RU2323227C2 - Polymeric modifying agents and pharmaceutical compositions - Google Patents

Abstract

FIELD: medicine, polymers, pharmacy.

SUBSTANCE: invention relates to a copolymer or its pharmacologically acceptable salt that comprises the following components as elemental links forming their: (a) one or some structural elemental links describes by the formula (I) given in the invention description, and (b) one or some structural links describes by the formula (II) given in the invention description. Disposition of these structural elements represented by the formulae (I) and (II) is chosen from the following sequences: (i) sequence with alternation "head-to-head"; (ii) sequence with alternation "head-to-tail"; (iii) mixed sequence with alternation "head-to-head" and "head-to-tail"; (iv) random sequence and taking into account that the ratio between structural links of the formula (I) and structural links of the formula (II) in indicated copolymer is in the range from 10:1 to 1:10. Also, the invention relates to a copolymer or its pharmacologically acceptable salt synthesized by addition of one or some links of carboxylic acid anhydride described by the formula (III) given in the invention description that comprises as elemental links: (a) one or some structural elemental links described by the formula (I), and (b) structural link comprising carboxylic acid anhydride link described by the formula (III) for one or some reactions chosen from the group consisting of: (i) hydrolysis; (ii) ammonolysis; (iii) aminolysis, and (iv) alcoholysis. Also, invention relates to a pharmaceutical composition used for prophylaxis or treatment of osseous metabolism disorder and comprising an acceptable excipient or carrier, at least one of above indicated copolymers or their pharmaceutically acceptable salts and at least one protein representing osteoclastogenesis inhibition factor (OCIF) or its analogue, or variant. Also, invention relates to a modifying agent comprising above said copolymers, to a complex between of one of above said copolymers and protein or its analogue, or variant, to a pharmaceutical composition comprising this complex. Also, invention relates to a method for time prolongation when OCIF is retained in blood stream after intake by a patient a complex between protein and at least one of above said copolymers. Also, invention relates to a method for treatment or prophylaxis of disorders of osseous metabolism involving intake by a patient the effective amount of complex comprising complex including OCIF or its analogue or variant and bound with at least one of the claimed copolymers. Also, invention relates to use of the complex comprising OCIF bound with at least one of the claimed copolymers designated for preparing a drug designated for prophylaxis or treatment of disorder of osseous metabolism and showing sensitivity to the protein effect. Modifying the protein, namely OCIF, by the claimed copolymers results to formation of complex possessing uniform properties being especially characterizing by reduced formation of disordered structure cross-linked with protein, improved retention of the protein activity and the excellent retaining protein in blood after intake of the indicated complex.

EFFECT: improved and valuable medicinal and pharmaceutical properties of agents.

110 cl, 13 tbl, 3 dwg, 40 ex

Description

Technical field

The present invention relates to a new copolymer, a pharmaceutical composition containing said copolymer, a protein modifier containing said copolymer, a complex of said copolymer and protein, a method for preventing or treating diseases using said complex, using said complex for the manufacture of a medicament for prevention or treatment diseases, and methods for the synthesis of these copolymers and these complexes.

State of the art

Modification of proteins using additional agents, such as polymers, have been commonly used to provide improved pharmaceutical properties, for example, improved stability and retention in the blood and reduced antigenicity [for example, see FM Veronese and JM Harris, "Peptide and Protein Pegylation", Advanced Drug Delivery Reviews 54 (4), 2002].

When modifying proteins using a polymer, one technique that has been used in the past involves the binding of a protein and a polymer modifier using a covalent bond (for example, see WO-A-97/23614). In other examples of modifying a protein using a polymer, such as when modifying a polymer with a drug, the drug is modified using a non-covalent bond. One such example is given in Japanese Patent Application (Kokai) No. Hei 11-302199, which describes that a grafted copolymer that includes a grafted chain of a non-ionic polymer and a backbone of a negatively charged polymer forms an inclusion complex with a substance capable of carrying a positive charge under physiological conditions, for example, a liposome or poly-L-lysine carrying a positive charge, which leads to improved retention in the blood. Another alternative is proposed in WO-A-99/02131, which describes that a protein and a water-soluble polymer can be mixed under specific conditions in the presence of an organic solvent to obtain controlled release microparticles.

Unfortunately, for a variety of reasons, a small number of these polymer protein modifiers have been particularly successful. One recent example of a polymer protein modifier that exhibits some improved properties includes a polymaleic acid derivative-based copolymer containing a polyoxyalkylene alkyl ether derivative as its constituent unit [see, for example, Japanese Patents Nos. 3035675 and 3271265]. These polymeric modifiers definitely exhibit improved binding to target proteins. However, significant problems still exist for such copolymers. It was found that their link of maleic anhydride binds to proteins non-specifically. The result is the formation of complexes between the copolymer and protein, showing heterogeneous properties depending on the conditions. In particular, it was found that these polymer modifiers tend to easily form disordered crosslinked structures with proteins, thereby forming bulk complexes, which causes excessive modification of the protein structure and, consequently, a decrease in the desired activity of the protein. In addition, the complexes between these polymer protein modifiers and the protein have been found to exhibit poor blood retention after ingestion.

Therefore, there is a need for a polymer modifier capable of producing a complex having homogeneous properties, in particular characterized by a reduced formation of disordered crosslinked structures with protein, better retention of protein activity and excellent retention of protein in the blood after administration of the specified complex.

SUMMARY OF THE INVENTION

Therefore, the aim of the present invention is to obtain a polymer modifier that can lead to a complex having homogeneous properties, in particular characterized by a reduced formation of disordered crosslinked structures with a protein, better retention of protein activity and excellent retention of protein in the blood after administration of this complex.

The inventors of the present invention conducted extensive studies of various protein modifiers and, as a result, achieved success in the preparation of new copolymers that are capable of forming complexes with proteins that have homogeneous properties, and significantly improve the retention of proteins of these complexes in the blood, which, therefore, leads to the completion of the present inventions.

Other objectives and advantages of the present invention will become apparent as you read the description.

Thus, the present invention provides a copolymer or its pharmacologically acceptable salt, which contain as constituting their elementary units

(a) one or more structural elementary units, which may be identical or different from each other and which are described by the following formula (I):

Where:

m is an integer in the range from 3 to 100,

Alk is an alkylene group containing from 1 to 6 carbon atoms, and

R ¹ and R ² are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, which optionally can be substituted by at least one substituent selected from the group consisting of hydroxy -groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which may optionally be substituted with substituents in an amount of from 1 to 5, selected from substituents A defined below, and

(b) one or more structural elementary units, which may be identical or different from each other and which are described by formula (II):

Where:

R ³ represents

hydroxyl group

alkoxy group containing from 1 to 6 carbon atoms, which optionally can be substituted by at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which optionally may be substituted with substituents in an amount of from 1 to 5, selected from substituents A defined below,

an aryloxy group containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A defined below, or

a group described by the formula —NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different from each other, and each of them is a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, which may optionally be substituted, at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which optionally can be substituted by 1 to 5 substituents selected from substituents A defined below ;

substituents A are selected from alkyl groups containing from 1 to 6 carbon atoms, alkoxy groups containing from 1 to 6 carbon atoms, halogen atoms, hydroxy groups, nitro groups and carboxy groups.

The present invention further provides a copolymer or a pharmacologically acceptable salt thereof, obtained by carrying out for one or more carboxylic acid anhydride units described by formula (III), in a copolymer which contains the elementary units forming it

(a) one or more structural elementary units, which may be identical or different from each other and which are described by the following formula (I):

Where:

m is an integer in the range from 3 to 100,

Alk is an alkylene group containing from 1 to 6 carbon atoms, and

R ¹ and R ² are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, which optionally can be substituted by at least one substituent selected from the group consisting of hydroxy -groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which may optionally be substituted with substituents in an amount of from 1 to 5, selected from substituents A defined below, and

(b) said one or more carboxylic acid anhydride units described by formula (III):

one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis;

substituents A are selected from alkyl groups containing from 1 to 6 carbon atoms, alkoxy groups containing from 1 to 6 carbon atoms, halogen atoms, hydroxy groups, nitro groups and carboxy groups.

The present invention also provides a pharmaceutical composition comprising at least one copolymer or a pharmacologically acceptable salt thereof of the present invention described above, in particular such a composition that also contains at least one protein or an analogue or variant thereof.

The present invention also provides a modifier capable of modifying a protein or an analogue or variant thereof, said modifier comprising a copolymer or a pharmacologically acceptable salt thereof of the present invention described above.

The present invention also provides a complex containing at least one protein or its analogue or variant, which is associated with at least one copolymer or its pharmacologically acceptable salt of the present invention described above.

The present invention also provides a pharmaceutical composition comprising an effective amount of a pharmacologically active substance together with a carrier or diluent for it, wherein said pharmacologically active substance is a complex containing at least one protein or its analogue or variant that bind at least , with one copolymer or a pharmacologically acceptable salt thereof of the present invention described above.

The present invention also provides a method for prolonging the time during which a protein or its analogue or variant is held in the blood stream after administration by a patient, as a result of complexation between said protein or its analogue or variant and at least one copolymer or its pharmacologically acceptable salt of the present inventions described above.

The present invention also provides a method of treating or preventing a disease in a patient that is susceptible to the action of a protein or its analogue or variant, comprising administering to said patient an effective amount of a complex containing said protein or its analogue or variant that bind to at least one copolymer or its pharmacologically acceptable salt of the present invention described above.

The present invention also provides the use of a complex containing a protein or its analogue or variant that binds to at least one copolymer or its pharmacologically acceptable salt of the present invention described above for the manufacture of a medicament for the prevention or treatment of a disease susceptible to the action of said protein or its analogue or variant.

The present invention also provides a method for producing a copolymer or a pharmacologically acceptable salt thereof containing elementary units as constituting them.

(a) one or more structural elementary units, which may be identical or different from each other and which are described by the following formula (I):

Where:

m, Alk, R ¹ and R ² are as defined above, and

(b) one or more structural elementary units, which may be identical or different from each other and which are described by formula (II):

Where:

R ³ is as defined above;

wherein said method comprises carrying out for one or more units of carboxylic acid anhydride described by formula (III) in a copolymer that contains elementary units as constituting it

(c) one or more structural elementary units, which may be identical or different from each other, and which are described by the above formula (I), and

(d) said one or more carboxylic acid anhydride units described by formula (III):

one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis, and (iv) alcoholysis.

The present invention also provides a method for producing a complex containing at least one protein or an analogue or variant thereof that is coupled to at least one copolymer or a pharmacologically acceptable salt thereof as defined above, wherein said method comprises carrying out a reaction between said a copolymer or a pharmacologically acceptable salt thereof and said protein or its analogue or variant under conditions conducive to the formation of said complex.

Brief Description of the Figures

Figure 1 shows the results of polyacrylamide gel electrophoresis in the presence of SDS (sodium dodecyl sulfate) for the complexes of the present invention poly (PEG ₅₀₀ -MA) a-OCIF (Osteoclastogenesis Inhibition Factor) under non-reducing conditions in Test Example 6 below:

(1) molecular weight markers

(2) Complex poly (PEG ₅₀₀ -MA) a-Na (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 1 (mass ratio)]

(3) Complex poly (PEG ₅₀₀ -MA) a-Na (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 2 (mass ratio)]

(4) Complex poly (PEG ₅₀₀ -MA) a-Na (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 3 (mass ratio)]

(5) Complex poly (PEG ₅₀₀ -MA) a-Na (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 4 (mass ratio)]

(6) Complex poly (PEG ₅₀₀ -MA) a-Na (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 5 (mass ratio)]

(7) Unmodified OCIF.

Figure 2 shows the additional results of polyacrylamide gel electrophoresis in the presence of SDS for the complexes of the present invention poly (PEG ₅₀₀ -MA) a-OCIF in non-reducing conditions, carried out in the following test example 6:

(1) molecular weight marker

(2) Complex poly (PEG ₅₀₀ -MA) a-Na (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 1 (mass ratio), OCIF concentration during incubation: 3.5 mg / ml]

(3) Poly (PEG ₅₀₀ -MA) a-Na complex (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 1 (mass ratio), OCIF concentration during incubation: 1.75 mg / ml]

(4) Poly (PEG ₅₀₀ -MA) a-Na complex (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 1 (mass ratio), OCIF concentration during incubation: 0.875 mg / ml]

(5) Poly (PEG ₅₀₀ -MA) a-Na complex (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 2 (weight ratio), OCIF concentration during incubation: 1.75 mg / ml]

(6) Poly (PEG ₅₀₀ -MA) a-Na complex (compound No. 9) -OCIF [OCIF: polymer modifier = 1: 4 (weight ratio), OCIF concentration during incubation: 0.875 mg / ml]

(7) Unmodified OCIF.

Figure 3 shows the results of polyacrylamide gel electrophoresis in the presence of SDS for prior art poly (PEG ₅₀₀ -MA) -OCIF complexes under non-reducing conditions carried out in Test Example 6 below:

(1) molecular weight marker

(2) Complex poly (PEG ₅₀₀ -MA) (AM-0530K) -OCIF [OCIF: polymer modifier = 1: 10 (mass ratio)]

(3) Complex poly (PEG ₅₀₀ -MA) (AM-0530K) -OCIF [OCIF: polymer modifier = 1: 2.5 (mass ratio)]

(4) Complex poly (PEG ₅₀₀ -MA) (AM-0530K) -OCIF [OCIF: polymer modifier = 1: 1 (mass ratio)]

(10) Unmodified OCIF.

DETAILED DESCRIPTION OF THE INVENTION

(1) As noted above, one aspect of the present invention provides a copolymer or a pharmacologically acceptable salt thereof, which contain elementary units as constituting them

(a) one or more structural elementary units, which may be identical or different from each other and which are described by the following formula (I):

Where:

m, Alk, R ¹ and R ² are as defined above, and

(b) one or more structural elementary units, which may be identical or different from each other and which are described by formula (II):

where R ³ is as defined above. Among these copolymers and their pharmacologically acceptable salts, preferred compounds include:

(2) the copolymer or its pharmacologically acceptable salt, corresponding to the position (1), where for the structural elementary units described by formula (I) and the structural elementary units described by formula (II), receive a configuration in the form of a sequence with alternating "head to head ”, A sequence with alternating“ head to tail ”or a mixed sequence with alternating“ head to head ”and“ head to tail ”;

(3) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (1), where for the structural unit units described by formula (I) and the structural unit units described by formula (II), the configuration is obtained in the form of a statistical sequence;

(4) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (3), where Alk represents an ethylene or trimethylene group;

(5) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (4), where Alk is an ethylene group;

(6) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (5), where m is an integer in the range from 3 to 50;

(7) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (6), where m is an integer in the range from 3 to 40;

(8) a copolymer or a pharmacologically acceptable salt thereof, according to (7), where m is an integer in the range from 6 to 16 or in the range from 28 to 38;

(9) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (8), where m is an integer in the range from 6 to 16;

(10) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (9), where R ¹ represents a hydrogen atom or a methyl group;

(11) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (10), wherein R ¹ represents a hydrogen atom;

(12) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (11), where R ² represents a hydrogen atom or a methyl group;

(13) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (12), wherein R ² is a methyl group;

(14) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (13), where R ³ represents a hydroxyl group, an alkoxy group containing from 1 to 6 carbon atoms, or a group described by the formula -NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(15) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (14), wherein R ³ is a hydroxyl group or an alkoxy group containing from 1 to 6 carbon atoms;

(16) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (15), containing at least one structural unit described by formula (II), in which R ³ represents an alkoxy group containing from 1 to 6 carbon atoms and optionally at least one structural unit described by formula (II) in which R ³ represents a hydroxyl group, where the ratio between the structural unit described by formula (II) in which R ³ represents a hydroxy group , and the urine elementary units described by formula (II), in which R ³ represents an alkoxy group containing from 1 to 6 carbon atoms, is in the range from 4: 6 to 0:10;

(17) a copolymer or a pharmacologically acceptable salt thereof corresponding to positions (15) or (16), where R ³ is an alkoxy group containing from 1 to 6 carbon atoms;

(18) a copolymer or a pharmacologically acceptable salt thereof corresponding to any one of the positions (15) to (17), wherein said alkoxy group containing from 1 to 6 carbon atoms is an ethoxy group;

(19) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (14), wherein R ³ is a hydroxyl group or a group described by the formula —NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different, and each is represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(20) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (19), containing at least one structural unit described by formula (II), in which R ³ represents a group described by formula -NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and optionally at least one structural unit described by formula (II), in which R ³ represents a hydroxyl group, where the relationship between the structural elementary units described by formula (II) in which R ³ represents a hydroxy group and the structural elementary units described by formula (II) in which R ³ represents a group described by formula -NR ⁴ R ⁵ is in the range from 5: 5 to 0:10;

(21) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (20), wherein the ratio is between structural units described by formula (II) in which R ³ is a hydroxy group and structural units described by formula (II), in which R ³ represents a group described by the formula —NR ⁴ R ⁵ , is in the range from 4: 6 to 0:10;

(22) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (19) to (21), where R ³ is a group described by the formula —NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(23) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (19) to (22), where the group described by the formula —NR ⁴ R ⁵ represents an amino group, a methylamino group or a dimethylamino group;

(24) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (23), wherein the group described by the formula —NR ⁴ R ⁵ represents an amino group;

(25) a copolymer or a pharmacologically acceptable salt thereof, according to (23), wherein the group described by the formula —NR ⁴ R ⁵ represents a dimethylamino group;

(26) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (14), wherein R ³ is a hydroxyl group;

(27) a copolymer or a pharmacologically acceptable salt thereof, according to (14), wherein R ³ is a 1-amino-2-propanol group;

(28) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (27), where the ratio between the structural unit described by formula (I) and the structural unit described by formula (II) is in the range from 10: 1 to 1:10;

(29) the copolymer or its pharmacologically acceptable salt corresponding to position (28), where the ratio between the structural element unit described by formula (I) and the structural unit described by formula (II) is in the range from 3: 1 to 1: 8;

(30) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (28), where the ratio between the structural unit described by formula (I) and the structural unit described by formula (II) is in the range from 2: 1 to 1: 2 or in the range from 1: 2 to 1: 6;

(31) the copolymer or its pharmacologically acceptable salt corresponding to position (28), where the ratio between the structural unit described by formula (I) and the structural unit described by formula (II) is 1: 1 or is in the range from 1 : 2 to 1: 4;

(32) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (31), where the average degree of polymerization is in the range from 5 to 200;

(33) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (32), wherein the average degree of polymerization is in the range from 5 to 50;

(34) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (33), wherein the average degree of polymerization is in the range of 5 to 20;

(35) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (32), wherein the average degree of polymerization is in the range from 20 to 30;

(36) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (32), wherein the average degree of polymerization is in the range from 30 to 40;

(37) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (1) to (31), where their Stokes radius is 9.3 nm or less;

(38) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (37), where their Stokes radius is 7.3 nm or less;

(39) a copolymer or a pharmacologically acceptable salt thereof corresponding to (38), where their Stokes radius is 6.2 nm or less;

(40) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (39), where their Stokes radius is 4.7 nm or less;

(41) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (40), where their Stokes radius is 3.1 nm or less;

(42) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (37), where their Stokes radius is in the range from 1.5 nm to 4.7 nm;

(43) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (37), where their Stokes radius is in the range from 3.1 nm to 6.2 nm; and

(44) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (1), wherein:

m is an integer in the range from 3 to 100,

Alk is an alkylene group containing from 1 to 6 carbon atoms,

R ¹ and R ² are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and

R ³ represents a hydroxyl group, an alkoxy group containing from 1 to 6 carbon atoms, which may optionally be substituted with one hydroxy group, or a group described by the formula —NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different from each other, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, which optionally can be substituted with one hydroxy group;

(45) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (1), where Alk is an ethylene group, R ¹ is a hydrogen atom, R ² is a methyl group, and m, R ³ is the ratio between the structural units described formulas (I) and (II) (the ratio defined by the composition) and, where appropriate, the ratio between the elementary units described by formula (II), where R ³ represents a hydroxy group, and the elementary units described by formula (II ) wherein R ³ represents Rupp other than hydroxy (the ratio defined by hydrolysis) is selected from the following:

(i) m is in the range from 6 to 16, R ³ is a hydroxy group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range from 30 to 40;

(ii) m is in the range from 28 to 38, R ³ is a hydroxy group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range from 10 to 15;

(iii) m is in the range from 6 to 16, R ³ is an amino group, the ratio defined by the composition is 1: 1, and the average degree of polymerization is in the range from 30 to 40;

(iv) m is in the range of 6 to 16, R ³ is a dimethylamino group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range of 30 to 40;

(v) m is in the range of 6 to 16, R ³ is a 1-amino-2-propanol group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range of 30 to 40;

(vi) m is in the range from 6 to 16, R ^{3 is} selected from ethoxy and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, and the ratio determined by hydrolysis, equal to 4: 6;

(vii) m is in the range from 28 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 10 to 15, and the ratio determined by hydrolysis, equal to 4: 6;

(viii) m is in the range from 28 to 38, R ³ is a dimethylamino group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range from 10 to 15;

(ix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, the ratio determined by hydrolysis is 3.1: 6.9, and the copolymer is a sodium salt;

(x) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, and the ratio determined by hydrolysis, equal to 1.4: 8.6;

(xi) m is in the range from 6 to 16, R ^{3 is} selected from dimethylamino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, the ratio determined by hydrolysis is 2.9: 7.1, and the copolymer is a sodium salt;

(xii) m is in the range of 6 to 16, R ³ is an amino group, the ratio given by the composition is 1: 2.4, and the average degree of polymerization is in the range of 20 to 30;

(xiii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.4: 9.6;

(xiv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 2.9: 7.1;

(xv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.9: 9.1;

(xvi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.5: 9.5;

(xvii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio defined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.3: 8.7;

(xviii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.9: 8.1;

(xix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.0: 9.0;

(xx) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.8: 9.2;

(xxi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 4.6: 5.4;

(xxii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.2: 8.8;

(xxiii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 2.0: 8.0;

(xxiv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.1: 8.9;

(xxv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 2.4: 7.6;

(xxvi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.9: 9.1;

(xxvii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.5: 8.5;

(xxviii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.7: 9.3;

(xxix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 4.5: 5.5;

(xxx) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.4: 8.6;

(xxxi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.7: 9.3;

(xxxii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.8: 9.2;

(xxxiii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.4: 8.6;

(xxxiv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 3.1, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.7: 9.3;

(xxxv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 0.9: 9.1;

(xxxvi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio specified by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified hydrolysis, equal to 1.9: 8.1;

(xxxvii) m is in the range from 6 to 16, R ^{3 is} selected from ethoxy and hydroxy groups, the ratio determined by the composition is approximately 1: 3, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis equal to 3.1: 6.9;

(xxxviii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius 9.3 nm or less;

(xxxix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is in the range from 3.1 to 6.2 nm;

(xl) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is in the range from 1.5 to 4.7 nm;

(xli) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 3.1 nm or less;

(xlii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 7.8 nm or less;

(xliii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 6.2 nm or less; and

(xliv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 4.7 nm or less.

(46) As already noted above, another aspect of the present invention provides a copolymer or a pharmacologically acceptable salt thereof, obtained by carrying out for one or more units of carboxylic acid anhydride described by formula (III) in a copolymer which contains elementary constituents thereof links

(a) one or more structural elementary units, which may be identical or different from each other and which are described by the following formula (I):

Where:

m, Alk, R ¹ and R ² are as defined above, and

(b) said structural unit containing a carboxylic acid anhydride unit described by formula (III):

one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis, and (iv) alcoholysis.

Among these copolymers and their pharmacologically acceptable salts, preferred compounds are:

(47) the copolymer or its pharmacologically acceptable salt corresponding to position (46), where for the structural unit described by formula (I) and the structural unit described by formula (III), the configuration in the sequence is alternated in the copolymer with the head alternating to head ”, a sequence with alternating“ head to tail ”or a mixed sequence with alternating“ head to head ”and“ head to tail ”;

(48) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (46), where, for a structural unit described by formula (I) and a structural unit described by formula (III), a configuration is obtained in the form of a statistical sequence;

(49) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (46) to (48), where Alk represents an ethylene or trimethylene group;

(50) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (49), where Alk is an ethylene group;

(51) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (46) to (50), where m is an integer in the range from 3 to 50;

(52) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (51), where m is an integer in the range from 3 to 40;

(53) a copolymer or a pharmacologically acceptable salt thereof, according to (52), where m is an integer in the range from 6 to 16 or in the range from 28 to 38;

(54) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (53), where m is an integer in the range from 6 to 16;

(55) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (46) to (54), where R ¹ represents a hydrogen atom or a methyl group;

(56) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (55), wherein R ¹ represents a hydrogen atom;

(57) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (46) to (56), where R ² represents a hydrogen atom or a methyl group;

(58) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (57), wherein R ² is a methyl group;

(59) a copolymer or its pharmacologically acceptable salt corresponding to any one position from (46) to (58), where the ratio between the structural elementary unit described by formula (I) and the structural elementary unit obtained as a result of carrying out for one or more structural elementary units described by formula (III), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is in the range from 10: 1 to 1: 10;

(60) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (59), wherein the ratio is between a structural unit described by formula (I) and a structural unit obtained by carrying out for one or more structural unit described by formula (III) ), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is in the range from 3: 1 to 1: 8;

(61) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (59), wherein the ratio is between the structural unit described by formula (I) and the structural unit obtained by carrying out for one or more structural unit described by formula (III ), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is in the range from 2: 1 to 1: 2 or in the range from 1: 2 to 1: 6;

(62) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (59), wherein the ratio is between a structural unit described by formula (I) and a structural unit obtained by carrying out for one or more structural unit described by formula (III) ), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is 1: 1 or is in the range from 1: 2 to 1: 4;

(63) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one position from (46) to (62), where the average degree of polymerization is in the range from 5 to 200;

(64) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (63), wherein the average degree of polymerization is in the range from 5 to 50;

(65) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (64), wherein the average degree of polymerization is in the range of 5 to 20;

(66) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (63), wherein the average degree of polymerization is in the range from 20 to 30;

(67) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (63), wherein the average degree of polymerization is in the range from 30 to 40;

(68) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (46), wherein:

m is an integer in the range from 3 to 100,

Alk is an alkylene group containing from 1 to 6 carbon atoms, and

R ¹ and R ² are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(69) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one of the positions (46) to (68), which can be obtained by ammonolysis of the carboxylic acid anhydride unit described by formula (III) in the copolymer;

(70) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (69), which can be obtained by ammonolysis using aqueous ammonia solution;

(71) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one of items (46) to (68), which can be obtained by aminolysis for the carboxylic acid anhydride unit described by formula (III) in a copolymer;

(72) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (71), which can be obtained by aminolysis using an aqueous solution of dimethylamine;

(73) a copolymer or a pharmacologically acceptable salt thereof, corresponding to any one of items (46) to (68), which can be obtained by alcoholysis of a carboxylic acid anhydride unit described by formula (III) in a copolymer; and

(74) a copolymer or a pharmacologically acceptable salt thereof, corresponding to (73), which can be obtained by alcoholysis using ethanol.

