect was accompanied by a decrease in cell density in these zones, respectively, by 36.2 ± 1.8% and 26.6 ± 1.8% (graph 2c in Fig. 6). By the 7th day, the density of cell infiltrate was restored and the area of T- and B-dependent spleen zones increased (graph 2b in Fig. 5). However, if the area of the T-dependent zone reached the initial level, the area of the B-dependent zone remained somewhat reduced (by 17.3 ± 5.6%). It is noteworthy that on the 10th day after the introduction of MT, the cell density in the B-dependent zone continued to increase to values exceeding the initial level by 11.7 ± 3.2%. This shift was accompanied by an increase in spleen mass, which by the indicated observation period exceeded the initial value by 23.2 ± 4.1% (graph 2a in Fig. 4). Starting from the 14th day of observation, the morphological features of the spleen in animals of this group corresponded to those in intact mice.

The introduction of the solvent did not lead to significant changes in the studied parameters and their dynamics compared with animals that received only MT.

In the group of animals treated with miliacin after MT, on the 4th day of observation, a significantly smaller decrease in spleen mass was noted (only 12.4 ± 4.5% compared with intact animals), the practical absence of reduction of the B-dependent area of the organ and less significant an indicator of a decrease in its T-dependent zone (by 63.0 ± 2.7%) than in mice of groups 2 and 3 (graphs 2a and 2b in Figs. 4 and 5). Against the background of the use of triterpenoid, less pronounced cellular devastation of the B-dependent zone (16.7 ± 3.3%) of the spleen was recorded than in mice that received only MT or MT with a solvent (graph 2c in Fig. 6). The protective effect of miliacin was also manifested in relation to the restriction of the cellular emptying of the T-dependent zone of the spleen (decrease in the number of cells by 12.2 ± 1.8%). In the subsequent periods of observation, the positive effect of mylacin on the morphological parameters of the spleen of animals treated with MT was not manifested in connection with the actual restoration of these parameters in mice of groups 2 and 3. Moreover, mylacin did not prevent an increase in the density of cell infiltrate in the B-dependent area of the organ (graph 2c) in Fig. 6) and did not affect the increase in spleen mass on the 10th day of observation (graph 2a in Fig. 4).

MT caused devastation of the red bone marrow, which was expressed in a sharp decrease in the content of myelocaryocytes, which amounted to 40.5 ± 5.7% on intact animals on the 4th day of observation (Table 3 and graphs 3a and 3b in Figs. 7 and 8). Starting from day 7, the recovery of the myelokaryocyte pool was recorded, which was completed by 10 days of observation (graph 3a in Fig. 7). The use of the solvent did not affect the severity of the cellular emptying of the red bone marrow, nor the subsequent dynamics of its recovery. In contrast, miliacin exerted a pronounced protective effect, expressed, first of all, in a less significant decrease in myelokaryocytes on the 4th day of observation (9.8 ± 3.5%) and a much faster recovery of this indicator by 7 days. Moreover, under the influence of miliacin, red bone marrow hyperplasia was observed on the 7th, 10th and 14th day of observation: the number of myelocaryocytes exceeded that in intact animals, respectively, 15.7 ± 3.7%, 16.7 ± 3.8% and 17.7 ± 1.7%.

Analysis of the relative content of lymphocytes on the 4th and 7th day of observation did not reveal a statistically significant decrease in animals treated with MT + solvent and animals treated with MT alone. In contrast, group 4 mice (MT + miliacin) showed a significant decrease in this indicator, amounting to 21.6 ± 6.6% on day 4 and 36.6 ± 5.8% of the corresponding indices on day 7 in intact animals. On the 10th day of observation, a decrease in the relative content of lymphocytes was noted in all groups of animals, and by the 14th day it was restored to the level determined in intact mice (graph 3b in Fig. 8).

