Medication for regenerative medicine

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to pharmacology and cell technologies. Medication, possessing regenerative activity, represents granulocytic colony-stimulating factor, immobilised on water-soluble polymers with molecular weight 400-4000 Da. Granulocytic colony-stimulating factor is immobilised on water-soluble polymers by means of ionising radiation - flow of accelerated electrons with energy 2.5 MeV, absorbed dose from 2 to 10 kGy and rate of dose gain 1.65 kGy/hour.

EFFECT: medication by claimed invention does not have immunogenicity, is highly efficient both in parenteral and peroral introduction.

10 tbl, 10 ex

 

The invention relates to medicine, specifically to pharmacology and cellular technologies, and can be used in regenerative medicine.

There are medications granulocyte colony-stimulating factor (G-CSF)with gemostimuliruyuschee mobilizing stem cells [1, 2], hepatoprotective [3] and cerebroprotective [4] effects.

Known drug pegylated (immobilized/chemically conjugated with polyethylene glycol) G-CSF - pegfilgrastim [5]. This tool is the closest to the technical essence and the achieved result and selected as a prototype.

The lack of preparations of G-CSF, including pegfilgrastim is their immunogenicity, which is determined by the protein nature of the connection and the only possible way - by parenteral introduction of this tool [2, 5]. In particular, their application may be accompanied by severe systemic and local allergic reactions of varying intensity (up to anaphylactic shock) [6, 7]. In addition, therapy of degenerative diseases using existing drugs G-CSF is not effective enough.

The problem solved by this invention is the creation of tools for regenerative medicine, no immunogenicity highly effective as parenteral, and at first the General introduction.

This object is achieved by immobilization of granulocyte colony-stimulating factor on media with ionizing radiation. As the carrier used biologically indifferent substance is a water - soluble polymer with a molecular weight of 400-4000 Yes.

The most preferred method of immobilization is the impact on the polymeric carrier and the biologically active compound with a directed stream of accelerated electrons with an energy of 2.5 MeV, the absorbed dose from 2 to 10 kGy, the speed of the set dose of 1.65 kGy/hour.

As the water-soluble polymer used polyethylene, gidroxiatilkrahmal, polyvinylpyrrolidone and other

New in the present invention is the creation of tools for regenerative medicine, which represents a granulocyte colony-stimulating factor, immobilizovannyi on the polymer carrier with a molecular weight of 400-4000 Yes using ionizing radiation, with gamma-stimulating mobilizing stem cells, hepatoprotective and cerebroprotective effects. The technical result of the invention is the lack of immunogenicity, and high efficiency means both parenteral and oral administration.

We use the original tool granulocyte colonies Kulibayeva factor, immobilized using ionizing radiation at low-molecular media, we developed and received jointly LLC "Scientific Future Management" (Novosibirsk), Institute of pharmacology, SB RAMS, Tomsk

It is known that a decrease in the immunogenicity of substances can be achieved by oral administration. However, the means protein, which is G-CSF, in the gastrointestinal tract are split under the influence of proteolytic enzymes, which makes this way of introducing them into the body ineffective. In this regard, the importance of the problem of creating tools for regenerative medicine, with a wide range of therapeutic effects, low risk of side effects and complications, including through the use of oral.

There are data on increased physical stability, solubility, and reduced immunogenicity and sensitivity to proteolytic enzymes proteins during their coverage of biologically indifferent polymer conjugation with certain holders (including immobilization using radiation effects) [8, 9]. However, information about the possibility of creating connections protein, including G-CSF, deprived allergenic (immunogenic) properties, as well as ways of creating drugs is their basis, high specific activity, characteristic of the native protein substances, and is equally effective when used parenterally and orally, to date, does not exist. It is known that the process of manipulation of heterogeneous substances at the molecular level, including the use of physical factors with high energy, can be accompanied by significant changes in the stereochemical structure of the original substance, and ultimately lead to modification of their properties, the nature of which, however, is unpredictable [10, 11]. However, currently, no data exists about the pharmacological benefits of the drugs obtained by immobilization of biologically active substances using ionizing radiation, before the connections formed by the conjugation of the same original substance, but by chemical means. That is not aware of the existence of qualitative differences of the final product is related to the manner of its production: the technology of chemical synthesis (which receive pegfilgrastim [2, 5]) and electron-beam technology immobilization (with which you are obtaining the offer). In addition, in world literature, in principle, there is no information about the possibility to increase the biological (farmacologicas the th) activity of protein regulatory molecules by immobilization of the active substance on the media, not associated with a change solely in their pharmacokinetics (due to changes in pharmacokinetic parameters due to the modification of G-CSF in the synthesis pegfilgrastim).