The present invention also uses the copolymers and their pharmacologically acceptable salts of the present invention to create a pharmaceutical composition, a modifier capable of modifying a protein, a complex, a method for prolonging the time during which a protein is held in a blood stream, a method for treating or preventing diseases, and using the complex of the invention for the manufacture a medicine intended for the treatment or prevention of diseases. Preferred examples of aspects of these inventions include:

(75) a pharmaceutical composition comprising a pharmaceutically acceptable diluent or carrier and at least one copolymer or a pharmacologically acceptable salt thereof of the present invention, corresponding to any one position from (1) to (74);

(76) a pharmaceutical composition according to (75), wherein said composition further comprises at least one protein or an analogue or variant thereof;

(77) a pharmaceutical composition corresponding to (76), wherein the protein or an analogue or variant thereof is a basic protein;

(78) a pharmaceutical composition according to (77), wherein the main protein is major fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet derived growth factor (PDGF) isolated from the brain neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocytic growth factor (HGF) or vascular endothelial growth factor (VEGF) or their analogues or variants;

(79) a pharmaceutical composition according to (77), wherein the major protein is an osteoclastogenesis inhibition factor (OCIF) or an analogue or variant thereof;

(80) a pharmaceutical composition according to (79), wherein said OCIF or an analogue or variant thereof is of a natural type or recombinant type OCIF;

(81) a pharmaceutical composition according to (79), wherein said OCIF or an analogue or variant thereof is a monomer or dimer;

(82) a pharmaceutical composition according to (79), wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE (non-reducing sodium polydeacrylamide gel electrophoresis) under non-reducing conditions, or a dimer Human OCIF, characterized by a molecular weight of approximately 120,000 as measured by SDS-PAGE under non-reducing conditions;

(83) a pharmaceutical composition according to (79), wherein said OCIF contains amino acids from - 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(84) a pharmaceutical composition corresponding to (79), wherein said OCIF contains amino acids from + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(85) a modifier capable of modifying the protein or its analogue or variant, wherein said modifier contains a copolymer or its pharmacologically acceptable salt corresponding to any one position from (1) to (74);

(86) a modifier capable of modifying the protein or its analogue or variant corresponding to position (85), where the protein is the main protein;

(87) a modifier capable of modifying a protein or its analogue or variant corresponding to position (86), where the main protein is the main fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet growth factor (PDGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF), or analogues or variants thereof;

(88) a modifier capable of modifying the protein or its analogue or variant corresponding to position (86), where the main protein is an osteoclastogenesis inhibition factor (OCIF) or its analogue or variant;

(89) a modifier capable of modifying the protein or its analogue or variant corresponding to the position (88), wherein said OCIF or its analogue or variant are of natural type or recombinant type OCIF;

(90) a modifier capable of modifying the protein or its analogue or variant corresponding to position (88), wherein said OCIF or its analogue or variant is a monomer or dimer;

(91) a modifier capable of modifying a protein or its analogue or variant corresponding to (88), wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer characterized by a molecular weight of approximately 120,000 as measured by SDS-PAGE under non-reducing conditions;

(92) a modifier capable of modifying the protein or its analogue or variant corresponding to position (88), wherein said OCIF contains amino acids from - 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(93) a modifier capable of modifying a protein or its analogue or variant corresponding to position (88), wherein said OCIF contains amino acids from + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(94) a complex containing at least one protein or its analogue or variant that bind to at least one copolymer or its pharmacologically acceptable salt corresponding to any one position from (1) to (74);

(95) a complex corresponding to position (94), where the protein is a basic protein;

(96) the complex corresponding to position (95), where the main protein is the main fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet-derived growth factor (PDGF), isolated from the brain neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF), or analogues or variants thereof;

(97) a complex corresponding to (95), wherein the main protein is an osteoclastogenesis inhibition factor (OCIF) or an analogue or variant thereof;

(98) a complex corresponding to (97), wherein said OCIF or an analogue or variant thereof is of a natural type or recombinant type OCIF;

(99) a complex corresponding to (97), wherein said OCIF or an analogue or variant thereof is a monomer or dimer;

(100) a complex according to (97), wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 according to SDS-PAGE measurement under non-reducing conditions;

(101) the complex corresponding to (97), wherein said OCIF contains amino acids from - 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(102) a complex corresponding to (97), wherein said OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(103) a pharmaceutical composition containing an effective amount of a pharmacologically active substance together with a carrier or diluent for it, where the specified pharmacologically active substance is present in the form of a complex corresponding to any one position from (94) to (102);

(104) a method for prolonging the time during which a protein or its analogue or variant is held in a blood stream after administration by a patient as a result of complexation between said protein or its analogue or variant and at least one copolymer or its pharmacologically acceptable salt corresponding to any one position from (1) to (74);

(105) a method corresponding to (104), wherein the protein is a basic protein;

(106) the method corresponding to (105), where the main protein is the main fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet-derived growth factor (PDGF), isolated from the brain neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocytic growth factor (HGF) or vascular endothelial growth factor (VEGF), or analogues or variants thereof;

(107) a method corresponding to (105), wherein the base protein is an osteoclastogenesis inhibition factor (OCIF) or an analogue or variant thereof;

(108) a method corresponding to (107), wherein said OCIF or an analogue or variant thereof is of a natural type or recombinant type OCIF;

(109) a method corresponding to (107), wherein said OCIF or an analogue or variant thereof is a monomer or dimer;

(110) a method corresponding to (107), wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 according to SDS-PAGE measurement under non-reducing conditions;

(111) the method corresponding to (107), wherein said OCIF contains amino acids from - 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(112) the method corresponding to (107), wherein said OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(113) a method for treating or preventing a disease in a patient that is susceptible to the action of a protein or its analogue or variant, comprising administering to said patient an effective amount of a complex containing said protein or its analogue or variant that bind to at least one copolymer or its pharmacologically acceptable salt corresponding to any one position from (1) to (74);

(114) a method corresponding to (113), wherein the protein is a basic protein;

(115) the method corresponding to (114), where the main protein is the main fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet-derived growth factor (PDGF), isolated from the brain neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocytic growth factor (HGF) or vascular endothelial growth factor (VEGF), or analogues or variants thereof;

(116) a method corresponding to (114), wherein the base protein is an osteoclastogenesis inhibition factor (OCIF) or an analogue or variant thereof;

(117) a pharmaceutical composition corresponding to (116), wherein said OCIF or an analogue or variant thereof is of a natural type or recombinant type OCIF;

(118) a method corresponding to (116), wherein said OCIF or an analogue or variant thereof is a monomer or dimer;

(119) a method corresponding to (116), wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 according to SDS-PAGE measurement under non-reducing conditions;

(120) the method corresponding to (116), wherein said OCIF contains amino acids from - 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(121) a method corresponding to (116), wherein said OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(122) a method corresponding to any one position from (116) to (121), wherein said disease is a metabolic disorder in the bones;

(123) the use of a complex containing a protein or its analogue or variant, which is associated with at least one copolymer or its pharmacologically acceptable salt corresponding to any one position from (1) to (74), for the manufacture of a medicinal product intended for prevention or treatment of a disease susceptible to the action of said protein or its analogue or variant;

(124) an application corresponding to (122), wherein the protein is a basic protein;

(125) the application corresponding to (123), where the main protein is the main fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet-derived growth factor (PDGF), isolated from the brain neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF), or analogs or variants thereof;

(126) an application corresponding to (123), wherein the main protein is an osteoclastogenesis inhibition factor (OCIF) or an analogue or variant thereof;

(127) an application corresponding to (126), wherein said OCIF or an analogue or variant thereof is of a natural type or recombinant type OCIF;

(128) an application corresponding to (126), wherein said OCIF or an analogue or variant thereof is a monomer or dimer;

(129) the application corresponding to (126), wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 according to SDS-PAGE measurement under non-reducing conditions;

(130) the application corresponding to (126), wherein said OCIF contains amino acids from - 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(131) the application corresponding to (126), wherein said OCIF contains amino acids from + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences; and

(132) an application corresponding to any one position from (126) to (131), where the disease is a metabolic disorder in the bones.

"Alkylene group containing from 1 to 6 carbon atoms," in the definition of the substituent Alk in the above formula (I), is a straight or branched alkylene group containing from 1 to 6 carbon atoms, such as methylene, methylmethylene, ethylene, propylene, trimethylene, tetramethylene, 1-methyltrimethylene, 2-methyltrimethylene, 3-methyltrimethylene, pentamethylene or hexamethylene group. Among these alkylene groups, straight or branched alkylene groups containing from 1 to 4 carbon atoms are preferred, ethylene or trimethylene groups are more preferred, and an ethylene group is most preferred.

An alkyl group in an “alkyl group containing from 1 to 6 carbon atoms, which may optionally be substituted with at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 atoms carbon, which may optionally be substituted with substituents in an amount of 1 to 5, selected from substituents A defined below, "in the definition of substituents R ¹ , R ² , R ⁴ , R ⁵ and substituents A in the above formulas (I) and (II ) is a straight or branched alk a yl group containing from 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1 -ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1 , 3-dimethylbutyl, 2,3-dimethylbutyl or 2-ethylbutyl group. Among these alkyl groups, straight or branched alkyl groups containing from 1 to 4 carbon atoms are preferred, methyl and ethyl groups are more preferred, and methyl group is most preferred.

The alkoxy group in the "alkoxy group containing from 1 to 6 carbon atoms, which may optionally be substituted by at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms which may optionally be substituted with 1 to 5 substituents selected from substituents A defined below, "in the definition of substituent R ³ and substituents A in the above formulas (I) and (II) is a substituent in which the above alkyl hydrochloric group containing from 1 to 6 carbon atoms linked to an oxygen atom. Examples of such an alkoxy group include straight or branched alkoxy groups containing from 1 to 6 carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, 2-methylbutoxy, neopentyloxy, n-hexyloxy, 4-methylpentyloxy, 3-methylpentyloxy, 2-methylpentyloxy, 3,3-dimethylbutyoxy, 2, 2-dimethylbutoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy and 2,3-dimethylbutoxy. Among these alkoxy groups, straight or branched alkoxy groups containing from 1 to 4 carbon atoms are more preferred, and the ethoxy group is most preferred.

The “halogen atom”, which is one of the above “substituents A”, is an optional substituent in the “alkyl group containing from 1 to 6 carbon atoms, which may optionally be substituted by at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A defined below, "in the definition of substituents R ¹ , R ² , R ⁴ and R ⁵ in the above formulas (I) and (II) and represents an optional substituent in the "alkoxy group containing from 1 to 6 carbon atoms, which optionally can be substituted by at least one substituent, selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A defined below, "in the definition of R ³ in the above formula (II), representing is a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; and preferably it is a fluorine atom or a chlorine atom.

"Aryl group containing from 6 to 14 carbon atoms", which is an optional substituent in the "alkyl group containing from 1 to 6 carbon atoms, which may optionally be substituted by at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A defined below, "in the definition of substituents R ¹ , R ² , R ⁴ and R ⁵ and neo the optional substituent in the "alkoxy group containing from 1 to 6 carbon atoms, which may optionally be substituted with at least one substituent selected from the group consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms, "in the definition of the substituent R ³ in the above formula (II), is an aromatic hydrocarbon group containing from 6 to 14 carbon atoms, and it can be, for example, a phenyl, indenyl, naphthyl, phenanthryl or anthril group. Preferably, it is a phenyl group.

“An aryloxy group containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A,” in the definition of substituent R ³ in the above formula (II), is an aryl group as defined above, which is bonded to an oxygen atom, and it may be, for example, a phenoxy, indenyloxy, naphthyloxy, phenanthryloxy or anthryloxy group. Preferably, it is a phenoxy group.

"An alkyl group containing from 1 to 6 carbon atoms, which is optionally substituted with at least one halogen atom," in the definition of the substituents R ¹ , R ² , R ⁴ and R ⁵ in the above formulas (I) and (II) represents an alkyl group containing from 1 to 6 carbon atoms described above, and may be, for example, a trifluoromethyl group, a trichloromethyl group, a difluoromethyl group, a dichloromethyl group, a dibromomethyl group, a fluoromethyl group, a 2,2,2-trifluoroethyl group, 2 , 2,2-trichloroethyl group, 2-bromoethyl group, 2-chloroethyl group, a 2-fluoroethyl group, 2-iodoethyl group, a 3-chloropropyl group, a 4-fluorobutyl group, a 6-iodohexyl group, a 2,2-dibromoethyl group or a pentafluoroethyl group. Preferably, it is a trifluoromethyl group, a trichloromethyl group, a difluoromethyl group or a pentafluoroethyl group; and most preferably it is a trifluoromethyl group.

Examples of the “alkyl group containing from 1 to 6 carbon atoms, which is optionally substituted with at least one hydroxy group,” in the definition of the substituents R ¹ , R ⁴ and R ⁵ in the above formulas (I) and (II) include hydroxymethyl group, 1-hydroxyethyl group, 1-hydroxypropyl group and 2-hydroxypropyl group.

"Alkoxy group containing from 1 to 6 carbon atoms, which is optionally substituted by at least one halogen atom," in the definitions of the substituent R ³ in the above formula (II) is an alkoxy group containing from 1 to 6 atoms carbon described above, which is substituted by at least one halogen atom described above, and may be, for example, a trifluoromethoxy group, trichloromethoxy group, difluoromethoxy group, dichloromethoxy group, dibromomethoxy group, fluoromethoxy group, 2 , 2,2-trifluoroethoxy group, 2,2,2-t a trichloroethoxy group, 2-bromoethoxy group, 2-chloroethoxy group, 2-fluoroethoxy group, 2-iodoethoxy group, 3-chloropropoxy group, 4-fluorobutoxy group, 6-iodohexyloxy group, 2,2- dibromethoxy group or pentafluoroethoxy group; preferably it is a C ₁ -C ₄ alkoxy group substituted with fluorine or chlorine atoms, such as a trifluoromethoxy group, trichloromethoxy group, difluoromethoxy group or pentafluoroethoxy group. More preferably, it is a trifluoromethoxy group.

Examples of the “alkoxy group containing from 1 to 6 carbon atoms, which is optionally substituted with at least one hydroxy group” in the definition of the substituent R ³ in the above formula (II) include a hydroxymethoxy group, a 1-hydroxyethoxy group , 1-hydroxypropoxy group; and 2-hydroxypropoxy group.

"An alkyl group containing from 1 to 6 carbon atoms, which is optionally substituted by at least one aryl group containing from 6 to 14 carbon atoms, which optionally can be substituted by 1 to 5 substituents selected from substituents A, "In the definition of substituents R ¹ , R ² , R ⁴ and R ⁵ in the above formulas (I) and (II) may be, for example, a benzyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, indenylmethyl group, 1-phenethyl group, 2-phenethyl group, 1-naphthylethyl group th, 2-naphthylethyl group, 1-phenylpropyl group, 2-phenylpropyl group, 3-phenylpropyl group, 1-naphthylpropyl group, 2-naphthylpropyl group, 3-naphthylpropyl group, 1-phenylbutyl group, 2-phenylbutyl group, 3-phenylbutyl group, 4-phenylbutyl group, 1-naphthylbutyl group, 2-naphthylbutyl group, 3-naphthylbutyl group, 4-naphthylbutyl group, 1-phenylpentyl group, 2-phenylpentyl group, 3-phenylpentyl group, 4-phenylpentyl group, 5-phenylpentyl group group, 1-phenylhex a methyl group, a 2-phenylhexyl group, a 3-phenylhexyl group, a 4-phenylhexyl group, a 5-phenylhexyl group or a 6-phenylhexyl group; preferably it is an alkyl group substituted with an aryl group containing from 6 to 10 carbon atoms, such as a benzyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, 1-phenethyl group, 2-phenethyl group, 1-naphthylethyl group, 2- a naphthylethyl group, a 1-phenylpropyl group, a 2-phenylpropyl group, a 3-phenylpropyl group or a 1-naphthylpropyl group; and more preferably it is a benzyl group.

“An alkoxy group containing from 1 to 6 carbon atoms, which is optionally substituted with at least one aryl group containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A , "In the definition of the substituent R ³ in the above formula (II), may be, for example, a benzoxy group, 1-naphthylmethoxy group, 2-naphthylmethoxy group, indenylmethoxy group, 1-phenethoxy group, 2-phenethoxy group , 1-naphthylethoxy group, 2-naphthylethoxy group, 1-phenylpropoxy group, 2-phenylpropoxy group, 3-phenylpropoxy group, 1-naphthylpropoxy group, 2-naphthylpropoxy group, 3-naphthylpropoxy group, 1-phenylbutoxy group, 2-phenylbutoxy group, 3-phenylbutoxy group, 4-phenylbutoxy group, 1-naphthylbutoxy group, 2-naphthylbutoxy group, 3-naphthylbutoxy group, 4-naphthylbutoxy group, 1-phenylpentoxy group, 2-phenylpentoxy group, 3-phenylpentoxy group, 4- phenylpentoxy group, 5-phenylpentoxy group, 1-phenylhexyloxy group, 2-phenylhexyloxy group, 3-phenylhexyloxy group, 4-phenylhexyloxy group, 5-phenylhexyl a xi group or 6-phenylhexyloxy group; preferably it is an alkyl group substituted with an aryl group containing from 6 to 10 carbon atoms, such as a benzoxy group, 1-naphthylmethoxy group, 2-naphthylmethoxy group, 1-phenethoxy group, 2-phenethoxy group, 1- a naphthylethoxy group, 2-naphthylethoxy group, 1-phenylpropoxy group, 2-phenylpropoxy group, 3-phenylpropoxy group or 1-naphthylpropoxy group; and more preferably, it is a benzoxy group.

Where R ³ is “an aryloxy group containing from 6 to 14 carbon atoms, which may optionally be substituted with 1 to 5 substituents selected from substituents A,” it is preferably an aryloxy group containing from 6 to 10 carbon atoms, which are optionally substituted with substituents in an amount of 1 to 3, selected from substituent group A; more preferably, it is a phenoxy group which is optionally substituted with 1 to 3 substituents selected from substituent group A; even more preferably, it is a phenoxy group optionally substituted with halogen atoms, alkyl groups containing from 1 to 6 carbon atoms, hydroxy groups or nitro groups in an amount of from 1 to 3; and most preferably, they are a phenoxy group or a p-nitrophenoxy group.

Where the copolymer of the present invention contains a basic group, the compound can be converted to its pharmacologically acceptable salt by reaction between some or all of these basic groups and an acid. In addition, the copolymers of the present invention contain acid carboxyl groups, and the copolymer can be converted into its pharmacologically acceptable salt by reaction between some or all of these carboxyl groups and the base.

Preferred examples of pharmacologically acceptable salts obtained when the main group is contained in the copolymers of the present invention include inorganic acid salts such as hydrohalic acid salts (eg, hydrochlorides, hydrobromides and hydroiodides), nitrates, perchlorates, sulfates and phosphates; organic acid salts, such as lower alkanesulfonates, in which their lower alkyl unit is as defined above (e.g. methanesulfonates, trifluoromethanesulfonates and ethanesulfonates), aryl sulfonates in which their aryl unit is as defined above (e.g., benzenesulfonate or p-toluenesulfonate), acetates, malates, fumarates, succinates, citrates, ascorbates, tartrates, oxalates and maleates; and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutaminates and aspartates. Particularly preferred are salts of hydrohalic acids.

Preferred examples of pharmacologically acceptable salts obtained by containing an acidic carboxyl group in the copolymers of the present invention include metal salts such as alkali metal salts (e.g. sodium salts, potassium salts and lithium salts), alkaline earth metal salts (e.g. calcium salts and magnesium salts ), metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts and cobalt salts; amine salts, such as inorganic amine salts (e.g., ammonium salts) and organic amine salts (e.g., tert-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycinealkyl ester salts, ethylene diamine salts, N-methylglucamine salts, guanidine salts , diethylamine salts, triethylamine salts, dicyclohexylamine salts, N, N'-dibenzylethylenediamine salts, chlorprocaine salts, procaine salts, diethanolamine salts, N-benzylphenethylamine salts, piperazine salts, tetramethylammonium salts and tris (hydroxymethyl) amine salts and amino acid salts such as glycine salts, lysine salts, arginine salts, ornithine salts, glutaminates and aspartates, Particularly preferred are alkali metal salts and alkaline earth metal salts.

In the present invention, the “structural unit” is defined as the minimum unit forming the copolymer of the invention, and is depicted when defining the above copolymer as the unit described by formula (I) or the unit described by formula (II). A “structural unit” is not a structure of a monomeric starting material used in a polymerization reaction in the synthesis of a copolymer of the present invention; more precisely, this is an elementary unit that is obtained from the specified monomeric starting material and which is present in the specified copolymer of the present invention.

In the present invention, the phrase “head-to-head sequence” means that for the structural units described by formulas (I) and (II), the configuration shown in the following formula is obtained:

In the present invention, the phrase “head-to-tail sequence” means that for the structural units described by formulas (I) and (II), the configuration shown in the following formula is obtained:

In the present invention, the copolymers and their pharmacologically acceptable salts may be alternating copolymers or random copolymers. Alternating copolymers are those in which the ratio of the structural elementary units described by formula (I) and the structural elementary units described by formula (II) is 1: 1, and for the structural elementary units described by formula (I) and structural elementary units described by formula (II), receive a configuration in the form of a sequence with alternating "head to head", a sequence with alternating "head to tail" or a mixed sequence with alternating "head to head" and "head to tail". Statistical copolymers are those in which for the structural elementary units described by formula (I) and the structural elementary units described by formula (II), they are configured in the form of a statistical sequence.

In the present invention, the “compositional ratio” is the average ratio between the number of structural unit units described by formula (I) and the number of structural unit units described by formula (II) in the copolymer of the present invention. If the copolymer is essentially an alternating copolymer, then the ratio specified by the composition will be 1: 1. However, if the copolymer is a random copolymer, the ratio may vary. In the copolymers of the present invention, the specified ratio is not particularly limited, and usually it can be in the range from 10: 1 to 1:10; preferably from 3: 1 to 1: 8, more preferably from 2: 1 to 1: 2 or from 1: 2 to 1: 6, and most preferably it is either 1: 1 or is in the range from 1: 2 to 1 :four. It should be noted that the values of the ratio specified by the composition inevitably vary to some extent, which is caused by the presence of minor changes in the starting materials, polymerization conditions, and the like. As a result, the ratios given by the composition are approximate values; variations for the values of the ratios specified by the composition given above up to ± 30% are still considered to fall within the scope of the indicated ratios.

The ratio defined by the composition for the copolymers of the present invention can be determined using known analytical methods. As a result of determining the content of carboxyl groups in the copolymer (mmol / g) by conductometric titration [determination requiring the preparation of the corresponding fully hydrolyzed copolymer (that is, R ³ is OH) or as a result of hydrolysis of the copolymer to be analyzed, or as a result of independent synthesis of the corresponding hydrolyzed copolymer] and, based on the knowledge of the formula weights for each of the structural elementary units of the specified copolymer, you can determine the ratio specified e composition using the following formula:

Rii / Ri = (C × FWi) / (2000-C × FWii),

where Ri represents the average number of structural elementary units (I), Rii represents the average number of structural elementary units (II), C represents the content of carboxyl groups in the copolymer (mmol / g), FWi represents the formula weight of the structural elementary unit (I) , a FWii is the formula weight of a structural unit (II).

In the General case, the molecular weight of the polymer is determined as the relative molecular weight in terms of the molecular weight of a standard compound, which includes a structure similar to the structure of the specified polymer, and is characterized by a known absolute molecular weight. Such an evaluation method is often used in determining the molecular weight of new copolymers, such as the copolymers of the present invention.

The average molecular weight of the copolymer of the present invention is a value measured by gel filtration chromatography using, as a standard compound, a polymer having a known absolute molecular weight. The nature of the gel, the elution conditions, and the polymer with known absolute molecular weight used for comparison can be appropriately selected by one skilled in the art using known techniques and generally available information, for example, see Comprehensive Polymer Science, pub. Pergamon Press (Oxford) 1989. The standard compound used for comparison purposes is preferably a polymer having similar structure and properties. In the case of the copolymers of the present invention, the side chain region is considered as a characteristic structure. Therefore, the standard compound used for comparison is preferably a polymer comprising a structure similar to the structure of the side chain region of the copolymer of the present invention, the average molecular weight of which is to be measured. The standard compound used is more preferably poly (ethylene glycol).

In the present invention, the “average degree of polymerization” is the average value of the degree of polymerization of the structural units in the copolymer of the present invention, that is, this is the average number of structural units in the specified copolymer. It is determined on the basis of the average molecular weight of the copolymer, the formula weights of each of the structural elementary units of the specified copolymer and the ratio specified by the composition for the structural elementary units in the specified copolymer. The "average degree of polymerization" of the copolymer of the present invention containing structural units I and II can be calculated using the following formula:

The average degree of polymerization = MS / (FWi × Ri + FWii × Rii),

where Ms is the average molecular weight of the copolymer, FWi and FWii are the formula weights for each of the structural unit I and II, respectively, and Ri and Rii are the fractions of the structural unit I and II in the copolymer, calculated from the ratio given by the composition ( the ratio given by the composition = Ri: Rii; Ri + Rii = 1).

In the above formula for determining the average degree of polymerization of the copolymer of the present invention, all parameters, such as the value of the ratio specified by the composition, for structural units, the average molecular weight of the copolymer and the formula weights of the structural units, can be determined as discussed above. In order to do this, it is necessary to obtain the corresponding copolymer subjected to complete hydrolysis, ammonolysis, aminolysis or alcoholysis (preferably the corresponding copolymer subjected to complete hydrolysis). Alternatively, if the starting copolymer used in the preparation of the copolymer of the present invention has a known composition, these values can be determined without the need for analysis. There is no particular limitation on the average degree of polymerization in the copolymers of the present invention. Usually it is in the range of 5 to 200; preferably it is in the range from 5 to 50; more preferably 5 to 20 or 20 to 30 or 30 to 40.

