Polymeric modifying agents and pharmaceutical compositions

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

 

The technical field

The present invention relates to a new copolymer, pharmaceutical compositions containing the specified copolymer, the modifier protein containing a specified copolymer, complex specified copolymer and protein, method of prevention or treatment of diseases using the specified complex, the use of this complex for the manufacture of a medicinal product intended for the prevention or treatment of diseases, and methods of synthesis of these copolymers and these complexes.

The level of technology

The modification of proteins with the use of additional agents, such as polymers, usually used for imparting improved pharmaceutical properties, such as improved stability and retention in the blood and reduced antigenicity [for example, see F. M. Veronese and J. M. Harris, "Peptide and Protein Pegylation", Advanced Drug Delivery Reviews 54(4), 2002].

When the modification of proteins using polymer one technique used in the past include binding protein and polymer modifier using covalent bonds (for example, see WO-A-97/23614). In other examples, the modification of a protein using a polymer, such as with the modification of using polymer drug, the drug is modified using a non-covalent link to the I. One such example is shown in Japanese patent application (Kokai) No. Hei 11-302199, which describes what the grafted copolymer, which includes the grafted chain nonionic polymer main chain is negatively charged polymer that forms an inclusion complex with a substance capable of carrying a positive charge under physiological conditions, for example, a liposome or poly-L-lysine, bearing a positive charge, resulting in improved retention in the blood. Another alternative is proposed in WO-A-99/02131, which describes the fact that protein and water-soluble polymer can be mixed in specific conditions in the presence of an organic solvent to obtain microparticles with controlled release.

Unfortunately, for many reasons were particularly successful small number of data of polymer modifiers of protein. One recent example of a polymeric modifier protein that shows some improved properties, includes a copolymer on the basis of the derived polimolekuly acid, containing as forming its elementary level derived polyoxyethyleneglycol ether [see, for example, Japanese patent numbers 3035675 and 3271265]. Data of polymer modifiers definitely demonstrate enhanced binding to target proteins. However, such copolymers still there is one problem. As was found, the level of maleic anhydride is associated with nonspecific proteins. The result is the obtaining of complexes between the copolymer and protein, showing inhomogeneous properties depending on the conditions. In particular, it was found that these polymeric modifiers tend to be easily education disordered proteins cross-linked structures, thus forming a volumetric complexes, which causes excessive modification of protein structure, and thus reduce the desired activity of the protein. In addition, the complexes between the polymer modifiers of protein and protein has been found to show poor retention in the blood after administration.

There is therefore a need in the polymer modifier that can result in complex with homogeneous properties, in particular characterized by low education disordered cross-linked structures with protein, the best preservation of the protein activity and excellent retention of protein in the blood after administration of specified property.

Summary of invention

Therefore, the aim of the present invention to provide a polymeric modifier, can result in complex with homogeneous properties, in particular the characteristics of arisugawa low education disordered cross-linked structures with protein, the best preservation of the protein activity and excellent retention of protein in the blood after administration of specified property.

The inventors of the present invention have conducted extensive studies of various modifiers of protein and as a result succeeded in getting the new copolymers which are capable of forming complexes with proteins, which have homogeneous properties, and significantly improve the retention in the blood proteins of these complexes, which thus leads to the committing of the present invention.

Other objectives and advantages of the present invention will become apparent when reading the description.

Thus, the present invention provides a copolymer or a pharmacologically acceptable salt, which contain as forming their elementary parts

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

where:

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

Alk represents alkylenes group containing from 1 to 6 carbon atoms, and

R1and R2are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 atoms of plastics technology : turning & the Yes, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below, and

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

where:

R3represents a

hydroxyl group,

alkoxy group containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below And,

aryloxy-group containing from 6 to 14 carbon atoms, which optionally may be substituted by substituents in an amount of from 1 to 5, selected from the substituents defined below A, or

the group has the formula-NR4R5where R4and R5are identical or different from each other, and each of them before the hat is a hydrogen atom or alkyl group, containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below;

substituents And 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 additionally provides a copolymer or a pharmacologically acceptable salt, obtained as a result of one or more parts of the anhydride of the carboxylic acids described by formula (III), in the copolymer, which contains as a form of elementary links

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

where:

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

Alk represents alkylenes group containing from 1 to 6 carbon atoms, and

R1and R2are identical or different and each p is ecstasy a hydrogen atom or alkyl group, containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below, and

(b) the one or more parts of the anhydride of the carboxylic acids described by formula (III):

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

substituents And 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 of the present invention described above, in particular, such a composition that also contains at least one protein or an analogue or variant.

The present invention also provides a modifier capable of modifying a protein or an analogue or variant, when the specified modifier contains a copolymer or it is pharmacologically acceptable salt of the present invention, above.

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

The present invention also provides a pharmaceutical composition comprising an effective amount of pharmacologically active substances together with a carrier or diluent for him, where the aforementioned pharmacologically active substance is a compound containing at least one protein or an analogue or variant that bind at least one copolymer or a pharmacologically acceptable salt of the present invention described above.

The present invention also provides a method of prolonging the time during which a protein or an analogue or variant are retained in the bloodstream after ingestion by the patient, in result of complex formation between protein or an analogue or variant and at least one copolymer or a pharmacologically acceptable salt of the present invention described above.

The present invention also provides a method of treatment or prevention in a patient of a disease which is susceptible to the action of the protein or its analog or VA is Ianto, including reception of the specified patient an effective amount of a complex containing the protein or an analogue or variant that bind at least one copolymer or a pharmacologically acceptable salt of the present invention described above.