The fact immobilization of granulocyte colony-stimulating factor on the media using ionizing radiation to a new technical result: the creation of funds with gemostimuliruyuschee mobilizing stem cells, hepatoprotective and cerebroprotective action, low immunogenicity, high-performance parenteral and oral (enteral) admission to a specialist is not obvious.

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

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

The experiments were performed on 259 outbred rats of either sex, weighing 200-250 g, mice-samz the x CBA/CaLac in the number 367 pieces, weighing 18-20 g, 54 Guinea pigs of either sex weighing 200-250 g Animals obtained from the nursery of the Department of experimental biomedical modeling, Institute of pharmacology, SB RAMS.

The present invention is illustrated by the following examples.

Example 1

A 10% aqueous solution of polyethylene glycol with a molecular weight of 400 Da was made by G-CSF (LLC "Scientific Future Management, Novosibirsk) to a final concentration of 10 mg G-CSF in 1 ml of 10% polyethylene glycol. The mixture was stirred and irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. The processing performed by the bremsstrahlung radiation generated by the accelerator ILU-6 with the electron energy of 2.5 MeV, the absorbed dose of 2 kGy, the speed of the set dose of 1.65 kGy/hour. Received final immobilizovannyi the drug in the form of a slightly opalescense solution. The output of the finished product is of 98.2%.

Example 2

5% aqueous solution of hydroxyethyl-starch with a molecular weight of 1.5 kDa was irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. The processing performed by the bremsstrahlung radiation generated by the accelerator ILU-10 with the electron energy of 2.5 MeV, the absorbed dose of 10 kGy, the speed of the set dose of 1.65 kGy/hour. In the irradiated solution was made by G-CSF (LLC "Scientific Future Management, Novosibirsk) to a final concentration of 10 mg G-CSF in 1 ml of 5% hydroxyethyl-starch.

The mixture was stirred 10 minutes and got the horse is hydrated immobilizovannyi the drug in the form of a slightly opalescense solution. Yield is 99.1%.

Example 3

10.0% aqueous solution of polyvinylpyrrolidone with a molecular mass of 4 kDa was made by G-CSF (LLC "Scientific Future Management, Novosibirsk) to a final concentration of 1 mg G-CSF in 1 ml of the mixture is Then stirred for 30 minutes and was irradiated with gamma radiation at a dose of 1.0 Mrad. The processing performed by the bremsstrahlung radiation generated by the accelerator ILU-10 with the electron energy of 2.5 MeV, the absorbed dose of 5 kGy, the speed of the set dose of 1.65 kGy/hour.

The drug is immobilized G-CSF was given in the form of a transparent solution. The yield is 98%.

Example 4

Comparative study of the allergenic (immunogenic) properties of drugs granulocyte colony-stimulating factor immobilized on polyethylene glycol with ionizing radiation (img-CSF) (obtained according to example 1), and pegfilgrastim ("NEULASTIM", Switzerland, Hoffmann-La Roche).

Preparations were examined in the same doses (determined by the content of G-CSF), which was determined to be optimal in relation to the normalization of blood counts in mice with cytotoxic myelosuppression, and 10 times its excess (the content of G-CSF 100 and 1000 µg/kg, respectively). Assessment of the allergenic properties of the drug was performed using the following tests: total reaction anaphylaxis reaction specifices the second agglomeration of cells, when intradermal and conjunctival testing on Guinea pigs, the reaction of delayed-type hypersensitivity in mice [12]. Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney.

For sensitization of Guinea pigs medicines were introduced: first, 1 times subcutaneously, then the same dose 2 times intramuscularly, every day, in the thigh. Anaphylactogenic properties of drugs G-CSF were detected by intracardiac injection on the 21st day after the last sensitizing injections. Allow testing intracardiac injection of the drug was carried out at a dose equal to the total sensitizing, i.e. to 300 mcg/kg served as Control desensibilisation Guinea pigs, which the drug was administered by intracardiac.

Accounting for the intensity of the anaphylactic reaction was performed using the formula

,

where AI - anaphylactic index;

n - number of animals, anaphylactic reaction which was fatal;

n1- the number of animals with significant manifestations of anaphylactic reactions;

n2- the number of animals with secondary manifestations of the reaction;

n3- the number of animals with weak manifestations of reaction;

N is the total number of stomach who's in the group.

For evaluation of the skin sensitizing properties of the drug in 20 days after the end of the sensitizing injections on the side surface of the body of Guinea pigs was vestigal wool, and each animal was injected intradermally drug img-CSF or pegfilgrastim in doses of 10 mg/animal (50 µg/kg) in 0.02 ml of physiological solution (dose not cause visual changes skin intact Guinea pigs).

Skin reaction to the drug was evaluated in points with regard to the severity of hyperemia and size of damaged area (average diameter in millimeters). Identified four degrees of hyperemia, which corresponded to a numeric indexes: strong (+++) - 1,0; moderate (++) - 0,66; weak (+) - 0,33; equivocal (+/-) is 0.17. Multiplying the diameter of the damaged area on the index of the corresponding degree of hyperemia allowed in a single index (points) to Express both the characteristics of the response.