In the copolymers of the present invention, the structural units described by formula (II) may include at least one structural unit described by formula (II), where R ³ is an optionally substituted alkoxy group containing from 1 to 6 carbon atoms optionally substituted aryloxy group or a group described by the formula —NR ⁴ R ⁵ , and optionally at least one structural unit described by formula (II), in which R ³ represents a hydroxyl group. The ratio between the structural element units described by formula (II) in which R ³ represents a hydroxy group and the structural element units described by formula (II) in which R ³ represents an optionally substituted alkoxy group, an optionally substituted aryloxy group or a group described by the formula —NR ⁴ R ⁵ is called the ratio given by hydrolysis. As a result of determining the level of carboxyl groups in the copolymer (mmol / g) by conductometric titration and based on knowledge of the formula weights for each of the structural elementary units of the copolymer and the ratio specified by the composition (see above), one can determine the ratio given by hydrolysis, N : And using the following formula:

A = 2000 × (Rii / Ri) -C × [FWi + FWii × (Rii / Ri)] H + A (Rii / Ri) × [C × (FW (A) ii-FWii) +1000]

where H: A is the ratio given by hydrolysis, Ri: Rii is the ratio given by composition, C is the content of carboxyl groups in the copolymer (mmol / g), FWi is the formula weight of the structural unit (I), FWii is a formula weight of the structural elementary unit (II), wherein R ³ is OH and FW (a) ii is the formula weight of the structural elementary unit (II), wherein R ³ represents an optionally substituted alkoxy group, neobyazat flax substituted aryloxy group, or a group described by formula -NR ⁴ R ^5.

Preferred ranges for the ratio determined by hydrolysis are in the range from 5: 5 to 0:10, from 4: 6 to 0:10, from 3: 7 to 0:10, from 2: 8 to 0:10 and from 1: 9 to 0:10. It should be noted that the values of the ratio specified by hydrolysis inevitably vary to some extent, which is caused by the presence of minor changes in the starting materials, reaction conditions, and the like. As a result, the ratios given by hydrolysis given in this application are approximate values; variations for the values of the ratios specified by hydrolysis given in this application, up to ± 30%, are still considered to fall within the scope of these ratios.

The size of the molecules of the copolymers of the invention can be measured using known analytical techniques, such as size exclusion chromatography, using proteins of known molecular size as standards (Examples 15 and 16 below provide examples of the application of such a technique). Using size exclusion chromatography, the size of the copolymer molecule is obtained in the form of its Stokes radius. In the present invention, there is no particular limitation on the size of the copolymer molecules. Typically, the Stokes radius of the copolymers of the present invention is 9.3 nm or less, preferably it is 7.3 nm or less, more preferably it is 6.2 nm or less, 4.7 nm or less or 3.1 nm or less, or it is in the range from 3.1 to 6.2 nm or in the range from 1.5 to 4.7 nm.

The copolymers and their pharmacologically acceptable salts of the present invention can be obtained by carrying out a suitable copolymerization reaction known to a person skilled in the art [see, for example, the Comprehensive Polymer Science published by Pergamon Press (Oxford) in 1989] using the starting monomers described the following formulas (IV) and (V):

(where m, Alk and R ¹ , R ² and R ³ are as defined above). In order to obtain a copolymer characterized by a particular desired average degree of polymerization or molecular weight, the resulting copolymer can be fractionated using gel filtration chromatography.

Monomers are known in the art, or can easily be obtained in accordance with a method well known to one skilled in the art, [see, for example, J. M. Harris, "Laboratory synthesis of polyethylene glycol derivatives", Rev. Macromol. Chem. Phys. C25, 326-373 (1985) and Japanese Patent No. 2621308]. The monomers described by formula (V) can easily be obtained by carrying out one or more reactions for maleic anhydride selected from the group consisting of (a) hydrolysis, (b) ammonolysis, (c) aminolysis and (d) alcoholysis.

Alternatively, the copolymer or a pharmacologically acceptable salt thereof can be obtained by carrying out for one or more carboxylic acid anhydride units described by formula (III) in a copolymer which contains the elementary units forming it

(a) one or more structural elementary units, which may be identical or different from each other, and which are described by the following formula (I):

Where:

m, Alk, R ¹ and R ² are as defined above, and

(b) said structural unit containing a carboxylic acid anhydride unit described by formula (III):

one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis, and (iv) alcoholysis. The initial copolymers containing elementary units described by formulas (I) and (III) are either well known in the art (for example, copolymers such as AM-0530K and AM-1510K can be obtained from NOF Corporation), or they are easily can be obtained in accordance with a method well known to those skilled in the art [see, for example, Yoshimoto et al., "Polyethylene glycol derivative-modified cholesterol oxidase soluble and active in benzene", Biochem. Biophys. Res. Comm. 148, 876-882 (1987), Japanese Patent No. 2621308, Japanese Patent Application Publication No. 2003-105040 and Japanese Patent Publication No. 2003-105003].

To obtain a copolymer characterized by a desired average degree of polymerization or a desired molecular weight, the resulting copolymer can be subjected to fractionation by gel filtration chromatography.

In the present invention, the copolymers of the present invention can be obtained by carrying out for one or more carboxylic acid anhydride units described by formula (III) one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis.

In the present invention, “hydrolysis” means a ring opening reaction for a carboxylic acid anhydride unit described by formula (III) under the influence of water to obtain a structural unit described by formula (II), wherein R ³ is a hydroxy group. There are no particular restrictions on the nature of the actual hydrolysis reaction as long as it is a method commonly used by those skilled in the art.

Examples of a suitable solvent used in the hydrolysis reaction include: water; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethyl ether; and amides, such as formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphoric triamide. Among these solvents, water or dioxane are preferred.

The reagent used is suitably water. If water is used as a solvent, then there is no need to add additional water. In addition to this, a base may be added to accelerate the reaction. Examples of such a base include: inorganic bases such as alkali metal carbonates (e.g. sodium carbonate, potassium carbonate and lithium carbonate), alkali metal hydrogen carbonates (e.g. sodium hydrogen carbonate, potassium hydrogen carbonate and lithium hydrogen carbonate), alkali metal hydrides (e.g. lithium hydride , sodium hydride and potassium hydride), alkali metal hydroxides (eg, sodium hydroxide, potassium hydroxide, barium hydroxide and lithium hydroxide), alkali metal fluorides (eg, sodium fluoride and potassium fluoride); and organic bases such as alkali metal alkoxides (e.g. sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium tert-butoxide and lithium methoxide), N-methylmorpholine, triethylamine, tripropylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methylpiperidine , pyridine, 4-pyrrolidinopyridine, picoline, 4- (N, N-dimethylamino) pyridine, 2,6-di (tert-butyl) -4-methylpyridine, quinoline, N, N-dimethylaniline, N, N-diethylaniline, 1 , 5-diazabicyclo [4.3.0] non-5-ene (DBN), 1,4-diazabicyclo [2.2.2] octane (DABCO) and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU ) Among these bases, organic bases are preferred, and pyridine is most preferred. In this regard, it should be noted that when the hydrolysis reaction proceeds satisfactorily in the absence of a base, there is no need to add a base.

The reaction temperature varies depending on the starting compound and the reagent, but usually it is in the range from 0 to 100 ° C, and preferably in the range from 20 to 60 ° C.

The reaction time varies depending on the reaction temperature, the starting compound, the reagent and the type of solvent used, but usually it is in the range from 10 minutes to 3 days, and preferably in the range from 6 hours to 24 hours.

After completion of the reaction, the desired hydrolysis reaction compound can be isolated from the reaction mixture using a conventional method known to one skilled in the art. For example, the resulting reaction mixture can be condensed using an ultrafiltration membrane, and then freeze-dried to obtain the desired reaction compound. Alternatively, the desired compound can be used in the form of a solution, without isolation, when it will need to be used to modify the proteins.

Thus, the desired compound obtained, if necessary, can be further purified using conventional techniques, such as, for example, gel filtration chromatography. To obtain a compound characterized by a particular desired average degree of polymerization or the desired molecular weight, the purified compound can be further fractionated using gel filtration chromatography.

In the present invention, “ammonolysis” means a ring opening reaction for a carboxylic acid anhydride unit described by formula (III) under the action of ammonia to give a unit described by formula (II), where R ³ represents an amino group. There are no particular restrictions on the nature of the actual ammonolysis reaction as long as it is a method commonly used by those skilled in the art.

Examples of a solvent suitable for use include: water; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethyl ether; and amides, such as formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphoric triamide. Among these solvents, water or dioxane are preferred. It should be noted that the reagent used can also be used as a solvent.

Examples of suitable reagents include gaseous ammonia and aqueous ammonia solution, and ammonia is preferred.

The reaction temperature varies depending on the starting compound and reagent, but usually it is in the range from 0 to 100 ° C, and preferably in the range from 10 to 40 ° C.

The reaction time varies depending on the reaction temperature, the starting compound, the reagent and the type of solvent used, but usually it is in the range from 10 minutes to 3 days, and preferably in the range from 6 hours to 24 hours.

After completion of the reaction, the desired ammonolysis reaction compound can be isolated from the reaction mixture using a conventional method known to one skilled in the art. To isolate the desired compound, the resulting reaction mixture can, for example, be processed as follows: (1) dialysis through a semipermeable membrane using an acid such as an aqueous solution of acetic acid to remove excess ammonia (dialysis is carried out under conditions under which the solution of the reaction mixture does not become acidic, when water is added as needed), followed by condensation using an ultrafiltration membrane, and then by freeze drying the condensate obtained; or (2) adding an aqueous solution of sodium hydroxide and an immiscible organic solvent such as diethyl ether, shaking the resulting mixture (shaking can be carried out two or more than two times as needed), and then an isolated aqueous layer containing the target compound, subjected to freeze drying to obtain the target compound. Alternatively, the desired compound can be used in the form of a solution, without isolation, when it will need to be used to modify the proteins.

Thus, the desired compound obtained, if necessary, can be further purified using conventional techniques, such as, for example, gel filtration chromatography. To obtain a compound characterized by a particular desired average degree of polymerization or the desired molecular weight, the purified compound can be further fractionated using gel filtration chromatography.

It should be noted that the target compound of the ammonolysis reaction may contain structural elementary units described by formula (II), where R ³ represents a hydroxy group, as a result of hydrolysis of some of the elementary units described by formula (III) in the starting compound under the action of water present in a solvent or reagent. It should also be noted that to improve storage, a base can be added to the target compound.

In the present invention, “aminolysis” means a ring-opening reaction for a carboxylic acid anhydride unit described by formula (III) under the action of an amine to give a unit described by formula (II), where R ³ is a group described by formula —NR ⁴ R ⁵ where R ⁴ and R ⁵ are as defined above. There are no particular restrictions on the nature of the actual aminolysis reaction as long as it is a method commonly used by those skilled in the art.

Examples of a solvent suitable for use include: water; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane and diethylene glycol dimethyl ether; and amides, such as formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphoric triamide. Among these solvents, water or dioxane are preferred. It should be noted that the reagent used can also be used as a solvent.

The reagent used obviously depends on the nature of the target group described by the formula —NR ⁴ R ⁵ . Commonly used amines include methylamine, dimethylamine, ethylamine, diethylamine, 2-hydroxyethylamine, di-2-hydroxyethylamine, n-propylamine, di-n-propylamine, isopropylamine, diisopropylamine, 1-amino-2-propanol and 2-hydroxyisopropylamine and their aqueous solutions. Among these reagents, preferred reagents include an aqueous solution of dimethylamine and 1-amino-2-propanol; most preferred is an aqueous solution of dimethylamine.

The reaction temperature varies depending on the starting compound and reagent, but usually it is in the range from 0 to 100 ° C, and preferably in the range from 10 to 40 ° C.

The reaction time varies depending on the reaction temperature, the starting compound, the reagent and the type of solvent used, but usually it is in the range of 10 minutes to 3 days, and preferably in the range of 6 hours to 36 hours.

After completion of the reaction, the desired aminolysis reaction compound can be isolated from the reaction mixture using a conventional method known to one skilled in the art. To isolate the desired compound, the resulting reaction mixture can, for example, be processed as follows: (1) dialysis through a semipermeable membrane using an acid such as an aqueous solution of acetic acid to remove excess ammonia (dialysis is carried out under conditions under which the solution of the reaction mixture does not become acidic, when water is added as needed), followed by condensation using an ultrafiltration membrane, and then freeze-drying BrAZ resultant condensate; or (2) adding an aqueous solution of sodium hydroxide and an immiscible organic solvent such as diethyl ether, shaking the resulting mixture (shaking can be carried out two or more than two times as needed), and then an isolated aqueous layer containing the target compound, subjected to freeze drying to obtain the target compound. Alternatively, the desired compound can be used in the form of a solution, without isolation, when it will need to be used to modify the proteins.

Thus, the desired compound obtained, if necessary, can be further purified using conventional techniques, such as, for example, gel filtration chromatography. To obtain a compound characterized by a particular desired average degree of polymerization or the desired molecular weight, the purified compound can be further fractionated using gel filtration chromatography.

It should be noted that the target compound of the aminolysis reaction may contain structural units described by formula (II), where R ³ is a hydroxy group, as a result of hydrolysis of some of the elementary units described by formula (III) in the starting compound under the action of water, present in a solvent or reagent. It should also be noted that to improve storage, a base can be added to the target compound.

In the present invention, “alcoholysis” means a ring opening reaction for a carboxylic acid anhydride unit described by formula (III) under the action of an alcohol or an aryl alcohol to produce a unit described by formula (II), wherein R ³ is an alkoxy or aryloxy group defined above. There are no particular restrictions on the nature of the actual reaction of alcoholysis as long as we are talking about the method commonly used by specialists in the relevant field.

Examples of the solvent used in the alcoholysis reaction include water; and amides, such as formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl-2-pyrrolidone, N-methylpyrrolidinone and hexamethylphosphoric triamide. Among these solvents, water is preferred. It should be noted that the reagent used can also be used as a solvent.

The reagent used obviously depends on the nature of the target alkoxy or aryloxy group. Typical examples of the alcohol used to produce the desired alkoxy group include methanol, ethanol, ethylene glycol, n-propanol, propylene glycol, 2-hydroxy-n-propanol and isopropanol and their aqueous solutions, while typical examples of the alcohol used to prepare desirable aryloxy groups include phenol and p-nitrophenol. Among these reagents, ethanol is preferred.

The reaction temperature varies depending on the starting compound and reagent, but usually it is in the range from 0 to 100 ° C, and preferably in the range from 10 to 40 ° C.

The reaction time varies depending on the reaction temperature, the starting compound, the reagent and the type of solvent used, but usually it is in the range of 10 minutes to 3 days, and preferably in the range of 6 hours to 36 hours.

After completion of the reaction, the desired compound of the alcoholysis reaction can be isolated from the reaction mixture using a conventional method known to one skilled in the art. For example, to remove excess alcohol, it is possible to condense the reaction mixture using an ultrafiltration membrane, add water, and then additionally condense the mixture two or more more than two times using an ultrafiltration membrane, and then the condensate obtained is subjected to freeze drying to obtain the target compound. Alternatively, the desired compound can be used in the form of a solution, without isolation, when it will need to be used to modify the proteins.

Thus, the desired compound obtained can, if necessary, be further purified using a conventional technique, such as, for example, using a gel filtration column. To obtain a compound characterized by a particular desired average degree of polymerization or the desired molecular weight, the purified compound can be further fractionated using gel filtration chromatography.

It should be noted that the target compound of the alcoholysis reaction may contain structural elementary units described by formula (II), where R ³ represents a hydroxy group, as a result of hydrolysis of some of the elementary units described by formula (III) in the starting compound under the action of water, present in a solvent or reagent. It should also be noted that to improve storage, a base can be added to the target compound.

Then, when a pharmacologically acceptable salt is a desired copolymer of the present invention, there is no particular limitation on the method for producing said salt as long as it is a method commonly used by those skilled in the art. For example, a pharmacologically acceptable salt can be obtained by dissolving the copolymer of the invention in an organic solvent, adding a base to the thus obtained solution, and then collecting the resulting salt, which precipitates.

To control the average molecular weight, the proportion of the alternating configuration and the ratio specified by the composition, a wide range of techniques can be used for the copolymers of the present invention.

It is well known that maleic anhydride monomer tends to alternate polymerization with comonomers, including polyoxyethylene allyl methyl diester [for example, see Comprehensive Polymer Science Vol. 4, Chain polymerization Part II, Ed. by G. C. Eastmond et al., pp. 377-422, Pergamon Press (1989); T. Yoshimoto et al., Biochemical Biophysical Research Communication Vol. 148, 876-882 (1987)]. The average molecular weight and polymer configuration can be adjusted by a person skilled in the art (for example, see T. Ohtsu and M. Kinoshita, Koubunshi Gousei no Jikkenhou pp. 125-154, Kagaku-Dojin 1972). In addition, despite the fact that maleic anhydride tends to polymerize alternating with comonomers, specialists in the relevant field are well aware of the possibility that, in the ratio specified by the composition, the fraction formed in the copolymer by the elementary units of maleic anhydride will exceed 50 % (mol.) (Japanese patents No. 2621308, No. 2701295, No. 2803265, No. 3271265, No. 3035675 and No. 3106265 and publication of Japanese patent applications No. 2003-105003 and No. 2003-105040).

In the General case, to obtain polymers with a high molecular weight and a greater proportion of the configuration with alternating polymerization, it is necessary to carry out under mild conditions, such as low temperature and low concentration of initiator. An increased concentration of monomer and a lower concentration of solvent, which reduces the relative concentration of the initiator, also result in an increased molecular weight and an increased proportion of the alternating configuration.

Alternatively, to obtain polymers with a reduced molecular weight and a reduced proportion of the alternating configuration, elevated temperature, increased initiator concentration, reduced monomer concentration and increased solvent concentration are favorable. In addition, under such relatively severe polymerization conditions and / or in the presence of maleic anhydride in an amount exceeding 50% (mol.) Of the total amount of monomers in the monomer mixture, in the ratio specified by the composition, the resulting copolymer has a fraction of elementary units of maleic anhydride may exceed 50% (mol.).

Some examples of polymerization conditions for polyoxyethylene allyl methyl diester and maleic anhydride are described in T. Yoshimoto et al., Biochemical Biophysical Research Communication Vol. 148, 876-882 (1987). In accordance with this article, polyoxyethylene allyl methyl diester, maleic anhydride, toluene and benzoyl peroxide (initiator) were mixed and polymerization was carried out under reflux at 80 ° C for 7 hours. The resulting polymer was characterized by a molecular weight of 13 kDa and had 8 PEG chains and 8 elementary units of maleic anhydride. The alternating configuration for the copolymer was proposed on the basis that the copolymer had a ratio defined by a composition of 1: 1, and that maleic anhydride monomer tended to copolymerize with alternating comonomers. If a copolymer having a reduced molecular weight and a reduced proportion of an alternating configuration is required, then an increased initiator concentration, a reduced monomer concentration and / or an increased solvent concentration will need to be used. For example, if toluene is used as a solvent in the polymerization, and the polymerization is carried out at 1 atmosphere, then the polymerization can be carried out at a temperature up to its boiling point of 110 ° C. In addition, in the case of another solvent whose boiling point exceeds the boiling point of toluene, an elevated temperature is preferred. In such cases, it will be appropriate to select the type of initiator, since each initiator has its own specific decomposition rate constant, and at certain elevated temperatures certain initiators will decompose. Numerous initiators with different degradation rates are already known in the art [for example, see Polymer Handbook, Third Edition, pp. II / 1-II / 65, Ed. by J. Brandrup and E. Immergut, John Wiley & Sons (1989)].

Other prior art documents, such as Japanese Patent Application Publication No. 2003-105003, also describe alternative solvents, initiators and reaction conditions resulting in a change in the ratio given by the composition, molecular weight and proportion of the configuration with alternating polyoxyethylene allyl methyl diester in copolymerization reactions and maleic anhydride. Xylene, for example, has a higher boiling point at 1 atmosphere (140 ° C) compared to toluene. It is also known that in some cases, the copolymerization reaction can be carried out without the use of any solvent, which eliminates the limitation on the polymerization temperature, determined by the boiling point of the solvent. Various alternative initiators, such as benzoyl peroxide, di-tert-butyl peroxide and tert-butyl peroxyisobutyrate and azo initiators such as azobisisobutyronitrile, are also described. It is also possible to reduce the average molecular weight of the copolymer as a result of using chain transfer reagents.

If in the ratio specified by the composition, for the copolymer, the proportion of elementary units of maleic anhydride in the total number of monomeric elementary units, exceeding 50%, is required, then in the monomer mixture it will be necessary to use a higher percentage of maleic anhydride monomer with or without the use of such relatively hard polymerization conditions.

When a pharmaceutically active ingredient is prepared using the copolymer of the present invention or its pharmacologically acceptable salt and protein or its analogue or variant, there are no special restrictions on the ratio between the copolymer and the protein or its analogue or variant until the resulting complex is characterized by the desired pharmaceutical activity. Typically, the weight ratio between the copolymer of the present invention or its pharmacologically acceptable salt and protein or its analogue or variant is in the range from 0.01 to 100: 1; preferably it is in the range from 0.1 to 50: 1; even more preferably, it is in the range from 1 to 10: 1; and most preferably it is in the range from 1 to 1.5: 1.

In practical use, a pharmaceutically active ingredient containing a copolymer of the present invention or a pharmacologically acceptable salt thereof and a protein or an analogue or variant thereof is usually prepared as a solution of a protein containing a copolymer or freeze-dried solution containing a copolymer of the present invention or a pharmacologically acceptable salt thereof and protein or its analogue or variant. In the latter case, the freeze-dried form of the active ingredient is dissolved immediately before it needs to be used. Alternatively, the pharmaceutically active ingredient may be prepared in kit form. In this case, the copolymer of the present invention or its pharmacologically acceptable salt and protein or its analogue or variant is stored in various containers, and then mixed to obtain the desired pharmaceutically active ingredient immediately before use. Among the various forms, a freeze-dried form is most preferred.

Then, when the copolymer of the present invention or its pharmacologically acceptable salt is used as a protein modifier, no particular restrictions are imposed in the form of any specific value on the ratio between the copolymer or its pharmacologically acceptable salt or protein or its analogue or variant until achieve the desired protein modification. Typically, the weight ratio between the copolymer of the present invention or its pharmacologically acceptable salt and protein or its analogue or variant is in the range from 0.01 to 100: 1; preferably it is in the range from 0.1 to 50: 1; even more preferably, it is in the range from 1 to 10: 1; and most preferably it is in the range from 1 to 1.5: 1.

Then, when the copolymer of the present invention is used as a protein modifier, there are no particular restrictions on the method for modifying a protein as long as it is a method commonly used for modifying a protein. For example, a protein or its analogue or variant can be modified as follows.

The solvent used in the modification method is an aqueous solution containing an electrolyte, which is usually used to dissolve the protein, and its pH value in the form of a specific value is not imposed until it falls into the range in which the modifier the protein can be negatively charged as a result of the dissociation of at least some carboxyl groups in the protein modifier of the present invention, and the protein or its analogue or variant can be positively charged . For example, the pH value can be set to a value in the range from 3 to the isoelectric point of the protein or its analogue or variant, and preferably it is set to the value in the range from 4 to 8. In this regard, it should be noted that the isoelectric point of the protein or its analogue or variant can be determined using electrophoresis.

The protein modifier is dissolved in the solvent described above, and then the solution thus obtained is added to an aqueous solution of the specified protein or its analogue or variant. If necessary, the resulting solution can be shaken to facilitate the reaction. If desired, the pH of the resulting mixture can be adjusted by adding acid and / or base. The ratio between the protein modifier of the present invention used and said protein or its analogue or variant of restrictions in the form of any specific value is not superimposed as long as it leads to the achievement of the desired modification of the protein. Typically, the ratio is in the range from 0.01 to 100 (wt.); preferably in the range of 0.1 to 50; more preferably in the range of 1 to 10; and most preferably it is in the range from 1 to 1.5: 1.

The reaction temperature for the modification method varies depending on the compound and reagent used, but usually it is in the range from 0 to 100 ° C, preferably in the range from 4 to 40 ° C, and most preferably in the range from 30 to 40 ° C.

The reaction time varies depending on the reaction temperature, the compound used, the reagent and the type of solvent used, but usually it is in the range of 5 minutes to 14 days, preferably in the range of 1 hour to 12 days, and more preferably in the range of 5 days to 10 days.

Sometimes during the process of modifying the protein, the conditions used are such that some structural elementary units described by formula (II) in the copolymer turn into elementary units described by formula (II) having a different essence. For example, when R ³ in the structural units described by formula (II) is a group other than a hydroxy group, the conditions used for the modification method are such that some of these R ³ groups are hydrolyzed to give structural units the units described by formula (II), where R ³ represents a hydroxy group.

As noted above, the complexes of the present invention contain at least one protein or its analogue or variant that bind to at least one copolymer of the present invention or its pharmacologically acceptable salt. In this complex, a protein or an analogue or variant thereof and a copolymer or a pharmacologically acceptable salt thereof are linked to each other via a chemical bond, such as a covalent bond (e.g., Schiff base formation, amide bond formation and ester bond formation), ionic bond or coordination bond, or via non-chemical bonds, such as hydrophobic interactions, hydrogen bonds, electrostatic interactions or affinity binding.

Preferably, size exclusion chromatography or SDS-PAGE under non-reducing conditions does not substantially reveal the complexes of the present invention in dissociated form. More preferably, the complexes of the present invention in dissociated form are not found to any significant extent by size exclusion chromatography and SDS-PAGE under non-reducing conditions. In addition, it is even more preferable that the degradation rate, which allows detecting the protein of the complex by ELISA (enzyme-linked immunosorbent assay) for the complexes of the present invention obtained by modifying the protein structure with copolymers and their pharmacologically acceptable salts, is very low (preferably, the degradation rate is 20% or less, more preferably 15% or less, and most preferably 10% or less).

In the present invention, a protein analogue is defined as a protein encoded by a cDNA molecule cloned from a cDNA library obtained from animal cells, body fluids, or tissues as a result of hybridization using protein cDNA under stringent conditions (60 to 70 ° C., 6 × SSC).

In the present invention, a protein variant is defined as a protein that is obtained by substituting, removing, adding or inserting one or more amino acids into the original protein, and which still detects at least a portion of the activity of the original protein at a detectable level.

Preferably, the protein or its analogue or variant is a basic protein. More preferably, the basic protein is basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet-derived growth factor (PDGF), brain derived neurotrophic factor (BDNF), nerve growth factor (NGF) , human growth hormone (HGH), hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF), or an analog or variant thereof. Most preferably, the basic protein is an osteoclastogenesis inhibition factor (OCIF) or an analogue or variant thereof.

OCIF, an analogue thereof, or a variant thereof used in the present invention may be of the natural type, or they may be of the recombinant type, and there are no particular restrictions on their origin. Natural-type OCIF means OCIF, which is obtained as a naturally-formed protein by extraction, purification and / or isolation from an organ, body fluid, cell culture or cell medium obtained from a human or non-human animal. Recombinant type OCIF, an analogue thereof, or a variant thereof is a recombinant protein obtained by extraction, purification and / or isolation of the indicated protein from the host body, commonly used in techniques such as a prokaryotic host cell (e.g. Escherichia coli ) or eukaryotic a cell, such as a cell line from a human body or non-human body, that changes under the action of a vector containing a polynucleotide that encodes OCIF, its analogue or variant thereof [eg, see recombin ntnye methods described in EP-A-0816380 (WO-A-96/26217) and WO-A-97/23614].

