RU2442589C1 - Method for stimulating myelogenesis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the invention relates to medicine, in particular, to haematology, and can be used for stimulation of myelogenesis. Pegylated hyaluronidase is administered with the dosage of 50 action units/kg 1 time a day for two days, additionally a medication containing D-glucuronic acid is intravenously administered once with the dosage of 2 mg/kg.

EFFECT: accelerated regenerative processes of haemopoietic tissues with additional administration of low doses of D-glucuronic acid, caused by its regulatory influence on hyaluronic acid metabolism of blood-forming precursors.

3 tbl, 1 ex, 1 dwg

Description

The invention relates to medicine, specifically to pharmacology and hematology.

The high incidence of blood system diseases resistant to modern drug therapy [1, 2] is the basis for the development of new methods for stimulating hematopoiesis.

A large number of methods for stimulating hematopoiesis are known.

The closest in technical essence and the achieved result of the prototype is a method of stimulating myelopoiesis using the drug immobilized hyaluronidase [3].

The disadvantage of this method is its lack of effectiveness.

The problem solved by this invention is to increase the efficiency of the method.

The problem is achieved by a technical solution, which consists in the simultaneous administration of orally pegylated hyaluronidase at a dose of 50 U / kg once a day for 2 days and once intravenously with D-glucuronic acid at a dose of 2 mg / kg.

New in the invention is the additional single administration of intravenous D-glucuronic acid at a dose of 2 mg / kg.

To date, the possibility of stimulating erythroid and granulomonocytic hematopoietic sprouts using pegylated hyaluronidase (peGD) has been shown [3]. In this case, the mechanism of action of the drug is to stimulate the proliferation and differentiation of hematopoietic cells under the influence of the low- and medium-molecular forms of hyaluronic acid (HA) intercellular matrix of hematopoietic tissue formed under the action of the enzyme [4].

The hemostatic effects of D-glucuronic acid are known, but only with its course administration in high doses (50 mg / kg) [5]. At the same time, the wide clinical use of this drug as a hemostatic stimulator in the indicated doses, given its acidity, frequency and severity of local irritating reactions, seems unlikely.

At the same time, it is known that the introduction of hyaluronidase preparations can be accompanied by a separation of the proliferation and differentiation of hematopoietic precursors [4, 6], and as a result, a limitation of the severity of hemostimulating effects, the potential of which can be significantly higher. In addition, a decrease in the ability of progenitor elements exposed to hyaluronidase to be engrafted during their transplantation was shown [7], as well as the possibility of preventing the formation of this phenomenon by incubating the graft in a solution containing D-glucuronic acid, which is a structural unit of HA monomer [8] .

However, the possibility of enhancing the haemostimulating effect of peGGD by additional administration of D-glucuronic acid, at doses significantly lower than therapeutically effective doses, is not known.

The fact of the combined use of peGGD and D-glucuronic acid with the achievement of a new technical result: the creation of an effective method of stimulating myelopoiesis by potentiating the specific hematotropic effects of each of the substances used is not obvious to a specialist. The experiment showed an unpredictable result.

The claimed essential features in the aggregate showed new properties that are not explicitly derived from the prior art in this field. The present invention can be used in experimental medicine with access to practical health care. An identical set of features was not found in the study of the state of the art in patent and medical literature.

Based on the foregoing, the claimed technical solution should be considered relevant criteria: "Novelty", "Inventive step", "Industrial applicability".

The method is as follows:

A pegylated hyaluronidase preparation is administered orally to a laboratory animal (mice) at a dose of 50 U / kg once a day for 2 days, and once, intravenously, D-glucuronic acid at a dose of 2 mg / kg.

The claimed dose and mode of administration of peGD and D-glucuronic acid are selected empirically and are optimal for obtaining the claimed technical result.

The proposed method was studied in experiments on CBA / CaLac mice in the amount of 244 pieces, weighing 18-20 g. Category 1 mice (conventional linear mice) were obtained from the nursery of the experimental biomedical modeling department of the Research Institute of Pharmacology SB RAMS (certificate is available).