Assessing these results, it should be noted that the absence of a decrease in the relative content of lymphocytes in animals of groups 2 and 3 occurred against the background of cellular depletion of bone marrow and, therefore, could not be the result of increased proliferation and differentiation of bone marrow lymphocyte precursors. The basis of this phenomenon is the migration of extra bone marrow lymphocytes into the red bone marrow, mainly of a thymic origin, which is necessary to stimulate suppressed hematopoiesis. Such migration is known as the reaction of lymphoid tissue under the influence of stress factors of a physical and chemical nature (including cytotoxic drugs) of nature that have a pronounced myeloinhibitory effect (Goldberg E.D., Dygay A.M., Karpova G.V. 1983). Myliacin limited this shift, reducing the severity of cellular depletion of bone marrow and thymus. The decrease in the relative content of lymphocytes on the 10th day of observation in group 4 reflects the number of own karyocytes, since by the indicated time the “migrant” lymphocytes in the red bone marrow are no longer detected (Goldberg E.D., Dygay AM, Zhdanov V.V., Khlusov I .A. 1999). The result of the restoration of the relative content of lymphocytes and red bone marrow hyperplasia in animals treated with MT + miliacin was a significant increase in the absolute number of lymphocytes by 14 days of observation (graph 3b in Fig. 8).

Thus, the antitumor drug methotrexate exhibits a pronounced lymphotoxic effect, characterized by cellular depletion of the bone marrow and lymphoid organs, a decrease in the corresponding areas of the distribution of lymphocytes and the density of cell infiltrate. This effect is more pronounced in the T-link of immunity (by the degree of decrease in the studied parameters and the duration of their depression). The plant-derived triterpenoid miliacin weakens the lymphotoxic effect of methotrexate and provides a more rapid restoration of morphological parameters in the central and peripheral organs of immunogenesis. The obtained results expand the understanding of the processes that determine the protective effects of miliacin in the body and substantiate the potential of this compound as an immunocorrector.

Literature

1. Avtandilov G.G. Morphometry in pathology. M .: Medicine, 1973. 248 p.

2. Goldberg E.D., Dygay A.M., Karpova G.V. The role of lymphocytes in the regulation of hematopoiesis. Tomsk 1983. 159 p.

3. Goldberg E.D., Dygay A.M., Zhdanov V.V., Khlusov I.A. Bull. exp biol. honey. 1999.V. 127. No. 5. S.484-494.

4. Ilyicheva T.N., Pronyaeva T.R., Schulz E.E. et al. microbiol. epidemiol. immunol. 2001. No2. S.53-56.

5. Kirillova A.V., Korneev G.I. // Morphology. 2003.V. 124. No. 5. S.54.

6. Kirillova A.V., Skachkov M.V., Panfilova T.V. and others. Epidemiology and vaccination. 2003. No.6. S.36-38.

7. Nuzov B.G. Stadnikov A.A., Borodin V.I. Ann injuries, and orthopedist. - 2001. - No. 2. - S.50-51.

8. Olifson L.E. Osadchaya N.D., Nuzov B.G. et al. Nutrition issues. 1991. No2. S.57-59.

9. Pavlova M.M. Study of the effect of the active steroid of millet (3-β-methoxy-Δ ¹⁸ -oleanene) in case of toxic liver damage CCl ₄ in the experiment: Abstract. diss ... cand. biol. sciences. - Orenburg, 1984. - P.21.

10. Panfilova T.V., Shtil A.A., Polosukhina E.R. et al. Bull. exp biol. honey. 2003.V. 136. No. 10. S.382-385.

11. Panfilova T.V., Shtil A.A., Frolov B.A. Bull. exp biol. honey. 2006.V.141. No. 6. S.633-635.

12. Yushkov V.V., Yushkova T.A., Kazyanin A.V. Immunocorrectors. Yekaterinburg. LLC "IRA UTK". 2002.255 s.

13. Behboudi S., Morem B., Villacress-Eriksson M. Clin. Exp. Immunol. - 1996 .-- Vol.105. - P.26-30.

14. Behboudi S., Morem B., Villacress-Eriksson M. Cytokine. - 1997 .-- Vol. 9. - P.682-687.

15. Dotsika E., Karagouni E., Sundquist B., Morgan A., Villacress-Eriksson M. Scan. J. Immunol. - 1997. - Vol. 45. - P.261-268.

16. Logambal S.M., Michael R. D. Indian J. Exp. Biol. 2000. Vol. 38. P.1092-1096.

17. Morem B., Villacress-Eriksson M., Lovgren-Bengtsson K. Dev. Biol. Stand. - 1998 .-- Vol. 92. - P.33-39.

Claims (1)

The use of miliacin as a means to limit the lymphotoxic effect of methotrexate.

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