On the 20th day after the end of the cycle sensitizing injections were performed conjunctival testing. The test consisted of instillation into the left eye of each animal 0,02 ml of physiological solution containing 10 µg of the drug, in the right eye saline solution in the same volume. Assessment of the conjunctiva of the eye was performed after 4 and 24 hours after exposure.

To confirm results is, received the above tests were used reaction alergodiagnostiki in vitro of RSAL (reaction specific agglomeration of cells). Working dose G-CSF for this reaction was 2.5 μg in 0.05 ml of blood (50 µg/ml).

In mice sensitization was performed using a full adjuvant's adjuvant (PAF). Preparations of G-CSF was administered once subcutaneously at the base of the tail in 0.06 ml PAF, which was taken in the ratio 1:1 by volume solution of the drug. The dose was 1000 mg/kg Control animals were injected with PAF in the same amount.

Through 5 days after the injection of the test and control mice in the tip of one of the hind paw was injected preparations of G-CSF at a dose of 20 μg/animal 0.04 ml of physiological solution (1000 µg/kg), another paw - saline. 24 hours after the second injection with engineering micrometer type MK measured the thickness of both legs. The degree of response was defined as the difference in the thickness of the experimental and control legs in millimeters.

During the experiment it was found that the reaction of Guinea pigs by intradermal injection of the drug img-CSF on such visual indicators, such as redness or swelling, did not differ from the respective figures of animals, which were injected distilled water, while with the introduction of pegfilgrastim skin reaction was on Severna more pronounced. Conjuncti-roll test and reaction alergodiagnostiki in vitro showed no signs of Aller-generouse action none of the preparations of G-CSF. At the same time when testing intracardiac administration of drugs img-CSF and pegfilgrastim was a statistically significant increase in anaphylactic index in the group of animals treated with pegfilgrastim, while with the introduction of img-CSF, AI did not differ from those in the control group (table 1).

In addition, the study allergenic (immunogenic) properties showed that intradermal testing injection img-CSF and pegfilgrastim not led to a change in the value of the reaction GST compared with a control level indicator after 4 and 24 hours after exposure (table 2).

Similar results were obtained when studying img-CSF, obtained by immobilization of G-CSF on hydroxyethyl-starch and dextran.

Table 1
Assessment anaphylactogenic properties of the img-CSF and pegfilgrastim in the experiment on Guinea pigs
The group of animalsIndicators
The reaction of the skin, pointsConjunctive, test scores The coefficient of RSALAI
Control0,78±0,0201,5±0,20,26
Pegfilgrastim0,89±0,03∗01,5±0,20,31
img-CSF0,79±0,0501,5±0,20,87
* - the reliability of differences in the rate from its value in the control at p<0,05

Table 2
Assessment of the sensitizing properties of the drug img-CSF in the experiment on mice (X±m)
The conditions of the experimentThe reaction GST, mm
The group of animalsSensitizing dose, ág/kgResolving dose, ág/kg4 hours24 hours
Control10000,073±0,030,08±0,01
Pegfilgrastim100010000,087±0,020,09±0,01
img-CSF100010000,090±0,0300,79±0,01

Thus, the drug is immobilized using ionizing radiation G-CSF does not immunogenic effect, manifested allergization of an organism, while pegfilgrastim has a mild allergic properties

Example 5

Experimental study of the immunotoxic properties of G-CSF, immobilized on polyethylene glycol with ionizing radiation (obtained according to example 2) [12]. As the comparison drug was used pegfilgrastim ("NEULASTIM, Switzerland, Hoffmann-La Roche).

The preparations were studied in mice at a subcutaneous dose of 100 µg/kg, which was defined as therapeutic (1 TD) and at a dose on the order above 1000 µg/kg (10 TD). Experimental evaluation of the immunotoxic properties of preparations of G-CSF was performed using the following tests: a preliminary assessment of immunotoxic the activity after a single dose; study of the effect of the drug on the weight and cellularity of the Central and peripheral organs of the immune system; evaluation of the impact of the drug on the phagocytic activity of peritoneal macrophages; study of the influence of the drug on the number of antibody productive cells (AFC) after immunization with sheep red blood cells (EB); study on the impact of drugs on spontaneous and induced mitogen proliferation of splenocytes.

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

Course introduction img-CSF used in doses had no effect on the weight and cellularity of the Central and peripheral organs of the immune system of experimental mice, the level of antibody productive cells after immunization of animals with sheep red blood cells, the proliferative response of lymphocytes. While pegfilgrastim has led to a decrease in the spontaneous proliferative activity of lymphocytes (by 5.8% from the background) when added to the culture at a dose of 5 ng/ml.

