RU2410116C2 - Medication of stimulation of stem cell production in bone marrow - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: medication contains activated form of antibodies to stem cell factor (anti SCF), obtained by multiple successive dilution and external impact - multiple vertical shaking of each obtained dilution in accordance with homeopathic technology, making for supersmall dose of said antibodies.

EFFECT: invention can be used as efficient medication for increasing number of stem cells in organism.

4 tbl, 4 ex

Description

The invention relates to medicine and can be used as an effective tool to increase the number of stem cells in the body.

From the prior art, preparations are known (granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, erythropoietin) that stimulate proliferation of stem cells (see Digay AM, Zhdanov V.V., Pozhenko N.S., Krivopalova G.N., Masycheva V. I., Pustoshilova N.M., Sinichkina S.A., Goldberg V.E. Acceleration of hematopoietic cell maturation under the action of recombinant granulocyte colony stimulating factor against the background of cytostatic myelosuppression Bull. Experimental biology and medicine. 2000, v.129, Appendix 1, p. 75 -79).

However, these substances also activate the processes of differentiation and mobilization of stem cells into peripheral blood, which ultimately leads to a decrease in the content of regeneratively competent cells in natural depots (bone marrow, adipose tissue, etc.). In this case, accordingly, the possibilities of a deep reserve of compensation involved in the processes of sanogenesis are limited. In addition, the preparations of these growth factors themselves have a number of undesirable side effects, which significantly limits their clinical use.

The invention is aimed at creating an effective tool to increase the content of regeneratively competent cells in the bone marrow with low toxicity and the possibility of prolonged use.

The solution to this problem is achieved by the fact that the drug for stimulating stem cell production in the bone marrow, according to the invention, contains an activated form of antibodies to the stem cell factor (anti-FSK), obtained by multiple serial dilution and external exposure - multiple vertical shaking of each received dilution according to homeopathic technology, which determines the smallest dose of these antibodies.

Obtained in accordance with the invention, the drug, which is an activated - potentiated form of antibodies to the stem cell factor in an ultra-low dose, allows to stimulate the production of stem cells and, practically, has no side effects, which is confirmed experimentally.

The drug is prepared as follows.

To obtain antibodies, a human or heterologous stem cell factor (including recombinant or genetic engineering) is used as an immunogen for immunization of laboratory animals (rabbits). The resulting antibodies are purified by affinity chromatography.

The method of obtaining polyclonal immune and monoclonal antibodies is described, for example, in the book: Immunological methods, ed. G. Fremel, M., Medicine, 1987, S. 9-33. The method of obtaining natural antibodies is described, for example, in the book. Natural antibodies to low molecular weight compounds. M.A. Myagkova. M., MGUL, 2001 (ISBN 5-8135-0058-8), pp. 70-114. The method of obtaining recombinant antibodies is described, for example, in the article Laffly E., Sodoyer R. Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

The isolated antibodies are subsequently diluted repeatedly and subjected to external influences, mainly vertical shaking, until the activated form is obtained in ultra-low doses, for example, according to the homeopathic potentiation technology (see, for example, Homeopathic medicines. Description and production manual, V. Schwabe, Moscow, 1967, pp. 12-38). In this case, a uniform decrease in concentration is achieved by successive dilution of 1 volume part of the starting substance (antibodies) in 9 volume parts (for decimal dilution D) or in 99 volume parts (for hundredth dilution C) of a neutral solvent with multiple vertical shaking of each dilution obtained and using mainly separate containers for each subsequent dilution - until the required dose of dilution (potency) is obtained.

External processing in the process of reducing the concentration can also be performed by ultrasound, electromagnetic or other physical effects.

A medicine prepared in this way is used mainly in dosage forms and dilutions accepted in homeopathic practice, in the form of alcohol or aqueous solutions or tablets (granules), obtained by soaking a neutral filler in the dosage form by saturation with the obtained potentiated solution, an activated form of antibodies.

To increase the therapeutic effect, the drug may contain a mixture of various homeopathic dilutions of antibodies in an activated - potentiated form.

