WO2009045123A1

WO2009045123A1 - Medicinal agent exhibiting hemopoiesis-stimulating and hepatoprotective actions

Info

Publication number: WO2009045123A1
Application number: PCT/RU2008/000252
Authority: WO
Inventors: Andrey Vladimirovich Artamonov; Petr Ivanovich Rodionov; Eugeny Danilovich Goldberg; Alexander Mikhailovich Dygai; Vadim Vadimovich Zhdanov; Gleb Nikolaevich Zyuzkov; Petr Petrovich Rodionov; Alexey Vyacheslavovich Stepanov; Evgeniy Ivanovich Vereshchagin
Original assignee: Company Limited 'concern O3'
Priority date: 2007-10-05
Filing date: 2008-04-22
Publication date: 2009-04-09
Also published as: RU2007137044A

Abstract

The invention relates to a medicinal agent exhibiting hemopoiesis-stimulating and hepatoprotective actions, comprising a granulocytic colony-stimulating factor which is immobilised on a water-soluble polymer carrier by the action of ionising radiation. Polyethylenglycol or hydroxyethyl starch having a molecular mass ranging from 400 to 4000 Da are used as a water-soluble polymer carrier. Electronic, gamma, X, laser or ultraviolet radiation is used in the form of a ionising radiation source.

Description

HEMOPOESITIMULATING AND HEPATOPROTECTIVE MEDICINAL PRODUCT

ACTION

5

Technical field

The invention relates to medicine, in particular to pharmacology and drugs, and can be used in the treatment of diseases associated with bone marrow depression and liver cirrhosis ιо at the initial stage of development.

State of the art

One of the most effective drugs for the treatment of diseases associated with bone marrow depression is granulocyte colony stimulating factor (G-CSF) (Vosé JM, 15 Armitage J.O. Slipal arrlicisopof of chemofortis // Chn. Opl. 13, N ° 4, p. 1023-1035). However, the known preparation has inherent drawbacks associated with the toxic effects of G-CSF upon parenteral administration (Vital T., Descottes J. Slipiculitus ocellulose used acomposites. Dosg Saf., 40, 1995, 1995. de Wit R., Verweij J., Vopeptbal M.E.A. -1398).

In particular, in addition to direct toxic effects, high immunogenicity of G-CSF is noted, which makes the prolonged use of this drug impossible.

Known methods for overcoming the immunogenicity of protein preparations are those in which glucocorticoid hormones or antihistamines are used for desensitization (Sentyakova T.N. Systemic lupus erythematosus, Novosibirsk, 2003, p. 192). However, these methods have certain disadvantages. First of all, the use of glucocorticoid hormones is accompanied by immunosuppression and an increased risk of infectious complications. In addition, this method cannot be implemented with prolonged use of protein preparations, for example, with diabetes.

Known drug Neupogen (Vidal Handbook. Medicinal 5 drugs in Russia: Handbook, M.:AstraFarmCervis, 1999, 1520s.) Which is a non-glycolized recombinant colony-stimulating human granolocyte factor intended for parenteral administration. However, this drug has a number of side effects due to its high immunogenicity and limited bioavailability.

Nevertheless, for clinicians, the prospect of using a drug based on G-CSF is so great that it becomes obvious the need for further improvement in terms of reducing toxicity and increasing bioavailability. Development

15 drugs that provide bioavailability of G-CSF when taken orally, aims to prevent dangerous complications observed in modern medicine with intravenous, intramuscular and subcutaneous injections.

SUMMARY OF THE INVENTION 0 The object of the invention is to develop a medicament based on a granulocyte colony stimulating factor that can be used not only for parenteral, but also for oral administration. At the same time, this drug should have 5 no toxic effects inherent in G-CSF.

The solution to this problem is achieved by immobilizing a therapeutically effective amount of G-CSF on a water-soluble polymer carrier under the action of ionizing radiation. As a source of ionizing radiation, you can use electron radiation (directed stream of accelerated electrons), gamma radiation, x-ray radiation, laser radiation and ultraviolet radiation. Appropriate ionization equipment and techniques are described in the following publications (Gopshar A.M. apd Auslepdeg VL Imbilizoprotezes on water-solvepulmegpremeprechim. .M.upd Auslepdeg VL ELECTROP BEAM IMMOBILIZATOPOF HUDROLUTIS FERMEPTS HAVIPG ROULUFUPSTIOPAL ARRLICATIOP. RAD. Prhus. Scheme.H. V. Grishiпp, S.E. Retrov, E.R. Gulaueva, L.A. Wagdapova, M.V. Arsh. Pharm. Res. 2001. V. 24, NOZ p. 229-233).