Thus, as a result of the research showed that immobilizovannyi using ionizing radiation G-CSF does not have immunotoxic properties.

Example 6

Experiments were carried out to study gemopoeticakimi activity img-CSF (poluchennogo is as in example 1). As a comparison was a drug pegfilgrastim ("NEULASTIM", Switzerland, Hoffmann-La Roche).

Research gemostimuliruyuschee properties were performed on mice CBA model mielosupression. To do this, all animals were injected with cyclophosphamide (CP) at a dose of 170 mg/kg Then mouse 1st group received pegfilgrastim subcutaneously, mouse, 2nd group - pegfilgrastim oral, animals of the 3rd group was subcutaneously injected img-CSF, and 4-th - img-CSF oral. All the drugs were injected in a dose of 100 mg/kg 2 times: on day 1 after administration of cytostatic and 5 days. Control animals were injected with saline in the respective modes. 7, 10 days experience with standard and cultural hematological methods defined peripheral blood and bone marrow, as well as the number of CFU-GM in the bone marrow [13]. Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney.

Experiments have established that oral administration of pegfilgrastim did not lead to changes of the studied parameters indicating the inefficiency of use per os of the drug G-CSF, immobilized on the carrier by chemical means. In other cases in peripheral blood was increased content of Palocco and segmented stoichiometric the silts and the number of CFU-GM, immature and Mature neutrophilic granulocytes in hematopoietic tissue. The most pronounced were the changes in the group of animals treated img-CSF subcutaneously (table 3). At the same time quite significant was the rise of the figures and animals treated immobilized G-CSF oral.

Table 3
Indicators granulomonocitarnyi Rostock blood with the introduction of cyclophosphamide (1), subcutaneous (2) and oral administration pegfilgrastim (3), subcutaneous (4) and oral (5) the introduction of img-CSF on the background modeling mielosupression, (X±m)
Time / dayStab neutrophils in the peripheral blood, G/lSegmented neutrophils in the peripheral blood, G/lImmature neutrophilic granulocytes in the bone marrow, ×106/hipMature neutrophilic granulocytes in the bone marrow, ×106/hipCFU-GM in the bone marrow, 106the myelokaryocytes
background0,3±0,022,44±0,23 1,12±0,043,64±0,215,11±0,21
5th10,0∗0,9 ħ 0.09∗0,56±0,03∗1,02±0,08∗12,68±0,8∗
20,03±0,01∗1,3±0,01∗0,99±0,031,69±0,02∗17,9±0,9∗
#####
30,0∗0,94±0,04∗0,58±0,02∗1,07±0,1∗11,8±1,0∗
40,06±0,01∗1,98±0,02∗1,34±0,01∗1,72±0,07∗29,7±1,2∗
#&#&#&##&
5 0,07±0,01∗1,56±0,03∗1,06±0,021,56±0,03∗26,4±1,01∗
#&####&
10th10,43±0,093,01±0,90,98±0,023,4±0,674,01+1,4
20,9±0,01∗3,76±0,07∗2,35±0,07∗5,9±0,1∗14,1±1,3∗
#####
30,37±0,12,96±0,71,1±0,033,57±0,445,2±0,7
41,5±0,06∗4,59±0,1∗3,14±0,1∗6,98±0,1∗18,6±2,1∗
#&#&#&#&#
51,3±0,04∗3,3±0,1∗2,97±0,1∗6,57±0,5∗16,3±1,07∗
#&####
* - the significance of change of the indicator from its background values at p≤0,05.
# - noted the significance of change of the indicator from its control value at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

Example 7

The study of mobilizing stem cells properties img-CSF were performed on intact mice CBA. Research has used the drug img-CSF, obtained according to example 2. As the comparison drug served pegfilgrastim ("NEULASTIM", Switzerland, Hoffmann-La Roche). The mode of administration was selected in preliminary experiments, the AK is the most effective. Mouse 1st group received pegfilgrastim subcutaneously, mouse, 2nd group - pegfilgrastim oral, animals of the 3rd group was subcutaneously injected img-CSF, and 4-th - img-CSF oral. All the drugs were injected in a dose of 100 μg/kg, 1 time a day for 5 days. On the 5th, the 7th day was determined by the number of mesenchymal stem cells (MSCS) and granulopenia-Erythro-megakaryocytic (SOME GEMM) precursor cells in the peripheral blood[1, 13, 14].

During the experiment it was found that parenteral use (but not oral) pegfilgrastim, as well as parenteral and oral administration of img-CSF resulted in a significant increase in the maintenance of stem cells in the peripheral blood. Moreover, the maximum values increase was observed when injecting img-CSF, and the change in the number of MSC and SOME HAM oral use of G-CSF was such percutaneously pegfilgrastim (table 4).