The effectiveness of stimulation of myelopoiesis was determined on the model of cytostatic myelosuppression in accordance with the guidelines for the study of hemostimulating activity of pharmacological substances [9] using standard and culture hematological methods on days 3, 5 and 8 after administration of cytostatic [10].

Example 1

The modeling of cytostatic myelosuppression was carried out by a single intraperitoneal injection of cyclophosphamide in the maximum tolerated dose (250 mg / kg) in 0.3 ml of physiological saline. Against the background of myelosuppression modeling, experimental animals were injected with the peGGD drug (LLC “Scientific Future Management”, Novosibirsk) once a day for 2 days at a dose of 50 PIECES / kg (prototype method) and the peGGD drug in the same regimen together with a single intravenous administration of D-glucuronic acid at a dose of 2 mg / kg (Sigma, USA).

The controls were groups of animals that were injected either once with intravenous D-glucuronic acid at a dose of 2 mg / kg, or 1 time per day for 2 days with physiological saline.

The introduction of peGD preparation on the background of modeling cytostatic myelosuppression was accompanied by stimulation of the regeneration of erythroid and granulocytic hematopoietic growths. There was an increase in the content of hematopoietic precursors (CFU-E, CFU-GM) and morphologically recognizable cellular elements of erythroid and granulomonocytic hematopoietic cells in the hematopoietic tissue (Tables 1, 2), as well as the number of mature erythroid and granulocytic cells in the peripheral blood (Table. 3).

In contrast, the administration of D-glucuronic acid at a dose of 2 mg / kg did not have a statistically significant effect on the parameters of the blood system (Table 1-3).

At the same time, the use of D-glucuronic acid against the background of the introduction of peGD was accompanied by a significant stimulation of hematopoiesis. Moreover, the rate and severity of hematopoietic tissue regeneration in this case significantly exceeded those when using only peGD. So, the number of CFU-E and CFU-GM in the bone marrow on the 5th day was 2.3 and 1.8 times, respectively, higher than similar parameters in animals treated with the prototype method.

The indicated changes in the state of the pool of parent elements were accompanied by a naturally earlier restoration of morphologically recognizable indicators of bone marrow (primarily immature and mature neutrophilic granulocytes, erythroid cells) (Table 2) and peripheral blood (reticulocytes, stab and segmented neutrophils and platelets) ( table 3).

Thus, the additional administration of low doses of D-glucuronic acid during therapy with pegylated hyaluronidase led to a significant acceleration of the regenerative processes of hematopoietic tissue. At the same time, the regulatory effect of D-glucuronic acid on the metabolism of the hematopoietic glycocalyx hyaluronic acid metabolism, apparently, was the mechanism of potentiation of the hemostimulating effects of peGGD.

Literature

1. Glaspy J.A. Hematopoietic management in oncology practice. Part 1. // Myeloid growth factors Oncology (Huntingt). - 2003. - Vol.17. - No. 11. - P.1593-1603.

2. Volkova M.A. Granulocyte colony-stimulating factor granocyte and its clinical use // Ter. archive. - 1998. - No. 4. - S.80-84.

3. Digay A.M., Artamonov A.V., Bekarev A.A. et al. Hemostimulating effects of immobilized hyaluronidase and mechanisms of their development in cytostatic myelosuppression // Bull. an experiment. biol. and medicine. - 2010. - No. 5. - S. 528-531.

4. Digay A.M., Zyuzkov G.N., Zhdanov V.V. et al. Regulation of progenitor cell functions using hyaluronidase // Vestnik RAMS. - 2009. - No. 11. - S.7-10.

5. Guryantseva L.A., Udut E.V., Simanina E.V. et al. Mechanisms of regulation of the blood system under the influence of haemostimulants on the background of cytostatic myelosuppression // Siberian Oncology Journal. - 2005. - No. 3. - S. 39-43.

6. Zyuzkov G.N., Zhdanov V.V., Digay A.M., Goldberg E.D. The role of hyaluronidase in the regulation of hematopoiesis // Bull. an experiment. biol. and medicine. - 2007. - No. 12. - S.690-695.

