EA020425B1 - Polyethyleneglycol covalent conjugate with granulocytic colony-stimulating human factor - Google Patents

Abstract

The invention provides medicine and biotechnology and relates to polyethyleneglycol covalent conjugate with granulocytic colony-stimulating human factor in which PEG is attached to tyrosine or histidine fragments of polypepetide by an azo group of general structurewhere n is an integral between 100 and 1200; A is an aminoacid of tyrosine or histidine fragment; G-CSF is a granulocytic colony-stimulating human factor. The Γ-KCΦ conjugate has a greater circulation time in blood stream compared to none-modified polypeptide, preserving a substantial potion of its activity.

Description

The invention relates to medicine and biotechnology, and relates to a covalent conjugate of polyethylene glycol with a granulocyte colony stimulating factor in humans, in which the polymer is attached to the polypeptide via an azo group.

Background of the Related Art

Granulocyte colony stimulating factor (G-CSF, O-C8P) is a polypeptide hormone that stimulates the formation of granulocyte colonies in cell culture. G-CSF stimulates the proliferation and differentiation of late progenitor cells into neutrophils. Granulocyte colony-stimulating factor is the main hematopoietic growth factor that regulates granulocytopoiesis, and is widely used in medicine in the treatment of neutropenia of various etiologies.

Indications for the use of O-C8P are hereditary, periodic or idiopathic neutropenia, in which the number of neutrophils is lower than or equal to 500 cells / μl (including in patients receiving cytostatic drugs for non-myeloid malignant neoplasms, as well as those who have a history of severe or recurring infection in the last 12 months), a decrease in the duration of the period of neutropenia and its clinical consequences in patients preparing for bone marrow transplantation, persistent neutropenia in pa ientov with advanced HIV infection (absolute neutrophil count 1,000 cells / mm and less), mobilization of peripheral blood stem cells (including after myelosuppressive therapy).

G-CSF preparations are known, for example Filgrastim, a methionylcolony-stimulating factor with a molecular weight of 18,800 Da, which is a non-glycosylated protein consisting of 175 amino acids (production: Masterklon, Russia). Nevertheless, the use of G-CSF in medicine is limited by a number of factors, among which the most significant is the lack of enzymatic stability in the body, which leads to the need to increase the frequency of administration of the drug, which in some cases is associated with increased side effects.

The average half-life of filgrastim from serum in healthy people and in patients with tumors is about 3.5 hours; clearance rate of approximately 0.5-0.7 ml / (min-kg). Only with continuous 24-hour iv infusions of filgrastim at a dose of 20 mcg / kg for 11-20 days, an equilibrium concentration in the blood is achieved.

In this regard, there is a need for chemical modification of the G-CSF molecule in order to overcome these disadvantages.

For the modification of various polypeptides it is known to use monomethoxypolyethylene glycol (mPEG), which is a neutral polyester with different molecular weights [I. Nikitin, T. N. Storozhakov Pegylated drugs: current status of the problem and prospects, in collection: Viral hepatitis: achievements and prospects. No. 3 (13) (2001), p. 3-8].

As a rule, in covalent conjugates, the polymer fragment is attached to the polypeptide via one of the free amino groups of the latter Κοζ1ο \ ν5ΐ <ί A., Cbag1sk 8.A., Natk GM. Osus1ortsp1 οί Rsdu1a1sb 1p1sgGsgop5 ίοτ 1Ns TgeaNpsSh οί Shots NsraIIk S. ΒιοΌγη ^ κ .; νοί. 15 (7) (2001), ρ. 419-429], and the preparation is a mixture of positional isomers, the chemical structure of which differs from each other in the place of attachment of the polymer to the polypeptide. Theoretically, the number of positional isomers in this case is equal to the number of free amino groups in the polypeptide, provided that they are available for the pegylating agent [Blokhin NP, Nikitin IG Peculiarities of the pharmacological dynamics and kinetics of pegylated α-interferon (40 kDa) Pegasis: new possibilities for the treatment of chronic hepatitis C. In Sat: Materials of the VII Russian Conference Hepatology today. RZHGGK. 2002, p. 6].