The most preferred method of immobilization is the impact on the polymer carrier and biologically active compound by a directed stream of accelerated electrons. The treatment is carried out by bremsstrahlung generated by an ILU-6 or ILU-10 accelerator with an electron energy of 2.5 MeB, an absorbed dose of 2 to 10 kGy, and a dose rate of 1.65 kGy / hour. As a water-soluble polymer, polymers traditional for immobilizing biologically active compounds are used, in particular, polyethylene glycol (PEG), hydroxyethyl starch, polyvinylpyrrolidone, dextrans, isoprenols can be used. Most preferably, polyethylene glycol of molecular weight 400 to 4000 Da is used as the water-soluble polymer.

Immobilization can be carried out either in one stage, when a reaction medium containing both a biologically active molecule and a polymer is irradiated, or in two stages, when the polymer is first activated, and then the second stage is carried out immobilization itself.

G-CSF immobilized by the action of ionizing radiation on a water-soluble polymer carrier can be used either directly for oral administration, or in the form of a pharmaceutical composition containing a physiologically acceptable solvent, for example water, and optionally other pharmaceutically acceptable additives traditionally used in the composition of pharmaceutical compositions.

The therapeutically effective amount of recombinant human G-CSF is determined depending on the specific purpose of the proposed medicinal product, the method of its administration, the severity of the disease and the condition of the patient and can be subcutaneous or intravenous: in humans

5 to 15 μg / kg for 3 to 10 days, in a mouse from 10 to 200 μg / kg for 3 to 10 days.

Examples of carrying out the invention.

The present invention is illustrated by the following examples. Example 1

A 10% aqueous solution of polyethylene glycol with a molecular mass of 1.5 kDa was irradiated with a stream of accelerated electrons at a dose of 1.5 Mrad. G-CSF was added to the irradiated solution to a final concentration of 10 mg G-CSF in 1 ml of 10% polyethylene glycol. The mixture was stirred for 10 minutes and the final immobilized preparation was obtained in the form of a slightly opalescent solution. The yield of the finished product is 98%. Example 2

A 10.0% aqueous solution of polyethylene oxide with a molecular weight of 0.4 kDa was irradiated with inhibitory gamma radiation at a dose of 1.0 Mrad. G-CSF was added to the irradiated solution to a final concentration of 1 mg G-CSF in 1 ml of 10% polyethylene glycol. The mixture was stirred for 30 minutes and received the preparation of immobilized G-CSF in the form of a clear solution. The yield of the finished product is 97%.

As a result of the use of biologically active drugs immobilized in this way, the formation of 5 antibodies is practically absent even with prolonged parenteral use. G-CSF immobilized in a similar way acquires the ability to be absorbed in the gastrointestinal tract when taken orally and to have a therapeutic effect in doses close to those used for parenteral administration. ι o Example 3

Experiments have been conducted to study the hematopoietic stimulating and mobilizing activity of the drug proposed in the present invention when administered in various ways compared to the standard preparation

15 non-immobilized recombinant G-CSF in a model of cytostatic myelosuppression.

Studies were performed on C57B1 / 6 mice. 63 individuals weighing 18-20 g were divided into 3 groups. Cyclophosphamide (CF) was administered to all animals at a dose of 170 mg / kg. Then, mice of the 1st group received 0 subcutaneous drug G-CSF Neupogen ("Hoffman-la Roche"), mice of the 2nd group - the drug proposed in the present invention (imG-CSF), subcutaneously, and animals of the 3rd groups - immG-CSF intragastrically. All preparations were administered at a dose of 100 μg / kg, starting from the day after the administration of cytostatic 5 for 5 days 1 time per day. On the 5th and 7th days of the experiment (counting from the moment of administration of CF) in the experimental and control mice, the total bone marrow cellularity (OKK) and the total number of leukocytes (OKJI) in the peripheral blood were determined by conventional methods. On bone marrow and blood smears were counted, respectively. b myelo- and hemogram (Laboratory research methods in the clinic: Handbook / edited by VV Menshikov. - M .: Medicine, 1987. - 368 s). After partial decapitation, the animals received blood in the amount necessary to isolate a sufficient number of mononuclear cells. The latter were used to determine the number of hematopoietic progenitor cells (CFU-GM) in peripheral blood by colony formation in a semi-viscous medium (Goldberg E.D., Dygay AM, Shakhov V.P. Methods of tissue culture in hematology. Tomsk: TSU Publishing House, 1992 , 272 s). Statistical processing of the obtained data was carried out by methods of variation statistics using Student's T-criterion (Lakin G.F. Biometrics.- M .: Higher School, 1973. -215 s).

