Agent possessing antidiabetic activity

FIELD: medicine, endocrinology, pharmacy.

SUBSTANCE: invention proposes an agent eliciting antidiabetic activity. Agent represents nonglycosylated recombinant human granulocyte colony-stimulating factor. It differs from native preparation by absence of glycosyl group and the presence of additional methionine residue by its N-end. Effect of agent is associated with mobilization and migration of bone marrow mesenchymal stem cells, homing into pancreas. This results to reparation of insulin-producing activity of organ and normalization of the peripheral blood glucose level after monotherapy with recombinant human granulocyte colony-stimulating factor.

EFFECT: valuable medicinal property of agent.

3 tbl, 2 dwg, 1 ex

 

The invention relates to the field of experimental medicine and can be used to create a new antidiabetic drug.

Today we know that deglycosylated form of recombinant human granulocyte colony-stimulating factor (Neupogen, filgrastim) possess a wide range of effects on different cell types, including hematopoietic precursor cells neutrophils, Mature neutrophils, some malignant cells, and can induce mobilization of stem cells from bone marrow into peripheral blood [1, 2].

We use the original tool deglycosylation form of recombinant human granulocyte colony-stimulating factor from E. coli was developed and received in nikti BAS SRC VB "Vector" together with the Institute of pharmacology of RAMS Siberian branch [3].

In its structure this compound is a protein consisting of 174 amino acids, with a molecular mass of 19.6 kDa.

We first identified its antidiabetic activity.

Previously it was shown the presence of anti-diabetic effect on the background of traditional integrated glucose-lowering therapy in drug Granocyte [4], which represents an acidic glycoprotein (produced by cells of the culture of ovarian Golden hamster), which is sportwave us means on the structure differs that it is not glycosylated and has an additional methionine residue at the N-end.

Given the above data, we hypothesized the presence of anti-diabetic activity in analog Granocyte - deglycosylation form of recombinant human granulocyte colony-stimulating factor derived from E. coli is more cost-effective and less time-consuming way.

The problem solved by this invention is the expansion of the means with antidiabetic activity.

New in the present invention is the use of replicationmanager recombinant human granulocyte colony-stimulating factor for effective treatment of experimental diabetes.

Diseases of the pancreas are very common worldwide and occupy a significant place in the structure of morbidity, mortality and disability of the population of our country. A special danger to the health and social significance of the pathology of this organ is diabetes mellitus. At present, methods of radical cure of this disease does not exist. Patients constantly, throughout life, to take hormone replacement therapy or other anti-diabetic drugs. However, thanks to modern the development of cell technologies have the possibility of searching for new therapies [2, 5], including diseases of the pancreas, is able by modifying the functions of endogenous stem cells to exert a therapeutic effect. According to the available representations of the non-recombinant human granulocyte colony-stimulating factor most effectively able to influence the functional properties of endogenous stem cells in its subcutaneous injection at a dose of 125 mg/kg for 5 days [2].

The essential features of the claimed showed together new properties that are not obvious to the expert and not derived explicitly from the prior art in this field. New features allow for effective therapy of diabetes, and the present invention can be used in medicine. Identical set of features not found in the study of the prior art in the patent and scientific and medical literature.

Based on the above you should consider the claimed solution meets the criteria of "Novelty", "Inventive step", "Industrial applicability".

The invention will be clear from the following description and the attached drawings:

figure 1 - histological preparation of pancreatic tissue containing the islet of Langerhans with the introduction of alloxan (21st day of the experiment), the color of the blood is Silin-eosin, HC. 600×; figure 2 - histological preparation of pancreatic tissue containing the islet of Langerhans with the introduction of alloxan against the application of replicationmanager recombinant human granulocyte colony-stimulating factor (21st day experience), color hematoxylin-eosin, HC. 600×.

The experiments were conducted on mice CBA/CaLac in the amount of 215 pieces, weighing 18-20 g Mouse 1 category (conventional linear mouse) were obtained from the nursery of the Department of experimental biomedical modeling, Institute of pharmacology, Siberian branch of the Russian Academy of medical Sciences (certificate available).