The present invention also provides the use of a complex containing protein or an analogue or variant that bind at least one copolymer or a pharmacologically acceptable salt of the present invention described above, for manufacturing a medicinal product intended for the prevention or treatment of diseases susceptible to the action of the protein or its analogue or variant.

The present invention also provides a method of producing a copolymer or a pharmacologically acceptable salt containing as forming their elementary parts

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

where:

m, Alk, R1and R2are defined above, and

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

where:

R3is a defined above;

while this method involves performing one or more parts of the anhydride of the carboxylic acids described by formula (III), in the copolymer, which contains as a form of elementary links

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

(d) the one or more parts of the anhydride of the carboxylic acids described by formula (III):

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

The present invention also provides a method of obtaining complex containing at least one protein or an analogue or variant that bind at least one copolymer or its pharmacologically acceptable salt, as defined above, with the specified method comprises conducting the reaction between the specified copolymer or its pharmacologically acceptable salt and the specified protein or an analogue or variant under conditions favorable for the formation of the specified property.

Brief description of figures

Figure 1 is played is the duty to regulate the results of polyacrylamide gel electrophoresis in the presence of SDS (sodium dodecyl sulfate) for complexes of the present invention poly(PEG 500-MA)a-OCIF (factor inhibiting osteoclastogenesis) in non conditions conducted in the following test example 6:

(1) molecular weight Markers

(2) a Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:1 (mass ratio)]

(3) a Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:2 (mass ratio)]

(4) the Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:3 (mass ratio)]

(5) a Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:4 (mass ratio)]

(6) Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:5 (mass ratio)]

(7) Unmodified OCIF.

Figure 2 shows the results of polyacrylamide gel electrophoresis in the presence of SDS for complexes of the present invention poly(PEG500-MA)a-OCIF in non conditions conducted in the following test example 6:

(1) molecular weight Marker

(2) a Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:1 (mass ratio), the concentration of OCIF during incubation: 3.5 mg/ml]

(3) a Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:1 (mass ratio), the concentration is of OCIF during incubation: 1.75 mg/ml]

(4) the Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:1 (mass ratio), the concentration of OCIF during incubation: 0,875 mg/ml]

(5) a Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:2 (mass ratio), the concentration of OCIF during incubation: 1.75 mg/ml]

(6) Complex of poly(PEG500-MA a-Na (compound No. 9)-OCIF [OCIF:polymer modifier=1:4 (mass ratio), the concentration of OCIF during incubation: 0,875 mg/ml]

(7) Unmodified OCIF.

Figure 3 shows the results of polyacrylamide gel electrophoresis in the presence of SDS for complexes of the prior art poly(PEG500-MA)-OCIF in non conditions conducted in the following test example 6:

(1) molecular weight Marker

(2) a Complex of poly(PEG500-MA) (AM-C)-OCIF [OCIF:polymer modifier=1:10 (mass ratio)]

(3) a Complex of poly(PEG500-MA) (AM-C)-OCIF [OCIF:polymer modifier=1:2,5 (mass ratio)]

(4) the Complex of poly(PEG500-MA) (AM-C)-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, which contain as obrazowe is their basic links

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

where:

m, Alk, R1and R2are defined above, and

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

where R3is a defined above. Among these copolymers and their pharmacologically acceptable salts are preferred compounds include:

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

(3) a copolymer or a pharmacologically acceptable salt corresponding to the position (1), where for elementary structural units described by formula (I), and elementary structural units described by formula (II), receive the configuration in the de statistical sequence;

(4) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (3), where Alk represents ethylene or trimethylene group;

(5) a copolymer or a pharmacologically acceptable salt corresponding to the position (4)where Alk represents ethylene group;

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

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

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

(10) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (9), where R1represents a hydrogen atom or methyl group;

(11) a copolymer or a pharmacologically acceptable salt corresponding to the position (10), where R1represents a hydrogen atom;

(12) a copolymer or a pharmacologically acceptable salt, suitable for the exercise of any one of items (1) to (11), where R2represents a hydrogen atom or methyl group;

(13) a copolymer or a pharmacologically acceptable salt, the corresponding position (12), where R2represents a methyl group;

(14) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (13), where R3represents a hydroxyl group, alkoxy group containing from 1 to 6 carbon atoms, or a group described by formula-NR4R5where R4and R5are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(15) a copolymer or a pharmacologically acceptable salt corresponding to the position (14), where R3represents a hydroxyl group or alkoxy group containing from 1 to 6 carbon atoms;

(16) a copolymer or a pharmacologically acceptable salt, the corresponding position (15)containing at least one structural elementary link described by the formula (II)in which R3represents an alkoxy group containing from 1 to 6 carbon atoms, and optionally at least one structural elementary link described by the formula (II)in which R3represents a hydroxyl group, where the ratio between the structural elementary is the disappearance, described by formula (II)in which R3represents a hydroxy group, and elementary structural units described by formula (II)in which R3represents 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 corresponding to the positions (15) or (16), where R3represents an alkoxy group containing from 1 to 6 carbon atoms;

(18) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (15) to (17)where the specified alkoxy group containing from 1 to 6 carbon atoms, represents ethoxy-group;

(19) a copolymer or a pharmacologically acceptable salt corresponding to the position (14), where R3represents a hydroxyl group or a group described by formula-NR4R5where R4and R5are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(20) a copolymer or a pharmacologically acceptable salt corresponding to the position (19), containing at least one structural elementary link described by the formula (II)in which R3represents a group described by formula-NR4R5where R4and R5ablauts the same 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 elementary link described by the formula (II)in which R3represents a hydroxyl group, where the ratio between the elementary structural units described by formula (II)in which R3represents a hydroxy group, and elementary structural units described by formula (II)in which R3represents a group described by formula-NR4R5is in the range from 5:5 to 0:10;

