EA019043B1 - A novel conjugate of granulocyte colony-stimulating factor (g-csf) with polyethylene glycol - Google Patents

Abstract

The present invention relates to pharmaceuticals and medicine, namely, to new physiologically active conjugates of granulocyte colony-stimulating factor (G-CSF) with the general formula (I)where n is integers from 681 to 1000; m is an integer ≥4; NαH-G-CSF is granulocyte colony-stimulating factor. The invention also relates to medicines containing the claimed conjugate of formula (I), pharmaceutical compositions, the use of conjugate of formula (I) for drugs and medicines having the activity with granulocyte colony stimulating factor as an active ingredient, a method of preventing and/or treating neutropenia, the container that comprises pharmaceutical composition or a medicine and a kit.

Description

The present invention relates to pharmaceuticals and medicine, in particular to new physiologically active conjugates of granulocyte colony stimulating factor (G-CSF), in particular to new conjugates of G-CSF with polyethylene glycol suitable for medical use, for example for the treatment of leukopenia, mainly various types of neutropenia in patients receiving myelosuppressive chemotherapy for cancer, for the treatment of myelodysplastic syndrome, improve tolerability of immunosuppressive drugs with per cage of bone marrow, improving the immune status of patients suffering from AIDS and other infections.

G-CSF is a hematopoietic growth factor that stimulates the proliferation, differentiation and maturation of granulocytes (Ms1ca1G. 1992). The introduction of exogenous G-CSF causes a rapid, specific and dose-dependent increase in neutrophils in the peripheral blood (\ Me11e e! A1., 1985; Naiypde e! A1., 1995).

The human G-CSF gene was cloned and expressed in bacterial and animal cells; as a result, various preparations of recombinant G-CSF were obtained and purified (No. 1a1a e! A1., 1986; 8onha e! A1., 1986; Kosha18i e! A1., 1987).

Two preparations of recombinant human G-CSF are known - filgrastim and lenograstim, obtained from E. soy cells and Chinese hamster cells, respectively. Filgrastim consists of 175 amino acids, has the same amino acid sequence as the natural protein, but contains an additional methionine residue at the Ν-terminus of the molecule. Lenograstim consists of 174 amino acids, its amino acid sequence is fully consistent with that of natural human G-CSF.

Medicines containing recombinant human G-CSF (rhG-G-CSF) are used in clinical practice for the treatment of various leukopenias, in particular neutropenia that occur after chemotherapy, idiopathic, congenital and cyclic neutropenia, for the treatment of severe infections in combination with antibiotics, and neutropenia in AIDS patients (Moypeikh, 2004).

In clinical use, the effectiveness of drugs containing rhG-CSF is limited by rapid absorption from subcutaneous tissues, relatively low stability, short half-life, high immunogenicity and toxicity (Mashkovsky, 2006; no \ νίί e! A1., 1996). As a result, the use of rhG-CSF requires daily administration and repeated monitoring of effectiveness, in this case, a clinical blood test with a leukocyte count.

The therapeutic efficacy of G-CSF preparations can be enhanced by the use of sustained release drugs in which the native protein molecule is chemically bonded to monomethoxy polyethylene glycol (mPEG). PEGylation of G-CSF leads to improved pharmacokinetics; increase half-life, decrease clearance, reduce fluctuations in blood concentration; a decrease in immunogenicity and toxicity, an increase in the activity of ίη у1уо, an increase in stability (MoBpeikh, 2003; Mopeich, 2004).

The biological properties of PEG-G-CSF conjugates are largely dependent on the molecular weight and type of activated mPEG used, the functional groups of which differ in their ability to modify various amino acid residues of the protein and in the type of chemical bonds formed with the protein (Koeik e! A1., 2002). In the preparation of PEGylated proteins, activated mPEGs that can bind to the free amino groups of proteins (succinimidyl carbonate, succinimidyl succinate, trasylate, etc.) found the widest application.

Various PEG-G-CSF conjugates are known in the art.

Patent I8 2007/0014762 describes the preparation of PEG-G-CSF conjugates using various activated mPEGs with a molecular weight of 5000 Da, in particular mPEG-succinimidyl butanoate, mPEG-succinimidyl propionate, mPEG-succinimidyl α-methylbutanoate. To obtain the conjugate, rhG-CSF obtained from CHO cells (Lenograstim) was used, containing several point substitutions of amino acids at different positions. The conjugation reaction was carried out at pH 7-9. The resulting PEG-G-CSF conjugate consisted of several positional isomers in which PEG bound to the epsilon-amino groups of lysines and to the alpha-amino group of the Ν-terminal amino acid. Purification of the resulting conjugate was carried out using ion exchange chromatography using 8P-Sepharose sorbent. Data on the activity and purity of the resulting conjugates in the patent are missing.

The disadvantages of the resulting conjugate are:

1) low molecular weight MPEG attached to the G-CSF;

2) the formation of several positional isomers in which PEG was bound to the G-CSF molecule through the free amino groups of various lysines and the Ν-terminal amino acid.

EP 0 401 384 discloses PEG-G-CSF conjugates obtained using MPEG linear and branched structures with a molecular weight of 4,500-1,000 Da activated with various reactive groups (succinimidyl carbonate, triazine chloride and polyoxyethylene diamine). The obtained PEG-G-CSF conjugates had a prolonged action of ίη у1уо, and the degree of prolongation depended on the molecular weight of the attached PEG. The half-life of the PEG (10000) -G-CSF conjugate and modified G-CSF conjugate was 7.05 and 1.79 h, respectively.

