RU2078581C1 - Method of leukopoiesis stimulation - Google Patents

Abstract

FIELD: medicine, oncology. SUBSTANCE: method involves a single subcutaneous administration of exogenic DNA obtained from sturgeon fish milt. This DNA is a powder of low-polymeric preparation containing 80% native DNA sodium salt, molecular mass - 270-500 x 10³ Da, at the following ratio nitrogen bases: adenine 29.0; thymine 27.0; guanine 22.0, and cytosine 20.0. Method stimulates bone marrow hemopoiesis significantly and can be used at myelodepression caused by cytostatic therapy. EFFECT: improved method of stimulation, absence of by-side effects.

Description

 The invention relates to medicine and, in particular, to oncology and can be used to stimulate leukopoiesis in myelodepression caused by cytostatic therapy.

 It is known that cytostatic therapy, which forms the basis of drug treatment for tumors, causes inhibition of hematopoiesis even in therapeutic doses / 5 /. In this case, toxic myelodepression is recorded in the peripheral blood as leukopenia, thrombocytopenia and further anemia / 2 /. The prevention of this complication is carried out by adjusting the doses of cytostatics and the appointment of agents that stimulate leukopoiesis. It is known the use for this purpose of splenin, leucogen, pentoxyl, zymosan, vitamins of various groups / 3 /. In the process of searching for new biologically active materials, the leukostimulating effect of nucleic acids was revealed / 1, 4 /. So there is a known method of stimulating leukopoiesis, including the introduction of a preparation of homologous deoxyribonucleic acid (DNA) obtained from the thymus and spleen of rats / 6 /. However, this method has several disadvantages, the most significant of which are the slow development of the leukocyte reaction and the lack of stimulation of bone marrow hematopoiesis.

 The essence of the invention lies in the fact that for the first time in the framework of the claimed method, the stimulation of leukopoiesis is carried out due to the sodium salt of DNA obtained from sturgeon milk, while the drug is administered once, three or five times subcutaneously in equi-therapeutic doses.

 Significant features of the proposal should be considered the use of sodium salt of DNA obtained from sturgeon milk, previously not used according to these indications, as well as the development of drug administration modes.

 To obtain the sodium salt of DNA, sturgeon milk is homogenized in an aqueous solution of sodium chloride and sodium citrate, washing the material, mixing with detergent, separating and enriching the liquid fraction, washing in permeate, and isolating purified bioactive material.

 The salt of DNA obtained as a result of the described process has the following properties. This is a white powder containing at least 80% by weight of sodium salt of native, low molecular weight, low polymer DNA obtained from sturgeon milk, not more than 1% by weight of proteins, not more than 17% by weight of moisture, the rest is sodium chloride.

DNA obtained from sturgeon milk has the following nucleotide composition:
Nucleotide Molar Product
adenine 29.0
thymine 27.0
guanine 22.0
cytosine 20.0
The sodium salt of DNA has a molecular weight of 270,500 • 10 ³ daltons and a hyperchromic effect of at least 37%
To disclose the invention, the following are the results of experimental studies on the stimulation of leukopoiesis with a DNA preparation under conditions of leukocytopenia caused by cytostatics.

 In the first series of experiments, studies were conducted on dogs of the English Beagle breed weighing 11.5 15.6 kg. Hematopoietic tissue aplasia was caused by the oral administration of myelosan at a dose of 15 mg / kg once per day of the "0" experiment. The sodium salt of DNA was injected subcutaneously in single doses of 10 mg / kg and 25 mg / kg twice on days 3 and 7 after the administration of myelosan. The total doses of DNA were 20 mg / kg and 50 mg / kg, respectively. Dogs in the control group received 10 ml of saline subcutaneously at the same time. On the 3rd, 7th, 14th, 21st, 28th, 42nd and 49th day of the experiment, the number of leukocytes in peripheral blood and the content of bone marrow karyocytes in sternal punctures were determined.

 The experimental results are presented in table 1.

 As can be seen from the presented results, the introduction of myelosan caused a sharp drop in the content of leukocytes in the peripheral blood and karyocytes in the bone marrow. In the control group, the content of leukocytes and karyocytes in animals remained low until the 28th day of the experiment and began to recover gradually, starting from 42 days, but until the end of the observation on the 49th day it did not reach the initial level.

 The introduction of the sodium salt of DNA significantly changed the picture of the toxic effect of the cytostatic and contributed to a more rapid restoration of the content of bone marrow cells and leukocytes. So, in both groups with the introduction of sodium salt of DNA in total doses of 20 and 50 mg / kg, a less deep leukocytopenia was observed.

 The tendency to restore the number of peripheral blood leukocytes was noted already from 21 days of experience, and by the end of the observation, the leukocyte level almost reached the initial level. A similar pattern was observed in the restoration of bone marrow karyocyte content.

 Thus, in this series of experiments on dogs, it was shown that the sodium salt of DNA in a total dose of 20 mg / kg and most clearly at a dose of 50 mg / kg contributes to the accelerated restoration of hematopoiesis during inhibition of hematopoiesis caused by the myelosan cytostatic.