(21) a copolymer or a pharmacologically acceptable salt corresponding to the position (20), where the ratio between the elementary structural units described by formula (II)in which R3represents a hydroxy group, and elementary structural units described by formula (II)in which R3represents a group described by formula-NR4R5is in the range from 4:6 to 0:10;

(22) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (19) to (21), where R3represents a group described by formula-NR4R5where R4and R5are identical or different and each represents a hydrogen atom or alkyl group, terzidou from 1 to 6 carbon atoms;

(23) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (19) to (22), where the group has the formula-NR4R5is an amino group, methylaminopropyl or dimethylaminopropyl;

(24) a copolymer or a pharmacologically acceptable salt corresponding to the position (23), where the group has the formula-NR4R5is an amino group;

(25) a copolymer or a pharmacologically acceptable salt corresponding to the position (23), where the group has the formula-NR4R5is dimethylaminopropyl;

(26) a copolymer or a pharmacologically acceptable salt corresponding to the position (14), where R3represents a hydroxyl group;

(27) a copolymer or a pharmacologically acceptable salt corresponding to the position (14), where R3represents 1-amino-2-propanolol group;

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

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

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

(32) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (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 corresponding to the position (32), where the average degree of polymerization is in the range from 5 to 50;

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

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

(36) a copolymer or f is macological acceptable salt, the corresponding position (32), where the average degree of polymerization is in the range from 30 to 40;

(37) a copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (31)where the Stokes radius is 9.3 nm or less;

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

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

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

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

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

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

(44) a copolymer or a pharmacologically acceptable salt corresponding to the position (1), where:

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

Alk represents alkylenes group containing from 1 to 6 ATO is s carbon

R1and R2are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and

R3represents a hydroxyl group, alkoxy group containing from 1 to 6 carbon atoms, which optionally may be substituted by one hydroxy group, or a group described by formula-NR4R5where R4and R5are 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 may be substituted by one hydroxy group;

(45) a copolymer or a pharmacologically acceptable salt corresponding to the position (1), where Alk represents ethylene group, R1represents a hydrogen atom, R2represents a methyl group, and m, R3the ratio between the elementary structural units described by formulas (I) and (II) (the ratio of specified composition) and, where appropriate, the relationship between elementary units described by formula (II), where R3represents a hydroxy group, and the basic units described by formula (II), where R3represents a group other than hydroxy (ratio set by hydrolysis), choose from digislide the future:

(i) m is in the range from 6 to 16, R3represents a hydroxy group, the ratio of the specified 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, R3represents a hydroxy group, the ratio of the specified 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, R3represents the amino group, the ratio of the specified 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, R3is dimethylaminopropyl, the ratio of the specified composition is 1:1, and the average degree of polymerization is in the range from 30 to 40;

(v) m is in the range from 6 to 16, R3represents 1-amino-2-propanolol group, the ratio of the specified composition is 1:1, and the average degree of polymerization is in the range from 30 to 40;

(vi) m is in the range from 6 to 16, R3choose from ethoxy - and hydroxy-groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, and the ratio specified by hydrolysis, 4:6;

(vii) m is in the range from 28 to 16, R3selected from amino and hydroxy groups, the ratio for avemue composition, is 1:1, the average degree of polymerization is in the range from 10 to 15, and the ratio specified by hydrolysis, 4:6;

(viii) m is in the range from 28 to 38, R3is dimethylaminopropyl, the ratio of the specified 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, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, the value specified by hydrolysis, as well 3,1:6,9, and the copolymer is a sodium salt;

(x) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, and the ratio specified by hydrolysis, is equal to 1.4:8,6;

(xi) m is in the range from 6 to 16, R3selected from dimethylamino - and hydroxy-groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, the value specified by hydrolysis, equal to 2.9:7,1, and the copolymer is a sodium salt;

(xii) m is in the range from 6 to 16, R3represents the amino group, the ratio of the specified composition is 1:2,4, and the average degree of polymerization is in the range is from 20 to 30;

(xiii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 0.4:9,6;

(xiv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 2.9:7,1;

(xv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 0.9:9,1;

(xvi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.5 to 9.5;

(xvii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.3:8,7;

(xviii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of sadava is my composition, it is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 1.9:8,1;

(xix) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 1.0:9,0;

(XX) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.8:9,2;

(xxi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, as well 4,6:5,4;

(xxii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio set by the hydrolysis is 1.2:8,8;

(xxiii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified hydro is Isom, is 2.0:8,0;

(xxiv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.1:8,9;

(xxv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 2.4:7,6;

(xxvi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 0.9:9,1;

(xxvii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 1.5:8,5;

(xxviii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.7:9,3;

(xxix) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the group asked composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, as well 4,5:5,5;

(xxx) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.4:8,6;

(xxxi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.7:9,3;

(xxxii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.8:9,2;

(xxxiii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.4:8,6;

(xxxiv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition, is equal to 1:3,1, average degree of polymerization is in the range from 20 to 30, and the ratio of the group asked by hydrolysis, is 0.7:9,3;

(xxxv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 0.9:9,1;

(xxxvi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 1.9:8,1;

(xxxvii) m is in the range from 6 to 16, R3choose from ethoxy - and hydroxy-groups, the ratio of the specified composition is approximately equal to 1:3, the average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, as well 3,1:6,9;

(xxxviii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 9.3 nm or less;

(xxxix) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 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, R3selected from amino and hydroxy groups, is compared, asked composition is 1:1, the ratio specified by hydrolysis, is equal to 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, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 3.1 nm or less;

(xlii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 7.8 nm or less;

(xliii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 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, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 4.7 nm or less.