There are no particular restrictions on the origin of OCIF, its analogues and its variants used in the present invention, and they can be obtained from a human or non-human animal body. Preferably, they can be obtained from a mammalian organism, such as a human, rat, mouse, rabbit, dog, cat, cow, pig, sheep or goat; or birds, such as chicken, goose, chicken or turkey. More preferably, they are obtained from mammalian organisms, and most preferably, they are obtained from the human body.

OCIF or an analogue thereof used in the present invention may be of the Monomeric type OCIF (for example, in humans, the monomer has a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions) or the dimer type (for example, in humans, the dimer is molecular mass approximately equal to 120,000 according to measurements by the SDS-PAGE method in non-reducing conditions) [see EP-A-0816380 (WO-A-96/26217)]. Preferably, it is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, and more preferably is a human OCIF dimer characterized by a molecular weight of approximately 120,000 as measured by SDS-PAGE under non-reducing conditions.

It is known that OCIF is translated into cells in the form of a polypeptide containing a signal peptide at its amine end, and that after it undergoes maturation as a result of processing, including removal of the specified signal peptide, [for example, see recombinant methods described in EP-A-0816380 (WO-A-96/26217) and WO-A-97/23614]. OCIF, an analogue thereof, or a variant thereof, used in the present invention includes both a polypeptide containing a signal peptide and its mature form. Preferred examples include a human OCIF containing a signal peptide comprising amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 of the sequence listing, and the mature form of a human OCIF that does not contain a signal peptide comprising amino acids +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences. Among these compounds, mature OCIF is particularly preferred.

It is also known that methionine can be added to such a mature form of OCIF, an analogue or variant thereof if the form is expressed as a recombinant protein in a host cell, especially in a prokaryotic host cell, such as Escherichia coli . This is achieved by adding a nucleotide triplet including an ATG (AUG) sequence to the end of the 5 ′ polynucleotide encoding the mature form of OCIF, an analogue or variant thereof, and inserting the resulting polynucleotide into a suitable expression vector. The desired mature protein having methionine at its amine end can then be expressed using a suitable host cell that has been altered by the indicated recombinant expression vector. In addition, one or more amino acids can be added to the specified protein at a position adjacent to the methionine at the amine end, as a result of the addition of additional nucleotide triplets adjacent to the ATG triplet added at the end of the 5 'polynucleotide encoding the mature form of OCIF, its analogue or its variant. The desired mature form of OCIF, its analogue or variant thereof, containing methionine at the amine end, can be purified and isolated from the culture of the altered host cell in accordance with a commonly used method. In addition, one or more amino acids can be inserted into the mature form of OCIF containing methionine at the amine end, its analogue or variant thereof in a position adjacent to the methionine and closer to the carboxy terminus compared to methionine.

In the present invention, an OCIF analogue denotes a protein encoded by a polynucleotide that exists in vivo in cells, body fluids and / or organs of a human or non-human animal, such as those exemplified above. Specific preferred examples of such analogs include OCIF2, OCIF3, OCIF4 and OCIF5 [see EP-A-0816380 (WO96 / 26217)]. Such OCIF analogs or their active fragments can be prepared by a method such as the following: RNA is extracted from a cell, organ, tissue or fluid from a human or non-human animal; using reverse transcriptase, the first cDNA strand is synthesized that is complementary to the indicated RNA, and then, using the first strand as a template, the second strand of the indicated cDNA is synthesized using DNA polymerase; the cDNA double chain thus obtained is inserted into a suitable commonly used expression vector; a suitable commonly used host cell is then altered by the action of the vector thus obtained; the host generating the desired peptide is then selected to use a hybridization technique, such as platelet hybridization or phage hybridization, using OCIF cDNA or a fragment thereof as a probe under stringent conditions [see EP-A-0816380 (WO-A-96/26217)]; and then, in conclusion, the desired OCIF analogue is expressed by a commonly used technique using the thus obtained host cell.

In the present invention, an OCIF variant denotes a protein that contains an amino acid sequence where, in the amino acid sequence of OCIF or its analogue, one or more amino acid residues are substituted, removed, added or inserted, and which still has at least some OCIF activity. Such OCIF variants can be obtained, for example, by the following method: substitution, deletion, addition and / or insertion of one nucleotide or several nucleotides in the nucleotide sequence encoding OCIF or its analogue using the polymerase chain reaction method (hereinafter, referred to as as PCR), a genetic recombination method, or a nuclease cleavage method using an exonuclease or endonuclease, such as a restriction enzyme; transforming a eukaryotic host cell, such as an animal cell, or a prokaryotic host cell, such as Escherichia coli , by the expression vector, where the resulting nucleotide encoding the desired OCIF variant is inserted; and then extraction, purification and / or isolation of the desired peptide from the protein-containing fraction obtained using the cell culture of the indicated transformed host in accordance with a method well known to those skilled in the art.

Processed forms of OCIF, in which a significant portion of the amino acid sequence is removed from the carboxy terminus of the OCIF polypeptide, are also known to retain at least some degree of OCIF activity [see, for example, EP-A-0816380 (WO-A- 96/26217) and WO-A-97/23614]. Such processed OCIF types retaining at least some activity of the complete OCIF polypeptide are also included in the OCIF variants of the present invention.

In addition, OCIF or its processed form, which is subjected to fusion with an immunoglobulin domain, such as an Fc domain (for example, a fusion polypeptide in which the Fc domain of human IgG is attached to the carboxy terminus of OCIF), and which retain at least some activity of the complete OCIF polypeptide is known (see WO-A-97/23614), and such fusion proteins are also included in the OCIF variants of the present invention.

Any naturally-formed OCIF or an analogue thereof, or a recombinant OCIF or an analogue or variant thereof may contain a sugar chain that is attached to the OCIF or its analogue or variant in the post-translational step. The naturally-formed OCIF or an analogue thereof containing a sugar chain can be obtained from cell cultures, tissues, organs, body fluids, or cell lines obtained from a human body or non-human animals using conventional techniques. Recombinant OCIF or an analogue or variant thereof containing a sugar chain can be obtained from a culture of a eukaryotic host cell transformed using a vector containing a nucleotide sequence encoding any OCIF or its analogue or variant, such as those described and exemplified. above. Examples of suitable host cells that can be used and which are capable of post-translationally modifying OCIF or an analogue or sugar chain attachment variant thereof include Chinese hamster ovary cells and COS cells [Yasuda, H. et al., Endocrinology, 139, 1329-1337 (1998)]. OCIF or an analogue or variant thereof containing such a sugar chain is suitable for use in the preparation of the complexes of the present invention. The sugar chain in OCIF or an analogue or variant thereof containing a sugar chain can be artificially modified (in particular by enzymes) using polymers, polysaccharides or modified polysaccharides.

The pharmaceutical composition of the present invention, which contains the copolymer of the present invention or its pharmacologically acceptable salt and protein or its analogue, can be a solution obtained in accordance with the method described above, or a composition obtained by freeze-drying the solution. Alternatively, such a pharmaceutical composition may be formulated in accordance with an alternative method, or it may be in the form of a kit. In the latter case, the copolymer of the present invention or its pharmacologically acceptable salt and protein or its analogue or variant is stored in various containers, and then mixed to obtain the desired pharmaceutical composition immediately before use.

The pharmaceutical compositions of the present invention optionally may further comprise a base. No restrictions are imposed on the base in the form of any specific base as long as it is the base commonly used in pharmaceutical compositions. Examples of such a base include: inorganic bases such as alkali metal carbonates (e.g. sodium carbonate, potassium carbonate and lithium carbonate), alkali metal hydrogen carbonates (e.g. sodium hydrogen carbonate, potassium hydrogen carbonate and lithium hydrogen carbonate), alkali metal hydrides (e.g. lithium hydride , sodium hydride and potassium hydride), alkali metal hydroxides (eg, sodium hydroxide, potassium hydroxide, barium hydroxide and lithium hydroxide), alkali metal fluorides (eg, sodium fluoride and potassium fluoride); and organic bases such as alkali metal alkoxides (e.g. sodium methoxide, sodium ethoxide, potassium methoxide, potassium ethoxide, potassium tert-butoxide and lithium methoxide), alkali metal mercaptans (e.g. sodium methyl mercaptan and sodium ethyl mercaptan), N-methylmorpholine, triethylamine, tripropylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinopyridine, picoline, 4- (N, N-dimethylamino) pyridine, 2,6-di (tert-butyl) -4-methylpyridine, quinine N, N-dimethylaniline, N, N-diethylaniline, 1,5-diazabicyclo [4.3.0] non-5 ene (DBN), 1,4-diazabicyclo [2.2.2] octane (DABCO) and 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU). Among these bases, alkali metal hydroxides are preferred, and sodium hydroxide is particularly preferred. In this regard, it should be noted that part or all of the bases may form salts with the copolymer of the present invention.

Examples of the pharmaceutical composition of the present invention include a complex between at least one substance selected from a copolymer of the present invention or a pharmacologically acceptable salt thereof, and a protein or its analogue or variant selected from bFGF (used to treat or prevent ischemic arteriopathic disease and non-scarring skin ulcers), EGF (used to treat or prevent peptic ulcer diseases, such as ulcerative colitis and prolonged dysfunction of the corneal epithelium), PDGF (using for treating wounds), BDNF and NGF (used to treat or prevent central nervous system diseases such as Parkinson’s disease and Alzheimer's disease), HGH (used to treat or prevent growth hormone deficiency, growth hormone secretion syndrome, Turner syndrome and cartilage dystrophy), HGF (used to treat or prevent diabetes mellitus, arteriosclerosis, such as cerebral infarction, and liver fibrosis) and VEGF (ischemic arteriopathic disease and peripheral occlusive lesion) x arteries).

One particularly preferred example of a pharmaceutical composition of the present invention comprises a complex between at least one substance selected from OCIF, its analogue and variant, and at least one substance selected from a copolymer of the present invention or a pharmacologically acceptable thereof salt. Such a pharmaceutical composition is particularly suitable for the prophylaxis or treatment of metabolic disorders in the bones. In the present invention, such metabolic disorders in the bones include any diseases that are characterized by a significant decrease in bone mass in a patient suffering from them, and in which it is necessary to suppress bone resorption or the rate of bone resorption in order to prevent or treat these diseases.

Bone metabolic disorders that can be treated or prevented using the pharmaceutical composition of the present invention include: primary osteoporosis (senile osteoporosis, postmenopausal osteoporosis and idiopathic juvenile osteoporosis); endocrine osteoporosis (hyperthyroidism, hyperparathyroidism, hyperadrenocorticism and acromegaly); osteoporosis with concomitant hypogonadism (hypopituitarism, Klinefelter syndrome and Turner syndrome); hereditary and congenital osteoporosis (imperfect osteogenesis, homocystinuria, Menkes syndrome and familial autonomic dysfunction); osteopenia due to a decrease in the load from its own weight or fixation and immobilization of the limbs; deforming osteodystrophy; osteomyelitis; infectious lesion due to bone reconstruction; hypercalcemia that occurs as a consequence of solid cancer (breast cancer, lung cancer, kidney cancer, and prostate cancer); hematological malignant diseases (multiple myeloma, lymphoma and leukemia); idiopathic hypercalcemia; hypercalcemia with concomitant hyperthyroidism or impaired renal function; osteopenia arising as a consequence of treatment with steroid drugs; osteopenia that occurs as a consequence of taking other drugs (for example, immunosuppressive drugs such as methotrexate and cyclosporin A, heparin and antiepileptic drugs); osteopenia with concomitant renal impairment; osteopenia associated with surgery or digestive diseases (for example, small bowel obstruction, large bowel obstruction, chronic hepatitis, gastric resection, primary biliary cirrhosis and cirrhosis); osteopenia due to various types of rheumatism, such as rheumatoid arthritis; osteoclasia and joint destruction caused by various types of rheumatism, such as rheumatoid arthritis; rheumatoid arthritis, a type of myelodysplastic trophopathy; osteoarthritis; Rareification of periodontal bone; bone cancer metastases (osteolytic metastases); bone necrosis or necrosis of bone cells associated with traumatic injury, Gaucher disease, sickle cell anemia, systemic erythematous lupus, or non-traumatic injury; osteodystrophy, such as nephrogenic osteodystrophy; osteopenia associated with hypophosphatasia or diabetes; osteopenia associated with alimentary dystrophy or malnutrition; and other osteopenia. In addition, cachexia due to the aforementioned solid cancer, bone cancer metastases (osteolytic metastases), or hematologic malignancies (see Japanese Patent Application (Kokai) No. 2000-178200) are also included in bone metabolism disorders of the present invention.

The pharmaceutical composition of the present invention described above, humans or animals other than humans, can safely be taken orally or non-orally. The dosage form of the pharmaceutical composition can be appropriately selected depending on the type of disease, the degree of disease and condition, age, gender and weight of the patient. For example, the pharmaceutical composition can be administered orally using the form of tablets, capsules, powders, granules or syrups; administered using an intravenous injection form, individually or in combination with a conventional adjuvant such as glucose, amino acids and the like, or using a separate form of intramuscular, subcutaneous, intradermal or intraperitoneal injection; administered transdermally using the form of a poultice; administered transnasally using the form of nasal drops; injected transmucosally or into the oral cavity using the form of a transmucosal preparation; or administered intrarectally using the suppository form. The compositions of these preparations can be formulated in the usual way using well-known adjuvants that are commonly used in the field of medicine, such as fillers, binders, disintegrants, lubricants, flavorings, solubilizers, suspending agents, colorants, pH adjusters, antiseptics, gelling agents, surface-sensitive active substances and substances for coating.

Then, when the pharmaceutical composition is obtained in the form of tablets, it is possible to use various carriers known in the art. Examples of such carriers include: fillers such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose and silicic acid; binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and polyvinylpyrrolidone; disintegrants such as dry starch, sodium alginate, powdered agar, powdered laminar, sodium bicarbonate, calcium carbonate, esters derived from polyoxyethylene sorbitan and fatty acids, sodium lauryl sulfate, stearic acid monoglyceride, starch and lactose; disintegration inhibitors such as sucrose, stearin, cocoa butter and hydrogenated oil; absorption accelerators such as quaternary ammonium bases and sodium lauryl sulfate; moisturizers such as glycerin and starch; absorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid; and lubricants, such as purified talc, stearates, powdered boric acid, and polyethylene glycol. In addition to this, if necessary, such a tablet can be coated in the form of a commonly used coating. Examples of such a coated tablet include a sugar coated tablet, a gelatin coated tablet, an enteric coated tablet, a film coated tablet, a two-layer tablet, and a multi-layer tablet.

Then, when the pharmaceutical composition is obtained in the form of a pill, it is possible to use various carriers known in the art. Examples of such carriers include: fillers such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, kaolin and talc; binders such as powdered gum arabic, powdered tragacanth, gelatin and ethanol; and disintegrants, such as laminaran based agar.

Then, when the pharmaceutical composition is obtained in the form of a suppository, it is possible to use various carriers known in the art. Examples of such carriers include polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and semi-synthetic glycerides.

Then, when the pharmaceutical composition is prepared as an injection, it is preferred that the composition in the form of a solution or suspension is sterilized and made isotonic with blood. Then, when such a composition is obtained in the form of a solution, emulsion, suspension or a substantially homogeneous aqueous solution, it is possible to use various diluents known in the art. Examples of such diluents include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and esters derived from polyoxyethylene sorbitan and fatty acids. In this case, the pharmaceutical composition may further comprise sodium chloride, glucose or glycerin in an amount sufficient to maintain isotonicity with respect to blood. In addition, the pharmaceutical composition may also contain commonly used solubilizers, buffers, emollients, pH regulators, stabilizers or solubilizing components. Such an injection can be freeze dried.

In addition to this, the pharmaceutical composition according to the invention, if necessary, may also contain colorants, preservatives, fragrances, flavorings, sweeteners or other medicinal product.

The amount of the complex between OCIF or its analogue or variant and the copolymer or its pharmacologically acceptable salt contained in the pharmaceutical composition of the present invention is not limited in the form of any specific value, but usually it is in the range from 0.1 to 70% ( wt.), and preferably in the range from 1 to 30% (wt.).

In the present invention, the dosage of the complex of OCIF or its analogue or variant with the copolymer or its pharmacologically acceptable salt contained in the pharmaceutical composition of the present invention depends on various factors, including the condition and age of the patient, the method of use and the form of the drug. In general, the amount administered to an adult at one time is in the range from the upper limit in the range of 1 to 50 mg / kg to the lower limit in the range of 0.001 to 0.1 mg / kg, where a range of 0.01 is preferred up to 1 mg / kg, and a range of from 0.1 to 1 mg / kg is more preferred.

The pharmaceutical composition of the present invention must be taken once every several months, once a month, once every several days, once a day, or several times a day, depending on the type of ingredient contained in the pharmaceutical composition, method of use and form medicinal product. Then, when the composition contains a complex of OCIF or its analogue or variant with a copolymer or its pharmacologically acceptable salt, intended for use in the treatment or prevention of metabolic disorders in the bones in accordance with the present invention, it must be taken once every few months, once a month, once every few days, once a day or several times a day, depending on the type of active ingredient contained in an agent intended for treatment or prevention metabolic disorders in the bones, method of use and form of the drug.

The copolymer of the present invention and its pharmacologically acceptable salts are suitable as a polymer modifier capable of producing a complex having homogeneous properties, in particular, reduced formation of disordered crosslinked structures with protein, better retention of protein activity and excellent retention of protein in the blood after administration of this complex. This makes it particularly suitable as a modifier for modifying the pharmaceutical properties of proteins having useful pharmaceutical activity.

Examples

The following examples, reference examples, and test examples are intended to further illustrate the present invention, and they are in no way intended to limit the scope of the invention. In the following examples, m and R ³ are the same as defined above for formulas (I) and (II), “rel. represents the ratio defined by the composition [that is, the ratio between the structural elementary units (I) and (II)], "cf. step. floor." represents the average degree of polymerization for the resulting copolymer, and "relative hydr." for the resulting copolymer, is the ratio given by hydrolysis [ie, the average ratio between the structural elementary units, where R ³ is —OH, and the structural elementary units, where R ³ differs from —OH].

Example 1

Obtaining hydrolyzate poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) g], where m = 6-16, R ³ = OH, resp. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 10: 0 (compound No. 1)

A copolymer of polyoxyethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ¹ = hydrogen, and R ² = methyl) and maleic anhydride, in which the polyoxyethylene side chain has an average molecular weight of approximately 500, and the average degree of polymerization for the main chain is in the range of 30 to 40 [AM-0530K manufactured by NOF Corporation (hereinafter referred to as “poly (PEG ₅₀₀ -MA)”)]. To 3 g of poly (PEG ₅₀₀ -MA) to dissolve the specified starting compound was added 50 ml of distilled water and the resulting solution was stirred at 40 ° C for 15 hours. The resulting solution was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (characterized by an ultimate molecular weight of 10000 manufactured by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 1.3 g of the desired compound poly (PEG ₅₀₀ -MA) g (compound No. 1) as an oily compound.

Poly (PEG ₅₀₀ -MA) g (compound No. 1) was purified using gel filtration as follows. 100 mg poly (PEG ₅₀₀ -MA) g (compound No. 1) was dissolved in 4 ml of 0.001 N. sodium hydroxide solution. The solution was divided into four batches and each batch of 1 ml was applied to a gel filtration column (PD-10 manufactured by Amersham-Pharmacia). The first 1 ml of eluent was discarded. After that, 1.5 ml of 0.001 N were applied to each column. sodium hydroxide solution and as a result of elution from the column, another 1.5 ml was obtained and discarded. Then, 2.5 ml of 0.001 N were applied to each column. a solution of sodium hydroxide and as a result of elution from the column, another 2.5 ml was obtained, and it was this fraction that contained the purified poly compound (PEG ₅₀₀ -MA) g (compound No. 1). The purified fractions from four columns were combined and received 10 ml of a purified solution of the target compound. The yield after the purification step (determined spectrophotometrically when measuring the optical density for poly (PEG ₅₀₀ -MA) g in the 210 nm region before and after purification) was fixed at 80% (80 mg of polymer), and the concentration in the purified solution was determined equal to 8 mg / ml

The level of carboxyl groups in the target compound was determined by conductometric titration as follows. 7.5 ml of a solution of the purified target compound obtained above (containing 60 mg of the target compound) was diluted to a volume of 50 ml using distilled water and the pH of the resulting solution was adjusted to 12 using a 1 M aqueous solution of sodium hydroxide. 0.1 M hydrochloric acid was added to the solution in 0.1 ml portions, and after each addition of hydrochloric acid, pH and conductivity of the solution were measured. Then, the level of carboxyl groups in the copolymer was calculated from the amount of 0.1 M hydrochloric acid added in the conductivity buffer region (i.e., in the region on the graphical dependence of the conductivity on the amount of added hydrochloric acid, in which the carboxyl groups of the copolymer of the target compound act as a buffer, wherein said region corresponds to a pH range of from about 10.5 to 3); the molar amount of hydrochloric acid used in the conduction buffer region is equal to the molar amount of carboxyl groups in the copolymer of the target compound. As a result, the level of carboxyl groups in 1 g of the target compound was determined to be 3.22 mmol. Hence, the ratio specified by the composition was calculated equal to 1: 1.07. This numerical value was used in the following examples, where the same starting compound was used.

Example 2

Obtaining hydrolyzate poly (PEG ₁₅₀₀ -MA) [poly (PEG ₁₅₀₀ -MA) g], where m = 28-38, R ³ = OH, resp. comp. = approximately 1: 1, cf. step. floor = 10-15, and rel. hydr. = approximately 10: 0 (compound No. 2)

A copolymer of polyoxyethylene allyl methyl diester (m = 28-38, Alk = ethylene, R ¹ = hydrogen, and R ² = methyl) and maleic anhydride, in which polyoxyethylene is characterized by an average molecular weight of approximately 1500 and an average degree, was used as the starting compound. polymerization for the main chain is in the range of 10-15 (AM-1510K manufactured by NOF Corporation) [hereinafter referred to as "poly (PEG ₁₅₀₀ -MA)"]. To 1.5 g of poly (PEG ₁₅₀₀ -MA), 25 ml of distilled water was added to dissolve the indicated starting compound, and the resulting solution was stirred at room temperature for 20 hours. At the end of this time period, the solution was condensed in the same manner as described in Example 1 above using an ultrafiltration membrane made of ether-based polyethersulfone (characterized by an ultimate molecular weight of 10,000 from Millipore Corporation, model number PBGC07610), and the resulting condensate was freeze-dried to obtain 0.8 g of the target compound poly (PEG ₁₅₀₀ -MA) g (compound No. 2) as an oily compound.

The level of carboxyl groups in the target compound poly (PEG ₁₅₀₀ -MA) g (compound No. 2) was measured in the same manner as described in Example 1 above, and as a result, the level of carboxyl groups in 1 g of the target compound was determined to be 1 63 mmol. Hence, the ratio specified by the composition was calculated equal to 1: 1.4. This numerical value was used in the following examples, where the same starting compound was used.

Example 3

Preparation of Ammonolysis Product Poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) a], where m = 6-16, R ³ = NH ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 0:10 (compound No. 3)

To 1 g of poly (PEG ₅₀₀ -MA) (AM-0530K manufactured by NOF Corporation) was added 9.5 g of an aqueous ammonia solution (ammonia concentration: 28% (w / w)) and thus obtained the solution was stirred at room temperature for 16 hours. At the end of this time period, the solution thus obtained was dialyzed through a regenerated cellulose membrane (characterized by an ultimate molecular weight in the range of 12000 to 14000, manufactured by Sanko Junyaku Co., Ltd., model number UC36-32-100) using for one day 1 liter of an aqueous solution of acetic acid with a concentration of 0.1% (wt.), and then it was further subjected to dialysis using 1 liter of water for 1 day. After updating 1 liter of water, dialysis was performed for an additional day. As a result of such dialysis, excess ammonia was removed from the product. The solution thus obtained was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (characterized by an ultimate molecular weight of 10,000 and manufactured by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 0, 99 g of the target compound poly (PEG ₅₀₀ -MA) a (compound No. 3) as an oily compound.

The level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) a (compound No. 3) was determined by conductometric titration in the same manner as described in Example 1 above, and as a result, the level of carboxyl groups in 1 g of the target compound determined equal to 1.55 mmol.

Under the reaction conditions used, it may turn out that the maleic anhydride residue will be able to undergo a ring opening reaction as a result of not only ammonolysis but also hydrolysis due to the presence of water in the reaction system. The ratio between residues of maleic anhydride subjected to ammonolysis and residues of maleic anhydride subjected to hydrolysis was calculated as follows.

In example 1, the level of carboxyl groups was determined in 1 g of poly (PEG ₅₀₀ -MA) g (compound No. 1). From this, a numerical value was obtained for the level of carboxyl groups for the case of complete hydrolysis of all maleic acid residues (3.22 mmol in 1 g of polymer). From this result, in the course of the calculations, the mass of poly (PEG ₅₀₀ -MA) g (compound No. 1) per mole of carboxyl group (1 / 3.22 × 10 ^-3 g) and the mass of poly (PEG ₅₀₀ -MA) g (compound No. 1) per mole of maleic acid residue obtained by opening the cycle [2 × mass of poly (PEG ₅₀₀ -MA) g (compound No. 1) per mole of maleic acid residue obtained by opening the cycle, since there are 2 carboxyl groups per one fully hydrolyzed maleic acid residue], resulting in 311 g and 621 g, respectively. From these values, we determined the mass of poly (PEG ₅₀₀ -MA) (i.e., the mass of the copolymer before hydrolysis) per gram of functional group and the mass of poly (PEG ₅₀₀ -MA) a (compound No. 3) obtained by adding to poly (PEG ₅₀₀ -MA) ammonia per gram of functional group. Specifically, the mass of poly (PEG ₅₀₀ -MA) (i.e., the mass of the copolymer before hydrolysis) per mole of maleic anhydride residue was obtained by subtracting the molecular weight of the water molecule (18 g) from the mass of the fully hydrolyzed copolymer, which resulted in a numerical value equal to 603 g. A mass of poly (PEG ₅₀₀ -MA) a (compound No. 3) per mole of carboxyl group was obtained by adding the molecular weight of an ammonia molecule (17 g) to the mass of poly (PEG ₅₀₀ -MA), which resulted in numerical value equal to 620 g. From this value in the calculation result (1 g / 620) determined the theoretical level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) a (compound No. 3), in which all residues of maleic anhydride were subjected to ammonolysis (i.e., in the absence of hydrolysis), and it was obtained equal to 1.61 mmol. From the level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) g (compound No. 1), in which all residues of maleic anhydride were hydrolyzed, the theoretical level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) a (compounds No. 3), in which all residues of maleic anhydride were subjected to ammonolysis, and the actual level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) a (compound No. 3), measured above (i.e., 1.55 mmol), was calculated the ratio between residues of maleic anhydride, subjected to ammonolysis in the target unity, and the total number of residues of maleic anhydride in the original compound and it was determined equal to 1.0. Thus, it was confirmed that essentially all residues of maleic anhydride were subjected to ammonolysis, and that virtually no hydrolysis occurred.