- 1 019043

The disadvantages of the obtained conjugates are:

1) succinimidyl carbonate derivatives of MPEG can bind to all sterically available free amino groups of the protein, as a result of which the resulting PEG-G-CSF conjugates consist of several positional isomers;

2) the triazine chloride derivatives of MPEG, in addition to free amino groups, are able to bind to functional groups of other amino acids - serine, tyrosine, threonine and histidine, which leads to the appearance of many isomers, some of which are characterized by an unstable bond. Moreover, triazine derivatives are not currently used due to their high toxicity (Uegopeke & Cancers !, 2005).

EP 0335423 discloses conjugates of G-CSF with a triazine chloride derivative of mPEG PEG with a molecular weight of 300-30000 Da. The obtained PEG-G-CSF conjugates had a prolonged action, their specific specific activity ranged from 11 to 60% of the activity of unmodified G-G-CSF.

The disadvantages of the obtained conjugates are:

1) low specific specific activity;

2) the use of triazine chloride derivatives of MPEG, resulting in the formation of a number of positional isomers, some of which are characterized by an unstable bond. Moreover, triazine derivatives are not currently used due to their high toxicity (Uegopeke & Cancers !, 2005).

As a prototype of the present invention, the PEG-G-CSF conjugate described in I8 5824784 (Example 2) was selected. The patent describes a reductive alkylation reaction of G-CSF using linear mPEG with a molecular weight of from 6000 to 25 activated by a propionaldehyde group (ABE-tREC). The PEGylation reaction was carried out at pH 5. Under these conditions, PEG addition occurred selectively through the alpha-amino group of the кон-terminal amino acid. According to the described method, a G-CSF conjugate with PEG with a molecular weight of 20 kDa was obtained, which is currently used in the clinic for the treatment of neutropenia of various etiologies - the drug Neulastim (firm. NoGGtapp-Ba Roeye. Switzerland). The obtained PEG-G-CSF conjugate was purified by ion exchange chromatography on a column with 8-Sepharose HP sorbent. The biological activity of the obtained purified PEG-G-CSF conjugate was 68% of the native G-CSF. The content of unmodified G-CSF in the resulting conjugate was not more than 5%. The pharmacokinetic parameters of the resulting conjugate were better than unmodified G-CSF. In animal experiments, it was shown that the introduction of the PEG-G-CSF conjugate led to an increase in the level of leukocytes in the blood, and the PEG-G-CSF conjugate had a more prolonged action compared to unmodified G-CSF. With a single administration of the PEG-G-CSF conjugate, the blood leukocyte content of the animals reached a maximum 1 day after administration, this level remained during the day and then the leukocyte level decreased to the initial level 4 days after administration. With the introduction of unmodified G-CSF, the maximum level of leukocytes in the blood of animals was observed 1 day after administration, then the content of leukocytes rapidly decreased.

The disadvantages of the resulting conjugate are:

1) low specific activity of the obtained conjugate, comprising 68% of the activity of unmodified G-CSF;

2) the presence of unmodified G-CSF in the conjugate.

The objective of the present invention was to obtain a new stable highly purified PEGylated G-CSF conjugate with higher activity, with more prolonged action, improved stability, with improved pharmacokinetic parameters, with an optimal combination of PEG molecular weight parameters and specific activity, with a high degree of purity suitable for the creation of a medicinal product for medical purposes, contributing to the expansion of the arsenal of drugs of a given orientation, f rmatsevticheskoy compositions based on the claimed conjugate.

The problem is solved by creating a new molecule of the functionally active PEG-G-CSF conjugate with G-CSF activity, in which a linear PEG molecule with a molecular mass of 30,000 Da is connected to the G-CSF molecule by a stable bond strictly through the alpha-amino group of the кон-terminal amino acid (methionine) so that the general formula of the PEG-G-CSF conjugate with linear mPEG is the following compound (I):

P '111 where η is an integer value from 681 to 1000;

t is an integer> 4;

G-CSF is a natural or recombinant polypeptide having G-CSF activity.

In the resulting conjugate, a linear PEG with a molecular weight of 30000-40000 Da is bound to alpha

- 2 019043 the amino group of the Ν-terminal amino acid G-CSF.

The selectivity of the modification of G-CSF strictly according to the alpha-amino group of the кон-terminal amino acid is achieved through the use of aldehyde-activated mPEGs of the general formula (II) and the PEGylation reaction at pH <5:

snDo-sn ₂ -sn ₂ Do4sn ₂ 4— s / θ (P) where η are integer values from 681 to 1000, so that the molecular weight of PEG is approximately 3000040000 Da;

t is an integer> 4;

The optimal ratio of the parameters of the molecular weight of PEG and specific hemostimulating activity is achieved through the use of activated mPEG (II) with a value of m> 4, which leads to a more effective interaction of the inventive conjugate with receptors and higher biological activity.

The decrease in immunogenicity, toxicity and improvement of pharmacokinetic parameters is achieved by increasing the molecular weight of the attached PEG.

New in comparison with the prototype is the following.

1. To obtain the conjugate PEG-G-CSF used aldehyde derivatives of activated mPEG of formula 2.

2. The structural formula of the conjugate PEG-G-CSF.

3. An increase in the molecular weight of the PEG-IFN conjugate due to the size of the attached PEG.

4. A higher level of specific specific activity.

5. Improved pharmacokinetic parameters.

The inventive conjugate PEG-G-CSF is a highly purified preparation with prolonged action, characterized by high specific biological activity (not less than 84-94% of unmodified G-CSF), protein purity of not less than 97%, high thermal stability, increased resistance to proteolytic enzymes with improved pharmacokinetic parameters.