(46) As mentioned above, another aspect of the present invention provides a copolymer or a pharmacologically acceptable salt, obtained as a result of one or more parts of the anhydride of the carboxylic acids described by formula (III), in the copolymer, which contains as arr is based on its elementary parts

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

where:

m, Alk, R1and R2are defined above, and

(b) the specified structural elementary link that contains a link anhydride carboxylic acids described by formula (III):

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

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

(47) a copolymer or a pharmacologically acceptable salt corresponding to the position (46), where the structural elementary level, described by formula (I), and structural elementary level, described by formula (III)in the copolymer get the configuration as a sequence of alternating "head to head", the sequence of alternating "head to tail" or mixed sequence with alternating "head to head" and "head to tail";

(48) a copolymer or a pharmacologically acceptable salt corresponding to the position (46), where the structural elementary level, described by formula (I), and structural cell battery (included) the container element, described by formula (III), receive the configuration in the form of statistical sequence;

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

(50) a copolymer or a pharmacologically acceptable salt corresponding to the position (49), where Alk represents ethylene group;

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

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

(55) a copolymer or a pharmacologically acceptable salt corresponding to any one position from (46) to (54), where R1represents a hydrogen atom or methyl group;

(56) a copolymer or a pharmacologically acceptable salt corresponding to the position (55), where R1the present is the focus of a hydrogen atom;

(57) a copolymer or a pharmacologically acceptable salt corresponding to any one position from (46) to (56), where R2represents a hydrogen atom or methyl group;

(58) a copolymer or a pharmacologically acceptable salt corresponding to the position (57), where R2represents a methyl group;

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

(60) a copolymer or a pharmacologically acceptable salt corresponding to the position (59), where the ratio between the elementary structural element described by formula (I), and elementary structural units derived from the result of one or more elementary structural units described by formula (III), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) the United States, is in the range from 3:1 to 1:8;

(61) copolym the p or its pharmacologically acceptable salt, the relevant items (59), where the ratio between the elementary structural element described by formula (I), and elementary structural units derived from the result of one or more elementary structural units described by formula (III), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) the United States, 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 corresponding to the position (59), where the ratio between the elementary structural element described by formula (I), and elementary structural units derived from the result of one or more elementary structural units described by formula (III), one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) the United States, is 1:1 or in the range from 1:2 to 1:4;

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

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

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

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

(68) a copolymer or a pharmacologically acceptable salt corresponding to the position (46)where:

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

Alk represents alkylenes group containing from 1 to 6 carbon atoms, and

R1and R2are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms;

(69) a copolymer or a pharmacologically acceptable salt corresponding to any one of the items from (46) to (68), which can be obtained as a result of the ammonolysis to link anhydride carboxylic acids described by formula (III)in the copolymer;

(70) a copolymer or a pharmacologically acceptable salt corresponding to the position (69), which can be obtained in the ammonolysis of using water-ammonia solution;

(71) a copolymer or a pharmacologically acceptable salt, is sootvetstvuyushie any one of the items from (46) to (68), you can get as a result of aminolysis to link anhydride carboxylic acids described by formula (III)in the copolymer;

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

(73) a copolymer or a pharmacologically acceptable salt corresponding to any one of the items from (46) to (68), which can be obtained by carrying out the alcoholysis to link anhydride carboxylic acids described by formula (III)in the copolymer; and

(74) a copolymer or a pharmacologically acceptable salt corresponding to the position (73), which can be obtained by carrying out the alcoholysis with 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 complex, the method of extending the time during which the protein is retained in the blood stream, the method of treatment or prevention of disease and the use of a complex invention for the manufacture of a medicinal product intended for the treatment or prevention of diseases. Preferred examples of the aspects of these inventions included the t:

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

(76) the pharmaceutical composition corresponding to the position (75)where the specified composition further comprises at least one protein or an analogue or variant;

(77) the pharmaceutical composition corresponding to the position (76)where the protein or an analogue or variant represent the main protein;

(78) the pharmaceutical composition corresponding to the position (77), where the main protein is a basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), factor inhibiting osteoclastogenesis (OCIF), platelet-derived growth factor (PDGF), isolated from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepaticotomy growth factor (HGF) or growth factor vascular endothelial (VEGF) or their analogues or variants;

(79) the pharmaceutical composition corresponding to the position (77), where the main protein is a factor inhibiting osteoclastogenesis (OCIF or analogue or variant;

(80) the pharmaceutical composition corresponding to the position (79)where the specified OCIF or analogue or variant include what I OCIF natural type or recombinant type;

(81) the pharmaceutical composition corresponding to the position (79)where the specified OCIF or analogue or variant represents a monomer or dimer;

(82) the pharmaceutical composition corresponding to the position (79)where the specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE (polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate) in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions;

(83) the pharmaceutical composition corresponding to the position (79)where the specified OCIF contains amino acids from 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(84) the pharmaceutical composition corresponding to the position (79)where the specified OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(85) a modifier capable of modifying a protein or an analogue or variant, when the specified modifier contains a copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (74);

(86) a modifier capable of modifying a protein or an analogue or variant corresponding positions and (85), where protein is the main protein;