In the application of RI 2009122976 (published on December 27, 2010) a conjugate of the three-branched PEG-G-KSF is proposed with the following general formula:

/, —O \ —Ζ — Υ — Θ-SZR —ο-ν / η and in which the binding ratio of the three-branched polyethylene glycol and G-CSF is 1: 1 (mol / mol) and the PEG has an average molecular weight of 200 to 45,000 Da where η is an integer from 1 to 1000; t is an integer from 10 to 1000;

Ζ represents (CH ₂ ) _k or (CH ₂ ) _k ONSO (CH ₂ ) _k as a linker G-CSF and PEG, where k is an integer from 1 to 6;

Υ is an amide bond formed by combining one of the IN ₂ -functional

- 1,020,425 groups in G-CSF and a functional group of the PEG derivative.

In the application I8 2008/0287659 (publ. 20.11.2008) disclosed pegylated mutein G-CSF, which is a mixture of covalent conjugates with different degrees of pegylation, having the General formula

where C is the mutein G-CSF;

K is lower alkyl; η is an integer from 420 to 550; t is an integer from 1 to 5.

Thus, the proposed mixture of conjugates is a mixture of various positional isomers of varying degrees of pegylation.

It is known that positional isomers of polypeptides have different biological activity (I8 2004/0223950, publ. 11.11. 2004). This creates the prerequisites for expanding the arsenal of such funds due to various options for modifying protein molecules. For example, changing the positions and methods of attaching PEG to a protein allows you to change the ratio of positional isomers in the final product.

One way to reduce the number of positional isomers of PEG attachment is preferably to the Ν-terminal amino group of the polypeptide using the difference values for _pKa Ν-terminal α-amino and ε-amino groups of lysine moieties of the protein.

So, in the international application \ UO 96/011953 (publ. 04.25.1996) a covalent conjugate of G-CSF with PEG is proposed, obtained by reductive alkylation of the термина-terminal amino group of the methoxypolyethylene glycol-aldehyde peptide in the presence of Ν; · ιί '.' ΝΒΗ ₃ at pH 5. However, the conjugation conditions in this patent does not fully eliminate the likelihood of adverse reactions, including attachment of PEG to the ε-amino groups of lysine moieties of which are, despite the high pK _a value, even at acidic pH deprotonated, and their number in the protein in unlike Ν-term total α-amino group, more than one.

Another direction of reducing the number of positional isomers is the addition of PEG to the thiol group of the cysteine fragment of the polypeptide.

So, in the patent \ UO 03/078461 (publ. March 25, 2003) a covalent conjugate is disclosed in which polyethylene glycol is bound to G-CSF in a 1: 1 ratio through a maleimide group with a thiol of the cysteine fragment of the polypeptide. However, the values of the relative activities of the conjugates given in the patent show that modification with thiol fragments has no significant advantages over conjugates formed by the amino groups of lysine.

Thus, the further development of new types of conjugates, including those in which the amino groups of the polypeptides are not affected, is a very promising task.

The closest in technical essence are considered physiologically active conjugates, in particular insulin, disclosed in I8 4179337 (publ. 12/18/1979), containing a fragment of paraphenylenediazonium in the molecule and having the structure of an amino derivative formula

where K represents PEC-O-CH2-, PEC-O-CH2-CH (OH) -CH2-O- or PEC-O-C (= O) -;

represents a peptide chain.

The disadvantages of such derivatives are the relatively large number of possible variants of positional isomers due to the addition of the polymer part to the amino groups of the polypeptides and their low stability in the body and, as a result, the possibility of uncontrolled structural changes and cleavage of reactive particles, in particular diazo compounds.

SUMMARY OF THE INVENTION

The inventors have unexpectedly found that PEG conjugates with G-CSF, in which the polymer is attached to the tyrosine or histidine fragments of the polypeptide by the azo group, having the general structure (I), acquire the ability of prolonged circulation in the blood and retain a significant part of the biological activity of unmodified G-CSF.

- 2 020425 where η is an integer in the range from 100 to 1200;

A is the amino acid fragment of tyrosine or histidine;

G-CSF - granulocyte colony stimulating factor.

In order to prove the structure of the conjugate as an azo compound, in particular, electronic absorption spectroscopy (ESP) is used, the spectral characteristics (intensity, width and position of the absorption band maxima) of the solutions of pegylated and unmodified G-CSF are compared and the patterns obtained are interpreted from the perspective of the color theory of organic compounds, optional , involving adequate methods of quantum chemical modeling.

The characteristics of the maxima of the absorption bands are given in table. one.