Example 4

Obtaining the reaction product between dimethylamine and poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) dma], where m = 6-16, R ³ = NMe ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 0:10 (compound No. 4)

To 10 g of poly (PEG ₅₀₀ -MA) (AM-0530K manufactured by NOF Corporation), 71 g of an aqueous solution of dimethylamine (dimethylamine concentration: 50% by weight) was added to dissolve it, and the resulting solution was stirred at room temperature within 20 hours. At the end of this time period, the solution thus obtained was dialyzed through a regenerated cellulose membrane (characterized by an ultimate molecular weight in the range of 12000 to 14000 and manufactured by Sanko Junyaku Co., Ltd., model number UC36-32-100) using within one day, 10 liters of an aqueous solution of acetic acid with a concentration of 0.1% (wt.), and then it was further subjected to dialysis using 10 liters of water for 1 day. After updating 10 liters of water, dialysis was performed for an additional day. As a result of such dialysis, excess dimethylamine was removed from the product. The solution thus obtained was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (having an ultimate molecular weight of 10000 and manufactured by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 6, 3 g of the target compound poly (PEG ₅₀₀ -MA) dma (compound No. 4) as an oily compound.

The level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) dma (compound No. 4) was determined in the same manner as described in the above Example 1, and it was determined to be 1.53 mmol. Using a calculation similar to that shown in Example 3 for the ammonolysis product, the theoretical level of carboxyl groups was established in 1 g of poly (PEG ₅₀₀ -MA) dma (compound No. 4), in which all residues of maleic anhydride were subjected to aminolysis, and it was determined equal to 1.54 mmol. From these results, the ratio between maleic anhydride residues subjected to aminolysis in the target compound and the total number of maleic anhydride residues in the starting compound was calculated and determined to be 1.0. Thus, it was confirmed that essentially all residues of maleic anhydride were subjected to aminolysis under the influence of dimethylamine, and that practically no hydrolysis occurred.

Example 5

Preparation of the reaction product between 1-amino-2-propanol and poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) ipa], where m = 6-16, R ³ = NH (CH ₂ CH (OH) CH ₃ ), resp. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 0:10 (compound No. 5)

To 1.5 g of poly (PEG ₅₀₀ -MA) (AM-0530K manufactured by NOF Corporation), 14 g of 1-amino-2-propanol were added to dissolve it, and the resulting solution was stirred at room temperature for 16 hours. At the end of this time period, 300 ml of distilled water was added to the solution, and glacial acetic acid was additionally added to neutralize the solution. The solution thus obtained was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (having an ultimate molecular weight of 10,000 and manufactured by Millipore Corporation, model number PBGC07610) to obtain 50 ml of condensate. 300 ml of distilled water was added to the condensate, and the resulting solution was again condensed in the same manner. This condensate dilution cycle using distilled water and subsequent re-condensation was repeated five times to eliminate excess 1-amino-2-propanol. The resulting condensate was subjected to freeze drying to obtain 1.3 g of the target compound poly (PEG ₅₀₀ -MA) ipa (compound No. 5) as an oily compound.

The level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) ipa (compound No. 5) was determined in the same manner as described in Example 1 above, and it was determined to be 1.55 mmol. Using a calculation similar to that shown in example 3 for the product of ammonolysis, the theoretical level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) ipa (compound No. 5) was established, in which all residues of maleic anhydride were subjected to aminolysis, and it was determined to be 1.47 mmol. From these results, the ratio between maleic anhydride residues subjected to aminolysis in the target compound and the total number of maleic anhydride residues in the starting compound was calculated and determined to be 1.0. Thus, it was confirmed that essentially all residues of maleic anhydride in the starting compound were subjected to aminolysis under the action of 1-amino-2-propanol, and that practically no hydrolysis occurred.

Example 6

The product of the alcoholysis reaction between ethanol and poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) ea], where m = 6-16, R ³ = OCH ₂ CH ₃ , rel. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 4: 6 (compound No. 6)

To 1.5 g of poly (PEG ₅₀₀ -MA) (AM-0530K manufactured by NOF Corporation), 25 g of absolute ethanol was added to dissolve it, and the resulting solution was stirred at room temperature for 16 hours. At the end of this time period, 300 ml of water was added and the resulting solution was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (characterized by an ultimate molecular weight of 10,000 and manufactured by Millipore Corporation, model number PBGC07610), obtaining 50 ml of condensate. 300 ml of distilled water was added to the condensate, and the resulting solution was again condensed in the same manner. This condensate dilution cycle using distilled water and subsequent re-condensation was repeated five times to eliminate excess ethanol. The resulting condensate was subjected to freeze drying to obtain 0.8 g of the target compound poly (PEG ₅₀₀ -MA) ea (compound No. 6) as an oily compound.

The level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) ea (compound No. 6) was determined in the same manner as described in the above Example 1, and it was determined to be 2.16 mmol. Using a calculation similar to that shown in Example 3 for the ammonolysis product, the theoretical level of carboxyl groups was established in 1 g of poly (PEG ₅₀₀ -MA) ea (compound No. 6), in which all residues of maleic anhydride were subjected to alcoholysis, and it was determined to be 1.47 mmol. From this result, the ratio between the residues of maleic anhydride, subjected to alcoholysis in the target compound, and the total number of residues of maleic anhydride in the starting compound was calculated and it was determined equal to 0.6. Thus, it can be seen that 60% of the maleic anhydride residues in the starting compound were subjected to alcoholysis, and the remaining 40% of the maleic anhydride residues were hydrolyzed.

Example 7

Preparation of Ammonolysis Product Poly (PEG ₁₅₀₀ -MA) [poly (PEG ₁₅₀₀ -MA) a], where m = 28-38, R ³ = NH ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 10-15, and rel. hydr. = approximately 4: 6 (compound No. 7)

To 1.5 g of poly (PEG ₁₅₀₀ -MA) (AM-1510K manufactured by NOF Corporation), 14.5 g of aqueous ammonia solution (ammonia concentration: 28% (wt.)) And thus obtained were added to dissolve it the solution was stirred at room temperature for 20 hours. At the end of this time period, 300 ml of distilled water was added to the solution, and glacial acetic acid was additionally added to neutralize this solution. The resulting solution was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (having an ultimate molecular weight of 10,000 and manufactured by Millipore Corporation, model number PBGC07610) to obtain 50 ml of condensate. 300 ml of distilled water was added to the condensate, and the resulting solution was again condensed in the same manner. This condensate dilution cycle using distilled water and subsequent re-condensation was repeated five times to eliminate excess ammonia. The resulting condensate was subjected to freeze drying to obtain 0.7 g of the target compound poly (PEG ₁₅₀₀ -MA) a (compound No. 7) as an oily compound.

The content of carboxyl groups in 1 g of poly (PEG ₁₅₀₀ -MA) a was determined in the same manner as that described in Example 1, and determined to be 1.12 mM. As in the case of poly (PEG ₅₀₀ -MA) ammonolysis from Example 3, it may turn out that the residues of maleic anhydride in the starting poly (PEG ₁₅₀₀ -MA) compound will be able to undergo both ammonolysis and hydrolysis. Therefore, the proportion of residues of maleic anhydride subjected to ammonolysis and the proportion of residues of maleic anhydride subjected to hydrolysis were calculated as follows.

In example 2, the level of carboxyl groups was determined in 1 g of poly (PEG ₁₅₀₀ -MA) g (compound No. 2). From this, a numerical value was obtained for the level of carboxyl groups for the case of complete hydrolysis of all maleic acid residues (1.63 mmol in 1 g of polymer). From this result, we calculated the mass of poly (PEG ₁₅₀₀ -MA) g (compound No. 2) per mole of carboxyl group (1 / 1.63 × 10 ^-3 g) and the mass of poly (PEG ₁₅₀₀ -MA) g (compound No. 2) per mole of maleic acid residue obtained by opening the cycle [2 × mass of poly (PEG ₁₅₀₀ -MA) g (compound No. 2) per mole of maleic acid residue obtained by opening the cycle, since there are 2 carboxylic groups per complete hydrolysis maleic acid residue], resulting in 613 g and 1227 g, respectively. From the mass of poly (PEG ₁₅₀₀ -MA) g thus obtained, and using an approach similar to that in Example 3 above, the theoretical level of carboxyl groups in 1 g of poly (PEG ₁₅₀₀ -MA) a (compound No. 7) was calculated in in which all residues of maleic anhydride were subjected to ammonolysis, and it was obtained equal to 0.82 mmol. From the data of the results and the actual level of carboxyl groups in poly (PEG ₁₅₀₀ -MA) a (compound No. 7) measured above (1.12 mmol), the ratio between the residues of maleic anhydride subjected to ammonolysis in the target compound was established as a result of calculations, and the total number of residues of maleic anhydride in the starting compound and it was determined equal to 0.6. Thus, it was confirmed that 60% of maleic anhydride residues in the starting poly compound (PEG ₁₅₀₀ -MA) were subjected to ammonolysis, and the remaining 40% of maleic anhydride residues were hydrolyzed.

Example 8

Obtaining the reaction product between dimethylamine and poly (PEG ₁₅₀₀ -MA) [poly (PEG ₁₅₀₀ -MA) dma], where m = 28-38, R ³ = NMe ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 10-15, and rel. hydr. = approximately 0:10 (compound No. 8)

To 1 g of poly (PEG ₁₅₀₀ -MA) (AM-1510K manufactured by NOF Corporation), 11 g of an aqueous solution of dimethylamine (characterized by a concentration of 50% (wt.)) Was added to dissolve it, and the resulting solution was stirred at room temperature in within 20 hours. At the end of this time period, 300 ml of distilled water was added to the solution, and glacial acetic acid was additionally added to neutralize the solution. The resulting solution was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (having an ultimate molecular weight of 10,000 and manufactured by Millipore Corporation, model number PBGC07610) to obtain 50 ml of condensate. 300 ml of distilled water was added to the condensate, and the resulting solution was again condensed in the same manner. This condensate dilution cycle using distilled water and subsequent re-condensation was repeated five times to eliminate excess dimethylamine. The resulting condensate was subjected to freeze drying to obtain the target compound poly (PEG ₁₅₀₀ -MA) dma (compound No. 8) as an oily compound.

The level of carboxyl groups in 1 g of poly (PEG ₁₅₀₀ -MA) dma (compound No. 8) was determined in the same manner as described in example 1, and it was calculated equal to 0.82 mmol. Using an approach similar to that used in example 3, the theoretical level of carboxyl groups in 1 g of poly (PEG ₁₅₀₀ -MA) dma, in which all residues of maleic anhydride were subjected to aminolysis, was determined to be 0.80 mmol during calculations. From these results, in the course of calculations, the relationship was established between the residues of maleic anhydride subjected to aminolysis in the target compound and the total number of residues of maleic anhydride in the starting compound and it was determined to be 1.0. Thus, it was confirmed that essentially all residues of maleic anhydride were subjected to aminolysis by dimethylamine.

Example 9

Obtaining complexes between the polymer modifiers of examples 1 to 8 and OCIF

Each of the polymer modifiers obtained in examples 1 to 8 was dissolved in physiological saline with phosphate buffer (PBS) at pH 6.0 (which is a solution obtained by mixing a solution containing 10 mm sodium hydrogen phosphate and 150 mm sodium chloride and a solution containing 10 mM sodium dihydrogen phosphate and 150 mM sodium chloride, in an appropriate ratio to obtain a buffer characterized by a pH value of 6.0), and solutions having a modifier concentration in the range of 1 to 20 mg / ml were obtained. For each solution of each modifier, in turn, 0.625 ml of the obtained polymer modifier solution and 0.625 ml of a solution containing purified, mature human OCIF (OCIF obtained as described in WO 96/26217 and EP 816380) were mixed [protein concentration: 2 mg / ml, medium: PBS (pH 6.0)], and the mixture thus obtained was allowed to stand at a temperature in the range from 4 ° C to 37 ° C for at least one hour to obtain a series of solutions [medium : PBS (pH 6.0)] containing OCIF modified using polymer modi fixatives from examples 1 to 8, in which the ratio between the modifier and OCIF in each solution was determined by the concentration of the added modifier solution and the reaction conditions. The modifier / OCIF weight ratios for some of the resulting complexes (and the conditions under which they were obtained) are shown in Table 6 in Test Example 2 below. The molecular size for each of the resulting complexes was measured as explained in the Test Example below. 11. The degree of detection of OCIF in complexes by ELISA was measured as described in Test Example 3 below.

In addition, for each of the polymer modifiers, an OCIF solution modified using a polymer modifier was prepared in exactly the same way, except that PBS with a pH of 7.4 was used as the medium.

Example 10

Obtaining the sodium salt of the product of poly (ammonolysis ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) a-Na], where m = 6-16, R ³ = NH ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 3.1: 6.9 (compound No. 9)

A copolymer of polyoxyethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ¹ = hydrogen, and R ² = methyl) and maleic anhydride, in which the polyoxyethylene side chain has an average molecular weight of approximately 500, and the average degree of polymerization for the main chain is in the range of 30 to 40 (AM-0530K manufactured by NOF Corporation having lot number M34529) [hereinafter referred to as “poly (PEG ₅₀₀ -MA)”]. To 10.1 g of the indicated starting poly compound (PEG ₅₀₀ -MA), 61 ml of a 0.5 M solution of ammonia / 1,4-dioxane were added, and the resulting solution was stirred at 25 ° C. for 20 hours. At the end of this time period, 200 ml of diethyl ether and 100 ml of a 0.2 M aqueous sodium hydroxide solution were added to the solution, and the mixture was vigorously shaken for approximately 3 minutes. After phase separation of the organic and aqueous layers, the lower layer was collected. 200 ml of diethyl ether and 60 ml of 1,4-dioxane were added to the selected layer, and the resulting solution was again vigorously shaken. After phase separation, the resulting lower layer was collected and then subjected to freeze drying to obtain 10.1 g of the desired compound sodium poly salt (PEG ₅₀₀ -MA) a (compound No. 9) as a yellow solid.

The level of carboxyl groups and the ratio specified by hydrolysis for the target polymer was determined as described in the following test example 1.

Example 11

Preparation of Ammonolysis Product Poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) a], where m = 6-16, R ³ = NH ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 1.4: 8.6 (compound No. 10)

To 1 g of poly (PEG ₅₀₀ -MA) (AM-0530K manufactured by NOF Corporation with lot number M34529) was added 9.5 g of an aqueous ammonia solution with a concentration of 28% (w / w) and the solution thus obtained was stirred at 25 ° C. for 4 hours. At the end of this time period, 250 ml of an aqueous ammonia solution with a concentration of 0.28% was added and the resulting solution was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (having an ultimate molecular weight of 10,000 and manufactured by Millipore Corporation, PBGC07610 model number), and the condensate thus obtained was freeze-dried to yield 0.8 g of the title compound poly (PEG ₅₀₀ -MA) a (№ compound 10) as an oily Cpd eniya.

The level of carboxyl groups and the ratio specified by hydrolysis for the target polymer was determined as described in the following test example 1.

Example 12

Obtaining the reaction product between dimethylamine and poly (PEG ₅₀₀ -MA) [salt poly (PEG ₅₀₀ -MA) dma-Na], where m = 6-16, R ³ = NMe ₂ , rel. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 2.9: 7.1 (compound No. 11)

To 5 g of poly (PEG ₅₀₀ -MA) (AM-0530K, batch number: M34529 obtained from NOF Corporation), 35 g of an aqueous solution of dimethylamine at a concentration of 50% (w / w) was added to dissolve it, and the resulting the solution was stirred at 25 ° C for 3 hours, after which it was further stirred at 4 ° C for 16 hours. At the end of this time period, 100 ml of a 0.1 M aqueous solution of sodium hydroxide was added and the resulting solution was freeze-dried to obtain 5.4 g of the target compound of the poly (PEG ₅₀₀ -MA) dma-Na salt (compound No. 11) as solid phase yellow.

The level of carboxyl groups and the ratio specified by hydrolysis for the target polymer was determined as described in the following test example 1.

Example 13

Obtaining hydrolyzate poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) g], where m = 6-16, R ³ = OH, resp. comp. = approximately 1: 1, cf. step. floor = 30-40, and rel. hydr. = approximately 10: 0 (compound No. 12)

To 1 g of poly (PEG ₅₀₀ -MA) (AM-0530K manufactured by NOF Corporation with lot number M34529), 17 ml of distilled water was added to dissolve it, and the resulting solution was stirred at 40 ° C. for 4 hours. At the end of this time period, 250 ml of an aqueous ammonia solution with a concentration of 0.28% (w / w) was added here, and the resulting solution was condensed using an ultrafiltration membrane made of ether-based polyethersulfone (characterized by a limiting molecular weight, equal to 10000, and manufactured by Millipore Corporation, model number PBGC07610). The condensate thus obtained was subjected to freeze drying to obtain 0.7 g of the target compound poly (PEG ₅₀₀ -MA) g (compound No. 12) as an oily compound.

The level of carboxyl groups and the ratio specified by hydrolysis for the target polymer was determined as described in the following test example 1.

Example 14

Obtaining complexes between the polymer modifiers of examples 10 to 13 and OCIF

Each of the polymer modifiers obtained in examples 10 to 13 was dissolved in physiological saline with phosphate buffer (PBS) at pH 7.0 (which is a solution obtained by mixing a solution containing 10 mm sodium hydrogen phosphate and 150 mm sodium chloride and a solution containing 10 mM sodium dihydrogen phosphate and 150 mM sodium chloride, in an appropriate ratio to obtain a buffer characterized by a pH value of 7.0), and solutions having a range of modifier concentrations ranging from 1 , 25 to 105 mg / ml. For each polymer modifier, in turn, at a volume ratio of 1: 1, the thus obtained solutions of polymer modifiers and a solution containing purified, mature human OCIF were mixed (OCIF obtained as described in WO 96/26217 and EP 816380) [concentration protein: 0.25 to 14 mg / ml, medium: PBS (pH 6.0)], and solutions were obtained characterized by different weight ratios between the modifier and OCIF. In the thus obtained solutions, the pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0 or 7.4 using a 1 M solution of hydrochloric acid or a 1 M aqueous solution of sodium hydroxide. Each of the solutions thus obtained was allowed to settle at 25 ° C. for a period of time ranging from 12 hours to one week, and a complex solution was obtained between the polymer modifier of the present invention and OCIF. The resulting solutions were stored at 4 ° C. The molecule size of the resulting complexes was measured as explained in the following test examples 6 and 11. The degree of detection of OCIF in the complexes by ELISA was measured as described in the following test example 8.

Example 15

Obtaining the sodium salt of the product of poly (ammonolysis ₅₀₀ -MA) [salts of poly (PEG ₅₀₀ -MA) _but -Na], characterized by an adjustable size of the molecule, where m = 6-16, R ³ = NH ₂ , rel. comp. = approximately 1: 1, cf. step. floor. shown below, and rel. hydr. = approximately 1.4: 8.6 (compounds nos. 13-19)

100 mg of the sodium salt of poly (PEG ₅₀₀ -MA) _a (compound No. 9) obtained in Example 10 was dissolved in 1 ml of physiological saline with phosphate buffer (PBS, characterized by a pH value of 7.4, obtained by stirring an aqueous solution containing 10 mM sodium hydrogen phosphate and 150 mm sodium chloride, and an aqueous solution containing 10 mm sodium dihydrogen phosphate and 150 mm sodium chloride, in an appropriate ratio). The solution thus obtained was fractionated by gel filtration chromatography. Two different sets of gel filtration conditions were used:

(1) A fractionation method using Superose 6 (hereinafter referred to as the SRF method)

Column: Superose 6 HR 10/30 Amersham Bioscience

Column temperature: 8 ° C

Mobile phase: PBS (pH 7.4)

Detection wavelength: 280 nm

Flow rate: 0.3 ml / min

The amount entered into the column: 100 μl

(2) The fractionation method using Superdex 200 (hereinafter referred to as the SDF method)

Column: Superdex 200 HR 16/60 Amersham Biosciences

Column temperature: room temperature

Mobile phase: PBS (pH 7.4)

Detection wavelength: 280 nm

Flow rate: 2 ml / min

The amount entered into the column: 5 μl

Polymer modifiers, the elution of which was carried out using the data of two fractionation methods for the elution time in the range from x to y minutes, was defined as poly (PEG ₅₀₀ -MA) _a -Na (SRF _xy ) and poly (PEG ₅₀₀ -MA) _a -Na (SDF _xy ), respectively.

The concentration of the polymer modifier in each fraction (aqueous solution) was determined by high performance liquid chromatography. The conditions used in high performance liquid chromatography were as follows:

Speaker: Shodex OHpak SB-806M HQ (acquired by SHOWA DENKO K. K.)

Protective column: Shodex OHpak SB-G (purchased from SHOWA DENKO K. K.)

Column temperature: 40 ° C

Mobile phase: an aqueous solution of 50 mm sodium hydrogen phosphate with a pH of 7.0 using 1 M hydrochloric acid

Detection Wavelength: 210 nm

Flow rate: 0.5 ml / min

The amount entered into the column: 50 μl

For poly (PEG ₅₀₀ -MA) _a -Na (SRF _xy ) and poly (PEG ₅₀₀ -MA) _a -Na (SDF _xy ) obtained respectively by fractionation in accordance with the SRF method and SDF method, an additional gel filtration was performed chromatography (SRF method) using the same elution conditions as described above for the first fractionation in accordance with the SRF method to estimate the size of the molecule for each of the samples after fractionation. Proteins characterized by known molecular sizes (purchased from Amersham Bioscience) were used as standard samples. In this regard, the molecular weight and size of the molecule for each of the proteins are shown in the corresponding catalog from which they were acquired.

The calculated molecular size for polymer modifiers obtained by fractionation is shown in Table 1 below.

Table 1 Molecular mass Stokes Radius (nm) Retention Time (min) Standard protein Thyroglobulin 669000 8.50 38.48 Ferritin 440000 6.10 44.85 Catalase 232000 5.22 48.77 Aldolase 158,000 4.81 50.11 Albumen 67000 3,55 51.85 Ovalbumin 43000 3.05 53.95 Chymotrypsinogen A 25,000 2.09 59.26 Ribonuclease A 13700 1,64 60.23 The modifier from example 15 Connection No. poly (PEG ₅₀₀ -MA) _a -Na (unfractionated) 9.3 or less ** 35-65 * 9 poly (PEG ₅₀₀ -MA) _a -Na (SRF _50-55 ), 23% yield 3.1-6.2 ** 45-55 * 13 poly (PEG ₅₀₀ -MA) _a -Na (SRF _55-60 ), yield 29% 1.5-4.7 ** 50-60 * fourteen poly (PEG ₅₀₀ -MA) _a -Na (SRF _60-65 ), 12% yield 3.1 or less ** 55-65 * fifteen poly (PEG ₅₀₀ -MA) _a -Na (SDF _46-52 ), yield 22% 7.8 or less ** 40-65 * 16 poly (PEG ₅₀₀ -MA) _a -Na (SDF _52-58 ), 22% yield 6.2 or less ** 45-65 * 17 poly (PEG ₅₀₀ -MA) _a -Na (SDF _58-64 ), 13% yield 3.1 or less ** 55-65 * eighteen poly (PEG ₅₀₀ -MA) _a -Na (SDF _60-70 ), 13% yield 3.1 or less ** 55-65 * 19 * The presence of a range for retention time indicates that the sample is characterized by molecular weight distribution.
** This value is calculated from a calibration curve obtained using the size of the molecule and retention time for each of the standard proteins. In cases where the lower limit is not shown, that is, when the Stokes radius is shown as “XX or less”, we can say that the sample contains a low molecular weight component that cannot be estimated using gel filtration chromatography carried out in conditions of this example.

Using this approach, various polymers (PEG ₅₀₀ -MA) _a -Na were obtained, characterized by unequal molecular sizes (compounds nos. 13-19). The average degree of polymerization for compounds Nos. 13, 14, 15, 16, 17, 18 and 19 was calculated as in the previous examples, and, as shown, it was <30, << 30, <<< 30, < 30, << 30, <<< 30 and <<< 30, respectively.

Example 16

Preparation of Ammonolysis Product Poly (PEG ₁₅₀₀ -MA) [poly (PEG ₁₅₀₀ -MA) _but ], characterized by an adjustable size of the molecule, where m = 28-38, R ³ = NH ₂ , rel. comp. = approximately 1: 1, cf. step. floor. below (compounds nos. 20-22)

Starting from [poly (PEG ₁₅₀₀ -MA) _a ] (compound No. 7) obtained in Example 7 above, used as the starting compound, and using the same approach as used in Example 15 above, the same the method itself, as in example 15, received poly (PEG ₁₅₀₀ -MA) _a (SRF _xy ). The elution conditions for SRF elution were the same as in Example 15. The yields of polymer modifiers obtained by fractionation are shown in Table 2 below.

table 2 The modifier from example 16 Connection No. poly (PEG ₁₅₀₀ -MA) _a , (unfractionated) 7 poly (PEG ₁₅₀₀ -MA) _a , (SRF _50-55 ), 12.2% twenty poly (PEG ₁₅₀₀ -MA) _a , (SRF _55-60 ), 13.1% 21 poly (PEG ₁₅₀₀ -MA) _a , (SRF _60-65 ), 16.4% 22

Using this approach, various polymers (PEG ₁₅₀₀ -MA) _а were obtained, characterized by unequal molecular sizes (compounds No. 20-22). The average degree of polymerization for compounds Nos. 20, 21 and 22 was calculated as in the previous examples, and, as shown, it was <10, << 10 and <<< 10, respectively.

Example 17

Obtaining complexes between the polymer modifiers of examples 15 and 16 and OCIF

The polymer-OCIF complexes of the present invention were obtained in the form of aqueous solutions using essentially the same preparative approach as in Example 14 above, using, as starting components, aqueous solutions of compounds Nos. 13 to 22 obtained in the above Examples 15 and 16 (medium: PBS characterized by pH 7.4) and an aqueous solution of purified, mature human OCIF (OCIF prepared as described in WO 96/26217 and EP 816380) (medium: PBS characterized by pH equal to 6.0). More specifically, in the case of each modifier, its solution in PBS (pH 7.4) with a concentration of 0.5 mg / ml (prepared as described in Example 9 above) and a solution of OCIF in PBS (pH 6.0) were mixed. ) with a concentration of 0.5 mg / ml with a volume ratio of 1: 1. The resulting reaction mixtures were allowed to settle at 25 ° C for 7 days. The size of the molecules of the resulting complexes was measured as explained in the following test example 11.

