RU2649817C2 - Means of hemostimulating action - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to pharmacology, namely to means of hemostimulating action. Means of hemostimulating action, which has the effect of proliferation and differentiation of the leukocyte germ of the blood in vivo, isolated from the homogenate of the ternary larvae, taken 10–12 days after inoculation of the honeycomb, ground in a meat grinder, filtered through a sieve, while the unfiltered residue is compressed, homogenized, mixed with the filtered liquid and frozen under certain conditions.

EFFECT: above described means allow to expand the range of means having a hemostimulating effect.

1 cl, 2 tbl

Description

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

Normalization of the level of leukocytes is an essential condition and a necessary step in the effective comprehensive treatment of cancer. Traditionally, it is carried out using combinations of various pharmacological agents. Most often, the degree of leukopenia determines the choice of drugs used to correct this negative effect of treatment. For example, with mild leukopenia (1-1.5 × 109 / l), leukopoiesis stimulants of various origins (leucogen, methyluracil) are used. For moderate leukopenia (0.5-1 × 109 / L) glucocorticoids are added and for severe leukopenia (<0.5 × 109 / L) combinations of antibiotics, glucocorticoids, leukopoiesis stimulants (leukogen, methyluracil) and colony-stimulating ones are already used factors (CSF). CSF has changed the mindset on the prevention and treatment of leukopenia. Such drugs as granocyte, leukomax, neupogen accelerate the maturation of leukocytes, increase their lifespan and release white blood cells from the bone marrow [Goldberg ED, Dygay A.M., Zhdanov VV The role of hematopoiesis-inducing microenvironment in the regulation of hematopoiesis in cytostatic myelosuppression // Tomck: STT. - 1999. - S. 33-84].

Disadvantages: the high cost of this series of drugs limits their use, the limited availability of the drug.

One of the drugs that are close in terms of the achieved result is lenograstim, one of the preparations of glycated granulocytic macrophage colony stimulating factor. Its disadvantages are the complex production and cleaning technology and, as a result, the high cost; a high frequency of side effects, as well as an injection type of administration, as the only possible for a protein preparation, which creates difficulties for the patient to take on his own. [Mironov A.N. Guidelines for preclinical studies of drugs. Part one. Grif and K // - 2012. - 944 p.].

The closest to the claimed decision on the purpose and combination of essential features is the study of the influence of the drone larva homogenate on the functional activity of hematopoietic progenitor cells in the in vitro system [Goryacheva K.V. et al. Investigation of the effect of the drone larva homogenate on the functional activity of hematopoietic progenitor cells in the in vitro system // Materials of the 82nd All-Russian Baikal Scientific and Practical Conference of Young Scientists and Students with International Participation dedicated to the 95th anniversary of ISMU and the 170th anniversary of birth I.I. Mechnikov "Actual issues of modern medicine." Irkutsk: INTSHT, 2015 (April 20-25, 2015). - S. 30-31], in which the stimulating effect of drone larva homogenate on the formation of granulocyte (leukocyte) colonies in vitro is disclosed, no in vivo studies have been performed.

The problem solved by the present invention is to expand the arsenal of effective and safe hemostatic agents.

The tool used was developed and obtained by FSBEI HE "Altai State University" Research Institute of Biological Medicine.

The task is achieved by the use of homogenate drone larvae (GLT) as a means of influencing the stimulation of proliferation and differentiation of leukocyte blood sprout.

The study revealed the fundamental possibility and high efficiency of controlling the ability of bone marrow nuclears to form granulocyte colonies of mesenchymal stem cells using GLT [Goryacheva KV, Keyno VV, Goryacheva MV, Shunk AA, Bondarev A.A ., Smirnov I.V. The study of the biological activity of animal species / Allegory and Immunology, Volume 16, No. 4, 2015. - Moscow: Publishing House "Medicine-Health". - 414 p.]. When applied in vivo, pronounced stimulation of proliferation and differentiation in granulocyte colonies was found.

GLT is prepared as follows.