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

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

(89) a modifier capable of modifying a protein or an analogue or variant corresponding to the position (88)where the specified OCIF or analogue or variant relate to OCIF natural type or recombinant type;

(90) a modifier capable of modifying a protein or an analogue or variant corresponding to the position (88)where the specified OCIF or analogue or variant represents a monomer or dimer;

(91) a modifier capable of modifying a protein or an analogue or variant corresponding to the position (88)where the specified OCIF is a monomer OCI person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions;

(92) a modifier capable of modifying a protein or an analogue or variant corresponding to the position (88)where the specified 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 an analogue or variant corresponding to the position (88)where the specified OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

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

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

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

(97) complex corresponding to the position (95), where the main protein is a factor inhibiting osteoclastogenesis (OCIF or analogue or variant;

(98) complex corresponding to the position (97)where the specified OCIF or analogue or variant relate to OCIF natural type or recombinant type;

(99) complex corresponding to the position (97)where the specified OCIF or analogue or variant represents a monomer or dimer;

(100) complex corresponding to the position (97)where the specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions;

(101) complex corresponding to the position (97)where the specified OCIF contains amino acids from 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(102) complex corresponding to the position (97)where the specified OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

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

(104) the way of extension of time within which a protein or an analogue or variant are retained in the bloodstream after ingestion by the patient as the result of complex formation between the specified protein or an analogue or variant and at least one copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (74);

(105) the method corresponding to the position (104), where the protein is the main protein;

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

(107) the method corresponding to the position (105), where the main protein is a factor inhibiting osteoclastogenesis (OCIF or analogue or variant;

(108) the way that meet the speaker position (107), where specified OCIF or analogue or variant relate to OCIF natural type or recombinant type;

(109) the method corresponding to the position (107)where the specified OCIF or analogue or variant represents a monomer or dimer;

(110) the method corresponding to the position (107)where the specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions;

(111) the method corresponding to the position (107)where the specified OCIF contains amino acids from 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

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

(113) a method of treatment or prevention of disease in a patient that is susceptible to the action of the protein or its analogue or variant, including reception of the specified patient an effective amount of the complex that contains the specified protein or an analogue or variant that bind at least one copolymer or a pharmacologically acceptable salt corresponding to any of the ne position from (1) to (74);

(114) the method corresponding to the position (113), where the protein is the main protein;

(115) the way corresponding to the position (114), where the main protein is a basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), factor inhibiting osteoclastogenesis (OCIF), platelet-derived growth factor (PDGF), isolated from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepaticotomy growth factor (HGF) or growth factor vascular endothelial (VEGF) or their analogues or variants;

(116) the method corresponding to the position (114), where the main protein is a factor inhibiting osteoclastogenesis (OCIF or analogue or variant;

(117) the pharmaceutical composition corresponding to the position (116)where the specified OCIF or analogue or variant relate to OCIF natural type or recombinant type;

(118) method corresponding to the position (116)where the specified OCIF or analogue or variant represents a monomer or dimer;

(119) the method corresponding to the position (116)where the specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured what Ecodom SDS-PAGE in non conditions;

(120) the method corresponding to the position (116)where the specified OCIF contains amino acids from 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(121) the method corresponding to the position (116)where the specified OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(122) a method that meets any one position from (116) $ (121), where the specified disease is a disorder of metabolism in the bones;

(123) the use of a complex containing protein or an analogue or variant which is associated at least one copolymer or a pharmacologically acceptable salt corresponding to any one of items (1) to (74), for manufacturing a medicinal product intended for the prevention or treatment of diseases susceptible to the action of the protein or its analogue or variant;

(124) the application corresponding to the position (122), where protein is the main protein;

(125) the application corresponding to the position (123), where the main protein is a basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), factor inhibiting osteoclastogenesis (OCIF), platelet-derived growth factor (PDGF), isolated from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepaticotomy growth factor (HGF) or facto the vascular endothelial growth (VEGF) or their analogues or variants;

(126) the application corresponding to the position (123), where the main protein is a factor inhibiting osteoclastogenesis (OCIF or analogue or variant;

(127) the application corresponding to the position (126)where the specified OCIF or analogue or variant relate to OCIF natural type or recombinant type;

(128) the application corresponding to the position (126)where the specified OCIF or analogue or variant represents a monomer or dimer;

(129) the application corresponding to the position (126)where the specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions;

(130) the application corresponding to the position (126)where the specified OCIF contains amino acids from 21 to + 380 of the sequence SEQ ID No. No. 1 list of sequences;

(131) the application corresponding to the position (126)where the specified OCIF contains amino acids + 1 to + 380 of the sequence SEQ ID No. No. 1 list of sequences; and

(132) the application corresponding to any one position from (126) $ (131), where the specified disease is a disorder of metabolism in the bones.

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

The alkyl group in the alkyl group containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below And in the definition of the substituents R1, R2, R4, R5and substituents in the above formulas (I) and (II) is a straight or branched alkyl group containing from 1 to 6 atoms angle of the ode, such as methyl, ethyl, n-sawn, ISO-propyl, n-bucilina, isobutylene, second-bucilina, tert-bucilina, n-pencilina, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-exilda, 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 the data alkyl groups, preferred are straight or branched alkyl group containing from 1 to 4 carbon atoms, more preferred are methyl and ethyl groups, and most preferred is methyl group.