The proposed drug has been studied in animal experiments. To confirm the positive effect of the claimed drug on the processes of restoring the number of parent cells in the bone marrow, studies were carried out on a model of myelosuppression caused by the introduction of cyclophosphamide. In this case, 239 male CBA / CaLac mice (conventional category 1 linear mice) were used. The animals were obtained from the nursery of the Research Institute of Pharmacology of the Siberian Scientific Center SB RAMS (certificate is available).

To simulate myelosuppression, cyclophosphamide at the maximum tolerated dose (250 mg / kg) was once administered intraperitoneally to experimental mice.

The inventive tool: a potentiated form of antibodies to stem cell factor (hereinafter referred to as anti-FSK) was administered 0.2 ml per os (1 time per day), starting from the 1st day after the introduction of cytostatic for 10 days. This dose for anti-FSK was established as the most effective in preliminary experiments. The control animals were injected with distilled water in a similar mode against cyclophosphamide. 10 mice kept under similar conditions served as the background.

Example 1

We studied changes in the state of the pool of granulomonocytopoiesis progenitor cells in the bone marrow under the action of anti-FSK against the background of cytostatic myelosuppression.

Studies were performed on 65 2-month old CBA / CaLac male mice.

Experimental animals (administering anti-FSK after cyclophosphamide) and control animals (distilled water after cyclophosphamide administration) were killed on the 3rd, 5th, 7th and 10th day after the start of the experiment (cyclophosphamide injection) by cervical vertebra dislocation under ether anesthesia. After aseptic isolation of the femur in the bone marrow of each mouse, the content of committed granulomonocytopoiesis precursors was determined by colony formation in a semi-viscous medium in vitro. In addition, the intensity of their proliferation by suicide using hydroxyurea and the differentiation activity by counting the maturation indices (the ratio of the number of clusters to the number of colonies grown in the same well) were evaluated. The results were processed by the method of variation statistics using Student's t-test.

The experiments showed that under the influence of cyclophosphamide on the 3rd day of the study, more than double inhibition of the colony-forming ability of the bone marrow develops (table 1). On the 5th day, a temporary increase in the number of granulocytomacrophageal precursors in the hematopoietic tissue was noted, which was significantly more pronounced in mice that were injected with an anti-FSK antibody preparation. The number of granulocytomacrophagal precursors was 1.5 times higher than the original. The value of this indicator in the indicated group, not only on the 5th, but also on the 3rd day of the experiment, significantly exceeded that in animals that received water after cytostatic. A subsequent drop in the colony forming ability of the hematopoietic tissue was observed in both control and experimental mice, but in the latter case it increased significantly by the 10th day, when the number of granulomonocytopoiesis precursors almost returned to the initial values (in contrast to control animals).

The study of the mechanisms underlying such differences in the state of the pool of hematopoietic precursors revealed a sharp increase in the proportion of DNA-synthesizing precursors of granulomonocytopoiesis under the influence of the preparation of SMD antibodies to FSK on the 5th day of the experiment, which followed a period of severe depression in their number (table 1). On the 7th day of observation, the proliferation rate of clonogenic myeloid elements significantly decreased in both studied groups. At the last stage of the experiment, the percentage of granulocyte-macrophage precursors in the S phase of the mitotic cycle was almost normal in all experimental mice.

Regarding the maturation intensity of granulocytomacrophageal precursors, there was no significant difference in this indicator between animals of both experimental groups throughout the study. Only the increase in the rate of differentiation of the parent cells in those cases when their content in the bone marrow was reduced relative to the initial level under conditions of insufficiently active proliferation was noteworthy (Table 1). Acceleration of maturation in such conditions is a compensatory phenomenon aimed at meeting the needs of the body in mature blood cells.

Example 2

We studied changes in the state of the pool of erythroid progenitor cells in the bone marrow under the action of anti-FSK against the background of cytostatic myelosuppression.

Studies were performed on 54 2-month old CBA / CaLac male mice.