Example 18

Preparation of Ammonolysis Product Poly (PEG ₅₀₀ -MA) [salts of poly (PEG ₅₀₀ -MA) _but -Na], characterized by an adjustable size of the molecule: (a) m = 6-16, R ³ = NH ₂ , rel. sost. = approximately 1: 2, and cf. step. pol. = 20-30, (compound No. 27), and (b) m = 6-16, R ³ = NH ₂ , rel. sost. = approximately 1: 1, and cf. step. gender = approximately 15 (compound No. 28)

Two polymers of the present invention poly (PEG ₅₀₀ -MA) _a (compound No. 27) and poly (PEG ₅₀₀ -MA) _a (compound No. 28) were prepared as follows. The starting compound for the first of these was poly (PEG ₅₀₀ -MA) (AM-0510K, manufactured using a technique similar to that described in Japanese Patent No. 2621308 and Japanese Patent Publications No. 2003-105040 and 2003-104003) - a copolymer of polyoxyethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ¹ = hydrogen, and R ² = methyl) and maleic anhydride in which the polyoxyethylene side chain has an average molecular weight of approximately 500 and an average degree of polymerization for the main the chain is in the range of 20 d about 30, while the ratio between the elementary units of the polyoxyethylene allyl diester and the elementary units of maleic anhydride is 1: 2, and the average molecular weight is approximately 6000 [number average molecular weight is approximately 6000, and the molecular weight distribution (Mw / Mn) is approximately equal 1.25]. The starting compound for the second of these was poly (PEG ₅₀₀ -MA) (AM-0510K, manufactured using a technique similar to that described in Japanese Patent No. 2621308 and Japanese Patent Publications No. 2003-105040 and 2003-104003) - copolymer of polyoxyethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ¹ = hydrogen, and R ² = methyl) and maleic anhydride, in which the polyoxyethylene side chain has an average molecular weight of approximately 500, the average degree of polymerization for the main chain is approximately 15 and the average molecular weight is approximately equal to 10000. Both starting compounds were subjected to ammonolysis using conditions essentially identical to the conditions used in example 10, and the target compounds were obtained salt poly (PEG ₅₀₀ -MA) _a -Na (compound No. 27) and poly salt (PEG ₅₀₀ -MA) _a -Na (compound No. 28). The level of carboxyl groups in the target polymer was determined as described in the following test example 1, and was found to be 2.73 mmol / g for compound No. 27 and 2.05 mmol / g for compound No. 28.

Example 19

Obtaining complexes between the polymer modifiers of example 18 and OCIF

The polymer-OCIF complexes of the present invention were obtained in the form of aqueous solutions using essentially the same preparative approach as in Example 14 above, using aqueous solutions of compounds No. 27 and 28 obtained in Example 18 above as starting components. More specifically, in the case of each modifier, its solution in PBS (pH 7.4) with a concentration of 5 mg / ml and a solution of mature human OCIF in PBS (pH 6.0) with a concentration of 5 mg / ml were mixed at a volume ratio of 1: one. The pH of the resulting mixture was adjusted to 5.5 using 1 M hydrochloric acid, and then the reaction mixture was left to settle at 25 ° C for 7 days. The size of the molecules of the resulting complexes was measured as explained in the following test example 11.

Example 20

Evaluation of the poly composition (PEG ₅₀₀ -MA) and obtaining and evaluating poly (PEG ₅₀₀ -MA) a (compounds No. 29-53) under various conditions

20 (1) Determination of compositional ratio for poly (PEG ₅₀₀ -MA)

Tests were carried out for several copolymers of polyoxyethylene allyl methyl diester and maleic anhydride poly (PEG ₅₀₀ -MA) with different compositions in order to determine the ratio between the elementary units of PEG-allyl methyl diester and maleic anhydride (the ratio specified by the composition) in them (various batches of AM-0510K made using a technique similar to that described in Japanese Patent No. 2621308 and Japanese Patent Publications No. 2003-105040 and 2003-104003), random copolymers of polyols diethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ¹ = hydrogen, and R ² = methyl) and maleic anhydride, in which the polyoxyethylene side chain has an average molecular weight of approximately 500 and an average degree of polymerization for the main chain ranges from 20 to 30. The number average molecular weight (M _n ) and the molecular weight distribution (M _w / M _n , where M _w is the weight average molecular weight) for each batch of tested copolymers are shown below. The molecular weight (MM) for each batch of poly (PEG ₅₀₀ -MA) was determined by gel filtration chromatography using poly (ethylene glycol) with a predetermined MM as a standard. Therefore, MM poly (PEG ₅₀₀ -MA), shown below, is not absolute MM, but is a relative MM, measured using PEG as a standard.

M3O538, M _n = 6431; M _w / M _n = 1.27

M3N549, M _n = 6360; M _w / M _n = 1.23

M3N550, M _n = 5891; M _w / M _n = 1.28

M3N569, M _n = 5897; M _w / M _n = 1.25

In order to determine the ratio determined by the composition for each of the copolymers, it was necessary to convert them into the corresponding sodium salts of hydrolyzed copolymers, that is, into the sodium salt of poly (PEG ₅₀₀ -MA) g. The hydrolysis conditions used were as follows.

To 1 g of poly (PEG ₅₀₀ -MA) (batch M3O538), 25 ml of 1,4-dioxane, 100 ml of ether and 42 ml of 0.1 N were added. aqueous NaOH solution. The resulting mixture was then vigorously shaken, and after it was allowed to settle, the thus obtained aqueous layer was collected. The thus obtained aqueous layer was stirred at 40 ° C. for 2 hours after filtration through filter paper (704 × 40 m / m obtained from Nihon Rikagaku-kiki Corporation). At the end of this time period, the resulting solution was freeze dried. The sodium salt of the hydrolyzed product obtained from the starting compound, namely the sodium salt of poly (PEG ₅₀₀ -MA) g (batch M3O538), was obtained as a yellow solid (yield 100%).

To 1 g of poly (PEG ₅₀₀ -MA) (batch M3N549) was added 12 ml of 1,4-dioxane and 9 ml of 1 N. aqueous NaOH solution. The resulting mixture was then stirred at room temperature for 24 hours. At the end of this time period, 1 ml of 1 N was added. aqueous NaOH and the resulting solution was subjected to freeze drying. The sodium salt of the hydrolyzed product obtained from the starting compound, namely the sodium salt of poly (PEG ₅₀₀ -MA) g (batch M3N549), was obtained as a yellow solid (yield 100%).

To 2 g of poly (PEG ₅₀₀ -MA) (batch M3N550) was added 5 ml of 1,4-dioxane and 9 ml of 1 N. aqueous NaOH solution. The resulting mixture was then stirred at 40 ° C. for 23 hours. At the end of this time period, the reaction mixture was freeze dried. The sodium salt of the hydrolyzed product obtained from the starting compound, namely the sodium salt of poly (PEG ₅₀₀ -MA) g (batch M3N550), was obtained as a yellow solid (yield 100%).

To 2 g of poly (PEG ₅₀₀ -MA) (batch M3N569) was added 5 ml of 1,4-dioxane and 9 ml of 1 N. aqueous NaOH solution. The resulting mixture was then stirred at 40 ° C. for 23 hours. At the end of this time period, the reaction mixture was freeze dried. The sodium salt of the hydrolyzed product obtained from the starting compound, namely the sodium salt of poly (PEG ₅₀₀ -MA) g, was obtained as a yellow solid (yield 100%).

The level of carboxyl groups for each of the sodium salts of poly (PEG ₅₀₀ -MA) g, thus obtained, was measured by conductometric titration as described above. The results are shown in table 3 below.

The sodium salt of poly (PEG ₅₀₀ -MA) g contains the following two monomeric elementary units:

If the number of monomeric elementary units of the maleic acid salt of sodium per 1 elementary unit of PEG-allylmethyl diester is designated as “a”, and the theoretical minimum repeating elementary unit will include one elementary unit of PEG-allylmethyl diester and “a” of sodium maleate, then then the formula mass (FM) of the minimum repeating elementary link will be obtained in accordance with equation 1:

FM [(PEG allyl methyl diester) ₁ + (maleic acid salt) _a ] = FM (PEG allyl methyl diester) + a [FM (maleic acid salt)] = 541 + 160a (equation 1)

The amount of carboxyl groups in the minimum repeating elementary unit of the sodium salt of poly (PEG ₅₀₀ -MA) g is 2a. Therefore, the level of carboxyl groups (C, mmol / g) in the minimum repeating elementary unit of the sodium salt of poly (PEG ₅₀₀ -MA) g is obtained in accordance with equation 2:

C = 2a / (541 + 160a) 1000 (equation 2)

The ratio between the number of elementary units of the sodium salt of maleic acid and the number of elementary units of the PEG-allylmethyl diester in the actual sodium salts of poly (PEG ₅₀₀ -MA) g is the same as the ratio in the minimum repeating elementary units. Therefore, the value of C is also the same in the minimum repeating elementary unit and in the actual sodium salts of poly (PEG ₅₀₀ -MA) g, the levels of carboxyl groups in which were determined by conductometric titration (see above). Thus, as a result of measuring the level of carboxyl groups and using equation 2, one can obtain the value of the ratio specified by the composition, “a,” namely, the number of elementary units of the maleic acid salt of maleic acid per 1 elementary unit of PEG allyl methyl diester [which corresponds to the composition of the monomeric compositions for poly (PEG ₅₀₀ -MA)]. The results are shown in table 3 below.

Table 3 Poly Party (PEG500-MA) The number of carboxyl groups (mmol / g) MA / PEG allyl methyl diester Poly sodium salt (PEG500-MA) g Number 1 M3O538 4.66 2.01 Number 2 M3N549 5.21 2.42 Number 3 M3n550 5.98 3.10 Number 4 M3N569 6.14 3.26

The ratio specified by the composition for poly (PEG ₅₀₀ -MA) g sodium salts was in the range from 1: 2 to 1: 3.3. Obviously, the ratio specified by the composition is the same in the cases of poly (PEG ₅₀₀ -MA) and sodium salt of poly (PEG ₅₀₀ -MA) g, so that the ratios specified by the composition of the monomer mixture for different initial poly (PEG) copolymers ₅₀₀ -MA), as was confirmed, ranged from 1: 2 to 1: 3: 3.

20 (2) Ammonolysis of poly (PEG ₅₀₀ -MA) under various conditions to obtain poly (PEG ₅₀₀ -MA) a (compounds No. 29-53) and the determination of the ratios specified by hydrolysis for these compounds

As a result of using the ratio specified by the composition (a) and the level of carboxyl groups for the sodium salts of poly (PEG ₅₀₀ -MA) g, the ratio specified by hydrolysis (i.e., the ratio between the elementary units of maleic anhydride in the starting polymer subjected to ammonolysis, and by these elementary units subjected to hydrolysis) can be calculated as follows. The formula of the sodium salt of the elementary unit of amidated maleic acid (sodium salt of maleic acid semiamide) is shown below:

In the same way as in the case of the above sodium salts of poly (PEG ₅₀₀ -MA) g, it can be assumed that the minimum elementary unit of the sodium salt of poly (PEG ₅₀₀ -MA) a includes one elementary unit of PEG-allylmethyl diester, ah elementary units of the sodium salt of maleic acid semiamide and a (1) elementary units of the sodium salt of maleic acid. The formula weight of the minimum elementary unit of the sodium salt of poly (PEG ₅₀₀ -MA) is obtained using the following equation 3:

FM [(PEG allyl methyl diester) ₁ + (maleic acid salt of maleic acid) _ah + (sodium salt of maleic acid) _{a (1-x)} ] = FM (PEG allyl methyl diester) + ah [FM (sodium salt of half-amide maleic acid)] + a (1-x) [FM (sodium salt of maleic acid)] = 541 + 137ax + 160a (1-x) = 541 + 160a-23ax (equation 3)

Since the level of carboxyl groups in the minimum elementary unit of the sodium salt of poly (PEG ₅₀₀ -MA) a is ax + 2a (1-x) = 2a-ax, the level of carboxyl groups (C, mmol / g) in the minimum elementary unit of sodium salts of poly (PEG ₅₀₀ -MA) and can be obtained from equation 4:

С = 10000 × (2а-ах) / (541 + 160а-23ах) (equation 4)

Thus, the ratio given by hydrolysis (1-x): x can be determined from equation 4 using the level of carboxyl groups (C, mmol / g), which can be obtained by conductometric titration of the sodium salt of poly (PEG ₅₀₀ -MA) a , and the value "a", which was obtained above.

Some examples of the preparation of poly (PEG ₅₀₀ -MA) a and the calculated ratio determined by hydrolysis for the poly (PEG ₅₀₀ -MA) a product are shown below.

(a) To 100 mg of poly (PEG ₅₀₀ -MA) was added a 0.5 M solution of ammonia / 1,4-dioxane (amounts are indicated in table 4 below) and the resulting solution was stirred at temperatures and times shown in table 4 below At the end of the reaction time, approximately 0.4 ml (in the range of 0.3 to 0.5 ml) of 1N was added to the reaction mixture. aqueous NaOH solution. The resulting solution was freeze-dried to obtain the poly sodium salt (PEG ₅₀₀ -MA) a as a solid phase.

"The degree of reaction for the amine component" for each of the sodium salts of poly (PEG ₅₀₀ -MA) a [that is, the percentage of elementary units of maleic acid anhydride in the starting polymer subjected to ammonolysis (100 · * x)] obtained in various reaction conditions shown in the following table 4.

Nos. 8-19, 25 and 27-37 are the target polymers poly (PEG ₅₀₀ -MA) a (compounds Nos. 29-52). Each of these polymers is statistical, and m = 6-16, R ³ = NH ₂ , the ratio specified by the composition = 1: 2.4, and the average degree of polymerization = 20-30.

As shown in table 4, all the reaction products of poly (PEG ₅₀₀ -MA) a (compounds No. 29-52) are characterized by very high "degrees of reaction for the amine component", which indicates that the degree of conversion for the amino group is very close to 100 % using various reaction conditions described above.

A few additional examples of alternative methods for producing poly (PEG ₅₀₀ -MA) a are described below:

(b) 0.44 g of poly (PEG ₅₀₀ -MA) was dissolved in 9 ml of dimethylformamide. After that, ammonia gas was bubbled into the solution at -50 ° C. The final amount of ammonia in the reaction mixture was 0.67 g. The reaction mixture thus obtained was sealed in a glass ampoule in a nitrogen atmosphere, and then stirred at room temperature for 24 hours. At the end of this time, ammonia gas was evaporated under reduced pressure thereafter. as a glass ampoule was opened. After that, the reaction mixture was added dropwise to 90 ml of diethyl ether. The precipitate thus obtained was collected and dried under reduced pressure. 0.28 g of the ammonium salt of poly (PEG ₅₀₀ -MA) a (compound No. 53) (designated as No. 41 in the above table 4) was obtained as a yellow powder. The polymer is statistical, and m = 6-16, R ³ = NH ₂ , the ratio specified by the composition = 1: 3.10, and the average degree of polymerization = 20-30.

(c) The ammonium salt of poly (PEG ₅₀₀ -MA) a was obtained by the same method as (b), using toluene instead of dimethylformamide.

(4) The ammonium salt of poly (PEG ₅₀₀ -MA) a was obtained by the same method as (b) using 1,4-dioxane instead of dimethylformamide.

Example 21

Obtaining the product of the alcoholysis reaction between ethanol and poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) ea-Na], where m = 6-16, R ₃ = OCH ₂ CH ₃ , rel. comp. = approximately 1: 3, cf. step. floor = 20-30, resp. hydr. = approximately 3.1: 6.9 (compound No. 54)

Poly (PEG ₅₀₀ -MA) (AM-0510K, batch M3N550, manufactured using a procedure similar to that described in Japanese Patent No. 2621308 and Japanese Patent Publications No. 2003-105040 and 2003-104003) was used as the starting compound. a copolymer of polyoxyethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ₁ = hydrogen, and R ₂ = methyl) and maleic anhydride in which the polyoxyethylene side chain has an average molecular weight of approximately 500, the average degree of polymerization for the main chain is in range e from 20 to 30, the relationship between elementary units polioksietilenallilmetilovogo diether and elementary units of maleic anhydride is about 1: 3, average molecular weight (M _n) is equal to 5891 and a molecular weight distribution (M _n / M _w) is 1, 28 (see Example 20 above). To 100 mg of the indicated starting compound, 1 ml of ethanol was added, and the resulting reaction mixture was left to stand for 16 hours at 40 ° C. At the end of this time period, 52 μl of 2.5 N was added. a solution of sodium hydroxide in ethanol. The mixture thus obtained was concentrated at 35 ° C. by evaporation, and then dried in vacuo to obtain the poly (PEG ₅₀₀ -MA) ea sodium salt (compound No. 54) as an oily substance. The ratio determined by hydrolysis was calculated using the method described in examples 3 and 6 above. Namely, by determining the level of carboxyl groups in the poly sodium salt (PEG ₅₀₀ -MA) ea (compound No. 54) and the corresponding poly sodium salt (PEG ₅₀₀ -MA) g, the ratio between the residues of maleic anhydride in the target compound subjected to alcoholysis and the total number of residues of maleic anhydride in the starting compound was calculated, and it was determined to be 0.69. Thus, it can be seen that 69% of maleic anhydride residues in the starting compound were subjected to alcoholysis, and the remaining 31% of maleic anhydride residues were hydrolyzed.

Example 22

Obtaining complexes between the polymer modifier of example 21 and OCIF

The polymer-OCIF complexes of the present invention were obtained as aqueous solutions using essentially the same preparative approach as in Example 14 above using an aqueous solution of compound No. 54 obtained in Example 21 above [concentration of compound No. 54: 17, 2 mg / ml; medium: PBS, pH 7.4 (which is a solution obtained by mixing an aqueous solution containing 10 mm sodium hydrogen phosphate and 150 mm sodium chloride, and an aqueous solution containing 10 mm sodium dihydrogen phosphate and 150 mm sodium chloride, in an appropriate ratio to obtain a buffer characterized by a pH value of 7.4)] and an aqueous solution of purified, mature human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380) (OCIF concentration: 4 mg / ml; medium : buffer containing 10 mm phosphate ion and 150 mm NaCl, pH 6.0). More specifically, to 1.328 ml of said OCIF solution with a concentration of 4 mg / ml was added 0.472 ml of a solution of compound No. 54 with a concentration of 17.2 mg / ml and a solution was obtained containing 4.5 mg / ml of compound No. 54 and 3 mg / ml OCIF The resulting reaction mixture was allowed to stand for 3 days at 4, 10 or 25 ° C until aqueous solutions of the desired complexes of the present invention were obtained. The dimensions of the complexes were measured in the test example 13 below.

Example 23

Obtaining the product of the alcoholysis reaction between ethanol and poly (PEG ₅₀₀ -MA) [poly (PEG ₅₀₀ -MA) ea], where m = 6-16, R ₃ = OCH ₂ CH ₃ , rel. comp. = approximately 1: 3, cf. step. floor = 20-30 (compound No. 55)

Poly (PEG ₅₀₀ -MA) (AM-0510K, batch M3N550, manufactured using a procedure similar to that described in Japanese Patent No. 2621308 and Japanese Patent Publications No. 2003-105040 and 2003-104003) was used as the starting compound. a random copolymer of polyoxyethylene allyl methyl diester (m = 6-16, Alk = ethylene, R ₁ = hydrogen, and R ₂ = methyl) and maleic anhydride, in which the polyoxyethylene side chain has an average molecular weight of approximately 500, the average degree of polymerization for main circuit naho ranges from 20 to 30, the ratio between the elementary units of the polyoxyethylene allyl methyl diester and the elementary units of maleic anhydride is approximately 1: 3, the number average molecular weight (M _n ) is 5891, and the molecular weight distribution (M _n / M _w ) is 1.28 (see Example 20 above). 0.5 ml of ethanol was added to 50 mg of the indicated starting compound, and the resulting reaction mixture was left to stand at 37 ° C for 24 hours. The target compound poly (PEG ₅₀₀ -MA) ea (compound No. 55) was obtained as a solution in ethanol [concentration of poly (PEG ₅₀₀ -MA) ea: 100 mg / ml].

Example 24

Obtaining complexes between the polymer modifier of example 23 and OCIF

The polymer-OCIF complexes of the present invention were obtained in the form of aqueous solutions using essentially the same preparative approach as in Example 14 above, using a solution of compound No. 55 in ethanol obtained in Example 23 above and an aqueous solution of purified, mature Human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380) (OCIF concentration: 5 mg / ml; medium: buffer containing 10 mM phosphate ion and 150 mM NaCl, pH 6.0). More specifically, 37.5 μl of a solution of compound No. 55 in ethanol was added to 0.5 ml of the indicated OCIF solution with a concentration of 5 mg / ml and the resulting reaction mixture was allowed to settle for 3 days at 25 ° C until the desired solutions were obtained complexes of the present invention. The dimensions of the complexes were measured in the test example 13 below.

Comparative Example 1

Obtaining a copolymer of monomethoxypolyethylene glycol-methylvinyl ether-maleic acid (PEG-PMVMA)

A grafted copolymer of monomethoxypolyethylene glycol methyl vinyl ether maleic acid (PEG-PMVMA) was prepared according to the method described in Example 2 of Japanese Patent Application (Kokai) No. Hei 11-302199.

Reference Example 2

Obtaining a complex containing a copolymer of monomethoxypolyethylene glycol-methylvinyl ether-maleic acid (PEG-PMVMA) and protein OCIF

Purified human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380), modified using PEG-PMVA prepared as described in Comparative Example 1 above, was obtained as a solution [medium: PBS (pH 6.0)] essentially by the same method as in Example 9. More specifically, 1 ml of OCIF solution was mixed [OCIF concentration: 2 mg / ml; medium: PBS (pH 6.0)] and 1 ml of PEG-PMVA solution [concentration of PEG-PMVA: 2 or 20 mg / ml; medium: PBS (pH 6.0)] and to obtain the target complex, the mixture was left to settle for 24 hours at 25 ° C.

Reference Example 3

Obtaining a complex containing a polymer modifier and OCIF protein using poly (PEG ₅₀₀ -MA) as a polymer modifier

To 28.4 μl of an aqueous solution of purified human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380) (polymer concentration: 3.5 mg / ml, medium: 0.5 M aqueous NaH ₂ PO solution ₄ , the pH of which was adjusted to 7.6 using a 5 M aqueous NaOH solution), 2.2 μl of a solution in dimethyl sulfoxide containing poly (PEG ₅₀₀ -MA) [AM-0530K manufactured by NOF Corporation (hereinafter, in the present) was added document referred to as “poly (PEG ₅₀₀ -MA)”] (polymer concentration: 35 to 350 mg / ml) and thus obtained solution (OCIF concentration: 3.2 mg / ml, concentration A poly (PEG ₅₀₀ -MA): 2.5 mg / ml or 6.3 mg / ml) was shaken at 25 ° C. for 40 hours. At the end of this time, the solution was diluted using PBS (pH 7.0) and received a solution of OCIF, modified using a polymer modifier, with an OCIF concentration of 0.25 mg / ml Thus, the resulting solution was stored at 4 ° C.

Test Example 1

The measurement of the level of carboxyl groups for the polymers of examples 10 to 13 by conductometric titration

The level of carboxyl groups for each of the polymers [poly (PEG ₅₀₀ -MA) a (compounds No. 9 and 10), poly (PEG ₅₀₀ -MA) dma (compounds No. 11) and poly (PEG ₅₀₀ -MA) g (compounds No. 12)] obtained in Examples 10 to 13 was determined by conductometric titration as follows.

First, each of the polymers was purified using gel filtration as follows. For each polymer, 100 mg of the polymer was dissolved in 4 ml of 0.001 n. sodium hydroxide solution. The solution was divided into four batches and each batch of 1 ml was applied to a gel filtration column (PD-10 manufactured by Amersham-Pharmacia). The first 1 ml of eluent was discarded. After that, 1.5 ml of 0.001 N were applied to each column. sodium hydroxide solution and as a result of elution from the column, another 1.5 ml was obtained and discarded. Then, 2.5 ml of 0.001 N were applied to each column. a solution of sodium hydroxide and as a result of elution from the column, another 2.5 ml was obtained, and it was this fraction that contained the purified compound. The purified fractions from four columns were combined to obtain a purified solution of the target compounds. The yield after the purification step (determined spectrophotometrically when measuring the optical density for poly (PEG ₅₀₀ -MA) g in the 210 nm region before and after purification) was fixed at 80%, and the concentration in the purified solution was determined to be 8 mg / ml.

For each of the solutions of the polymer to be purified, an aliquot (2.5 to 7.5 ml) was made up to 50 ml using distilled water or a 0.001 M aqueous solution of sodium hydroxide, and then a 1 M aqueous solution of sodium hydroxide was added to the resulting solution. to bring the pH to 12. Then, 0.1 M hydrochloric acid was added to the solution either in 0.1 ml portions or continuously at a rate of 0.1 ml / min. In the first case, pH and conductivity were measured after each addition, and in the second case, their measurement was carried out every 15 seconds. After that, the level of carboxyl groups in the polymer was calculated from the amount of 0.1 M hydrochloric acid added in the buffer region of conductivity (corresponding to a pH range in the range of about 10 to 5.5). The results are shown in table 5 below.

Table 5 Example Number Type of polymer (and compound no.) The level of carboxyl groups (mmol / g polymer) The degree of passage of ammonolysis / aminolysis (%) Hydrolysis Ratio 10 poly (PEG ₅₀₀ -MA) a (9) 2.10 69 3.1: 6.9 eleven poly (PEG ₅₀₀ -MA) a (10) 1.83 86 1.4: 8.6 12 poly (PEG ₅₀₀ -MA) dma (11) 2.02 71 2.9: 7.1 13 poly (PEG ₅₀₀ -MA) g (12) 3.21 - 10: 0

In the case of the polymers of Examples 10 to 12, the ratio between the residues of maleic anhydride in the starting compound, subjected to ammonolysis or aminolysis, and the residues of maleic anhydride, subjected to hydrolysis, in each case were determined by calculation as follows. As shown in the above table 5, the level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) g (compound No. 12) is 3.21 mmol. Based on these values, we determined the mass of poly (PEG ₅₀₀ -MA) (i.e., the mass of the copolymer before hydrolysis) per gram of functional group and the mass of poly (PEG ₅₀₀ -MA) a (compounds Nos. 9 and 10) obtained as a result adding ammonia to poly (PEG ₅₀₀ -MA), per gram of functional group. Specifically, the mass of poly (PEG ₅₀₀ -MA) (i.e., the mass of the copolymer before hydrolysis) per mole of maleic anhydride residue was obtained by subtracting the molecular weight of the water molecule (18 g) from the mass of the fully hydrolyzed copolymer, which yielded numerical a value of 605 g. A mass of poly (PEG ₅₀₀ -MA) a (compounds Nos. 9 and 10) per mole of carboxyl group was obtained by adding the molecular weight of an ammonia molecule (17 g) to the mass of poly (PEG ₅₀₀ -MA), which resulted in a numerical value equal to 622 g. Exodus from this value, as a result of calculation (1 g / 622), the theoretical level of carboxyl groups was determined in 1 g of poly (PEG ₅₀₀ -MA) a (compounds nos. 9 and 10), in which all residues of maleic anhydride were subjected to ammonolysis ( is, in the absence of hydrolysis), and it has been found to be 1.61 mmol. In the same way, by adding the molecular weight of the dimethylamine molecule to the mass of poly (PEG ₅₀₀ -MA), we determined the mass of poly (PEG ₅₀₀ -MA) dma (compound No. 11) obtained by adding dimethylamine to poly (PEG ₅₀₀ -MA), per mole of carboxyl group, which yielded a numerical value of 650 g. Based on this value, the theoretical level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) dma was determined in which all residues of maleic anhydride were subjected aminolysis under action it dimethylamine, and, as has been established, it is 1.54 mmol.