The alkoxy group in the alkoxy group containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below And in the definition of substituent R3and substituents in the above formulas (I) and (II) is a Deputy, in which the above alkyl group containing from 1 to 6 atoms angle of the ode, associated with the oxygen atom. Examples of such alkoxy groups include straight or branched alkoxy group containing from 1 to 6 carbon atoms, such as methoxy, ethoxy-, n-propoxy, isopropoxy, h-butoxy, isobutoxy-, second -, butoxy-, tert-butoxy-, n-pentyloxy, isopentylamine-, 2-methylbutoxy, neopentylene, h-hexyloxy-, 4-methylpentane-, 3-methylpentane-, 2-methylpentane-, 3,3-dimethylbutene-, 2,2-Dimethylbutane-, 1,1-Dimethylbutane-, 1,2-Dimethylbutane-, 1,3-Dimethylbutane - and 2,3-Dimethylbutane group. Among the data alkoxy groups, more preferred are straight or branched alkoxy group containing from 1 to 4 carbon atoms, and most preferred is ethoxy-group.

"Halogen atom", which is one of the above-mentioned "substituents A, represents optional substituent in the "alkyl group containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below And in the definition of the substituents R1, R2, R4and R5in the above formula is x (I) and (II) represents an optional substituent in the "alkoxy group, containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below And in the definition of substituent R3in the above formula (II)represents a fluorine atom, a chlorine atom, a bromine atom or an iodine atom; and preferably a fluorine atom or a chlorine atom.

"Aryl group containing from 6 to 14 carbon atoms, which represents an optional substituent in the "alkyl group containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, 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 the substituents in an amount of from 1 to 5, selected from the substituents defined below And in the definition of the substituents R1, R2, R4and R5and an optional substituent in the "alkoxy group containing from 1 to 6 carbon atoms, which optionally may be substituted by at least one Deputy, selected from GRU the dust, consisting of hydroxy groups, halogen atoms and aryl groups containing from 6 to 14 carbon atoms" in the definition of substituent R3in the above formula (II)represents an aromatic hydrocarbon group containing from 6 to 14 carbon atoms, and may be, for example, phenyl, indenolol, naftilos, phenanthroline or antilley group. Preferably it is phenyl group.

"Aryloxy-group containing from 6 to 14 carbon atoms, which optionally may be substituted by substituents in an amount of from 1 to 5 substituents selected from A," in the definition of substituent R3in the above formula (II) is an aryl group as defined above, which is associated with the oxygen atom, and it may be, for example, phenoxy, inderalici, naphthyloxy, phenantroline or antioxi group. Preferably it is phenoxy-group.

"An alkyl group containing from 1 to 6 carbon atoms, which is optionally substituted by at least one halogen atom" in the definition of the substituents R1, R2, R4and R5in the above formulas (I) and (II) represents an alkyl group containing from 1 to 6 carbon atoms, as described above, and may be, for example, triptorelin group, trichlorethylene group, deformation the th group, dichloromethylene group, dibromoethylene group, permatile group, 2,2,2-triptorelin group, 2,2,2-trichlorethylene group, 2-bromatology group, 2-chloraniline group, 2-foretelling group, 2-itatelnou group, 3-chloropropylene group, 4-terbutaline group, 6-idexilu group, 2,2-dibromoethylene group or panafcortelone group. Preferably it is triptorelin group, trichlorethylene group, deformational group or panafcortelone group; and most preferably it is triptorelin group.

Examples of "alkyl group containing from 1 to 6 carbon atoms, which is optionally substituted by at least one hydroxy group" in the definition of the substituents R1, R4and R5in the above formulas (I) and (II) include hydroxymethylene group, 1-hydroxyethylene 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 definition of substituent R3in the above formula (II) represents an alkoxy group containing from 1 to 6 carbon atoms described above, which is substituted by at least one halogen atom described above, and may be, for example, triptime the C-group, trichlormethane group, deformedarse group, dichloromethoxy group, dibromethane group, formatosi group, 2,2,2-triptoreline group, 2,2,2-trichloroethane group, 2-bromoethoxy group, 2-chloroethoxy group, 2-floratone group, 2-iodoxy group, 3-chloropropoxy group, 4-forbooks-group, 6-itemselect group, 2,2-dibromethane group or pentaborate group; preferably it is C1-C4alkoxy group substituted by fluorine atoms or chlorine, such as triptoreline group, trichlormethane group, deformedarse group or pentaborate group. More preferably it is triptoreline-group.

Examples of "alkoxy group containing from 1 to 6 carbon atoms, which is optionally substituted by at least one hydroxy group" in the definition of substituent R3in the above formula (II) include hydroxyethoxy group, 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 may be substituted by substituents in an amount of from 1 to 5 substituents selected from A," in the definition of the substituents R1, R2, R4and R5in p iudenich above formulas (I) and (II) may be, for example, benzyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, intermational group, 1-fenetylline group, 2-fenetylline group, 1-naphthylethylene group, 2-naphthylamine group, 1-phenylpropanol group, 2-phenylpropanol group, 3-phenylpropene group, 1-naphtylamine group, 2-naphtylamine group, 3-afterproperties group, 1-phenylbutyl group, 2-phenylbutyl group, 3-phenylbutyl group, 4-phenylbutyl group, 1-nativesignal group, 2-nativesignal group, 3-nativesignal group, 4-nativesignal group, 1-phenylmethylene group, 2-phenylmethylene group, 3-phenylmethylene group, 4-phenylmethylene group, 5-phenylpentane group, 1-phenylhexane group, 2-phenylhexane group, 3-phenylhexane group, 4-phenylhexanoic group, 5-phenylhexanoic group or 6-phenylhexane group; preferably it is an alkyl group, substituted aryl group containing from 6 to 10 carbon atoms, such as benzyl group, 1-naphthylmethyl group, 2-naphthylmethyl group, 1-penicilina group, 2-penicilina group, 1-naphthylethylene group, 2-naphthylethylene group, 1-phenylpropionate group, 2-phenylpropionate group, 3-phenylpropionate group or 1-naphtylamine group; and more preferably it is a benzo is supplemented flax group.