Table 1

Characteristics of the peaks of the absorption bands of pegylated and unmodified G-CSF (water, pH 4.5, 11 cm)

The appearance of absorption bands in pegylated G-CSF with maxima at 339 and 400 nm can be explained, for example, from the standpoint of the color theory of organic compounds, by local electronic transitions in chromophore systems not coupled by conjugation of diarylazo groups formed as a result of covalent addition of the PEG agent to tyrosine and histidine links of G-KSF. The broadening of the bands is due to the presence of several positional isomers with close energies of electronic-vibrational transitions.

The achievement of the technical result, which consists in increasing the circulation time in the bloodstream of pegylated G-CSF, is confirmed in a series of studies on a mouse model (Table 2).

table 2

The time to reach the maximum concentration (T _max ) and half-life (T _1/2 ) of G-CSF and its conjugate in accordance with the invention in the blood of mice after sc injection

A drug TSCH 1 * 1/2 / h G-CSF 3.9 + 0.3 3.9 ± 1, b PEG-G-KSF 9.8 ± 1.4 15.4 ± 3.1

The data show that the conjugate G-CSF in accordance with the invention has a longer circulation time in the bloodstream compared with unmodified protein.

The results of the study of the biological activity of the conjugates in accordance with the present invention are given in table. 3.

Table 3

The biological activity of pegylated and unmodified G-CSF

A drug IU / mg Residual activity,% G-CSF KO * one hundred PEG-G-KSF 2,4-U ⁷ 24

Thus, the conjugate in accordance with the present invention retains a substantial portion of the biological activity of unmodified G-CSF.

- 3,020,425

In accordance with the invention, the conjugate G-SCF with a high degree of purity can be obtained by a method comprising the steps in which:

a) methoxypolyethylene glycol aminobenzoic acid ester

where η takes values from 10 to 1200, diazotized with nitrite of an alkaline or alkaline earth metal in an aqueous or aqueous-organic medium at a temperature of from -2 to 30 ° C or organic nitrite in an environment of a polar organic solvent, miscible with water, at a temperature of -40 to 30 ° C, the molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid from 1.1: 1 to 1000: 1 and the molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid from 3: 1 to 10000: 1, followed by removal of TCA nitrite and produce activated pegiliruyuschego agent - ([metoksipolietilenglikoloksi] carbonyl) benzenediazonium:

where η - takes the above values;

b) an activated pegylating agent without isolation from the reaction mixture is introduced into the azo coupling reaction with G-CSF in a molar ratio of PEG-agent to G-CSF 1-10: 1, respectively, in an aqueous or aqueous organic medium with a pH from 7.0 to 10, 0 at a temperature of 0 to 30 ° C; upon reaching the degree of conversion of at least 70%, the reaction is stopped by adding a low molecular weight azo component to the reaction mass to obtain a mixture of conjugates of varying degrees of pegylation, an unmodified polypeptide and a blocked PEG agent;

c) the resulting mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions with the release of monopegylated G-CSF.

When implementing the inventive method provides an increase in the yield of pegylated G-CSF and an increase in the time of its circulation in the bloodstream.

The increase in the yield of pegylated G-CSF is achieved by quantitatively converting the PEG agent into the active form immediately before conjugation, excluding the degradation of the active group — the diazogroup during transportation and storage.

An increase in the circulation time of pegylated G-CSF in the bloodstream is possible due to the ability of tyrosine and histidine units of the polypeptide to enter into the azo coupling reaction with active diazo compounds with the formation of biologically active azo derivatives of G-CSF.

If necessary, increasing the stability of the pegylating agent during storage is achieved by using organic nitrite for activation of the pegylating agent in a polar organic solvent that mixes unlimitedly with water, which additionally allows expanding the temperature range of the diazotization reaction.

In a preferred embodiment of the invention, in step a), the diazotization is carried out with sodium nitrite in an aqueous solution of hydrobromic acid, and the excess nitrite is removed with sulfamic acid.

In a further preferred embodiment of the invention, in step a) the diazotization is carried out with tert-butyl nitrite in the presence of HCl in tetrahydrofuran.

In a further preferred embodiment of the invention, in step b), a borate-carbonate buffer solution is used to create and maintain the pH, the degree of conversion of G-CSF is 75100%, and tyrosine is used as the low molecular weight azo component.