7. Hui Zhu, Noboru Mitsuhashi, Andrew Klein e.a. The Role of the Hyaluronan Receptor CD44 in Mesenchymal Stem Cell Migration in the Extracellular Matrix // Stem Cells. 2006 - Vol.24. - N.4. - P.928-935.

8. Patent (RU) for the invention No. 2392947 "Method for producing cellular material for transplantation during myelosuppression", 2010. Authors: Dygay A.M., Zyuzkov GN, Zhdanov VV and etc.

9. Digay A.M., Zhdanov VV, Goldberg V.E. and other guidelines for the study of hemostimulating activity of pharmacological substances // Vedomosti Scientific Center for Expertise and State Control of Medicines. - 2002. - No. 1 (9). - S. 29-32.

10. Goldberg E.D., Dygay A.M., Shakhov V.P. Methods of tissue culture in hematology. - Tomsk: Publishing house of TSU, 1992. - S.130-145.

Table 1 Dynamics of CFU-E and CFU-GM content in the bone marrow of CBA \ CaLac mice (for 10 ⁵ non-adherent myelokaryocytes) after administration of distilled water (1), 2 mg / kg D-glucuronic acid (2) and 50 IU / kg pegylated hyaluronidase (peGGD) (3) and together D-glucuronic acid with peGGD (4) on the background of modeling cytostatic myelosuppression, (X ± m) Study time, days CFU-E CFU-GM Background 6.75 ± 0.73 4.50 ± 0.38 3rd one 8.40 ± 0.30 * 8.35 ± 0.44 * 2 8.58 ± 0.23 * 8.70 ± 0.96 * 3 14.1 ± 1.7 * # 12.38 ± 0.82 * # four 18.3 ± 0.23 * # $ 17.00 ± 0.53 * # $ 5th one 7.56 ± 0.48 6.63 ± 0.78 * 2 7.59 ± 0.51 7.75 ± 0.76 * 3 10.73 ± 0.65 * # 15.89 ± 0.45 * # four 24.8 ± 1.3 * # $ 28.5 ± 0.5 * # $ 10th one 5.8 ± 0.8 4.25 ± 0.53 2 5.75 ± 0.7 4.4 ± 0.29 3 7.7 ± 0.3 # 6.1 ± 0.80 * four 8.10 ± 0.42 * # 8.2 ± 0.7 * # * - the differences are significant in relation to the background at p <0.05; # - the differences are significant in relation to the group receiving distilled water against the background of cyclophosphamide administration at p <0.05; $ - differences are significant with the group of animals that received only peGD at p <0.05.

table 2 Dynamics of bone marrow hematopoiesis in CBA \ CaLac mice (× 10 ⁶ thigh) after administration of distilled water (1), 2 mg / kg D-glucuronic acid (2) and 50 IU / kg of pegylated hyaluronidase (peGD) and, together, D-glucuronic acid with peGD (4) on the background of modeling cytostatic myelosuppression, (X ± m) Study time, days Immature neutrophilic granulocytes Mature neutrophilic granulocytes Eosinophilic granulocytes Monocytes Erythroid cells background 1.0 ± 0.05 3.22 ± 0.19 0.57 ± 0.05 0.34 ± 0.08 1.85 ± 0.32 3rd one 0.03 ± 0.01 * 0.10 ± 0.04 * 0 0.25 ± 0.05 0.17 ± 0.04 * 2 0.03 ± 0.02 * 0.15 ± 0.02 * 0 0.20 ± 0.14 0.14 ± 0.05 * 3 0.14 ± 0.02 * # 0.14 ± 0.04 * 0 0.42 ± 0.07 0.36 ± 0.03 * # four 0.15 ± 0.02 * # 0.17 ± 0.03 * 0 0.51 ± 0.03 * 0.44 ± 0.02 * # $ 5th one 1.5 ± 0.21 * 2.8 ± 0.24 * 0.20 ± 0.08 0.96 ± 0.1 * 1.22 ± 0.1 2 1.6 ± 0.06 * 2.6 ± 0.29 * 0.09 ± 0.05 * 0.98 ± 0.08 * 1.28 ± 0.26 3 2.31 ± 0.02 * # 3.4 ± 0.10 * # 0.22 ± 0.05 1.34 ± 0.15 * 1.99 ± 0.32 # four 3.49 ± 0.05 * # $ 5.7 ± 0.37 # $ 0.2 ± 0.20 2,55 ± 0,25 * # $ 2.78 ± 0.2 * # $ 10th one 1.6 ± 0.08 * 4.5 ± 0.3 * 0.56 ± 0.25 0.40 ± 0.09 1.4 ± 0.04 2 0.80 ± 0.41 4.7 ± 0.5 * 0.58 ± 0.10 0.61 ± 0.17 1.39 ± 0.03 3 3.6 ± 0.08 * # 6.41 ± 0.4 * # 0.53 ± 0.7 0.99 ± 0.31 2.36 ± 0.1 * # four 4.3 ± 0.03 * # $ 7.32 ± 0.08 * # $ 0.57 ± 0.14 1.07 ± 0.06 * # 3.4 ± 0.07 * # $ * - the differences are significant in relation to the background at p <0.05; # - the differences are significant in relation to the group receiving distilled water against the background of cyclophosphamide administration at p <0.05; $ - differences are significant with the group of animals that received only peGD at p <0.05.