Experimental animals (administering anti-FSK after cyclophosphamide) and control animals (distilled water after cyclophosphamide administration) were killed on the 3rd, 5th, 7th and 10th day after the start of the experiment (cyclophosphamide injection) by cervical vertebrae dislocation under ether anesthesia. After isolation of the femur in the bone marrow of each mouse, the content of committed erythropoiesis precursors was determined by colony formation in a semi-viscous medium in vitro. In addition, the intensity of proliferation of erythroid progenitors by suicide using hydroxyurea and the activity of their differentiation by counting the maturation indices (the ratio of the number of clusters to the number of colonies grown in the same well) were evaluated. The results were processed by the method of variation statistics using Student's t-test.

The content of erythroid precursors in the bone marrow decreased under the action of cyclophosphamide on the 3rd day of the study by more than half compared with the initial level (table 2). On the 5th day, as well as from the side of granulocytomacrophageal precursors, an “abortive” (temporary) increase in the number of erythroid precursors in the hematopoietic tissue was noted. At the same time, the yield of the corresponding colonies in mice injected with an anti-FSK antibody preparation almost reached the background level. In the indicated group, on the 5th day of the experiment, the value of this indicator significantly exceeded that in animals that received water after cytostatic. The subsequent drop in colony forming ability of the hematopoietic tissue was replaced by its significant increase under the action of anti-FSK by the 10th day, when the number of erythropoiesis precursors was double that in control animals.

The study of the intensity of proliferation of hematopoietic precursors revealed a pronounced increase in the proportion of DNA-synthesizing erythropoiesis precursors under the action of the anti-FSK preparation on the 5th day of the experiment, which followed a period of decrease in their number (table 2). On the 7th day of observation, the rate of division of clonogenic erythroid elements in this group significantly exceeded that in intact animals, normalizing in the future.

There was no significant difference in the intensity of maturation of erythroid precursors between animals of the experimental and control groups throughout the study.

Thus, the anti-FSK preparation demonstrated a rather pronounced ability to increase the content of the parent cells of various hematopoiesis cells in the bone marrow under conditions of myelosuppression caused by cyclophosphamide. These properties of the drug are caused, as it turned out, by the stimulation of proliferation of committed hematopoietic precursors.

Example 3

We studied changes in the content of stromal precursor cells in bone marrow and peripheral blood under the action of anti-FSK against the background of cytostatic myelosuppression.

The experiments were conducted on CBA / CaLac mice in the amount of 66 animals, weighing 18-20 g.

Experimental animals (administration of cyclophosphamide and then anti-FSK) and control (administration of cyclophosphamide and distilled water) were killed on the 3rd, 5th, 7th and 10th day after the start of the experiment (cyclophosphamide injection) by partial decapitation under ether anesthesia. In the bone marrow and in the peripheral blood of mice of all groups, the content of committed stromal precursors (CFU-F) was determined by colony formation in a semi-viscous medium in vitro.

The results were processed by the method of variation statistics using Student's t-test.

When examining the state of the bone marrow pool of cells giving rise to stromal elements, a significant drop in their number was detected after cyclophosphamide was administered in both experimental groups over almost the entire observation period, with the exception of 3 days. At this time, the number of CFU-F in mice that were fed after cytostatic with both anti-FSK and water significantly exceeded the initial values (table 3). Further, the decrease in the number of stromal precursors in the bone marrow of animals of the experimental group was not as pronounced as in the control, significantly exceeding the values of this indicator in the comparison group on the 5th, 7th and 10th days of the study.

The dynamics of the content of stromal precursors in peripheral blood was characterized as a whole by a 2-3-fold increase in the number of circulating CFU-F throughout the experiment. The indicated phenomenon turned out to be most pronounced in both groups on the 5th day of the experiment (table 3). The discovered phenomenon, along with a decrease in the content of cells with similar properties in the bone marrow, speaks in favor of their mobilization into peripheral blood under the action of a cytostatic agent. However, the fact that under the influence of the anti-FSK preparation in the early stages of the study, the CFU-F content in the hematopoietic tissue nevertheless increased, indicates the drug’s ability to stimulate the production of the corresponding cells.