In a further preferred embodiment of the invention, in step c), the mixture is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions from 0.02 to 1.0 M NaCl.

At the stage a) diazotization of methoxypolyethylene glycol ether of aminobenzoic acid is carried out by adding nitrite of an alkali or alkaline-earth metal in an acidic aqueous or aqueous-organic medium at a temperature of from -2 to 30 ° C.

In the first embodiment of diazotization, alkali or alkaline earth metal nitrite is used in an acidic aqueous or aqueous-organic medium. The most preferred diazotization temperature range is from 0 to 5 ° C. An acidic medium is created using organic acids, for example, using acetic acid or its halogen derivatives, such as chloroacetic, trichloroacetic, bromoacetic, tribromoacetic, trifluoroacetic acids, as well as citric or tartaric acids, or inorganic acids, for example, hydrochloric, hydrobromic, sulfuric or phosphoric acids, as well as a mixture of organic and / or inorganic acids. The molar ratio of nitrite to methoxypolyethylene glycol aminobenzoic acid ester is from 1.1: 1 to 1000: 1, preferably 4,020,425, preferably from 1.1: 1 to 10: 1. The molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid is from 3: 1 to 10000: 1. The reaction is preferably carried out in the presence of a diazotization catalyst, which is used bromide ions introduced into the reaction mixture in the form of hydrobromic acid or its soluble salts, for example alkali metal bromides. It is most preferable to create an acidic medium with a solution of hydrobromic acid.

In the second embodiment, diazotization is carried out using organic nitrite in an environment of a polar organic solvent, unlimitedly miscible with water, at a temperature of from -40 to 30 ° C. The most preferred diazotization temperature range is from -20 to 0 ° C. Preferred organic nitrites are butyl nitrites or amyl nitrites, more preferably tert-butyl nitrite. The molar ratio of nitrite to methoxypolyethylene glycol ether of aminobenzoic acid is from 1.1: 1 to 1000: 1, preferably from 1.1: 1 to 10: 1. An acidic medium in a polar organic medium is created with solutions of HC1 or HBr in an aliphatic ether, for example diethyl ether, or a cyclic ether, for example dioxane or tetrahydrofuran. The molar ratio of acid to methoxypolyethylene glycol ether of aminobenzoic acid is from 3: 1 to 10000: 1.

Upon completion of diazotization, the activated PEG agent can be stored at a reduced temperature for no more than 2 hours in an aqueous or aqueous organic medium or for no more than 24 hours in a polar organic medium without significant loss of its ability to combine. The term reduced temperature means a temperature of from -2 to 5 ° C in the case of using an aqueous or aqueous-organic medium and from -40 to 0 ° C in the case of using a polar organic medium. Before using the activated PEG agent for the pegylation of G-CSF, it is necessary to remove excess nitrite ions, for which urea or sulfamic acid is added to its solution. Alternatively, azides of alkali or alkaline earth metals are used.

At stage b), the pegylation of G-CSF is achieved by the reaction of azo coupling of a diazotized methoxypolyethylene glycol ester of aminobenzoic acid with G-CSF in a neutral or slightly alkaline aqueous or aqueous-organic medium at a temperature of from 0 to 30 ° С. The most preferred pH range for pegylation is from 9 to 10. The pH is maintained using a suitable buffer solution, for example a borate-carbonate buffer solution. The choice of solution is within the competence of the average person skilled in the art. The molar ratio of the diazotized methoxypolyethylene glycol ester of 4-aminobenzoic acid to G-CSF is from 1: 1 to 10: 1, most preferably from 3: 1 to 8: 1.

The pegylation process is monitored by size exclusion or reverse phase HPLC. Upon reaching the desired degree of conversion of the polypeptide, the pegylation reaction is stopped by adding a low molecular weight azo component to the reaction mass. For this purpose, phenolic substances or their esters, substances having the nature of aromatic amines, or substances having a heterocyclic nature, in which the heterocycle is capable of acting as an azo component in the azo coupling reaction, are used as low molecular weight azo constituents. Most preferred are tyrosine and histidine, more preferably tyrosine. Preferably, the degree of conversion of G-CSF calculated by HPLC analysis is 75-100%.

The isolation of pegylated G-CSF from the reaction mixture is carried out by conventional methods of ion exchange chromatography, sequentially using buffer solutions with increasing ionic strength. The concentration of G-CSF is determined by HPLC or spectrophotometrically using the corresponding A ₂₈₀ value for a solution with a polypeptide concentration of 1 mg / ml.