Table 3 Peripheral blood counts in CBA \ CaLac mice after administration of distilled water (1), 2 mg / kg D-glucuronic acid (2) and 50 IU / kg of pegylated hyaluronidase (peGD) (3) and together D-glucuronic acid with peGD ( 4) against the background of modeling cytostatic myelosuppression, (X ± m) Study time, days Reticulocytes, in ppm Band neutrophils, g / l Segmented neutrophils, g / l Platelets, T / L Background 9.3 ± 0.2 0.30 ± 0.02 2.34 ± 0.07 654.7 ± 23.4 3rd one 10.4 ± 0.14 * 0.01 ± 0.01 * 0.11 ± 0.02 * 544.60 ± 31.1 * 2 10.89 ± 0.33 * 0.01 ± 0.01 * 0.14 ± 0.01 * 584.6 ± 36.7 3 14.7 ± 0.5 * # 0.01 ± 0.01 * 0.19 ± 0.02 * 507.9 ± 14.7 * four 16.8 ± 0.59 * # $ 0.01 ± 0.01 * 0.16 ± 0.02 * 577.1 ± 23.0 3rd one 23.7 ± 1.1 * 0.06 ± 0.04 * 1.5 ± 0.43 * 477.8 ± 6.4 * 2 22.6 ± 1.3 * 0.08 ± 0.03 * 1.62 ± 0.29 * 493.0 ± 37.9 * 3 35.7 ± 0.9 * # 0.33 ± 0.04 # 2.8 ± 0.26 # 487.3 ± 17.8 * four 42.4 ± 2.08 * # $ 0.94 ± 0.21 * # $ 3.71 ± 0.11 * # $ 794.5 ± 21.8 * # $ 10th one 16.2 ± 1.0 * 1.5 ± 0.2 * 4.23 ± 0.53 * 708.4 ± 17.1 2 15.7 ± 0.7 * 1.72 ± 0.1 * 4.37 ± 0.7 * 869.6 ± 94.3 * 3 17.4 ± 0.7 * 1.6 ± 0.3 * 6.3 ± 0.12 * # 1084.7 ± 42.8 * # four 20.1 ± 0.9 * # $ 1.97 ± 0.1 * 7.43 ± 0.45 * # $ 1256.7 ± 21.8 * # $ * - the differences are significant in relation to the background at p <0.05; # - the differences are significant in relation to the group receiving distilled water against the background of cyclophosphamide administration at p <0.05; $ - differences are significant with the group of animals that received only peGD at p <0.05.

Claims (1)

A method of stimulating myelopoiesis, which consists in administering a preparation of pegylated hyaluronidase at a dose of 50 U / kg once a day for 2 days, characterized in that the preparation containing D-glucuronic acid is additionally injected once at a dose of 2 mg / kg.