For each of the polymers from examples 10 to 12, the ratio specified by hydrolysis (the percentage of the starting compound subjected to hydrolysis compared with ammonolysis or aminolysis) and the degree of passage of the reaction of ammonolysis or aminolysis (%) were determined from the level of carboxyl groups in 1 g poly (PEG ₅₀₀ -MA) g (compound No. 11), in which all residues of maleic anhydride were subjected to hydrolysis, of the theoretical level of carboxyl groups in 1 g of poly (PEG ₅₀₀ -MA) a (compounds No. 9 and 10), which all residues are maleic of anhydride were subjected to ammonolysis, the theoretical carboxyl group content level in 1 g of poly (PEG ₅₀₀ -ma) DMA (№ compound 11), where all maleic anhydride residues have been subjected to aminolysis by the action of dimethylamine, and the actual measured levels of carboxyl groups in 1 g of each of the polymers from examples 10 to 12 in accordance with the measurements according to the method of conductometric titration described above. The results are shown in the above table 5.

Test Example 2

Assessment of the retention of OCIF-modifier complexes in the blood by the example of a rat

Each of the obtained samples described in Example 9, Comparative Example 2, and unmodified human OCIF purified (OCIF was prepared as described in WO 96/26217 and EP 816380) was properly diluted using PBS (pH 6.0) as so that the concentration of OCIF was 0.25 mg / ml. Each of the diluted samples thus obtained was injected through a vein into the tail of a Wistar female rat of five weeks old (having a body weight of approximately 100 g and which was not given food for one day) so that the dose of OCIF would be 0 5 mg / kg (2 ml / kg by volume). After 6 hours after administration, 200 μl of blood was taken from the rat heart, and after that, the concentration of OCIF in the blood serum was measured by ELISA, with the conditions described in Test Example 3 below.

The concentration of OCIF in serum as measured after administration of each sample is shown in Table 6 below.

Table 6
Serum OCIF concentration after intravenous administration of each OCIF sample Modifier (and compound number) Modifier / OCIF (mass ratio) Serum OCIF concentration 6 hours after administration (ng / ml) Stirring Conditions * Unmodified OCIF - 25 ± 19 - PEG-PMVMA one 101 ± 26 - 10 361 ± 33 - poly (PEG ₁₅₀₀ -MA) g (2) 10 502 ± 70 - poly (PEG ₁₅₀₀ -MA) a (7) 10 1029 ± 30 - poly (PEG ₁₅₀₀ -MA) dma (8) one 2145 ± 721 - poly (PEG ₅₀₀ -MA) g (1) 10 750 ± 80 - 2,5 434 ± 92 - poly (PEG ₅₀₀ -MA) a (3) 10 3416 ± 440 - 10 2445 ± 195 37 ° C 2,5 3428 ± 27 - 2,5 484 ± 92 1 hour one 3004 ± 158 - one 2275 ± 130 pH 7.4 one 3786 ± 461 40 hours one 777 ± 153 4 ° C 0.5 2951 ± 512 - poly (PEG ₅₀₀ -MA) dma (4) one 1014 ± 331 - * The basic conditions with stirring were an OCIF concentration of 1 mg / ml, pH 6.0, 16 hours and 25 ° C. Table 6 lists only conditions that differ from the data.

From Table 6 above, it can be easily seen that each of the complexes between the polymer modifier and the protein of the present invention significantly improves the retention of the protein in the blood in comparison with the retention of the protein when only one is introduced. From a comparison of the prior art complex between PEG-PMVMA and a protein (described in Japanese Patent Laid-open No. Hei 11-302199 and obtained in Comparative Example 2 above) and the complexes of the present invention characterized by the same mass ratio between modifier and protein (1 or 10), it can also be seen that the complexes between the modifier and the protein of the present invention achieve significantly improved retention of protein in the blood in comparison with the complex of the prior art iki between PEG-PMVMA and protein.

Test Example 3

Assessment of OCIF detection by ELISA

One of the problems encountered in the case of protein modifiers of the prior art is that the binding of modifiers to a protein causes a modification of the protein structure and / or protein screening due to the formation of bulk complexes. In order to test the complexes of the OCIF-modifier of the present invention, the ELISA method was used to determine the degree of detection of each of the complexes obtained in accordance with the description in Example 9 and Comparative Example 2 on the basis of unmodified, purified, human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380). The ELISA method was implemented as follows.

OI-19 human OCIF monoclonal antibody (prepared according to the method described in EP0974671) was dissolved in a 0.1 M sodium hydrogen carbonate solution (pH 9.6) to give a solution having an OI-19 concentration of 10 μg / ml . 100 μl of the thus obtained OI-19 solution was placed in each well of a 96-well immunoplate (manufactured by Nunc) and the plates were allowed to stand at 4 ° C. overnight. At the end of this time period, 50% Block Ace (available from Snow Brand Milk Products Co., Ltd.) was added to each well for blocking, and then the plates were washed three times using PBS (wash buffer) containing 0.1 % Tween 20. Purified human OCIF (OCIF prepared as described in WO 96/26217 and EP 816380) was dissolved in the primary reaction buffer (i.e., 0.2 M Tris buffer solution including hydrochloric acid (pH 7 , 4) containing 40% Block Ace, 0.1% Tween 20 and 10 μg / ml mouse IgG) to obtain standard solutions with different Concentrated OCIF. 100 μl of each of the thus obtained solutions with different concentrations of OCIF was added to each well, the plates were shaken at room temperature for 2 hours, and then each well was washed six times using wash buffer. Thereafter, a 10,000-fold dilution of POD-OI-4 (i.e., an antibody labeled with peroxidase and recognizing OCIF prepared as described in EP 0974671) was performed using a secondary reaction buffer (i.e., 0.1 M Tris buffer a solution comprising hydrochloric acid (pH 7.4) containing 25% Block Ace, 0.1% Tween 20 and 10 μg / ml mouse IgG), 100 μl of the resulting solution was added to each well, the plates were shaken at room temperature for 2 hours, and then each well was washed six times. Once this was done, 100 μl of substrate solution (TMB soluble reagent available from Scytek) was added to each well and the plates were shaken at room temperature for a period of 10 to 15 minutes. Thereafter, 100 μl of reaction-terminating reagent (containing TMB stop buffer available from Scytek) was added to each well, and the plates were gently shaken. At the end of this time period, an optical density measurement was carried out at 450 nm for each well using a microplate reader (MEML 001 manufactured by Molecular Device Corporation). Based on these results, a calibration curve was obtained in the form of the dependence of the optical density on the concentration of OCIF. After obtaining this calibration curve, the procedure was repeated for each of the test complexes between OCIF and the modifier, while 100 μl of each of the solutions containing the test complex was added to each well, the reaction with POD-OI-4 was carried out in the same way as the specified above, and after that, for each well, the optical density at a wavelength in the region of 450 nm was measured using a microplate reader. Comparison with the optical densities obtained using the calibration curve made it possible to obtain the concentration of OCIF detected by ELISA for each of the complexes for which measurements were performed.

For each sample, the degree of non-detection of OCIF by ELISA was calculated, and the results are shown in Table 7 below. The degree of non-detection of OCIF by ELISA was determined using the following equation:

Degree = [1- (OCIF concentration measured by ELISA) / (OCIF concentration measured by Lowry)] × 100

In the above equation, the Lowry method for measuring the concentration of OCIF in the complexes was determined as described in Japanese Patent Application No. 2002-190407. This made it possible to obtain a measure of the total concentration of OCIF in the complexes. The degree of non-detection of OCIF in the complex by ELISA is a measure of the change in OCIF conformation caused by complexation with a modifier. A low degree of non-detection is evidence that OCIF in the complex can readily bind to both OI-19 and OI-4 anti-OCIF antibodies, which thus demonstrates the presence of slight modification or its absence for the OCIF structure in the complex.

Table 7
The degree of non-detection for the OCIF sample by ELISA Modifier (and compound number) Modifier / OCIF (mass ratio) The degree of non-detection of OCIF by ELISA (%) (based on unmodified OCIF) OCIF - - PEG-PMVMA one 25 10 39 poly (PEG ₁₅₀₀ -MA) g (2) 10 13 poly (PEG ₁₅₀₀ -MA) a (7) 10 fifteen poly (PEG ₁₅₀₀ -MA) dma (8) 10 0 poly (PEG ₅₀₀ -MA) g (1) 10 17 poly (PEG ₅₀₀ -MA) a (3) 10 0 2,5 0 poly (PEG ₅₀₀ -MA) dma (4) one 12

Based on the results shown in Table 7 above, it can be seen that in the case of complexes between the polymer modifier and the protein of the present invention, the degree of non-detection of OCIF by ELISA by modifying the protein structure was significantly reduced in comparison with the high degree obtained in the case of the complex of the previous level technique between PEG-PMVMA and the protein obtained in the above test example 2.

Thus, based on the results of test examples 2 and 3, it was confirmed that the decrease in sensitivity in detecting a protein caused by excessive modification of the protein in the case of complexes between the polymer modifier and the protein of the present invention is very small, and in addition, the blood retention for protein in said complexes are significantly better than those achieved with prior art complexes.

Test Example 4

Blood OCIF

Each of the samples obtained as described in Example 9 and purified human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380) was properly diluted using PBS (characterized by a pH in the range of 6 , 0 to 7.4) so that the concentration of OCIF is in the range from 0.1 to 1 mg / ml. Each of the diluted samples thus obtained was then injected through the saphenous vein of the leg or subcutaneously into the back of a female Cynomolgus monkey aged six or seven years (having a body weight in the range of 2 to 4 kg and which was not given food for one day) as follows so as to administer a dose of OCIF in the range of 0.1 to 1 mg / kg (1 ml / kg by volume). After a predetermined period of time after administration in the range of five minutes to one month, 500 μl of blood was taken from her hip blood vessel and the concentration of OCIF in the blood serum was measured by the ELISA method described in test example 3 above.

Test Example 5

Bone density measurement

Each of the samples obtained as described in Example 9 and purified human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380) was properly diluted using PBS (characterized by a pH in the range of 6 , 0 to 7.4) so that the concentration of OCIF is in the range of 0.7 to 3.5 mg / ml. After that, an adjuvant was obtained using killed Mycobacterium butyricum cells and liquid paraffin, and it was injected into the skin of the root of the tail of a female Lewis rat aged 5 to 10 weeks to stimulate the development of arthritis in said rat. Two weeks after the adjuvant was injected into the rat, each of the obtained samples was injected through the tail vein or subcutaneously in the back so that the dose of OCIF would be in the range of 1.4 to 7 mg / kg (2 ml / kg by volume). Three weeks after the adjuvant injection, the rat was dissected to extract both femurs and its bone density was measured.

Test Example 6

Estimation of the size of a molecule by polyacrylamide gel electrophoresis in the presence of SDS under non-reducing conditions

The molecule size for each of the complexes between the polymer modifier and OCIF obtained as described in Example 14 and Comparative Example 3 and purified human OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380) was evaluated by the method SDS-PAGE in non-reducing conditions as follows.

To 10 μl of each of the test samples [which, if necessary, were diluted using physiological saline with phosphate buffer (PBS (pH 7.0), which is a buffer solution obtained by mixing a solution containing 10 mm sodium hydrogen phosphate and 150 mm sodium chloride and a solution containing 10 mM sodium dihydrogen phosphate and 150 mM sodium chloride, at a suitable volume ratio to obtain a buffer characterized by a pH value of 7.0) so that the protein concentration is 250 μg / ml ] 5 μl of NuPAGE LX (lithium dodecyl sulfate) sample buffer (trademark) (obtained from Invitrogen Life Technology) and 5 μl of purified water were added and each of the resulting solutions was heated at 95 ° C for 7 minutes . At the end of this time period, the polyacrylamide gel for electrophoresis in the presence of SDS (3-8% Tris-acetate gel having a thickness of 1 mm and manufactured by NOVEX) was added the full amount of the reaction mixture and a voltage of 150 V was applied to the gel using power supply unit (PhoreStar Pro manufactured by Anatech). After electrophoresis was completed, the protein on the gel was stained using Coomassie Blue in accordance with a method well known to those skilled in the art.

As can be seen from figures 1 and 2, OCIF, modified using the polymer modifier of the present invention, was stably detected as a substance having a molecular weight exceeding the molecular weight of unmodified OCIF (120 kDa) at all ratios of the components when mixed (primary band in the region from 130 to 150 kDa and a secondary band in the region of 180 to 200 kDa were detected based on the marks of molecular weight markers), and, in addition, no complex characterized by a mole was detected a mass of more than 210 kDa. Similar results (not shown) were also obtained for other polymer modifiers of the present invention, which were products of aminolysis and alcoholysis. On the other hand, as can be seen from FIG. 3, in the case of OCIF modified using a conventional poly polymer modifier (PEG ₅₀₀ -MA) obtained in comparative example 3, the amount of crude complex obtained increases significantly as the proportion of modifier increases.

This result demonstrated that, in comparison with the structurally rather similar polymer modifier poly (PEG ₅₀₀ -MA) of the prior art, the use of the polymer modifier of the present invention can significantly suppress the formation of crude complexes, which are not preferred from the point of view of pharmaceuticals, and obtain a complex between the polymer a modifier and a protein that has stable properties regardless of the ratio of components when mixed.

Test Example 7

Assessment of the activity of poly (PEG ₅₀₀ -MA) and in the formation of a covalent bond

The reactivity of each of the polymer modifiers poly (PEG ₅₀₀ -MA) a (compound No. 9) and poly (PEG ₅₀₀ -MA) g (compound No. 12) with respect to tetramethylrodaminocaverine, which is a fluorescent substance characterized by the presence of an amino group and molecular weight of 514.62 (available from molecular Probes, Inc.) (hereinafter, herein referred to as "Rho-NH _2") and activity modifiers during the formation of the covalent bond was evaluated by comparing the reactivity of the two n limernyh modifiers as follows.

To 18.9 μl of PBS (pH 6.0) containing 1.08 mg / ml Rho-NH ₂ , 3.8 μl of solutions of each of poly (PEG ₅₀₀ -MA) g (compound No. 12) (prepared as as described in Example 13 above) and poly (PEG ₅₀₀ -MA) a (compound No. 9) [polymer concentration: 21 mg / ml, medium: PBS (pH was adjusted to 9.5 using 1 M NaOH)] (obtained as described in the above example 10). Thus, the resulting solutions were left to settle at 25 ° C for 3 days. At the end of this time period, the reaction mixtures were fractionated by gel filtration chromatography (column: PD-10 manufactured by Amersham Biotech, mobile phase: purified water) as follows. 0.5 ml of the reaction mixture was applied to the gel filtration column and 0.5 ml of the column was discarded. 2 ml of distilled water was added to the column, and another 2 ml was obtained and discarded as a result of elution. An additional 2 ml of distilled water was added to the column, and an additional 2 ml was obtained by elution, and this fraction contained a polymer fraction. Data on the number of Rho-NH ₂ contained in the polymer fractions obtained by fluorescence spectrometry (excitation wavelength: 544 nm, fluorescence wavelength: 571 nm, medium: purified water to adjust the pH to 3), carrying out calculation of the ratio between the amount of Rho -NH ₂ contained in each complex between the polymer and Rho-NH ₂ , and the total amount of Rho-NH ₂ in the reaction mixture, that is, the degree of binding between Rho-NH ₂ and the polymer.

The results are shown in table 8 below.

Table 8
The reactivity of the polymer modifier The degree of binding between Rho-NH ₂ and the polymer * (%) Rho-NH ₂ + poly (PEG ₅₀₀ -MA) a 11.5, 12.6 Rho-NH ₂ + poly (PEG ₅₀₀ -MA) g 0.8, 1.6 Rho-NH ₂ alone 0, 0 * This test was performed twice.

From Table 8 above, it can be easily seen that for the reaction mixture between the poly (PEG ₅₀₀ -MA) a polymer modifier (compound No. 9) and Rho-NH _{2, there} is a rather high level of Rho-NH ₂ detected in the polymer fraction. This, this case differs from the reaction mixture between the poly (PEG ₅₀₀ -MA) g polymer modifier (compound No. 12) and Rho-NH ₂ , for which there is only a low level of Rho-NH ₂ detected in the polymer fraction. If only Rho-NH ₂ was introduced into the column, then no elution of Rho-NH ₂ occurred in the “polymer fraction”. These results indicate that the polymer modifier poly (PEG ₅₀₀ -MA) a (compound No. 9) of the present invention is strongly associated with the amino group Rho-NH ₂ . On the other hand, the degree of binding for the polymer modifier poly (PEG ₅₀₀ -MA) g (compound No. 12) of the present invention is small, presumably indicating that this polymer modifier is not strongly bound to the amino group Rho-NH ₂ .

Test Example 8

Assessment of OCIF detection by ELISA

The ELISA method was used to determine the degree of detection of each of the complexes between OCIF and the polymer modifier obtained in accordance with the description in Example 14 above and Comparative Example 3, based on unmodified human purified OCIF (OCIF was prepared as described in WO 96/26217 and EP 816380). The ELISA method was implemented as follows.

OI-19 human OCIF monoclonal antibody (prepared according to the method described in EP0974671) was dissolved in a 0.1 M sodium hydrogen carbonate solution (pH 9.6) to give a solution having an OI-19 concentration of 10 μg / ml . 100 μl of the thus obtained OI-19 solution was placed in each well of a 96-well immunoplate (manufactured by Nunc) and the plates were allowed to stand at 4 ° C. overnight. At the end of this time period, 50% Block Ace (available from Snow Brand Milk Products Co., Ltd.) was added to each well for blocking, and then the plates were washed three times using PBS (wash buffer) containing 0.1 % Tween 20. Purified human OCIF (OCIF prepared as described in WO 96/26217 and EP 816380) was dissolved in a primary reaction buffer (i.e., 0.2 M Tris buffer solution including hydrochloric acid (pH 7, 4) containing 40% Block Ace, 0.1% Tween 20 and 10 μg / ml mouse IgG) to obtain standard solutions with different GOVERNMENTAL concentrations of OCIF. 100 μl of each of the thus obtained solutions with different concentrations of OCIF was added to each well, the plates were shaken at room temperature for 2 hours, and then each well was washed six times using wash buffer. Thereafter, a 10,000-fold dilution of POD-OI-4 (i.e., an antibody labeled with peroxidase and recognizing OCIF obtained as described in EP 0974671) was performed using a secondary reaction buffer (i.e., 0.1 M Tris buffer a solution including hydrochloric acid (pH 7.4) containing 25% Block Ace, 0.1% Tween 20 and 10 μg / ml mouse IgG), 100 μl of the resulting solution was added to each well, the plates were shaken at room temperature in for 2 hours, and then each well was washed six times. Once this was done, 100 μl of substrate solution (TMB soluble reagent available from Scytek) was added to each well and the plates were shaken at room temperature for a period of 10 to 15 minutes. Thereafter, 100 μl of reaction-terminating reagent (containing TMB stop buffer available from Scytek) was added to each well, and the plates were gently shaken. At the end of this time period, an optical density measurement was carried out at 450 nm for each well using a microplate reader (MEML 001 manufactured by Molecular Device Corporation). The OCIF concentration in each standard OCIF sample was calculated based on a calibration curve obtained using OCIF solutions having a known concentration.

Optical densities were measured for each of the samples of the test complex and, in accordance with the explanations in the above test example 3, the OCIF concentrations calculated from the calibration curve and the degree of non-detection of OCIF by ELISA were calculated. The results obtained are shown in table 9 below.

Table 9
The degree of non-detection for the OCIF sample by ELISA Modifier (and compound number) Modifier / OCIF (mass ratio) The degree of non-detection of OCIF by ELISA (%) (based on unmodified OCIF) OCIF individually - - poly (PEG ₅₀₀ -MA) g (12) one 8 Poly (PEG ₅₀₀ -MA) a-Na salt (9) one 0 0.75 0 0.5 0 0.25 0 poly (PEG ₅₀₀ -MA) 7.8 98 1.9 89 0.78 60

From table 9 above, it can be easily seen that for each of the complexes between the polymer modifier and the protein of the present invention obtained in Example 14, the degree of non-detection of OCIF by ELISA due to protein modification was significantly reduced compared to the complex between the polymer and protein of the previous level the technique obtained in comparative example 3, for which the degree of non-detection of OCIF by ELISA was very large.

Based on these results, it becomes clear that the complexes between the polymer modifier and the protein of the present invention cause only a very slight decrease in detection sensitivity by ELISA, which may be due to excessive modification of the protein and / or the formation of a bulk complex.

Test Example 9

Assessment of blood retention on the example of a rat

Each of the obtained samples described in Example 14 and the unmodified human purified OCIF (OCIF were prepared as described in WO 96/26217 and EP 816380) were properly diluted using PBS (pH 7.0) so that the concentration OCIF was 0.25 or 0.025 mg / ml. Each of the diluted samples thus obtained was injected through the femoral vein to a Wistar female rat of five weeks old (having a body weight of approximately 100 g and which was not given food for one day) so that the dose of OCIF would be 0.5 or 0.05 mg / kg (2 ml / kg by volume). After 6 hours after administration, 200 μl of blood was taken from the jugular vein of the rat, and then the concentration of serum OCIF was measured by the ELISA method described above.

The concentration of OCIF in serum as measured after administration of each sample is shown in Table 10 below.

Table 10
Serum OCIF concentration after intravenous administration of each OCIF sample Modifier (and compound number) Modifier / OCIF (mass ratio) Dosage (mg / kg) Serum OCIF concentration 6 hours after administration (ng / ml) OCIF individually - 0.5 eighteen poly (PEG ₅₀₀ -MA) g (12) one 0.05 127 Poly (PEG ₅₀₀ -MA) a-Na salt (9) one 0.05 603 one 0.5 5549 0.75 0.5 4770 0.5 0.5 4292 0.25 0.5 3020

As evident from the above table 10, the complex between the polymer modifier and the protein of the present invention provides a significant improvement in the retention of the specified protein in the blood in comparison with the case of the introduction of the protein alone.

Based on the above results, it becomes apparent that the complex between the polymer modifier and the protein of the present invention can be stably obtained under a wide range of conditions, and that the complex provides a significant improvement in the retention of the complex protein in the blood after administration. Therefore, the complex is probably extremely useful in the field of pharmaceuticals and biochemistry.

Test Example 10

Assessment of blood retention on the example of a rat

For each of the samples obtained in example 17 and example 19, the evaluation was carried out by the same method as in example 9. The concentration of OCIF in the blood serum when measured after taking each sample is shown in table 11 below.

Table 11
Serum OCIF concentration after intravenous administration of each OCIF sample Example Number Modifier (and compound number) Fraction Modifier / OCIF (mass ratio) Dosage (mg / kg) Serum OCIF concentration 6 hours after administration (ng / ml) 17 poly (PEG ₅₀₀ -MA) _a -Na fourteen SRF55-60 one 0.05 437 fourteen 2,5 0.05 460 fifteen SRF60-65 one 0.05 478 19 SDF60-70 one 0.5 4255 17 poly (PEG ₁₅₀₀ -MA) _a 21 SRF55-60 one 0.5 256 21 2,5 0.05 334 19 poly (PEG ₅₀₀ -MA) _a 27 Unfractionated one 0.5 6138 28 Unfractionated one 0.5 6713

From the above table 11 it is easy to see that all tested polymer modifiers of the present invention, characterized by different molecular sizes, provide a significant improvement in the retention of protein in the blood in comparison with the case of the introduction of the protein alone.

Test Example 11

Size estimation of a molecule by size exclusion chromatography

The molecule size for each of the complexes between the polymer modifier and OCIF obtained in Example 9, Example 14, Example 17 and Example 19 was evaluated by size exclusion chromatography. Test conditions are shown in table 12 below.

Table 12
Size exclusion chromatography conditions Chromatography apparatus: Explorer 10S (Amersham Biotech)
Column: Superdex 200 HR10 / 30 (Amersham Biotech)
Mobile phase: saline with phosphate buffer (8 mM Na ₂ HPO ₄ , 15 mM KH ₂ PO ₄ , 145 mM NaCl, 0.5 g / l NaN ₃ )
Analysis temperature: 4 ° C
Detection wavelength: 280 nm
Mobile phase flow rate: 0.6 ml / min

The retention times of the following standard proteins under the above conditions are shown in Table 13 below.

Table 13
Retention time for each standard protein Type of protein Molecular weight (kDa) Stokes Radius (nm) Retention Time (min) Ferritin 440 6.10 18.41 Aldosa 158 4.81 22.48 Ovalbumin 43 3.05 25.19 Ribonuclease 13.7 1,64 29.56

Based on the size exclusion chromatography results, the Stokes radius for the uncomplexed OCIF was determined to be 5.63 nm, and the Stokes radius for each polymer modifier-OCIF complex of the present invention was determined as follows:

The complexes obtained in example 9:

Complexes poly (PEG ₅₀₀ -MA) g (compound No. 1) -OCIF (Stokes radii in the range from 6.13 nm and 7.32 nm),

Complexes poly (PEG ₅₀₀ -MA) a (compound No. 3) -OCIF (from 6.12 nm and 6.54 nm),

Complex poly (PEG ₅₀₀ -MA) dma (compound No. 4) -OCIF (6.39 nm),

The complex poly (PEG ₅₀₀ -MA) IPA (compound No. 5) -OCIF (6.26 nm),

Complex poly (PEG ₅₀₀ -MA) ea (compound No. 6) -OCIF (6.44 nm),

Complex poly (PEG ₁₅₀₀ -MA) g (compound No. 2) -OCIF (from 6.44 nm to 6.71 nm),

The complex poly (PEG ₁₅₀₀ -MA) a (compound No. 7) -OCIF (from 6.40 nm to 6.47 nm),

Complex poly (PEG ₁₅₀₀ -MA) dma (compound No. 8) -OCIF (6.55 nm).