Also included in the present invention are pharmaceutical compositions containing, as an active ingredient, the claimed PEG-G-CSF conjugate in an effective amount, these compositions may also include pharmaceutically acceptable carriers, buffering agents (organic and inorganic acids and their salts, for example citric acid buffers , succinate, tartaric acid, fumarate, gluconic, oxalic acid, lactic acid, acetate buffers), stabilizers (polyhydric sugar alcohols, amino acids, organic sugars or sp oral sugars, inositol, PEG, amino acid polymers, sulfur-containing reducing agents such as urea, glutathione, thioglycerol, etc., low molecular weight polypeptides, proteins such as human serum albumin, immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, monosaccharides such as mannose, xylose, fructose, glucose, disaccharides such as lactose, maltose, sucrose, trisaccharides such as raffinose, and polysaccharides such as dextran), preservatives (phenol, benzyl alcohol, meta-cryzole, me tilparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkane halides, alkyl parabens), antioxidants (methionine, vitamin E, ascorbic acid), isotonizing agents (polyhydric sugar alcohols, sodium chloride, sorbitol, mannitol, arabitol, etc.) nonionic surfactants and detergents (polysorbates, polyoxamers, polyols, tween), cosolvents, fillers (starch), chelating agents (e.g. EDTA). These pharmaceutical compositions can be applied in various forms, for example, lyophilisate, aqueous form (injections, aerosols, drops, etc.).

Also, the present invention includes drugs based on the inventive conjugate, in particular, the drugs used to treat leukopenia, mainly various types of neutropenia.

The inventive pegylated G-CSF, as well as pharmaceutical compositions and medicines based on it, can be used to treat neutropenia in patients receiving myelosuppressive chemotherapy for cancer, to treat myelodysplastic syndrome, to improve tolerability of immunosuppressive drugs in bone marrow transplantation, and to improve immune status in patients suffering from AIDS and other infections, in particular for antifungal therapy, in particular for the treatment of systemic or invasive vnogo candidiasis.

A conjugate of formula (I) with an optional pharmacologically acceptable excipient, diluent and / or pharmaceutically acceptable excipients is administered intravenously, subcutaneously, intramuscularly or in any other suitable way. The route of administration varies depending, for example, on symptoms and age, the frequency of administration and the interval between administrations vary depending on the disease and its severity or on the purpose (therapeutic or prophylactic

- 3 019043 use), the effective amount of the active principle of PEG-G-CSF is selected taking into account the above factors.

To obtain the inventive PEG-G-CSF conjugate, a highly purified recombinant human G-CSF (filgrastim) manufactured by ZAO Biocad and butyraldehyde derivatives of MPEG of formula (II) with a molecular weight of 30,000-40000 Da was used.

The conjugation reaction was carried out at a pH below 5.0 in the presence of a reducing agent at a temperature equal to or below 20 ° C. The PEG / protein molar ratio was 2.5-5 / 1. The formation of the PEG-G-CSF conjugate was monitored by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (DDS) under reducing conditions and reverse phase high performance chromatography (RP HPLC). MonoPEG-G-CSF was purified from reaction products unmodified G-CSF and from undesirable forms of PEG-G-CSF containing two or more PEG molecules per protein molecule by chromatography on cation exchange sorbents. MonoPEG-IFN conjugate was eluted with a gradient of sodium chloride concentration from 0.05 to 0.2 M in a buffer solution with a pH below 5. The purified monoPEG-G-CSF preparation was dialyzed against 1050 mM buffer solution with a pH of 4-5, salts were added, or polysaccharides, or polyhydric alcohols, or polyvinylpyrrolidones, or monosaccharides, or amino sugars, or proteins, or amino acids and non-ionic detergents and were stored in plastic or glass vials with a siliconized surface at a temperature of 4 ± 2 ° C.

To characterize the obtained monoPEG-G-CSF conjugate, its purity, homogeneity, physicochemical, biological and pharmacokinetic parameters were studied in comparison with unmodified G-CSF.

The present invention is illustrated by figures of a graphic image, which presents:

FIG. 1. Kinetics of the formation of the conjugate PEG-G-CSF.

FIG. 2. Reverse-phase HPLC of the PEG-G-CSF conjugate on a Bushteyu C4 column (4.6x4, bx 150 mm).

FIG. 3. Gel-filtration HPLC of the PEG-G-KSF conjugate on a TBC S30003 \ UH column (30 cmx7.8 mm).

FIG. 4. Electrophoretic analysis of the conjugate PEG-G-CSF in comparison with unmodified GCSF.

FIG. 5. MLBO1 mass spectra of modified G-CSF (A) and PEG-G-CSF (B) conjugate.

FIG. 6. Comparison of the mass spectra of tryptic peptides G-CSF (A) and the conjugate PEG-G-CSF (B).

FIG. 7. The study of thermal stability of PEG-G-CSF (green line) and unmodified GCSF (red line) at a temperature of 50 ± 2 ° C.

FIG. 8. The study of the stability of the conjugate PEG-G-CSF (green line) and unmodified G-CSF (red line) when treated with trypsin.

FIG. 9. Determination of immunoreactivity of the PEG-G-CSF conjugate (green line) in comparison with unmodified G-CSF (red line).

FIG. 10. Pharmacokinetics of the PEG-G-CSF conjugate (green line) compared with unmodified G-CSF (red line).

Examples of specific performance of the method of obtaining PEG-G-CSF and study of its properties are given below.