Assessment of endocrine apparatus of the pancreas of animals produced for registration of glucose in peripheral blood and morphological study of the body.

The glucose content was determined in the morning on an empty stomach on the 11th and 21st day of the experience with the help of glucometer "Optilite" (Hungary) and the attached strips "Optilite test strip".

For morphological studies 8, 11, 15, 21 St day of the experiment, the part of the pancreas adjacent to the spleen were fixed in 10% formalin solution and embedded in paraffin. Deparaffinization slices with a thickness of 5 μm were stained with hematoxylin and eosin. The sections were defined area of 10 consecutive islets of Langerhans by the method of graphical computer analysis, positivel is in them, the total number of cells, the number polnotsennyh cellular elements and calculate the content of cells per unit area of the island and the percentage polnotsennyh cells.

With the aim of studying the mechanism of action of the product studied the status of the various pools of stem cells: the number mechanimals stem cells (MSCS) in bone marrow and peripheral blood on the 8th day of the experiment [6] and the dynamics of the content of regional stem cells in the pancreas in 8, 11, 15, 21 St day. The number of regional stem cells in the pancreas was determined by its cultivation of the cell suspension in a 24-hole tablets in the culture medium of the following composition: 80% medium DMEM, 20% mainactivity fetal calf serum, 10 g/l glucose, 500 mg/l L-glutamine, 50 mg/l gentamicin, 5000 IU/l heparin, 50 mg/l insulin, 10 ng/ml of growth factor, stem cells, 30 ng/ml epidermal growth factor, 10 ng/ml leasingbereich factor, 10 ng/ml interleukin-6, 10 ng/ml fibroblast growth factor, and incubated in CO2-incubator at 37°C, 5% CO2and 100% humidity for 21 days. Then using a binocular microscope MBS-9 (CA. 56×) counted the number of multicellular (>30 cells) ainoaoaony formations - count of clonogenic units.

Analysis was performed by the method of variation statistics using the-W t-test t-test and non-parametric U-test, Wilcoxon-Mann-Whitney.

Example 1.

Experimental model of diabetes mellitus served as chronic alloxan diabetes. Mice diabetes simulated subcutaneous injection of 1-water alloxan according to the following scheme: within 4 days daily 300 mg/kg, then again at the same dose on day 7 after the last injection in a volume of 0.2 ml/mouse. Experimental animals 3 days after the start of the simulation diabetes subcutaneously 1 time a day for 5 days was administered 125 mg/kg replicationmanager recombinant human granulocyte colony-stimulating factor, dissolved in 0.2 ml of solvent. Control animals received the same pattern in the equivalent volume was administered distilled water.

In the experiment, the introduction of alloxan led to Pinoso significant part of the cells of the islets of Langerhans observed in all study periods. In addition, it was expressed by the phenomenon of edema and hyperemia of the endocrine system with the development of significant lymphomacrophagal infiltration of the tissue (table 1; figure 1). A natural reflection of the morphological changes of the pancreas was a significant increase of glucose in peripheral blood and the development of hyperglycemia (table 2)

In this course introduction replicationmanager recombinant human granulocyte colony-stimulating factor p the following simulation alloxan diabetes resulted in a significant reduction in the percentage of polnotsennyh cells in the islets of Langerhans 11, The 15th day of experience on the background of reducing the total number of cells per unit area of the island result in a significant reduction in the infiltration of the islets (see table 1; figure 2). Described pathomorphological picture was accompanied by normalization of glucose levels in the blood (see table 2)

Table 1

The dynamics of content polnotsennyh cells in the islet of Langerhans in % (a) and the total number of cells per unit area of the island (B), (X±m)
The time frame of the study (day)Control (alloxan diabetes)Used tool
AndBAndB
the intact group3,88±0,230,75±0,043,88±0,230,75±0,04
8th16,18±0,89*0,75±0,0314,32±1,23*0,79±0,06
11th14,35±0,94*0,77±0,038,44±0,49*#0,61±0,02#
15th12,8±1,06*0,74±0,037,9±0,23*#0,83±0,12
21st11,8±1,97*0,9±0,0713,39±0,55*0,77± 0,12
* The reliability of relatively intact animals at p<0,05.