The experiments made it possible to establish that under the influence of all G-CSF preparations in the peripheral blood, the content of stab and segmented neutrophils increased on the 5th and 7th days of the experiment (compared with mice that were administered only CF). This phenomenon was maximally expressed with the introduction of the drug G-CSF obtained by standard technology (Neupogen). The increase in these indicators was also quite significant in animals treated with subcutaneous immobilized G-CSF, which was accompanied by an increase in blood levels of eosinophils, monocytes and lymphocytes on the 7th day. With the introduction of imGG-CSF, the blood count also significantly increased (to a slightly lesser extent and without an increase in OKL, as in the two previous groups), the content of stab- and segmented neutrophilic granulocytes (table 1). In the bone marrow, granulation of locytopoiesis was also observed, and the increase in the number of mature neutrophils (compared with animals that were administered only CF) was statistically significant in all three experimental groups on the 5th day of the experiment. On the 7th day, this indicator remained increased only in mice treated with IMH- CSF subcutaneously (table 2).

As for the ability to induce the release of hematopoietic precursors into the blood, cyclophosphamide increased the CFU-GM content in circulation by 3 times already on the 5th day of the experiment (this is a known effect of this cytostatic agent). The use of the studied drugs enhanced the indicated effect of CF on the 5th day, moreover, reliably - of the standard preparation G-CSF and imG-CSF administered per os (Table 3).

The results obtained indicate the presence of G-CSF immobilized according to the present invention with hemostatic activity both when it is administered subcutaneously and intragastrically, and the ability to cause stem cells to enter the blood, more pronounced when the drug is administered orally.

Example 4. We studied changes in the morphological and functional state of liver tissue under the influence of imG-CSF and Neupogen in a model of chronic toxic liver damage.

The experiments were conducted on 120 outbred rats weighing 250-300 g. In rats, hepatitis was caused by intragastric administration of a 50% solution of CCl ₄ (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 of chronic toxic liver damage, the animals were divided into 3 equal groups. Rats of the 1st group for 5 days were subcutaneously injected with 100 μg / kg of Neupogen, dissolved in 0.5 ml of solvent. The first administration was carried out the day after the last administration of carbon tetrachloride. Animals of the 2nd group were intragastrically injected with imG-CSF at a dose of 100 μg / kg for 5 days. Animals of the control group were injected with the same scheme in an equivalent volume distilled water.

To study the state of the liver, rat death was assessed, biochemical studies of serum levels of aspartate and alanine aminotransferases (AcAT, AlAT) on days 14 and 28 were performed, 5 as well as a morphological study of the liver on day 40 of the experiment. The activity of serum enzymes was determined by conventional methods using a semi-automatic biochemical analyzer from Sormau and standard kits for it. Blood for the study was obtained from the femoral artery through a catheter. On histological ι about liver preparations stained with hematoxylin and eosin, the number of cells of the infiltrate was determined using an Avtandilov ocular network containing 25 test points. In 20 fields of view, the number of cells reaching the test points of the grid was counted. The relative area of infiltration was calculated as the ratio

15 grid points per infiltrate cell to all grid points in 20 fields of view. The area of connective tissue was determined using computer graphics processing. For this, on the standard area of the liver section (consecutive microphotographs of 10 fields of view made by a digital video camera 0 "Digital miсrо", with the program for transferring the image to a computer company "Elekard", Tomsk), the area of the structures stained with picrofuxin was measured, and the percentage of the selected standard area was calculated .

The results were processed by the method of variation 5 statistics using Student's t-criterion (Lakin G.F. Biometrics.- M .: Higher School, 1973. - 215 s).