Example 1. Obtaining purified rhG-CSF (filgrastim).

Isolation and purification of rhG-CSF was performed as described in patent UE 2278870.

Example 2. Obtaining the conjugate PEG-G-CSF.

To 2300 ml of a buffer solution (50 mM sodium acetate, pH 5.0 ± 0.2) containing 2500 mg of purified rhG-CSF obtained according to claim 1, 50 ml of a 1 M sodium cyanoborohydride solution was added, mixed and 10 g of dry butyraldehyde derivative of monomethoxy PEG (mPEG) with a molecular weight of 30,000 Da. The sample was incubated for 22 hours with constant stirring at a temperature of 20 ± 2 ° C. At different time intervals, 50 μl aliquots were taken from the reaction mixture and the kinetics of the formation of the PEG-G-CSF conjugate were analyzed using polyacrylamide gel electrophoresis (EPA) in the presence of sodium dodecyl sulfate (DDS) under reducing conditions. For this, 1/3 of the buffer containing 125 mM Tris-HCl, pH 6.8, 20% glycerol, 3% DDS, 15% 2-mercaptoethanol, 0.005% bromophenol blue was added to the sample and heated for 3 min in boiling water bathhouse. Samples in a volume of 5 μl were introduced into the wells of the prepared PAAG plates and electrophoresis was performed in 12.5% PAAG in the presence of DDS. At the end of electrophoresis, the gel was stained with Coomassie E-250. The kinetics of PEG-G-CSF conjugate formation is shown in FIG. 1. At the time when the content of PEG-G-CSF was more than 70%, the reaction mixture was diluted 3 times with 10 mM sodium acetate buffer, pH 4.8 ± 0.2.

Example 3. Purification of the conjugate monoPEG-G-CSF.

The diluted solution obtained in Example 2 was applied to a column with a cation exchange sorbent (CM-Sepharose, 300 ml), equilibrated with 10 mM sodium acetate buffer, pH 4.8 (buffer A), with

- 4 019043 at a rate of 10 ml / min. The sorbent column was washed with 1500 ml of buffer A. The PEG-G-CSF conjugate bound to the sorbent was eluted with 6000 ml of buffer A containing a concentration gradient of 1C1 from 0 to 0.2 M at a rate of 5 ml / min, collecting 50 ml fractions . In the obtained fractions, the optical density was measured at 280 and 260 nm, the protein concentration was determined, and analysis was performed using reverse phase HPLC and EF in PAG, as described in Example 2. Fractions containing the mono-PEGylated PEG-G-CSF conjugate with a purity of more than 95%, combined, dialyzed against 10 volumes of 1.6 mm sodium acetate buffer, pH 4.0 ± 0.2, sterile filtration was performed and stored at a temperature of 4 ± 2 ° C.

Example 4

Determination of the purity of the PEG-G-KSF conjugate using the method of reverse phase high performance liquid chromatography (RP HPLC)

The PEG-G-CSF conjugate obtained according to claim 3 was diluted with 20 mM sodium acetate buffer, pH 5.0, to a concentration of 0.3 mg / ml, and 100 μl of the obtained sample was added to a column of 8utte1gu C4 (4.6x150 mm). The study was carried out on a Vgsskhs chromatograph (Ca1eg8) at 214 nm. From the results presented in FIG. 2, it follows that the PEG-G-CSF conjugate is a high purity preparation. It can be seen from the presented chromatogram that from the Zutteiu C4 sorbent the protein elutes with one symmetrical peak, the proportion of which is 98.85%. Minor UV-absorbing impurities, eluting before and after the major peak of the protein, amount to <1.15%. Thus, from the presented data it follows that according to reverse phase HPLC, the purity of the inventive PEG-G-CSF conjugate is> 98%.

Example 5

Determination of the purity of the PEG-G-KSF conjugate using gel filtration (exclusion) high performance liquid chromatography (HPLC).

The PEG-G-CSF conjugate obtained according to claim 3 was diluted with 20 mM sodium acetate buffer pH 5.0 to a concentration of 0.3 mg / ml and 100 μl of the obtained sample was added to a T8K O30008 \ UH column (30 cmx7.8 mm ) The study was carried out on a Wgehech chromatograph (Ca1eg8) at 220 nm. The results of the study are presented in FIG. 3. It can be seen that the PEG-G-KSF sample under investigation exits the column with one clearly pronounced symmetrical peak of 98.47%. From the results presented in FIG. 3, it follows that the claimed conjugate PEG-G-CSF does not contain free unmodified G-CSF. The content of high molecular weight aggregates in the inventive conjugate does not exceed 1.53%. The purity of the inventive PEG-G-KSF conjugate according to GF HPLC is more than 98%.

Example 6

Determination of the level of endotoxins in the PEG-G-CSF conjugate.

The content of bacterial endotoxins (BE) in the PEG-G-CSF conjugate sample obtained according to claim 3 was determined ίη νίΐτο using the LAL test (gel-thrombus test modification) in accordance with the requirements of the General Pharmacopoeia Monograph 42-0002-00. In the work we used a diagnostic multicomponent kit of the company Az8OS1a1e8 οί CARE SOI, 1ps. RAL reagent ROOKET® with a sensitivity of 0.03 EU / ml, control endotoxin standard (KSE, 0.5 μg in a vial), water for the LAL test. From the results presented in table. 1, it can be seen that the content of BE in the sample of the PEG-G-CSF conjugate is less than 3 units of endotoxin (eE) per 1 mg of protein, which is much lower than the level of BE allowed for medications based on recombinant proteins.