"Alkoxy 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 may be substituted by substituents in an amount of from 1 to 5 substituents selected from A," in the definition of substituent R3in the above formula (II) can be, for example, benzoxa group, 1-aftermatket group, 2-aftermarket group, intermetalic group, 1-venetucci group, 2-venetucci group, 1-naphthylacetic group, 2-naphthylacetic group, 1-phenylpropoxy group, 2-phenylpropoxy-group, 3-phenylpropoxy group, 1-naftemporiki group, 2-naftemporiki-group, 3-naftemporiki group, 1-phenylmethoxy group, 2-phenylmethoxy-group, 3-phenylmethoxy group, 4-phenylmethoxy group, 1-natterbox group, 2-natterbox-group, 3-natterbox group, 4-natterbox group, 1-phenylmethoxy group, 2-phenylmethoxy-group, 3-phenylmethoxy group, 4-phenylmethoxy-group, 5-phenylmethoxy group, 1-phenylhexane group, 2-phenylhexane-group, 3-phenylhexane group, 4-phenylhexanoic-group, 5-phenylhexanoic-group or 6 phenylhexanoic group; preferably it is an alkyl group, substituted aryl group containing from 6 to 10 carbon atoms, such as benzoxa group, 1-aftermatket group, 2-naphthas is metoxy group, 1 venetucci group, 2-venetucci group, 1-naphthylacetic group, 2-naphthylacetic group, 1-phenylpropoxy group, 2-phenylpropoxy-group, 3-phenylpropoxy-group or 1-naftemporiki group; and more preferably it is benzoxa-group.

Where R3is a "aryloxy-group containing from 6 to 14 carbon atoms, which optionally may be substituted by substituents in an amount of from 1 to 5 substituents selected from As," they preferably is aryloxy-group containing from 6 to 10 carbon atoms, which is optionally substituted by substituents in an amount of from 1 to 3, selected from the group of substituents; more preferably it is phenoxy-group, which is optionally substituted by substituents in an amount of from 1 to 3, selected from the group of substituents; more preferably it is phenoxy group, optionally substituted by 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 phenoxy-group or p-nitrophenoxy-group.

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

Preferred examples of the pharmacologically acceptable salts obtained by the content in the copolymers of the present invention of the basic group include salts of inorganic acids, such as salts of halogen acids (for example, hydrochloride, hydrobromide and hydroiodide), nitrates, perchlorates, sulfates and phosphates; organic acid salts such as lower alkanesulphonic, in which the lower alkyl chain is a defined above (for example, methanesulfonate, triftoratsetata and econsultancy), arylsulfonate, in which aryl chain is a defined above (for example, bansilalpet or p-toluensulfonate), acetates, malaty, fumarate, succinate, citrates, ascorbates, tartratami, oxalates and maleate; and salts of amino acids such as salts of glycine, lysine salt, arginine salt, ornithine salts, glutaminate and aspartate. Particularly preferred are salts of halogen acids.

Preferred examples of the pharmacologically acceptable salts obtained by the content in the copolymers infusion is his invention of the acid carboxyl group, include metal salts, such as alkali metal salts (e.g. sodium salts, potassium salts and lithium salts), salts of alkaline earth metals (e.g. calcium salts and magnesium salts), salts of metals, such as aluminum salts, iron salts, zinc salts, salts of copper, Nickel salts and cobalt salts; salts of amines, such as inorganic salts of amines (e.g., ammonium salts) and organic salts of amines (for example, salts of tert-octylamine, salt dibenzylamine, salt of the research, glucosamine salt, salt phenylglycylamino of ester, salt of ethylene diamine, salt, N-methylglucamine salt of guanidine, salts diethylamine, salt, triethylamine salt dicyclohexylamine, salts of N,N'-dibenziletilendiaminom, salt chloroprocaine, salts of procaine, salt, diethanolamine, salts of N-benzylpenicillin, salt, piperazine salt of Tetramethylammonium and salt of Tris(hydroxymethyl)aminomethane; and salts of amino acids such as salts of glycine, lysine salt, arginine salt, ornithine salts, glutaminate and aspartate. Particularly preferred are alkali metal salts and salts of alkaline earth metals.

In the present invention "elementary structural link" is defined as the minimum basic element constituting the copolymer of the invention, and it is portrayed in the determination of the above copolymer as the elementary level, describes the shape of the Oh (I) or elementary level, described by formula (II). "Elementary structural link" is not a structure of Monomeric substances are used in the polymerization reaction in the synthesis of the copolymer of the present invention; more specifically, it is an elementary level, which is obtained from the specified monomer initial substance and which is present in the specified copolymer of the present invention.