Based on these results, it becomes apparent that each of the complexes of the invention is characterized by a Stokes radius that is approximately 1 nm higher than the corresponding value for uncomplexed OCIF in physiological saline with phosphate buffer used as the mobile phase. In addition, in each of the samples, the peak obtained due to the presence of unmodified OCIF was not recorded, which indicates the stability of the complexes.

The Stokes radii for other complexes were determined under conditions similar to those described above. The results were as follows:

The complexes obtained in example 14:

Complexes obtained under conditions: OCIF concentration of 5 mg / ml; the concentration of the polymer modifier in the range from 1.25 mg / ml to 5 mg / ml; pH 7.4; 25 ° C; 36 h; medium in the form of physiological saline with phosphate buffer (phosphate concentration 10 mM; NaCl concentration 150 mM), as shown, are characterized by Stokes radii in the range from 6.2 nm to 6.5 nm. In addition, in each of the samples, the peak obtained due to the presence of unmodified OCIF was not recorded, which indicates the stability of the complexes.

The complexes obtained in example 17:

Complexes obtained under incubation conditions: OCIF concentration of 0.5 mg / ml; the concentration of the polymer modifier 0.5 mg / ml; pH 6.0; 25 ° C; 168 h; medium in the form of physiological saline with phosphate buffer (phosphate concentration 10 mM; NaCl concentration 150 mM), as shown, are characterized by Stokes radii in the range from 6.1 nm to 6.7 nm. In addition, in each of the samples, the peak obtained due to the presence of unmodified OCIF was not recorded, which indicates the stability of the complexes.

The complexes obtained in example 19:

Complexes obtained under incubation conditions: OCIF concentration of 5 mg / ml; the concentration of the polymer modifier 5 mg / ml; pH 5.5; 25 ° C; 168 h; medium in the form of physiological saline with phosphate buffer (phosphate concentration 10 mM; NaCl concentration 150 mM), as shown, are characterized by Stokes radii in the range from 6.3 nm to 6.8 nm. In addition, in each of the samples, the peak obtained due to the presence of unmodified OCIF was not recorded, which indicates the stability of the complexes.

The increase in the Stokes radii for the modifier-OCIF complexes of the present invention in comparison with the uncomplexed OCIF can be attributed to the modification of OCIF by the polymer modifiers of the present invention.

Test Example 12

Evaluation of blood retention on an example of a rat and evaluation of the degree of detection of OCIF by ELISA

Tests to determine blood retention and the degree of detection of OCIF by ELISA were performed for the complex obtained in example 22 (complex between compound No. 54 and OCIF) (incubation temperature: 25 ° C) in the same manner as described in the above examples Tests 2 and 3. The equivalent dose for OCIF was set at 0.1 mg / kg. Serum OCIF concentration was found to be 189 ng / ml 6 hours after intravenous injection of the complex. Thus, it was confirmed that the complexes obtained using the poly (PEG ₅₀₀ -MA) ea sodium salt (compound No. 54) in Example 22 above show excellent blood retention. In addition, the decrease in sensitivity when detecting OCIF by ELISA due to excessive modification of the protein in the complex under the action of a modifier was found to be very small.

Test Example 13

Estimation of the size of the complex

The size of the molecules for the complexes obtained in examples 22 (complex between compound No. 54 and OCIF) and 24 (complex between compound No. 55 and OCIF) was evaluated by gel filtration chromatography as described in Test Example 11 above. It was found that the size of the molecules of these complexes demonstrates a high level of uniformity. In the case of complexes between compound No. 54 and OCIF formed at 4 ° C, 10 ° C and 25 ° C, the Stokes radii were shown to be 6.0 nm, 6.0 nm and 6.0 nm. In the case of complexes between compound No. 55 and OCIF, the Stokes radii were 6.4 nm. Thus, each of the complexes obtained in examples 22 and 24 had a large Stokes radius compared to unmodified human OCIF purified (Stokes radius: 5.63 nm).

Drug example

The solution containing the complex obtained under sterile conditions in the same manner as described in Example 14 above was freeze-dried and a freeze-dried preparation was obtained.

Claims (113)

1. The copolymer or its pharmacologically acceptable salt, which contain as constituting their elementary units (a) one or more structural elementary units, which may be identical or different from each other, and which are described by the following formula (I)

where m is an integer in the range from 3 to 100, Alk is an alkylene group containing from 1 to 6 carbon atoms, and R ¹ and R ² are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and (b) one or more structural units that may be identical or different from each other, and which are described by formula (II)

where R ³ represents hydroxyl group alkoxy group containing from 1 to 6 carbon atoms, which optionally may be substituted by an aryl group containing from 6 to 14 carbon atoms, an aryloxy group containing from 6 to 14 carbon atoms, which may optionally be substituted with a nitro group, or a group described by the formula —NR ⁴ R ⁵ , where R ⁴ and R ⁵ are identical or different from each other, and each of them is a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, which optionally can be substituted by a hydroxy group , moreover, the location of the structural units represented by formula (I), and the structural units represented by formula (II), is selected from the following options (i) - (iv): (i) the location of the indicated structural units of formula (I) and formula (II) in the form of a sequence with alternating "head to head"

(ii) the location of the structural units of formula (I) and formula (II) in the form of a sequence with alternating "head to tail"

(iii) the location of the indicated structural units of formula (I) and formula (II) in the form of a mixed sequence with alternating "head to head" and "head to tail"

and (iv) the location of these structural units of formula (I) and formula (II) in the form of an arbitrary sequence; and where the ratio between the indicated structural units represented by formula (I) and the structural units represented by formula (II) in the specified copolymer is in the range from 10: 1 to 1:10. 2. The copolymer or its pharmacologically acceptable salt according to claim 1, where Alk represents an ethylene or trimethylene group. 3. The copolymer or its pharmacologically acceptable salt according to claim 2, where Alk represents an ethylene group. 4. The copolymer or its pharmacologically acceptable salt according to claim 1, where m is an integer in the range from 3 to 50. 5. The copolymer or its pharmacologically acceptable salt according to claim 4, where m is an integer in the range from 3 to 40. 6. The copolymer or its pharmacologically acceptable salt according to claim 5, where m is an integer in the range from 6 to 16 or in the range from 28 to 38. 7. The copolymer or its pharmacologically acceptable salt according to claim 6, where m is an integer in the range from 6 to 16. 8. The copolymer or its pharmacologically acceptable salt according to claim 1, where R ¹ represents a hydrogen atom. 9. The copolymer or its pharmacologically acceptable salt according to claim 1, where R ² represents a methyl group. 10. The copolymer or its pharmacologically acceptable salt according to claim 1, where R ³ represents a hydroxyl group or alkoxy group containing from 1 to 6 carbon atoms. 11. The copolymer or its pharmacologically acceptable salt of claim 10, containing at least one structural unit described by formula (II), in which R ³ represents an alkoxy group containing from 1 to 6 carbon atoms, and optionally at least at least one structural unit described by formula (II) in which R ³ represents a hydroxyl group, where the ratio between the structural units described by formula (II) in which R represents a hydroxy group and the structural units described by formula (II) ) in which R ³ represents an alkoxy group containing from 1 to 6 carbon atoms is in the range from 4: 6 to 0:10. 12. The copolymer or its pharmacologically acceptable salt of claim 10, where R ³ represents an alkoxy group containing from 1 to 6 carbon atoms. 13. The copolymer or its pharmacologically acceptable salt of claim 10, where the specified alkoxy group containing from 1 to 6 carbon atoms, is an ethoxy group. 14. The copolymer or its pharmacologically acceptable salt of claim 10, where R ³ represents a hydroxyl group or a group described by the formula -NR ⁴ R ⁵ where R ⁴ and R ⁵ are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms. 15. The copolymer or its pharmacologically acceptable salt according to 14, containing at least one structural unit described by formula (II), in which R ³ represents a group described by the formula -NR ⁴ R ⁵ where R ⁴ and R ⁵ are identical or different, and each of them represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and optionally at least one structural unit described by formula (II), in which R ³ represents a hydroxyl a group where the relationship between the structural units described and formula (II), wherein R ³ represents a hydroxy group and repeating units described by formula (II), wherein R ³ represents a group -NR ⁴ R-described formula ^5, is in the range from 5: 5 to 0:10. 16. The copolymer or its pharmacologically acceptable salt according to clause 15, where the ratio between the structural units described by formula (II), in which R ³ represents a hydroxy group, and the structural units described by formula (II), in which R ³ represents a group described by the formula —NR ⁴ R ⁵ is in the range from 4: 6 to 0:10. 17. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.14-16, where R ³ represents a group described by the formula -NR ⁴ R ⁵ where R ⁴ and R ⁵ are identical or different, and each of them represents an atom hydrogen or an alkyl group containing from 1 to 6 carbon atoms. 18. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.14-16, where the group described by the formula -NR ⁴ R ⁵ represents an amino group, methylamino group or dimethylamino group. 19. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.14-16, where the group described by the formula -NR ⁴ R ⁵ represents an amino group. 20. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.14-16, where the group described by the formula -NR ⁴ R ⁵ represents a dimethylamino group. 21. The copolymer or its pharmacologically acceptable salt according to claim 1, where R ³ represents a hydroxyl group. 22. The copolymer or its pharmacologically acceptable salt according to claim 1, where R ³ represents a 1-amino-2-propanol group. 23. The copolymer or its pharmacologically acceptable salt according to claim 1, where the ratio between the structural unit described by formula (I) and the structural unit described by formula (II) is in the range from 3: 1 to 1: 8. 24. The copolymer or its pharmacologically acceptable salt according to claim 1, where the ratio between the structural unit described by formula (I) and the structural unit described by formula (II) is in the range from 2: 1 to 1: 2. 25. The copolymer or its pharmacologically acceptable salt according to claim 1, where the ratio between the structural unit described by formula (I) and the structural unit described by formula (II) is in the range from 1: 2 to 1: 6. 26. The copolymer or its pharmacologically acceptable salt according to claim 1, where the structural unit of the formula (I) and the structural unit of the formula (II) are arranged in an arbitrary sequence, and the ratio between the structural unit described by the formula (I) and the structural unit described formula (II) is 1: 1. 27. The copolymer or its pharmacologically acceptable salt according to claim 1, where the structural unit represented by the formula (I) and the structural unit represented by the formula (II) are arranged in an arbitrary sequence, and the ratio between the structural unit described by the formula (I) , and the structural unit described by formula (II) is in the range from 1: 2 to 1: 4. 28. The copolymer or its pharmacologically acceptable salt according to claim 1, where the average degree of polymerization is in the range from 5 to 200. 29. The copolymer or its pharmacologically acceptable salt according to p, where the average degree of polymerization is in the range from 5 to 50. 30. The copolymer or its pharmacologically acceptable salt according to clause 29, where the average degree of polymerization is in the range from 5 to 20. 31. The copolymer or its pharmacologically acceptable salt according to p, where the average degree of polymerization is in the range from 20 to 30. 32. The copolymer or its pharmacologically acceptable salt according to p, where the average degree of polymerization is in the range from 30 to 40. 33. The copolymer or its pharmacologically acceptable salt according to claim 1, where their Stokes radius is 9.3 nm or less. 34. The copolymer or its pharmacologically acceptable salt according to clause 33, where their Stokes radius is 7.3 nm or less. 35. The copolymer or its pharmacologically acceptable salt according to clause 34, where their Stokes radius is 6.2 nm or less. 36. The copolymer or its pharmacologically acceptable salt according to clause 35, where their Stokes radius is 4.7 or less. 37. The copolymer or its pharmacologically acceptable salt according to clause 36, where their Stokes radius is 3.1 nm or less. 38. The copolymer or its pharmacologically acceptable salt according to clause 33, where their Stokes radius is in the range from 1.5 to 4.7 nm. 39. The copolymer or its pharmacologically acceptable salt according to clause 33, where their Stokes radius is in the range from 3.1 to 6.2 nm. 40. The copolymer or its pharmacologically acceptable salt according to claim 1, where Alk represents an ethylene group, R ¹ represents a hydrogen atom, R ² represents a methyl group, am, R, the ratio between the structural units described by formulas (I) and ( II), (the ratio defined by the composition) and, where appropriate, the ratio between the units described by formula (II), where R ³ represents a hydroxy group, and the elementary units described by formula (II), where R ³ represents a group different from hydroxyl (ratio, ask my hydrolysis), choose from the following: (i) m is in the range of 6 to 16, R ³ is a hydroxy group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range of 30 to 40; (ii) m is in the range of 28 to 38, R ³ is a hydroxy group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range of 10 to 15; (iii) m is in the range from 6 to 16, R ³ is an amino group, the ratio defined by the composition is 1: 1, and the average degree of polymerization is in the range from 30 to 40; (iv) m is in the range from 6 to 16, R is a dimethylamino group, the ratio defined by the composition is 1: 1, and the average degree of polymerization is in the range from 30 to 40; (v) m is in the range of 6 to 16, R ³ is a 1-amino-2-propanol group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range of 30 to 40; (vi) m is in the range from 6 to 16, R ^{3 is} selected from ethoxy and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, and the ratio determined by hydrolysis is 4 : 6; (vii) m is in the range from 28 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 10 to 15, and the ratio determined by hydrolysis is 4 : 6; (viii) m is in the range from 28 to 38, R ³ is a dimethylamino group, the ratio given by the composition is 1: 1, and the average degree of polymerization is in the range from 10 to 15; (ix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, the ratio determined by hydrolysis is 3, 1: 6.9, and the copolymer is a sodium salt; (x) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, and the ratio determined by hydrolysis is 1 4: 8.6; (xi) m is in the range from 6 to 16, R ^{3 is} selected from dimethylamino and hydroxy groups, the ratio determined by the composition is 1: 1, the average degree of polymerization is in the range from 30 to 40, the ratio determined by hydrolysis is 2, 9: 7.1, and the copolymer is a sodium salt; (xii) m is in the range of 6 to 16, R ³ is an amino group, the ratio given by the composition is 1: 2.4, and the average degree of polymerization is in the range of 20 to 30; (xiii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.4: 9.6; (xiv) m is in the range from 6 to 16, R is selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis is 2.9: 7.1; (xv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.9: 9.1; (xvi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.5: 9.5; (xvii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.3: 8.7; (xviii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.9: 8.1; (xix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.0: 9.0; (xx) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.8: 9.2; (xxi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 4.6: 5.4; (xxii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.2: 8.8; (xxiii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 2.0: 8.0; (xxiv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.1: 8.9; (xxv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 2.4: 7.6; (xxvi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.9: 9.1; (xxvii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.5: 8.5; (xxviii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.7: 9.3; (xxix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 4.5: 5.5; (xxx) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.4: 8.6; (xxxi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.7: 9.3; (xxxii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.8: 9.2; (xxxiii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.4: 8.6; (xxxiv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 3.1, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.7: 9.3; (xxxv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 0.9: 9.1; (xxxvi) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 2.4, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis, equal to 1.9: 8.1; (xxxvii) m is in the range from 6 to 16, R ^{3 is} selected from ethoxy and hydroxy groups, the ratio determined by the composition is approximately 1: 3, the average degree of polymerization is in the range from 20 to 30, and the ratio determined by hydrolysis is 3.1: 6.9; (xxxviii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 9 3 nm or less; (xxxix) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is in the range from 3.1 to 6.2 nm; (xl) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is range from 1.5 to 4.7 nm; (xli) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 3 1 nm or less; (xlii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 7 8 nm or less; (xliii) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 6 2 nm or less; and (xliv) m is in the range from 6 to 16, R ^{3 is} selected from amino and hydroxy groups, the ratio determined by the composition is 1: 1, the ratio determined by hydrolysis is 1.4: 8.6, and the Stokes radius is 4 7 nm or less. 41. The copolymer or its pharmacologically acceptable salt, obtained by introducing one or more units of carboxylic acid anhydride described by formula (III) into a copolymer that contains one or more structural units, which may form its elementary units, (a) identical or different from each other, and which are described by the following formula (I)

where m is an integer in the range from 3 to 100, Alk is an alkylene group containing from 1 to 6 carbon atoms, and R ¹ and R ² are identical or different, and each of them represents a hydrogen atom or an alkyl group, and (b) said structural unit containing a carboxylic acid anhydride unit described by formula (III)

in one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis, and (iv) alcoholysis. 42. The copolymer or its pharmacologically acceptable salt according to paragraph 41, where the structural unit described by formula (I) and the structural unit described by formula (III) in the copolymer are oriented in the form of a sequence with alternating "head to head" sequence with alternating "head to tail" or a mixed sequence with alternating "head to head" and "head to tail". 43. The copolymer or its pharmacologically acceptable salt according to paragraph 41, where the structural unit described by formula (I) and the structural unit described by formula (III) in the copolymer are oriented in the form of a statistical sequence. 44. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, where Alk represents an ethylene or trimethylene group. 45. The copolymer or its pharmacologically acceptable salt according to item 44, where Alk represents an ethylene group. 46. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, where m is an integer in the range from 3 to 50. 47. The copolymer or its pharmacologically acceptable salt according to item 46, where m is an integer in the range from 3 to 40. 48. The copolymer or its pharmacologically acceptable salt according to clause 47, where m is an integer in the range from 6 to 16 or in the range from 28 to 38. 49. The copolymer or its pharmacologically acceptable salt according to p, where m is an integer in the range from 6 to 16. 50. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, where R ¹ represents a hydrogen atom. 51. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, where R ² represents a methyl group. 52. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, where the ratio between the structural unit described by formula (I) and the structural unit obtained by introducing one or more structural units described by formula (III), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is in the range from 10: 1 to 1:10. 53. The copolymer or its pharmacologically acceptable salt according to paragraph 52, where the ratio between the structural unit described by formula (I) and the structural unit obtained by introducing one or more structural units described by formula (III), in one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis, and (iv) alcoholysis, is in the range from 3: 1 to 1: 8. 54. The copolymer or its pharmacologically acceptable salt according to paragraph 52, where the ratio between the structural unit described by formula (I) and the structural unit obtained by introducing one or more structural units described by formula (III) into one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is in the range from 2: 1 to 1: 2 or in the range from 1: 2 to 1: 6. 55. The copolymer or its pharmacologically acceptable salt according to paragraph 42, where the ratio between the structural unit described by formula (I) and the structural unit obtained by introducing one or more structural units described by formula (III), in one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis, and (iv) alcoholysis, is 1: 1. 56. The copolymer or its pharmacologically acceptable salt according to item 43, where the ratio between the structural unit described by formula (I) and the structural unit obtained by introducing one or more structural units described by formula (III), in one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) alcoholysis, is in the range from 1: 2 to 1: 4. 57. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, where the average degree of polymerization is in the range from 5 to 200. 58. The copolymer or its pharmacologically acceptable salt according to clause 57, where the average degree of polymerization is in the range from 5 to 50. 59. The copolymer or its pharmacologically acceptable salt according to clause 57, where the average degree of polymerization is in the range from 5 to 20. 60. The copolymer or its pharmacologically acceptable salt according to clause 57, where the average degree of polymerization is in the range from 20 to 30. 61. The copolymer or its pharmacologically acceptable salt according to § 58, where the average degree of polymerization is in the range from 30 to 40. 62. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, which is obtained by ammonolysis of a fragment of a carboxylic acid anhydride of the formula (III) copolymer. 63. The copolymer or its pharmacologically acceptable salt according to item 62, upon receipt of which an aqueous solution of ammonia was used by ammonolysis. 64. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, which is obtained by aminolysis of a fragment of a carboxylic acid anhydride of the formula (III) copolymer. 65. The copolymer or its pharmacologically acceptable salt according to item 64, upon receipt of which an aqueous solution of dimethylamine was used by aminolysis. 66. The copolymer or its pharmacologically acceptable salt according to any one of paragraphs.41-43, which is obtained by alcoholysis of a fragment of the carboxylic acid anhydride of the formula (III) copolymer. 67. The copolymer or its pharmacologically acceptable salt according to item 64, upon receipt of which ethanol was used by alcoholysis. 68. A pharmaceutical composition for the prophylaxis or treatment of bone metabolic disorders, comprising a pharmaceutically acceptable diluent or carrier, at least one protein or its analogue or variant, and at least one copolymer or its pharmacologically acceptable salt according to any one of claims. 1-67, wherein said protein, or its analogue or variant, is an osteoclastogenesis inhibition factor (OCIF), or its analogue or variant. 69. The pharmaceutical composition according p, where the specified OCIF, or its analogue or variant, is a natural or recombinant type of OCIF. 70. The pharmaceutical composition according p, where the specified OCIF, or its analogue or variant, is a monomer or dimer. 71. The pharmaceutical composition of claim 68, wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 as measured by SDS-PAGE in non-reducing conditions. 72. The pharmaceutical composition according p, where the specified OCIF contains amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 73. The pharmaceutical composition according p, where the specified OCIF contains amino acids from +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 74. A modifier capable of modifying a protein, or an analogue or variant thereof, wherein said modifier contains a copolymer or a pharmacologically acceptable salt thereof according to any one of claims 1-67. 75. A modifier capable of modifying a protein, or its analogue or variant, according to claim 74, wherein the protein is a basic protein. 76. A modifier capable of modifying a protein or its analogue or variant according to claim 75, wherein the main protein is the main fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor, (OCIF), platelet growth factor ( PDGF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepatocytic growth factor (HGF) or vascular endothelial growth factor (VEGF), or an analogue or variant thereof. 77. A modifier capable of modifying a protein or its analogue or variant according to claim 75, wherein the basic protein is an osteoclastogenesis inhibition factor (OCIF), or its analogue or variant. 78. A modifier capable of modifying a protein or its analogue or variant according to Claim 77, wherein said OCIF or its analogue or variant is a natural type or recombinant type OCIF. 79. A modifier capable of modifying a protein or its analogue or variant according to claim 75, wherein said OCIF, or its analogue or variant, is a monomer or dimer. 80. A modifier capable of modifying a protein or its analogue or variant according to claim 77, wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer characterized by a molecular weight of approximately 120,000 as measured by SDS-PAGE under non-reducing conditions. 81. A modifier capable of modifying a protein, or its analogue or variant, according to claim 77, wherein said OCIF contains amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 82. A modifier capable of modifying a protein, or its analogue or variant, according to claim 77, wherein said OCIF contains amino acids from +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 83. The complex containing at least one protein, or its analogue or variant, which are associated with at least one copolymer or its pharmacologically acceptable salt according to any one of claims 1-67. 84. The complex of Claim 83, wherein the protein is a basic protein. 85. The complex of claim 84, wherein the main protein is major fibroblast growth factor (bFGF), epidermal growth factor (EGF), osteoclastogenesis inhibition factor (OCIF), platelet growth factor (PDGF), brain derived neurotrophic factor (BDNF ), nerve growth factor (NGF), human growth hormone (HGH), hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF), or an analogue or variant thereof. 86. The complex of claim 84, wherein the major protein is an osteoclastogenesis inhibition factor (OCIF), or an analogue or variant thereof. 87. The complex of claim 86, wherein said OCIF, or an analogue or variant thereof, refers to a natural type or recombinant type OCIF. 88. The complex of claim 86, wherein said OCIF, or an analog or variant thereof, is a monomer or dimer. 89. The complex of claim 86, wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 as measured by SDS -PAGE in non-reducing conditions. 90. The complex of claim 86, wherein said OCIF contains amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 91. The complex of claim 86, wherein said OCIF contains amino acids from +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 92. A pharmaceutical composition for the prophylaxis or treatment of metabolic disorders in the bones, containing an effective amount of a pharmacologically active substance together with a carrier or diluent for it, where the specified pharmacologically active substance is present in the form of a complex according to any one of claims 86-91. 93. A method for extending the time during which an osteoclastogenesis inhibition factor (OCIF), or an analogue or variant thereof, is retained in a blood stream after administration by a patient as a result of complexation between said protein, or its analogue or variant, and at least one copolymer , or a pharmacologically acceptable salt thereof, according to any one of claims 1-67. 94. The method of claim 93, wherein said OCIF, or an analogue or variant thereof, refers to a natural type or recombinant type OCIF. 95. The method of claim 93, wherein said OCIF, or an analogue or variant thereof, is a monomer or dimer. 96. The method of claim 93, wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 as measured by SDS -PAGE in non-reducing conditions. 97. The method according to p, where the specified OCIF contains amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 98. The method according to paragraph 93, where the specified OCIF contains amino acids from +1 to +380 sequence SEQ ID No. No. 1 list of sequences. 99. A method of treating or preventing metabolic disorders in bones, comprising administering to a patient an effective amount of a complex containing a complex comprising an osteoclastogenesis inhibition factor (OCIF), or an analogue or variant thereof that are associated with at least one copolymer, or pharmacologically acceptable salt according to any one of claims 1 to 67. 100. The method for the prevention or treatment of metabolic disorders in bones according to claim 99, wherein said OCIF, or an analogue or variant thereof, is a natural type or recombinant type OCIF. 101. A method for the prevention or treatment of metabolic disorders in bones according to claim 99, wherein said OCIF, or an analogue or variant thereof, is a monomer or dimer. 102. The method for the prevention or treatment of metabolic disorders in bones according to claim 99, wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight approximately equal to 120,000 as measured by the SDS-PAGE method under non-reducing conditions. 103. The method for the prevention or treatment of metabolic disorders in bones according to claim 99, wherein said OCIF contains amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 104. The method for the prevention or treatment of metabolic disorders in bones according to claim 99, wherein said OCIF contains amino acids from +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 105. The use of a complex containing a protein, or its analogue or variant, which is associated with at least one copolymer, or its pharmacologically acceptable salt, according to any one of claims 1-67, for the manufacture of a medicinal product intended for the prevention or treatment of metabolic disorders substances in the bones that are susceptible to the action of the specified protein, or its analogue or variant, where the specified protein is an osteoclastogenesis inhibition factor (OCIF), or its variant or analogue. 106. The application of claim 105, wherein said OCIF, or an analogue or variant thereof, refers to a natural type or recombinant type OCIF. 107. The application of claim 105, wherein said OCIF, or an analog or variant thereof, is a monomer or dimer. 108. The application of claim 105, wherein said OCIF is a human OCIF monomer having a molecular weight of approximately 60,000 as measured by SDS-PAGE under non-reducing conditions, or a human OCIF dimer having a molecular weight of approximately 120,000 as measured by SDS -PAGE in non-reducing conditions. 109. The application of claim 105, wherein said OCIF contains amino acids from -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences. 110. The application of claim 105, wherein said OCIF contains amino acids from +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences. Priority on points: 03.24.2003 according to claims 1-10, 12-14, 17-22, 26, 28, 30, 41-51, 57, 62-67; 10.16.2003 according to claims 11, 16, 55, 59; 03/24/2004 according to claims 15, 23-25, 27, 29, 31-40, 52-54, 56, 58, 60-61, 68-110.

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