Table 1

The content of bacterial endotoxins in the conjugate PEG-G-CSF

The drug conjugate PEG-G-KSF The content of bacterial endotoxins (eE / mg protein) PEG-G-KSF obtained according to item 3. <3.0

Example 7

Electrophoretic analysis of the conjugate PEG-G-CSF in comparison with unmodified G-CSF.

A sample of the conjugate PEG-G-CSF obtained according to claim 3, was analyzed by electrophoresis in SDS page, as described in claim 2. Electrophoresis was performed with a load of 40 μg of protein per well under non-reducing conditions. Proteins in the gel were stained with Coomassie B-250 dye. At the same time, electrophoresis of unmodified G-CSF was performed. The PEG-G-CSF conjugate was represented by one high molecular weight band (Fig. 4, track 1). From the electrophoregrams of the PEG-G-CSF conjugate and unmodified GCSF shown in FIG. 4, it can be seen that the PEG-G-CSF conjugate has a higher molecular weight than G-CSF (Fig. 4, compare tracks 1 and 2). It should be noted that the exact molecular weight for PEGylated proteins cannot be determined by the EF method, since the addition of a hydrophilic PEG molecule to a protein significantly increases the Stokes radius of the complex formed. As a result, the progress of the PEG-protein complex in the gel slows down and its molecular weight is much higher than the sum of the molecular weights of the unmodified protein and PEG.

- 5 019043

Example 8

Determination of the molecular weight of the PEG-G-CSF conjugate in comparison with unmodified G CSF by mass spectrometry.

μl of a sample of the PEG-G-CSF conjugate obtained according to claim 3, and 0.3 μl of a solution of 2,5-dihydroxybenzoic acid (L1bg1sy. 10 mgml ^-1 in 20% acetonitrile in water with 0.5% TFA) were mixed and dried in air . In a similar way, a sample of unmodified G-CSF was prepared.

Mass spectra were obtained on an Intayeh II VK.IKEK ΜΑΓΌΙ time-of-flight mass spectrometer. (Germany) equipped with a UV laser (N6). Mass spectra were obtained in the mode of positive ions, in the linear mode, the error in measuring the average mass does not exceed 10-15 Da.

The results of mass spectrometric analysis of unmodified G-CSF and the PEG-G-CSF conjugate in the t / ζ range from 10000-50000 are shown in FIG. 5.

From FIG. 5A, the G-CSF mass spectrum contains the main peak corresponding to a singly charged [M] ⁺ ion with a molecular weight of 18816 Da.

In the mass spectrum of the PEG-G-CSF conjugate shown in FIG. 5B, a diffuse main peak is shown corresponding to a singly charged [M] ⁺ ion with a molecular weight of 49,600 Da. Peak blur is associated with the heterogeneity of monomethoxyPEG preparations. For the PEG-G-CSF conjugate, the measured t / ζ value at the peak maximum (49600 Da) coincides with the calculated data on the sum of the molecular weights of G-CSF (19295 Da) and the attached PEG (30000 Da).

Example 9

Localization of the PEGylation site in the PEG-G-CSF conjugate.

To 5 μl of a sample of the PEG-G-CSF conjugate obtained according to claim 3, 5 μl of a solution of modified trypsin (Rgoted) in 0.05 M ΝΗ ₄ ΗΟΘ ₃ with a concentration of 15 μgml ^{-1 was} added. The hydrolysis was carried out for 16 h at 37 ° C, then 10 μl of 0.5% TFA in a 10% solution of acetonitrile in water was added to the solution and thoroughly mixed.

In a similar way, a sample of unmodified IFN was prepared. The resulting solutions were used to obtain MABI1 mass spectra.

The localization of the PEG binding site with the G-CSF molecule was determined by comparing the mass spectra of the tryptic hydrolyzate of the PEG-G-CSF conjugate and unmodified G-CSF. In the tryptic hydrolyzate of the PEG-G-CSF conjugate, there should be no peptide corresponding to the portion of the protein through which the modification occurred. In the table. 2 shows the mass spectra of experimental tryptic peptides for unmodified G-CSF and the PEG-G-CSF conjugate. The table shows that the mass spectra of the tryptic peptides of unmodified G-CSF practically coincide with the mass spectra of the tryptic peptides of the PEG-G-CSF conjugate (Table 2), but there is no peptide with a molecular weight of 1432.7 in the tryptic digest of the PEG-G-CSF conjugate present in the tryptic digest of unmodified G-CSF. The experimentally obtained mass spectra of tryptic peptides for unmodified G-CSF and the PEG-G-CSF conjugate are shown in FIG. 6, from which it is seen that in the tryptic digest of the PEG-G-CSF conjugate, there is no peptide with a molecular weight of 1432.7. According to the analysis of the theoretical masses of the tryptic hydrolyzate of G-CSF, the peak with t / ζ 1432.7 corresponds to the Ν-terminal protein peptide (Table 2). Its absence in the tryptic digest of the PEG-G-CSF conjugate indicates a modification of the кон-terminal peptide, which, having become heavier by the mass of PEG, is outside the studied region of the spectrum. The binding of PEG to the G-CSF molecule occurred at the only free amino group present in this peptide at the amino group of the Ν-terminal methionine.