The second series of experiments was carried out on mice with hybrids F ₁ (CBAXC ₅₇ Bl) males weighing 18 to 20 g. Cytopenia was caused by administering cyclophosphamum intraperitoneally once at doses of 200 and 275 mg / kg. The sodium salt of DNA was injected subcutaneously once at doses of 150 and 30 mg / kg, as well as 3, 5, 7 days after the administration of cyclophosphamide. On days 1, 3, 5, 7, 10, 12, 14, 17, 21, and 25, the total number of leukocytes in peripheral blood was determined using a Goryaev camera. The experimental results are presented in table 2.

 As can be seen from the results presented in table 2, the maximum cytopenic effect with the introduction of cyclophosphamide develops by 3 days, the restoration of the number of cells is noted by 7 9 days. However, on day 12-13 a second, less profound decrease in the number of leukocytes is observed. With the introduction of DNA at a dose of 150 mc / kg on days 3, 5 and 7, the maximum stimulating effect is achieved. Moreover, 2 3 days after the first injection of DNA, the number of cells returns to the background level before the introduction of myelosan, and by 7 10 days the number of cells reaches a maximum value exceeding this level. It is important to note that, unlike other hemocorrectors, DNA does not cause a compensatory drop in the number of cells after hemostimulation.

 In general, the analysis of experimental materials allows us to draw the following conclusions: 1. in contrast to the prototype, when using the inventive method, the leukocyte count in the peripheral blood is quickly restored and the number of karyocytes in the bone marrow is increased; 2. To obtain a stimulating effect, one injection of the DNA preparation is sufficient. The best effect is achieved with three or five times the use of the drug with an interval of 48 hours; 3. the claimed method, in contrast to the prototype does not cause a compensatory decrease in the number of cells after hemostimulation; 4. when using the method did not reveal side effects and complications.

Example N1. The experiment was conducted on 9 English Beagle dogs (6 males and 3 females) weighing 11.5 15.6 kg. Hematopoietic tissue aplasia was caused by oral administration of myelosan at a dose of 15 mg / kg once. The sodium salt of DNA was injected subcutaneously twice on days 3 and 7 at single doses of 10 mg / kg and 25 mg / kg after the administration of myelosan. The total course doses of DNA were 20 mg / kg and 50 mg / kg. Animals of the control group received 10 ml of saline subcutaneously at the same time. The number of leukocytes (in thousand per 1 mm ^{3 of} blood) according to the days of the study in one experimental animal was: with the introduction of DNA in a total dose of 20 mg / kg on day 3 11.2; on day 7, 5.9; on day 14 3.9; on day 21 3.1; on day 28 3.9; on day 42 6.9; on day 49, 9.9; with the introduction of DNA in a total dose of 50 mg / kg on day 3, 9.6; on day 7, 6.9; on day 14 4.3; on day 21 4.5; on day 28 5.95; on day 42, 8.95; on the 49th day 10.95.

The number of karyocytes in sternal punctate (in thousand per mm ^{3 of} punctate) in one experimental animal amounted to: with the introduction of DNA in a total dose of 20 mg / kg on day 3 51.0; on day 7 7.2; on day 14 10.8; on day 21 12.9; on day 28 13.3; on day 42 25.5; on day 49 32.1; with the introduction of DNA in a total dose of 50 mg / kg on day 3, 48.5; on day 7 11.2; on day 14 12.3; on day 21 13.7; on day 42, 27.4; on day 49 44.7.

 Thus, the presented example illustrates the restoration of the number of leukocytes in peripheral blood and bone marrow cells with the introduction of sodium salt of DNA in the claimed mode.

Example N2. The experiment was performed on 50 mice hybrids F ₁ (CBAXC ₅₇ Bl ₆₎ male weighing 18 '20 cytopenia caused by administration of cyclophosphamide intraperitoneally once at a dose of 200 mg / kg. The sodium salt of DNA was introduced in two schemes: once at doses of 30 and 150 mg / kg 3 days after the cytostatic and 3 times with an interval of 48 hours in a single dose of 150 mg / kg, starting 3 days after the cytostatic. The total dose in the latter case was 450 mg / kg.

 Thus, the presented example illustrates the stimulation of leukopoiesis with the introduction of the sodium salt of DNA in a model of cytopenia obtained by cytostatics.

 A comparison of the results of the studies with literature data on the issue under study allowed us to characterize the claimed method as follows (see table. 3).

 In general, it should be noted that the claimed method in comparison with the known prior art has the following advantages: 1. when using the method, not only a quick recovery of the number of leukocytes in the peripheral blood is performed, but also an increase in the number of bone marrow karyocytes, which is practically not found in analogues and prototype; 2. unlike other DNA preparations, the method does not cause mutagenic changes in the body; 3. the method does not cause a compensatory drop in the number of cells after hemostimulation.

 Thus, the claimed method significantly expands the arsenal of methods for the correction of leukopoiesis in case of leukocytopenia caused by cytostatics and, apparently, will find wide application not only in oncology, but also radiation medicine, medicine of extreme conditions.

Claims (1)

A method for stimulating leukopoiesis, including the introduction of DNA, characterized in that exogenous DNA obtained from sturgeon milk is used as DNA in the form of a low polymer powder containing at least 80% native sodium salt, DNA with a molecular weight of 270,500 • 10 ³ D, at molar ratios of nucleotides adenine 29.0, thymine 27.0, guanine 22.0, cytosine 20.0, and the drug is administered once, 2 3 times after 48 hours or 5 times after 24 hours subcutaneously in equitherapeutic doses.

Images