13,4±1,0∗
Table 4
The content of stem cells in the peripheral blood of mice after subcutaneous (1) and oral administration pegfilgrastim (2), subcutaneous (3) and oral (4) the introduction of img-CSF, (X±m)
Time / dayCFU-G IS MM, 105mononuclearMSC in the bone marrow, 106mononuclear
background4,7±0,219,0±2,0
5th19,8±1,1∗29,0±3,1∗
24,3±0,3918,4±2,2
17,4±2,0∗39,0±3,4∗
&&
410,3±0,92∗36,0±3,7∗
&
7th114,3±0,9∗31,0±2,4∗
25,2±0,7515,0±3,1
L19,7±1,7∗39,0±3,3∗
&&
437,2±2,4∗
&
* - the significance of change of the indicator from its background values at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

Example 8

We have studied changes of the functional state of the liver under the action of img-CSF and pegfilgrastim ("NEULASTIM", Switzerland, Hoffmann-La Roche) for modeling chronic toxic hepatitis.

The experiments were carried out on outbred rats weighing 250-300 g In rats hepatitis caused the intragastric administration of a 50% solution SSC (hepatotropic poison) in olive oil at a dose of 2 ml/kg for 3 weeks 2 times a week (6 times).

At the end of the simulation chronic hepatic toxicity animals were divided into 3 equal groups. Rats of the 1st group for 5 days) was subcutaneously injected with 100 µg/kg pegfilgrastim dissolved in 0.5 ml of solvent. The first introduction was carried out the next day after the last injection of tetrachlorophenol. Animals of the 2nd group was subcutaneously injected img-CSF at a dose of 100 µg/kg in those who tell 5 days. Rats of the 3rd group was administered intragastrically img-CSF at a dose of 100 µg/kg for 5 days. Animals of the control group according to the same scheme in the equivalent volume was injected with saline. Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney.

To study the state of the liver was assessed by the death of rats, conducted biochemical studies content in serum aspartate and alanineaminotransferase (AST, Alt) on the 40th day, as well as morphological examination of the liver on the 40th day experience. The enzyme activity of blood serum were determined by standard methods using semi-automatic biochemical analyzer company Cormay and standard sets to it. Blood for the study was obtained from the femoral artery through the catheter. On histological preparations of liver stained with hematoxylin and eosin, determined the number of cells infiltrate through the eyepiece grid Avtandilov containing 25 test points. In 20 fields of view were counted number of cells that fall on the test point grid. The relative area of infiltration was calculated as the ratio of grid points per cell infiltration, all grid points in 20 fields of view. The area of connective tissue was determined using a computer the computer graphics processing. For this purpose, standard square cut liver (serial micrograph of 10 fields of view, made by micrometeorology Digital micro", with the program transfer images to a computer firm "Elecard", Tomsk) measured the area of structures, painted pikrofusin, and calculated the percentage to the selected standard square.

The experiments showed that the death of rats in the groups of animals treated with the preparations of G-CSF was significantly lower than in control (35,6%). While subcutaneous injection img-CSF death of rats was not observed. Biochemical analysis of serum revealed an increase in the activity of Alt, AST and alkaline phosphatase on the 40th day of the experience after the beginning of the introduction of the CCl4in the control group (table 5). At the same time pegfilgrastim practically no effect on the enzyme content in the blood serum. However, both parenteral and oral use img-CSF was accompanied by a significant decline in their activity.

Table 5
Influence of drugs on the biochemical parameters of male rats with chronic CCl4hepatitis (1) percutaneously pegfilgrastim (2), subcutaneous (3) and oral (4) the introduction of img-CSF, 40 day experiment, X±m
GroupAlatASTAlkaline phosphatase
(ákat/l)(ákat/l)(E/l)
background0,22±0,010,24±0,03231,0±23,4
10,69±0,03∗0,70±0,04∗415,9±43,6∗
20,48±0,1∗0,66±0,03∗349,5±36,7∗
#
30,24±0,020,28±0,02227,5±34,3
#&#&#&
40,21±0,020,34±0,06239,3±41,2
#&#&#&
* - the significance of change of the indicator from its background values at p≤0,05.
# - noted the significance of change of the indicator from its control value at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

When the histological study of the liver in the control group of rats was observed a marked violation of the lobular structure of the body. The drugs were visible field of granulation tissue, replacing the dead hepatocytes, in which there was a tumor vessels and hepatic ducts. In the surviving hepatocytes was observed pronounced globular fatty degeneration. At the same time, there was a significant regenerative hypertrophy of liver cells. Introduction pegfilgrastim or img-CSF in the simulation CCl4-hepatitis preserved lobular structure of the liver, and fatty degeneration of hepatocytes was mostly atomized. In the experimental animals was observed less pronounced infiltration of portal tracts.