Table 2 Experimental mass spectra of tryptic peptides of the conjugate PEG-G-CSF and unmodified G-CSF in comparison with theoretical values

Experimentally Discovered Peptides [M + H ⁺ ] ⁺ Theoretical peptides G-CSF Konyogat PEG-G-KSF Weight [M + N * G Peptide Sequence 1432.7 1432.75 1-14 MTRORAZZRCHZZR 747.4 747.4 747.36 18-23 CEFUK 1129.6 1129.6 1129.61 20-29 ΕφνΚΚΙφΟϋΟ 1359.7 1359.7 1359.75 42-53 Bbc 2527.3 2527.3 2527.3 54-78 SHLR9 / LR1.88SR80 953.6 953.6 952.56 55-63 1.St1R \ UAR1.8 818.4 818.4 817.41 61-68 РЬ88СР8О 2033.1 2033.1 2033.10 72-90 EAOZZDENZAYEU 2104.1 2104.1 2104.1 75-93 SEZRENZOGYUOOE 1241.7 1241.7 1241.68 158-167 ФЙЕЬЕ..

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Example 10

Determination of the specific specific activity of the PEG-G-CSF conjugate in comparison with unmodified rhG-CSF.

The PEG-G-CSF conjugate obtained according to claim 3 was diluted to a concentration of 0.2 ng / ml with medium ΚΡΜΙ (ATCC cat. No. 30-2001). The resulting sample in a volume of 100 μl was added to a row of wells of a 96-well plate and a series of serial two-fold dilutions were prepared. Then, 100 μl of a suspension of Μ-ΝΕ 8-60 cells were added to each well and cultured in a CO ₂ incubator for 50. Then, 20 μl of Alamar Blue dye (or a similar one, for example, MTT) was added to each well and incubated under the same conditions 14 h. The increase in the number of cells was determined spectrophotometrically by increasing the optical density at a wavelength of 545 and 630 nm. Similarly, the specific activity of modified G-CSF was investigated. As a reference standard, we used a solution of the international standard sample of G-CSF activity (1-81 1n1ethiop1 81apyag1 88/502, dgapiosocialis8i8iapid Gas1og, iitap, ΌΝΑΌΝΑ-Χηνΐά. 10000 ΐυ / atr). The specific biological activity of the studied drugs (B ₀ ) in IU / mg was determined by the calculation method according to the formula:

where B ₀ is the specific biological activity of PEG-G-CSF or G-CSF (IU / mg); A is the specific biological activity of PEG-G-CSF or G-CSF (IU / ml); C - protein content in a solution of PEG-G-CSF or G-CSF (mg / ml).

Table 3

Specific specific activity of PEG-G-CSF and unmodified G-CSF

A drug Specific Specific Activity (IU / mg) PEG-G-KSF conjugate obtained according to claim 3 (0.845 ± 0.091) x10® Unmodified G-CSF (1.00 ± 0.081) x10®

Example 11

The study of thermal stability of the PEG-G-CSF conjugate compared with unmodified G CSF.

A sample of the PEG-G-CSF conjugate prepared according to claim 3 was diluted with 5 mM sodium acetate buffer, pH 4.0, containing 140 mM No. C1, to a protein concentration of 0.28 mg / ml, and placed in a water bath at a temperature of 50 ± 2 ° C and at various time intervals investigated the appearance of turbidity in the protein solution by measuring the optical density at 340 nm. A similar study was conducted of the thermal stability of unmodified G-CSF. Incubation of proteins at a temperature of 50 ± 2 ° C leads to the formation of insoluble aggregates; as a result, the turbidity of the protein solution increases, which can be measured by determining the optical density of the solution at 340 nm. In FIG. Figure 7 presents the results of the study, from which it is seen that the turbidity of the unmodified G-CSF preparation increases with increasing incubation time, while the PEG-G-CSF preparation remains stable throughout the entire observation time. From the data obtained it follows that the thermal stability of the inventive PEG-G-CSF conjugate is much higher than that of unmodified G-CSF.

Example 12

Investigation of the proteolytic stability of the PEG-G-CSF conjugate during trypsin treatment compared with unmodified G-CSF.

A sample of the PEG-G-CSF conjugate obtained according to claim 3 was diluted to a protein concentration of 0.6 mg / ml and 150 μl of 1 M Tris-HC1, pH 8.5 was added so that the final pH of the sample was 7.4, was added 2 μl of 0.5 M CaCl ₂ to a final concentration of 1 mM was stirred and 15 μl of trypsin (Rgoteda, Oo1y) was added to a final concentration of 0.02 μg / ml. Similarly, samples containing unmodified G-CSF were prepared. Samples were incubated at 37 ° C. At certain time intervals, 100 μl aliquots were taken and the EP was performed in PAGE in the presence of DDS, as described in paragraph 1. After electrophoresis, the gels were stained with Coomassie K-250 dye and scanned. The area of the main strip was determined using the computer program Oe-1Rto Apa1u8eg. The area of the main peak in the initial (untreated trypsin) samples was taken as 100%. In FIG. 8 presents data on the sensitivity of the PEG-G-CSF and G-CSF conjugate to trypsin treatment. It can be seen that with an increase in the trypsin treatment time, the amount of PEG-G-CSF and G-CSF decreases, however, the claimed PEG-G-CSF conjugate is more resistant to trypsin in comparison with unmodified G-CSF.

Example 13

The study of the immunoreactivity of the conjugate PEG-G-CSF in comparison with unmodified GCSF.

To determine the immunoreactivity, the binding of the claimed PEG-G conjugate was investigated.