Thus, the state of the fibroblast precursor pool is characterized by their mobilization to peripheral blood under the influence of cyclophosphamide, and the decrease in CFU-F in the bone marrow is compensated by the SMD antibody to FSK due to an increase in their production.

Example 4. We studied changes in the content of mesenchymal stem cells in the bone marrow and peripheral blood under the action of anti-FSK against the background of cytostatic myelosuppression.

The experiments were conducted on 54 CBA / CaLac mice weighing 18-20 g.

Experimental animals (administration of cyclophosphamide and anti-FSK) and control animals (administration of cyclophosphamide and distilled water) were killed on the 3rd, 5th, 7th and 10th day after the start of the experiment (cyclophosphamide injection) by partial decapitation under ether anesthesia. In the bone marrow (on the 3rd, 5th, 7th and 10th day) and in the peripheral blood (on the 3rd day), the mice of both compared groups determined the content of mechanochemical stem cells (MSCs) according to the method described by In't Anker PS, in own modification.

The results were processed by the method of variation statistics using Student's t-test. The incidence of MSCs in bone marrow and peripheral blood was determined using a generalized linear model for the distribution of Poisson. Correspondence of the data of the limiting dilutions of the one-dimensional Poisson model was assessed by linear log-log regression. In this case, the theoretical fraction of negative wells µ _{i was} distributed as µ _i = exp (-fx _i ), where f is the frequency of occurrence of MSCs, xi is the number of cells deposited in the well.

Studies have shown that from the side of the parent cells of the connective tissue, the quantitative determination of which was carried out by the method of limiting dilutions in culture, there were changes similar to those in the department of committed precursors. The content of mesenchymal stem cells in the bone marrow increased only on the 3rd day of the experiment in animals of both compared groups. In the subsequent follow-up periods, the number of MSCs decreased, however, the value of this indicator in the group with anti-FSK was almost three times higher than that in the control on the 5th, 7th and 10th days of the study (table 4). The study of cells capable of long-term maintenance of the culture of stromal mechanocytes did not reveal a significant difference in their content in the peripheral blood of mice of the compared groups on the 3rd day of the study (table 4). This fact indicates that MSCs are not as sensitive to the mobilizing effect of cyclophosphamide as their more differentiated descendants.

Thus, the effect of the anti-FSK preparation on the state of the pool of mesenchymal stem cells is similar to that of committed precursors of stromal mechanocytes (CFU-F), but its effect is less pronounced than with respect to CFU-F.

The results show that the proposed tool effectively provides stimulation of progenitor-stem cells of different lines of differentiation and maturity, but practically does not activate their maturation and does not cause mobilization in peripheral blood, and have no side effects, which makes it possible to use it for a long time.

Table 1 The dynamics of the content of granulomonocytopoiesis precursor cells (CFU-GM, per 10 ⁵ nuclears) in the bone marrow, the proportion of CFU-GM in the S phase of the mitotic cycle (%), and their maturation rate (differentiation coefficient) in CBA / CaLacY mice after the introduction of cyclophosphamide (CF) and the preparation of SMD antibodies to SCF (1) or CF and distilled water (2) (X ± m) Study time, days CFU - GM Share in the S phase of the mitotic cycle Maturation Background 10.67 ± 1.26 57.95 ± 4.63 2.37 ± 0.29 3rd one 5.33 ± 0.42 * # 10.58 ± 5.06 * 3.79 ± 0.38 * 2 4.0 ± 0.37 * 21.67 ± 7.38 * 5.2 ± 1.15 * 5th one 16.5 ± 1.73 * # 56.17 ± 4.56 2.17 ± 0.25 2 9.83 ± 1.35 44.58 ± 6.2 3.13 ± 0.47 7th one 6.0 ± 0.58 * 27.25 ± 6.97 * 4.75 ± 0.52 * 2 6.5 ± 0.67 * 19.18 ± 7.57 * 4.4 ± 0.53 * 10th one 8.67 ± 0.33 # 52.8 ± 1.36 3.69 ± 0.26 * 2 5.83 ± 0.31 * 51.32 ± 2.54 3.12 ± 0.24 Note: * - the significance of differences with background is noted at p <0.05
# - the significance of differences with control group 2 (water) was noted at p <0.05