A framework with drone wax is placed in the nest of bee colonies, after 7 days, the detuning of the honeycomb and its inoculation with the uterus are monitored until larva pupation. After seeding with the uterus of the entire honeycomb, sealed drone larvae are selected, removed from the honeycomb frame and allowed for processing after 10-12 days. Honeycombs are cut with a knife from the frames, chopped in a meat grinder, the mass is filtered through a sieve with a mesh of 0.8-1.0 mm. The unfiltered residue is placed in a manual screw press, pressed, homogenized. The filter and the liquid are mixed, poured into a container and frozen at a temperature not lower than -10 ° C, and a GLT is obtained, which is characterized by the following properties: a slightly yellow liquid with the smell of propolis and honey.

When “whitewash” honeycombs appears in the nest of bee colonies, a frame with drone wax is placed. The frame is placed between the stern and extreme brood frame. After 7 days, the control of the detuning of the honeycomb and sowing of its uterus to pupation of the larva is carried out. The start date of oviposition is recorded in the apiary. After sowing the entire honeycomb with the uterus, the drone honeycomb is removed from the nest of bee colonies and is put into processing after 10-12 days. At this time, drone larvae on such a honeycomb were sealed, but they still did not have the rudiments of the eyes, wings, legs (purple spots on the body of the larva). It is favorable that at this moment the histolysis of larval tissues and the formation of tissues and organs of an adult insect (imago) begin.

Instead of the extracted drone honeycomb, a new frame with drone wax is set. If the bee family is sufficiently developed (it takes more than 12 frames of Dadan, 8-10 frames of brood), then setting up the second frame with drone wax is possible. The second frame is placed on the opposite side of the nest - this procedure will allow you to get a larger amount of drone larva homogenate.

The limiting factor in the detuning of drone cells and their inoculation with the uterus is the presence of a drone bribe in nature. In the absence of pollen intake within 3 days, the uterus inoculates drone cells, and if such a period drags on for more than 6 days, then bees can destroy the eggs in the drone cells and eat the drone larvae. To prevent the death of drones, they feed the bees or place hives in more favorable areas with the largest number of plants that bloom.

Active construction of drone honeycombs and their inoculation with the uterus is observed with a stable, steady bribe of nectar and pollen - the gain of the control hive is 0.5-1.0 kg.

In the conditions of the southwestern part of the Altai Territory, the period of construction and sowing of drone honeycombs lasts until June 20, but in some years it is possible to extend this period until July 10. In the late period of the production of drone brood, after June 15-20, and when a strong bribe from clover manifests itself, nectar and honey may enter the drone larvae homogenate.

The extraction of the cell with the drone brood is transferred to the laboratory, where the honeycombs are cut with a knife from the frames. Cut honeycombs are chopped in a meat grinder. The ground mass is filtered through a sieve with a mesh of 0.8-1.0 mm. The unfiltered residue is placed in a manual screw press, where it is pressed. After pressing, the filtrate and liquid are mixed, poured into a container and frozen at a temperature not lower than -10 ° С. The remainder during pressing - cake - is also frozen. The resulting product is characterized by the following properties: slightly yellow liquid, with the smell of propolis and honey.

Instead of frames with drone wax, you can use building frames - store frames. Bees on the lower bar of the storeframe build a drone honeycomb, which is cut 10-12 days after the eggs are laid by the uterus. Using this framework reduces the cost of producing HLT.

The study of hemostimulating activity was carried out in accordance with (Mironov, A.N. Guidelines for preclinical studies of drugs. Part One. Grif and K // - 2012. - 944 p.).

GLT was administered intragastrically to a group of mice via a probe.

1. The experiment is carried out on white outbred mice, weighing 22-25 g. The animals of the first group are injected with cyclophosphamide at a dose of 80 mg / kg in 0.2 water for injection intraperitoneally.

2. Animals of the second group were injected with cyclophosphamide at a dose of 80 mg / kg in 0.2 water for injection intraperitoneally once, and the animals received homogenate of the drone larva intragastrically, with a volume of 0.2 ml, for 5 days.

3. The third group was intact. In the intact group, blood and bone marrow were taken from the femur to determine the normal hemopoiesis function of bone marrow and peripheral blood.

In mice of the first two groups, blood and bone marrow were taken on days 3, 5, 8 and 10 for 7 mice from each group. Bone marrow was harvested after euthanasia of mice with ether anesthesia.