In General, the relative area of infiltration was significantly lower than in the control group. In addition, the preparations of G-CSF significantly reduced and the area of connective tissue (6). The most pronounced HEPA-toprotecting effect occurred when p is cognom the introduction of the claimed img-CSF. However, a significant therapeutic activity of the drug possessed and by oral administration, and such was comparable to parenteral using pegfilgrastim.

Table 6
Influence of drugs on the biochemical parameters of male rats with chronic CCl4-hepatitis percutaneously pegfilgrastim, subcutaneous and oral administration of img-CSF, 21 and 40 days of experiment, X±m
GroupThe relative area of infiltration (%)The relative size Conn. tissue (%)
background2,34±0,2of 3.78±0,3
H2O (control)27,48±4,67∗16,5±0,42∗
Pegfilgrastim10,0±1,16∗#7,44±0,17∗#
Them G-CSF subcutaneously3,7±0,97∗#&3,56±0,4#&
Them G-CSF oral9,4±0,67∗#&6,12±0,09∗#&
* - the significance of change of the indicator from its background values at p≤0,05.
# - noted the significance of change of the indicator from its control value at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

Thus, immobilizovannyi using ionizing radiation G-CSF has pronounced hepatoprotective properties of both parenteral and oral administration. While the mechanism of its action, obviously, are the stimulation and mobilization of stem cells from bone marrow and their further deterministic hominem in hepatic tissue and trim retireval in tissue-specific elements [1, 3].

Example 9

In the experiment on mice conducted research cerebroprotective effects img-CSF on the model of hypoxic encephalopathy. Assessment of the condition of the Central nervous system of animals produced by recording the neuropsychiatric status: conditional reflex activity and orienting-exploratory behavior of animals in the open field.

Encephalopathy was simulated using thermal chambers with a volume of 500 ml Mouse POM whom come back to the heat chamber, the lid was closed, and the mouse remained there until atonal convulsive seizure or respiratory failure, defined visually, for 10-15 seconds. After removing from oven treatment and recovery of spontaneous breathing, after 5-10 minutes, the mouse was again placed in the heat chamber before the onset of atonal state (generalized convulsive seizure or respiratory arrest). Mouse 1st group received pegfilgrastim subcutaneously ("NEULASTIM", Switzerland, Hoffmann-La Roche), mouse 2-nd group - oral pegfilgrastim ("NEULASTIM", Switzerland, Hoffmann-La Roche), animals of the 3rd group was subcutaneously injected img-CSF, and 4-th - img-CSF oral. All the drugs were injected in a dose of 100 μg/kg, 1 time a day for 5 days, starting immediately after exposure. Control animals were injected with saline in the respective modes. After 1 day after modeling encephalopathy in animals produced a conditioned reflex of passive avoidance, checking safety reflex and orienting-exploratory behavior in the open field was carried out on 14, 21 St day experience. Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney.

During the experiment hypoxic exposure resulted in the formation of encephalopathy. Thus, the observed statistics is automatic significant increase in total motor activity of mice in the open field, the number of horizontal movements and the increase of the coefficient of asymmetry of movements. At the same time, there was a sharp fall in the level of play of the conditioned reflex of passive avoidance and spontaneous mortality of animals throughout the observation period, reaching for the 21 St day 35,7%.

Therapeutic effect pegfilgrastim manifested only when it is subcutaneous injection, while the drug img-CSF had a therapeutic effect in parenteral and oral assignment. In both cases (the introduction of img-CSF) cerebroprotective effect was higher than that pegfilgrastim when its hypodermic use. Furthermore, subcutaneous injection img-CSF completely abolished the signs of encephalopathy (table 7, 8).

Table 7
The conditioned-reflex activity and mortality of mice CBACaLac with encephalopathy caused by hypoxia containment (1), after subcutaneous administration pegfilgrastim (2), oral administration pegfilgrastim (3), subcutaneous (4) and oral (5) introduction to them G-CSF after a stay of animals in the chambers, in the services. units, (X±m)
The timeframe of the study dayThe proportion of dead animals, % The playback level of reflex1
14thIR00,9+0,07
122,73∗0,41±0,12∗
212,3∗#0,7±0,08#
323,1∗0,4±0,1∗
47,4∗0,87±0,04#&
514,1∗0,69±0,97#
21stIR00,8±0,13
135,7∗0,1±0,1∗
214,1∗0,6±0,02#
J34,6∗0,12±0,12∗
48,1∗0,86±0,05#&
514,1∗0,65±0,07#
1- the proportion of animals with stored reflex
IR - relevant indicators in intact animals
* - the reliability of differences in the rate from its value in the intact animals at p≤0,05.
# - noted the significance of change of the indicator from its control value at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