table 2 The dynamics of the content of erythropoiesis precursor cells (CFU-E, per 10 ⁵ nuclears) in the bone marrow, the proportion of CFU-E in the S phase of the mitotic cycle (%), and their maturation rate (differentiation coefficient) in CBA / CaLacY mice after the introduction of cyclophosphamide (CF) and the preparation of SMD antibodies to SCF (1), or CF and distilled water (2) (X ± m) Study time, days CFU-E Share in the S phase of the mitotic cycle Maturation Background 12.5 ± 0.81 37.98 ± 3.76 2.1 ± 0.19 3rd one 5.33 ± 0.42 * 16.5 ± 5.92 * 2.42 ± 0.24 2 4.5 ± 0.22 * 8.0 ± 3.58 * 2.39 ± 0.29 5th one 10.33 ± 0.56 40.83 ± 3.52 2.2 ± 0.08 2 8.0 ± 1.03 * 33.43 ± 6.15 2.79 ± 0.26 7th one 7.0 ± 0.86 * 49.67 ± 4.88 2.96 ± 0.15 * 2 7.67 ± 1.12 * 42.45 ± 7.25 3.23 ± 0.26 * 10th one 10.5 ± 1.06 # 34.72 ± 5.29 2.93 ± 0.3 * 2 5.5 ± 0.34 * 26.02 ± 4.0 3.18 ± 0.48 * Note: * - the significance of differences with background is noted at P <0.05
# - the significance of differences with control group 2 (water) was noted at P <0.05

Table 3 Dynamics of CFU-F content in the bone marrow and peripheral blood of CBA / CaLac mice (per 250 thousand mononuclear cells) after administration of cyclophosphamide (CF) and the preparation of SMD antibodies to SCF (1) or CF and distilled water (2) (X ± m) Study Dates Groups Bone marrow Peripheral blood Background 23.83 ± 1.28 13.33 ± 1.09 3 one 42.83 ± 1.17 * 28.50 ± 0.96 * # 2 41.17 ± 1.14 * 8.50 ± 0.96 * 5 one 14.50 ± 1.23 * # 36.00 ± 2.85 * 2 5.83 ± 0.65 * 32.00 ± 2.16 * 7 one 7.83 ± 1.33 * # 26.25 ± 1.11 * 2 1.83 ± 0.54 * 28.25 ± 0.25 * 10 one 6.67 ± 1.17 * # 35.83 ± 1.96 * 2 0.67 ± 0.33 * 30.25 ± 3.68 * Note: * - the significance of differences with the background is noted (P <0.05)
# - the significance of differences with control group 2 (water) was noted at P <0.05

Table 4 Dynamics of the content of mesenchymal stem cells (per 10 mononuclear cells) in the bone marrow and peripheral blood of CBA / CaLac mice after administration of cyclophosphamide (CF) and the preparation of SMD antibodies to SCF (1) or CF and distilled water (2) (X ± m) Study time (days) Bone marrow Peripheral blood background 113 ± 12 53 ± 6 3rd one 219 ± 87 73 ± 16 2 157 ± 39 60 ± 0 5th one 81 ± 9 # 2 32 ± 7 * 7th one 38 ± 7 * # 2 12 ± 2 * 10th one 32 ± 7 * # 2 7 ± 2 * Note: * - the significance of differences with background is noted at P <0.05
# - the significance of differences with control group 2 (water) was noted at P <0.05

Claims (1)

A medicine for stimulating stem cell production in the bone marrow, characterized in that it contains an activated form of antibodies to stem cell factor (anti-FSK), obtained by repeated serial dilution and external exposure - multiple vertical shaking of each dilution obtained using homeopathic technology, which causes ultra-small dose of said antibodies.