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CSF with monoclonal immunoglobulins against human G-CSF. The determination was carried out by the enzyme immunoassay using a set of reagents RgoSop O-S8B (Protein circuit LLC). The PEG-G-CSF conjugate obtained according to claim 3 was diluted with 10 mM Tris-HC1 buffer, pH 7.2, containing 140 mM No. 1C1 and 1% bovine serum albumin, to a concentration of 1 μg / ml, and 100 μl of the obtained sample introduced into the wells of microtiter plates previously sensitized with monoclonal antibodies against G-CSF. After incubation at a temperature of 37 ° C (60 min), monoclonal antibodies to an independent epitope labeled with biotin were introduced into the wells. The resulting immune complex was detected using streptavidin labeled with horseradish peroxidase. To develop a color reaction, 100 μl of a substrate solution (200 μg / ml tetramethylbenzidine (81 dt) and 0.1% H ₂ O ₂ in 0.05 M TB, pH 7.2 was added to the wells of the plates and incubated until a blue color developed. The reaction was stopped by adding 50 μl of a 2 M sulfuric acid solution to the wells, the results were taken into account by measuring the optical density at a wavelength of 405 nm on a Mi1J8eap EX multichannel spectrophotometer. R The results of the comparative determination of immunoreactivity shown in Fig. 9 shows that the immunoreactivity of the inventive PEG-G-CSF conjugate is 25% of the immunoreactivity of unmodified G-CSF.

Example 14

Determination of the stability of the PEG-G-CSF conjugate during storage.

Aliquots were taken from the sample obtained according to claim 3 into sterile tubes with caps, such as Eppendorf. The protein concentration in the PEG-G-CSF conjugate was 1 mg / ml. Samples were placed in a refrigerator at a temperature of 6 ± 2 ° C. At certain time intervals, samples were taken and the specific specific activity and homogeneity of the preparation were analyzed using GF HPLC and EF in PAGE in the presence of DDS under reducing conditions, as described in paragraph 1. From the data table. Figure 4 shows that when stored at 6 ± 2 ° C, the PEG-G-CSF conjugate is stable for at least 24 months.

Table 4

The stability of the inventive conjugate PEG-G-KSF at a temperature of 6 ± 2 ° C

Options Shelf life (months) 0 6 12 eighteen 24 Activity (IU / mg) (0.84 ± 0.09) x 10 ⁸ (O / 75 ± OD5) x1O & (0.9a0.10) x10 ⁸ (0.80 ± 0.15) x1 O ⁸ (0.86 ± 0> 07) xU 8 Homogeneity, by GF HPLC (%) > 95 > 95 > 95 > 95 > 95 H homogeneity, by EF in PAGE (%) > 98 > 98 > 98 > 98 > 98

Example 15

A study of the pharmacokinetics of the PEG-G-CSF conjugate.

A sample of the PEG-IFN conjugate obtained according to claim 3 was administered intraperitoneally to 1SC male mice. weighing 20 g at a dose of 5 μg / mouse. In parallel to another group of mice, unmodified G CSF was administered. At certain intervals, blood was collected from animals using retroorbital puncture. Blood serum was obtained by the standard method. The presence of G-CSF in serum was determined by enzyme-linked immunosorbent assay (ELISA). For the production of ELISA, a sandwich variant of solid-phase ELISA using mouse monoclonal immunoglobulins to various G-CSF epitopes of a person was used. Monoclonal murine antibodies were sorbed in the wells of a microtiter plate at a concentration of 2.5 μg / ml in 20 mM Tris-HCl buffer (TB), pH 9.0. Sorption of antibodies was carried out at a temperature of 4 ° C during the night. Nonspecific binding sites were blocked by adding 200 μl of TB, pH 7.4, containing 1% bovine serum albumin (BSA) and 0.05% tween20. Then, 100 μl of test serum diluted with TB containing 1% BSA and 0.05% Tween-20 in a ratio of 1: 2, 1: 4, and 1: 8 was added to the wells. In parallel, well-known concentrations of a standard sample of PEG-G-CSF in the range from 25 to 600 ng / ml in TB buffer containing 1% BSA and 0.05% Tween-20 were added to the wells for constructing a calibration curve. Incubation took place at a temperature of 37 ° C for an hour. Then, monoclonal antibodies to human G-CSF labeled with biotin and streptavidin labeled with peroxidase were successively added to the wells. To detect the resulting complex

- 0109043 used a 100 mm solution of sodium acetate buffer, pH 5.0, containing 0.015% hydrogen peroxide and 0.2 mg / ml tetramethylbenzidine. To stop the reaction, a solution of 2 M H ₂ 8O _{4 was used} . The concentration of G-CSF in serum was determined by the calibration curve taking into account the dilution of the test serum.

When analyzing the pharmacokinetics of unmodified G-CSF to construct a calibration curve, the well-known concentrations of a standard sample of G-CSF in the range from 6.25 to 200 ng / ml in TB buffer containing 1% BSA and 0.05% Tween-20 were added to the wells. The pharmacokinetics of the inventive PEG-G-CSF conjugate compared with unmodified G-CSF are shown in FIG. 10. From the results it follows that the inventive conjugate PEG-G-CSF has a significant prolonged effect. With the introduction of native G-CSF, its content in the blood of animals reached a maximum 1 h after administration and then decreased very rapidly. With the introduction of the PEG-IFN conjugate, the maximum content of IFN in the blood of animals was observed after 6 hours (Fig. 10) and then there was a slow decrease in the content of G-CSF in the blood for 140 hours. Based on the results obtained (Fig. 10), the main pharmacokinetic parameters for the inventive conjugate PEGG-KSF. The results showed that absorption from the injection site, volume of distribution, clearance of the PEG-G-CSF conjugate were significantly slowed down in comparison with unmodified G-CSF, which ensured a long circulation of the inventive PEG-G-CSF conjugate in the blood (more than 140 hours).