Blood sampling was performed as follows: the mouse was euthanized with diethyl ether, then decapitated, the tube was moistened with a 10% solution of ethylenediamine-tetraacetic acid, then blood was added from the decapitated mouse to it, and it was gently mixed.

To calculate the total number of red blood cells (OKE), 4 ml of physiological solution was poured into a new tube, 0.02 ml of blood was added to the Sali capillary, everything was gently shaken, and kept for five minutes. Goryaev’s camera was refueled, held for one minute. The total number of red blood cells (OKE) was calculated in 5 * 16 square squares along the diagonal. The obtained value was divided by 100, the number of red blood cells = N * 10 ⁶ per mouse was obtained.

Next, the total leukocyte count (OKL) was calculated. 0.02 ml of blood was added to a test tube to 0.4 ml of 3% acetic acid with a Sali capillary. Stirred, seasoned, Goryaev’s chamber, kept for 1 minute. OKL was counted in 25 large squares, the obtained value was divided by 20. Received N * 10 ⁶ leukocytes per mouse.

To study the bone marrow, the femur of the mouse was isolated, it was cleaned of soft tissues, the condyle was opened, on the other hand, the tip of this bone was cut off, and the central channel was dissected with 1 ml of a 3% solution of acetic acid. Bone marrow was suspended by syringe through a needle. Then, 0.05 ml of HLT was taken from this suspension using a micropipette, 0.45 ml of a 3% solution of acetic acid was added, and the total number of karyocytes (OCC) was counted in 5 large squares diagonally. The resulting number was divided by 8 and the number of myelocaryocytes N * 10 ⁶ per hip was obtained.

The obtained results were processed by the statistical method of variation series using the nonparametric Mann-Whitney test. The difference of the compared means was considered reliable if the confidence indicator (P) was less than 0.05.

The results of the study of bone marrow hematopoiesis function are presented in table 1, the OKC indices for the group that received the first five days of HLT during administration of cyclophosphamide are higher than for the group that received cyclophosphamide alone (P <0.005). An increased value of OCC is observed throughout the entire experiment and reaches its maximum on the 8th day (P> 0.005). The most pronounced difference between the groups was on the tenth day, when in the group that received only cyclophosphamide, there was a significant decrease in the TOC, and in the second group, the TOC indicator slightly changed compared to the eighth day (P <0.005).

The acceleration of the restoration of bone marrow cellularity with the subsequent increase in the number of mature cells in the peripheral blood after administration of the studied substance against the background of cytostatic myelosuppression should be considered as a manifestation of a hemostimulating effect in the studied drug.

Table 2 shows that the introduction of GLT on the third day in the second group led to a decrease in OKL lower than in the myelosuppression group (P> 0.005), but already on the fifth day this value exceeded the indicators of the first group (P> 0.005). The GLT group reached its maximum on the tenth day, when the amount of OKL was significantly higher (P <0.005) than in the intact one. In the group that received only cyclophosphamide, an increase in OKL was also observed, but to a lesser extent (P <0.005) compared with the second group, and in this group the indicators did not exceed the intact value.

During the experiment it was found that: the drone larva homogenate has a pronounced stimulating effect in relation to the proliferation of karyocytes in the bone marrow. This confirms an increase in OCC; an increase in OKL indicates that GLT has an effect on the stimulation of proliferation and differentiation of leukocyte blood cells. Moreover, the effect of the drone larva homogenate on the hematopoietic tissue regeneration processes in vivo and the possibility of stimulating hematopoiesis due to the activation of the parent blood cells are not known. The experiment showed unpredictable results.

The fact of the use of a drone larva homogenate with the achievement of a new technical result, consisting in the stimulation of hematopoiesis, is not obvious to a specialist. New properties do not follow explicitly from the prior art in this field and are not found in the patent and scientific literature. The present invention can be used in medicine.

Claims (1)

A hemostimulating agent having the effect of proliferation and differentiation of a leukocyte blood germ in vivo, isolated from a homogenate of drone larvae taken 10-12 days after inoculation with a uterus of honeycombs, crushed in a meat grinder, filtered through a sieve with a mesh of 0.8-1.0 mm, while the unfiltered residue is pressed, homogenized, mixed with the filtered liquid and frozen at a temperature not lower than -10 ° C.