Table 8
Dynamics of indices of orienting-exploratory behavior in mice of CBACaLac in an open field with encephalopathy caused by hypoxia containment (1), after subcutaneous administration pegfilgrastim (2), oral administration pegfilgrastim (3), subcutaneous (4) and oral (5) introduction to them G-CSF after a stay of animals in the chambers, in the services. units, (X±m)
The timeframe of the study dayThe asymmetry factorTotal locomotor activity is here Horizontal locomotor activity
14thIR0,49±0,0615,4±2,458,0±1,74
10,75±0,05∗36,86±8,1∗26,43±equal to 4.97∗
20,58±0,06#19,2±1,06#9,4±1,9#
0,76±0,03∗34,84±2,7228,71±2,11
40,37±0,04#&14,7±1,6#&12,4±1,5#
50,41±0,05#17,3±0,97#13,2±1,7#
21stIR0,56±0,0720,7±3,3112,6±2,21
10,75±0,03∗32,57±3,34∗23,86±of 4.38∗
20,62±0,04 26,59±2,317,2±1,3
30,74±0,02∗34,3±2,9∗22,4±2,7∗
40,54±0,02#&19,7±1,7#&11,7±2,4#&
50,61±0,12#21,8±a 3.87#15.62 wide,±2,19#
* - the reliability of differences in the rate from its value in the intact animals at p≤0,05.
# - noted the significance of change of the indicator from its control value at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

In addition, using cultural methods on the 7th day of the experiment was determined the content of neural precursors in paraventrikulyarnoe brain areas [4]. During the research it was found a significant increase in animals treated with pegfilgrastim subcutaneously subcutaneously and orally img-CSF. The highest values were reached change Yes the nogo option, it is in the groups of animals, which was introduced img-CSF (table).

Table 9
The content of neural precursors in paraventrikulyarnoe brain areas of mice with encephalopathy (1), subcutaneous (2) and oral (3) introduction pegfilgrastim, subcutaneous (4) and oral (5) introduction to them G-CSF after a stay of animals in the chambers, (X±m)
The timeframe of the study dayNeural precursor cells, 105the nuclears
7thbackground4,2±0,07
14,7±0,1
27,8±0,2∗#
34,12±0,08
412,6±1,1∗#&
59,78±1,3∗#
* - the significance of change of the indicator from its background values at p≤0,05.
# - noted the significance of change of the indicator from its control value at p≤0,05.
& is marked reliability of differences indicator of mice treated img-CSF from control values in animals treated with pegfilgrastim subcutaneously at p≤0,05.

Thus, the preparation is named G-CSF had a pronounced therapeutic effect in respect of post-hypoxic encephalopathy, and its therapeutic effect was significantly superior to the effect of therapy with the drug analogue - pegfilgrastim. The img-CSF was associated with the stimulation of regeneration mechanisms "deep" reserve defined functional activity of endogenous stem cells.

Example 10

In two variants studied the effects of G-CSF on granulocyte precursors (the main target cells G-CSF [13]. Analysis was performed by the method of variation statistics using t-test, t-test and non-parametric U-test, Wilcoxon-Mann-Whitney.

In the first scenario, non-stick bone marrow cells (at a concentration of 105the myelokaryocytes/ml) were placed in polveskoy of 1%methylcellulose culture medium of the following composition: 50% medium RPMI, 20% inactivated fetal calf serum, 6 g/l glucose, 280 mg/l L-glutamine, 50 mg/l gentamicin. Then in one case was added 5 ng/ml pegfilgrastim, and the other 5 ng/ml to them is-CSF, and incubated in CO2-incubator at 37°C, 5% CO2and 100%humidity for 7 days. In the control was added an appropriate volume of 10%) R-RA peg. Then made calculations granulocyte colonies (CFU-G), reflecting the content of the main target cells G-CSF[13].

While significantly more pronounced increase in CFU-G was observed when adding img-CSF (table 10). However, long-term cultivation of cellular material response CFU-G could be associated with more severe physical stability of immobilized using ionizing radiation G-CSF in model biological environment. To study the severity of the direct effects of G-CSF on progenitor cells, associated exclusively with the level (degree) activation under the influence of their specific receptors, experiments were carried out another design.

For this non-stick bone marrow cells (at a concentration of 105the myelokaryocytes/ml) were placed into the culture medium of the following composition: 90% RPMI medium, 10%" inactivated fetal calf serum, 6 g/l glucose, 280 mg/l L-glutamine, 50 mg/l gentamicin. Then in the first case was added 30 ng/ml pegfilgrastim, and in another 30 ng/ml img-CSF and incubated in CO2-incubator at 37°C, 5% CO2and 100%of humidity for 1 day. In control to allali corresponds to the amount of 10% R-RA peg.