Example 16

Comparative characteristics of the inventive PEG-G-CSF conjugate in comparison with the PEG-G-CSF conjugate described in the prototype method.

The structure, the main physicochemical and pharmacokinetic parameters of the inventive PEG-G-GSF conjugate obtained according to claim 3 are compared in comparison with the PEG-G-CSF conjugate described in the prototype method (Table 5). The data presented in table. 5, indicate a significant improvement in the properties of the inventive PEG-IFN conjugate in comparison with the conjugate from the prototype method.

Table 5 The main physico-chemical and pharmacokinetic parameters of the inventive PEG-G-CSF conjugate in comparison with the PEG-G-CSF conjugate described in the prototype method (patent I8 5824784)

Options Konyugg The inventive conjugate PEG- G-CSF 1t PEG-G-KSF The conjugate described in the prototype method Patent 118 5,824,784 G-CSF filgrastim filgrastim PEG structure Linear Linear Molecular mass PEG (kDa) 30-40 6-25 The structural formula of the conjugate PEG-GKSF N ₃ C - MY-S-S8P H ₃ C1 l 1 ° ./x G ° Ί. ^ -Me (- <3-C5g Specific activity,% of unmodified G-CSF 84-94 68 Purity according to the EF in PAG (%) one hundred ** PF cleanliness HPLC (%) > 98 ** GF cleanliness > 98 Well

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HPLC (%) The content of bacterial endotoxins (eE / mg) <3 * * * Pharmacokinetic parameters The ratio of A1DS PEG-G-KSF / G-KSF eighteen **

* The structural formula of the PEG-G-CSF conjugate obtained by the prototype method is given by the link \ UNO Aegid 1pRottayop, 2002, ν. 16, N. 1,

102, L1 47.

** Data not shown.

Example 17. The solution for subcutaneous administration of 0.6, 1.0, 2.0, 3.0 mg / ml

The subcutaneous solution used as a medicine contains, as an active ingredient, PEG or a recombinant human granulocyte colony stimulating factor (PEG-G-CSF) and additional pharmaceutically acceptable components in the following ratio.

Active substance:

PEGylated recombinant human granulocyte colony stimulating factor (PEG-G-CSF) mg - 0.6-3.0.

Excipients:

Polysorbate 20, mg - 0.04;

Mannitol, mg - 50;

Sodium acetate trihydrate, mg - 0.23;

Glacial acetic acid to pH 4.0;

Water for injection up to 1 ml.

Example 18

A method of packaging a finished drug, for example, an aqueous solution containing PEG-G-CSF, obtained according to claim 3.

1.0 ml each in syringes made of neutral glass of hydrolytic class I, with soldered needles, covered with protective caps, elastic or rigid, sealed with tips on pistons made of butyl rubber laminated with fluoropolymer.

Or 1.0 ml in neutral glass vials of hydrolytic class I, corked with fluorresin or butyl rubber tubes with Teflon coating, crimped with aluminum caps.

Example 19

A kit containing a medicament, for example, an aqueous solution containing PEG-G-CSF as the active ingredient obtained in accordance with claim 3.

A kit containing 1 syringe complete with a piston in a blister strip of polymer film, together with instructions for use, which are placed in a pack of cardboard.

Or a set, 1 bottle in a blister strip packaging from a polymer film together with instructions for use, which are placed in a pack of cardboard

Literature.

Mashkovsky M.D., Medicines, Vol. 15, Moscow: New Wave, 2006 - 1206 p.

Patent I8 7655766.

Patent EP 0 401 384.

Patent EP 0335423.

Patent I8 5824784.

Patent VI 2278870.

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Claims (14)

CLAIM 1. The conjugate of granulocyte colony stimulating human factor with monomethoxypolyethylene glycol of the formula (I) SNs / o SI. CH ₂ ; OCH ₂ Un-G-KSF ⁽¹⁾ , where n is an integer from 681 to 1000; w is an integer> 4; No. H-G-CSF is a granulocyte colony stimulating factor. 2. The conjugate according to claim 1, in which the average molecular weight of the units of polyethylene glycol is from 30 to 40 to Yes. 3. The conjugate according to claim 2, where the molecular weight of the polyethylene glycol units is 30 kDa. 4. The conjugate according to claim 1, in which w is an integer from 4 to 6. 5. The conjugate according to claim 1, in which the кон-terminal group is represented by a methionine residue (Me!). 6. A pharmaceutical composition having granulocyte colony stimulating factor activity, comprising the conjugate of general formula (I) according to any one of claims 1 to 5 in an effective amount and pharmaceutically acceptable additives. 7. The pharmaceutical composition according to claim 6, intended for use as a medicine for the treatment of neutropenia of various origins. 8. The pharmaceutical composition according to claim 6, including the conjugate of the general formula (I) in an effective amount, polysorbate, mannitol, sodium acetate trihydrate, acetic acid, water for injection up to 1 ml. 9. The pharmaceutical composition of claim 8 with a pH of 3-5. 10. The pharmaceutical composition of claim 8 with a pH of 4. 11. A drug having granulocyte colony stimulating factor activity, comprising a conjugate of the general formula (I) according to any one of claims 1-5. 12. The drug according to claim 11, intended for the treatment of neutropenia of various origins. 13. The use of the conjugate of formula (I) according to any one of claims 1 to 5 for the manufacture of a medicament having granulocyte colony stimulating factor activity. 14. A method for the prevention or treatment of neutropenia, comprising administering an effective amount of a conjugate of formula (I) according to any one of claims 1 to 5. PEGylation time (hour)