Information confirming the possibility of carrying out the invention

Further, the invention will be illustrated by the following examples, confirming the possibility of its implementation with the achievement of the technical result indicated in the description.

Example 1. Obtaining pegylated G-CSF.

To a solution of 0.5 μmol G-CSF cooled to 3 ° C in a borate-carbonate buffer with a pH of 9.5, a cooled solution of 2.5 μmol 4 - ([methoxypolyethylene glycol] carbonyl) benzenediazonium (M.m. 30 kDa) is added, maintaining The pH of the reaction mixture is 9.5 ± 0.3. The reaction mixture was stirred for approximately 2 hours while cooling, monitoring the progress of reverse phase HPLC transformations (Kgotaki 300-5C4, 250x4.6 mm column, spectrophotometric detection at 220, 280, 340 and 400 nm, gradient elution: from 30% aq. Acetonitrile + 0.2% TFA to 80% aq. Acetonitrile + 0.2% TFA). Upon reaching the degree of conversion of G-CSF equal to 70-80%, a tyrosine solution is poured, stirred for 10 minutes and the pH is adjusted to 5.0-6.5 with acetic acid. Next, the reaction mixture containing a mixture of dipipegylated, monopegylated and unmodified G-CSF, as well as a blocked pegylating agent, is separated by ion exchange chromatography with an increase in the ionic strength of the buffer elution solutions from 0.02 to 1.0 M NaCl. The yield of purified pegylated G-CSF

39% (counting on G-CSF). ESP (water, pH 4.5, 11 cm), X _max , nm ^{(Δ |} “'>: 219 (36.4), 279 (6.6), 339 (5.9), 400 (3.8 )

Example 2. Determination of the circulation time of pegylated G-CSF in the blood in a mouse model.

- 5,020,425

5 mcg of pegylated G-CSF in accordance with the invention is subcutaneously injected into male CBA mice in accordance with the invention, after which blood is collected on the first day 2 hours after injection and then every 24 hours for 10 days. As control using unmodified G-CSF, which is introduced according to the same scheme. Blood samples are incubated for 45 min at 37 ° C, after which the thrombus is separated and re-incubated at 4 ° C, the resulting serum is centrifuged and stored at -65 ° C until testing. The content in blood serum (pg / ml) is determined using the ELISA kit Nitap 0-C8P EGA Κίΐ, then the time to reach the maximum concentration and the half-life are calculated. The data are presented in table. 2.

Example 3. Determination of the activity of pegylated G-CSF.

The human myeloid cell line Hb-60 (ATCC CCb-240) was cultured in medium ΚΡΜΙ 1640, in which 10% PB§ was added. The cultured cells are suspended at a density of about 2.2 x 10 ⁵ cells / ml and DMSO (dimethyl sulfoxide with a purity suitable for culturing, §1§ta) is added at a final concentration of 1.25% (v / v). Then, 90 μl of the cell suspension are plated in each well of a 96-well plate (Sotdsd / 96-well plate with evaporation at low temperature), after which the plate is left until a density of approximately 2x10 ⁴ cells per well and cultured in an incubator at 37 ° C in the atmosphere 5% CO ₂ for 72 hours

Each sample was diluted with medium ΚΡΜΙ 1640 to an identical concentration of 10 μg / ml, and then diluted twice with medium ΡΡΜΙ 1640 19 times. These serial two-fold dilutions are separately added to each well containing Hb-60 cells in a volume of 10 μl, so that the concentration of each sample was initially 1 μg / ml. Then the cells were cultured in an incubator at 37 ° C for 72 hours.

Hb-60 cell proliferation was analyzed using reagent 96 (Ce11 Thiet 96TM, Ca £ 0.0400, Rgoteda) to determine cell titer and an increasing number of cells was determined by measuring the optical density at 670 nm. The measurement results are given in table. 3.

Claims (1)

CLAIM A conjugate of polyethylene glycol with a granulocyte colony-stimulating factor, in which the polymer is attached to tyrosine or histidine fragments of the polypeptide via an azo group having the general structural formula where n is an integer in the range from 100 to 1200; A is the amino acid fragment of tyrosine or histidine; G-CSF - granulocyte colony stimulating factor.