The number of G-CSF and time of incubation were selected in preliminary studies, on the basis of which it was concluded that the concentration of G-CSF and dates of inactivated cells with drugs allow you to evaluate the level of activation of the receptor for G-CSF, since further increase in the content of the preparations of the cytokine in the culture medium is no longer able to increase the degree of response of hematopoietic precursors (full saturation is involved all the receptor apparatus of the cells), and the period of maximum biological activity of these substances in specified conditions exceed 1 day.

Then the cells were washed by centrifugation, placed them in polveskoy methylcellulose medium and incubated in CO2-incubator at 37°C, 5% CO2and 100% humidity for 7 days. Then counted the number of granulocyte colonies.

During the study, as in the previous case, was shown to significantly higher stimulating img-CSF in relation progenitor cells (table 10, (B).

Thus, G-CSF, immobilizovannyi on the media using ionizing radiation, has a much higher specific activity than pegfilgrastim, including those not related to physical strength ven the ATA in the biological environment.

The obtained results allow to conclude that the more pronounced the effectiveness img-CSF in vivo is associated with the increasing realization of the potential of receptor-mediated activation functions progenitor cells.

In General, based on the obtained data suggest that the remedy representing granulocyte colony-stimulating factor, immobilizovannyi water-soluble form by ionizing radiation, has expressed gemostimuliruyuschee mobilizing stem cells, hepatoprotective and cerebroprotective activities - regenerative activity. The effectiveness of this tool significantly higher than that in G-CSF conjugated (immobilized) with the carrier by chemical means (pegfilgrastim). Moreover, the technology of obtaining this tools allow you to use it effectively in Hematology and regenerative medicine not only dropped, but inside.

Literature

1. Goldberg ED, Digi A.M., Zhdanov V., Sushkov GN. and other Pharmacological aspects of regenerative medicine. // Bul. the experimental. Biol. and medicine. - 2008. - Annex 2. - P.14-21.

2. Tricot g, Barlogie Century, Zangari M, van Rhee F. e.a. Mobilization of peripheral blood stem cells in myeloma with either pegfilgrastim or filgrastim following chemotherapy. Haematologica. 2008 Nov; 93(11): 1739-42. Epub 2008 Aug 25.

3. Patent (RU) for the invention №2295971 "Method of therapy e is Sperimentale chronic toxic hepatitis", 2007 (publ. 27.03.2007,, bull. No. 9). Authors: Goldberg ED, Digi A.M., Zhdanov V., Sushkov GN. and other

4. Patent (RU) for the invention №2284060 "experimental therapy encephalopathy", 2006, (publ. 20.09.2006,, bull. No. 26). Authors: Goldberg ED, Digi A.M., Sushkov G.N., Zhdanov V., Suslov N

5. Piedmonte D.M., Treuheit M.J. Formulation of Neulasta (pegfilgrastim). Adv Drug Deliv Rev. - 2008. Jan 3; 60(1):50-8.

6. Anderson J.A. Allergic reactions to drugs and biologic agents. JAMA. - 1992 - vol.268. - p.2845-2857.

7. De Swarte R.D., Drug allergy. In: Patterson R. e.a. Allergic Diseases Diagnosis and Management, 4th ed. Philadelphia, Pa^ J.B. Lippincott. - 1993 - p.396-551.

8. Veronese, F.M., Mero A. The impact of PEGylation on biological therapies. BioDrugs. 2008; 22(5):315-29.

9. Vereschagin E.I., Khan Do-Hung, et al. Radiation Technology in the Preparation of Polyethylene Oxide Hydrophilic Gels and Immobilization of Proteases for Use in Medical Practice. // Arch. Pharm. Res. - 2001. - V.24. - N3. - P.229-233.

10. Seyfulla RD, Timofeev A.B., Ordzhonikidze SG and other Problems of the use of nanotechnology in pharmacology. // Experimental and clinical pharmacology. - 2008. No. 1. - P.61-69.

11. Y.M. Yevdokimov Spatially ordered forms of DNA and its complexes is the basis for the creation of nanoconstructs for medicine and biocenology. // Nanotechnologies in Russia. - 2006. - №1-2. - S-264.

12. Manual on experimental (preclinical) study of new pharmacological substances. / Ed Rugarama. - 2 ed. - M.: JSC "Publishing house of Medicine, 2005. - P.54-69.

13. Goldberg ED, Digi A.M., Shah V.P. Methods of tissue culture in Hematology. - Tomsk, Publishing house of Tomsk state University, 1992. - 272 S.

14. 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.

Vehicle with regenerative activity, and represents immobilizovannyi water-soluble polymers with a molecular weight of 400-4000 Yes granulocyte colony-stimulating factor, wherein the granulocyte colony-stimulating factor immobilization on polymers using ionizing radiation is a stream of accelerated electrons with an energy of 2.5 MeV, the absorbed dose from 2 to 10 kGy and speed dialing dose of 1.65 kGy/h



 

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