The experiments showed that the death of rats at the end of the study in the control group was 22.3%, with the appointment of Neupogen - 15%, and the preparation imGH-CSF - 16.2% of initial number of animals. The revealed differences from the control were statistically significant. Biochemical studies of blood serum revealed an increase in AlAT activity on days 14 and 28, and AcAT on the 14th day of the experiment after the start of CCU administration in the control group (table 4). In rats treated with Neupogen, only the concentration of AlAT in serum on the 28th day of the experiment was significantly lower than in animals that were injected with a solvent. Normalization of the activity of transaminases in the blood of rats treated with imG-CSF occurred earlier and was more pronounced than with the use of neupogen (table 4).

In the study of histological preparations of the liver, in the control group of rats, a marked violation of the lobular structure of the organ was noted. The preparations showed fields of granulation tissue replacing dead hepatocytes, in which neoplasms of the vessels and hepatic ducts occurred. In preserved hepatocytes, pronounced large-drop fatty degeneration was observed. At the same time, significant regenerative hypertrophy of the liver cells took place. The relative area of infiltration on the 40th day of the experiment was 25.40 ± 1, 2%, and connective tissue - 5.75 ± 0.42% (table 5). The introduction of Neupogen or imG-CSF during the modeling of CCl ₄ hepatitis preserved the lobular structure of the liver, and the fatty degeneration of hepatocytes was predominantly small-droplet. In experimental animals, although less significant, but still significant infiltration of the portal tracts was noted, while the infiltrate, as a rule, did not penetrate into the lobules. In general, the relative area of infiltration was significantly (43.3%) lower than in the control group. In addition, the drug imG-CSF, like Neupogen, significantly reduced the area of connective tissue (l, 54 ± 0.18% on the 40th day) (table 5). Thus, using biochemical and morphological methods, the drug imG-CSF revealed a significant hepatoprotective activity comparable to that of the recombinant G-CSF (Neupogen) preparation. This drug had a pronounced anti-inflammatory and antisclerotic effect.

Table 1

Dynamics of the total number of leukocytes and the content of their individual morphological forms in the peripheral blood (xlO ⁹ / L) in C57 / B1 mice after administration of immobilized and standard preparations of G-CSF in various ways

(neupogen) on the background of cyclophosphamide (CF). X ± m

about

* - differences from intact control are significant (P <0.05)

# - differences from CF are significant (P <0.05)

$ - differences from CF + neupogen reliable (P <0.05)

table 2

The dynamics of the total number of myelocaryocytes and the content of their individual morphological forms in the bone marrow (xl0 ⁶ / thigh) of C57 / B1 mice after the administration of immobilized and standard preparations of G-CSF (neupogen) against cyclophosphamide (CF) in various ways. X ± m y

ϋύ

≡

S

Uj o

* - differences from intact control are significant (P <0.05)

# - differences from CF are significant (P <0.05)

$ - differences from CF + neupogen reliable (P <0.05)

Table 3

The content of granulocyte-macrophage precursors (CFU-GM) in the peripheral blood (5x10 ⁵ mononuclear cells) in C57 / B1 mice after the administration of various types of immobilized and standard (neupogen) G-CSF preparations against cyclophosphamide (CF), X ± m

* - differences from intact control are significant (P <0.05)

# - differences from CF are significant (P <0.05)

$ - differences from CF + neupogen reliable (P <0.05) Table 4

Changes in the biochemical blood parameters of rats with chronic CCL ₄ hepatitis under the influence of imG-CSF and prototype, X ± m

* - the significance of the difference between the indicator and background values is noted at p <0.05;

# - the significance of the difference between the indicator and control values at p <0.05 is noted.

Table 5

The effect of immG-CSF and prototype (neupogen) on the liver morphology of rats with chronic hepatitis on the 40th day after the introduction of CCL ₄ (X ± m)

Claims

CLAIM

1. A drug with hematopoietic and hepatoprotective effects, including granulocyte colony stimulating factor, characterized in that it contains

5 a therapeutically effective amount of a granulocyte colony-stimulating factor immobilized on a water-soluble polymer carrier under the influence of ionizing radiation.

2. The drug under item l, characterized in that it contains as a water-soluble polymer carrier polyethylene glycol or hydroxyethyl starch of molecular weight from 400 to 4000 Da.

3. The drug according to claim 1, characterized in that electron radiation is used as ionizing radiation,

15 gamma radiation, x-ray radiation, laser radiation or ultraviolet radiation.

0 5