# Marked reliability relative to animals with alloxan diabetes at p<0,05

Table 2

The level of glucose in peripheral blood, mmol/l (X±m)
The time frame of the study (day)Control (alloxan diabetes)Used tool
the intact group3,16±0,39
11th17.11 per bbl±1,15*4,6±0,72#
21st13,03±0,9*equal to 4.97±0,98#
* The reliability of relatively intact animals at p<0,05.

# Marked reliability relative to animals with alloxan diabetes at p<0,05

In the study of possible mechanisms of action identified high efficiency replicationmanager recombinant human granulocyte colony-stimulating factor in experimental therapy of diabetes in the control group was found no change from bone marrow and circulating pools true (mesenchymal) stem cells. Moreover, which is the attachment of the experiment it was noted the drastic decrease of the content of regional stem cells in the pancreas (table 3). However, the study of possible stimulation data compensatory-adaptive mechanisms in the simulation of diabetes by administering replicationmanager recombinant human granulocyte colony-stimulating factor revealed a significant increase in MSCS in peripheral blood (up to 325,0% of the background on the 8th day) while reducing the number of data elements in the bone marrow. In General, these shifts in the state of the pool of stem cells hematopoietic tissue and peripheral blood, indicating mobilization and migration of MSC, was accompanied by their hominem in the pancreas, which manifested itself in the increasing number of regional stem cells on the 11th day of experience in the appointment replicationmanager recombinant human granulocyte colony-stimulating factor (see table 3).

Table 3

The number of mesenchymal stem cells in the bone marrow, 10 myelokaryocytes (A), in peripheral blood, 106the mononuclear cells (B), and dynamics of the content of regional stem cells in the pancreas, 105the nuclears (In), (X±m)
The time frame of the study (day)Control (alloxan diabetes)Use the used tool
AndBInAndBIn
the intact group7±24±215,17±1.37±24±215.17±1,3
8th10±25±28,83±0,54*4±2#13±3*#8,0±0,52*
11th--6,17±0.87 x--a 9.5±0,76*#
15th--8,17±1,25*--6,83±0,3*
21st--6,5±0,76*--6,17±0,31*
* The reliability of relatively intact animals at p<0,05.

# Marked reliability relative to animals with alloxan diabetes at p<0,05

Thus, non-recombinant human granulocyte colony-stimulating factor allows for effective treatment of diabetes through the mobilization of mesenchymal stem cells from bone marrow, accompanied by an increase in the content of regional stem cells in p is zheludochno gland, with subsequent repair of the insulin-producing apparatus body and normalization of glucose levels in the peripheral blood.

Literature

1. Mashkovsky PPM Medicines. - 15-ed. Rev., Corr. and extra - M.: OOO "Publishing house New wave", 2005. - S.

2. Sushkov G.N., Suslov NI, Digi A.M., Zhdanov V., Goldberg ED Role of stem cells in adaptation to hypoxia and mechanisms of neuroprotective action of granulocyte colony-stimulating factor // Cell technologies in biology and medicine. - 2005. No. 4. - S-208.

3. Patent (RU) for the invention №2201962 from 10.04.2003 "Method of production of recombinant granulocyte colony-stimulating factor, human".

4. Patent (RU) for the invention №2240134 on 20.11.2004 "Method of treatment of diabetes mellitus".

5. Goldberg ED, Digi A.M., Zhdanov CENTURY Modern views on the issue of stem cells and their potential use in medicine // Cell technologies in biology and medicine. - 2005. No. 4. - P.184-199.

6. In't Anker P.S., Noort W.A., Scherjon SA e.a. Mesenchymal stem cells in human second-trimester bone marrow, liver, lung, and spleen exhibit a similar immunophenotype but a heterogeneous multilineage differentiation potential // Haematologica. - 2003. - Vol.88. - P.845-852.

Application replicationmanager recombinant human granulocyte colony-stimulating factor as a means of having anti-diabetic activity.



 

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