In the present invention, the phrase "sequence "head to head"" indicates that elementary structural units described by formulas (I) and (II)obtain the configuration shown in the following formula:

In the present invention, the phrase "sequence "head to tail" means that for elementary structural units described by formulas (I) and (II)obtain the configuration shown in the following formula:

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

In the present invention the ratio of the specified composition,represents the average ratio between the number of elementary structural units described by formula (I), and the number of elementary structural units described by formula (II), the copolymer of the present invention. If the copolymer is essentially alternating copolymer, then the ratio of the specified composition is 1:1. However, if the copolymer is a statistical copolymer, this ratio may vary. In the copolymers of the present invention to the specified value special limitation is imposed, 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 1:2 up to 1:6, and it is most preferable or equal to 1:1, or is in the range from 1:2 to 1:4. It should be noted that the values of the ratio defined by composition, inevitably, to some extent, vary, which is caused by the presence of minor changes in the original substances, the conditions of polymerization and the like. In the above correlation, asked composition, are approximate values; variations for the values of the ratios set by the composition of the above quantity up to ±30% are still considered to fall within the above ratios.

The ratio of the specified composition, for the copolymers of the present invention can be determined using known analytical techniques. By determining the content of carboxyl groups in the copolymer (mmol/g) by the method conductometric titration [definition, which requires receipt of the fully hydrolyzed copolymer (i.e., R3is a HE) or as a result of hydrolysis of the copolymer being analysed, or by an independent synthesis of the corresponding hydrolyzed copolymer], and from a knowledge of the formula mass for each of the elementary structural units of the specified copolymer, it is possible to determine the ratio of the specified composition, use what Itanium following formula:

Rii/Ri=(C×FWi)/(2000-C×FWii),

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

In General the molecular weight of the polymer determine the relative molecular mass in bringing to the molecular mass standard connection, which includes a structure similar to that of the specified polymer, and is characterized by a known value of the absolute molecular weight. This method of assessment is often used when determining the molecular weight of new copolymers, such as 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 polymer compound characterized by a known absolute molecular weight. The nature of the gel, elution conditions and the polymer with known absolute molecular weight, used for comparison, a specialist in the relevant field can allegedin way to choose using known techniques and publicly available information, for example, see "Comprehensive Polymer Science", pub. Pergamon Press (Oxford) 1989. The standard connection used for comparison purposes, is preferably a polymer having similar structure and properties. In the case of the copolymers of the present invention the area of the side chains is seen as a characteristic structure. Therefore, the standard connection used for comparison, is preferably a polymer including a structure similar to that of the area of the side chains of the copolymer of the present invention, the average molecular weight which you want to measure. Used standard connection preferably is poly(ethylene glycol).

In the present invention, the average degree of polymerization" represents the average degree of polymerization of the elementary structural units in the copolymer of the present invention, that is, it is the average number of elementary structural units in the specified copolymer. It is determined on the basis of the average molecular weight of the copolymer, the formula mass of each of the elementary structural units of the specified copolymer and the ratio of specified composition for elementary structural units in the specified copolymer. "The average degree of polymerization" copolymer of the present invention containing a structural element of the turning parts I and II, can be calculated using the following formula:

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

where MC is the average molecular weight of the copolymer, FWi and FWii represent the formula mass for each of the elementary structural units I and II, respectively, and Ri and Rii are the share of elementary structural units I and II in the copolymer calculated from the ratio of the specified composition (the ratio of specified composition=Ri:Rii; Ri+Rii=1).

In the above formula to determine the average degree of polymerization of the copolymer of the present invention, all parameters, such as the value of the specified composition for elementary structural units, the average molecular weight of the copolymer of formula masses of elementary structural units, can be defined as discussed above. In order to do this, you must obtain the appropriate copolymer subjected to complete hydrolysis, ammonolysis, aminals or alcoholysis (preferably corresponding copolymer subjected to complete hydrolysis). Alternatively, if the original copolymer used in the preparation of the copolymer of the present invention, is characterized by a known composition of these values can be determined without proper analysis. The average grade is polymerization in the copolymers of the present invention especial limitation is imposed. Usually it is in the range from 5 to 200; preferably it is in the range from 5 to 50; more preferably from 5 to 20 or 20 to 30 and from 30 to 40.

In the copolymers of the present invention elementary structural units described by formula (II)may include at least one structural elementary link described by the formula (II), where R3represents an optionally substituted alkoxy group containing from 1 to 6 carbon atoms, optionally substituted, aryloxy group or a group described by formula-NR4R5and optionally at least one structural elementary link described by the formula (II)in which R3represents a hydroxyl group. The ratio between the elementary structural units described by formula (II)in which R3represents a hydroxy group, and elementary structural units described by formula (II)in which R3represents an optionally substituted alkoxy group, optionally substituted, aryloxy group or a group described by formula-NR4R5, called the value set by hydrolysis. By determining the content of carboxyl groups in the copolymer (mmol/g) by the method conductometric titration and from a knowledge of the formula mass for each of the structural is elementarnykh links of the specified copolymer ratio, asked composition (see above), we can determine the value specified by hydrolysis, E.g. using the following formula:

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

where N:represents the ratio specified by hydrolysis, Ri:Rii represents the ratio of defined composition, represents the level of the content of carboxyl groups in the copolymer (mmol/g), FWi represents the formula weight of structural elementary level (I), FWii is a formula mass of elementary structural level (II), where R3is a HE, and FW(A) (ii represents the formula weight of structural elementary level (II), where R3represents an optionally substituted alkoxy group, optionally substituted, aryloxy group or a group described by formula-NR4R5.

Preferred ranges for the ratio specified 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 1:9 to 0:10. It should be noted that the values of the ratio set by hydrolysis, inevitably, to some extent, vary, which is caused by the presence of minor changes in the original substances, the river is high the reaction, and the like. In the ratio specified by the hydrolysis described in this application are approximate values; variations for the values of the ratios set by the hydrolysis described in this application, a value of up to ±30% are still considered to fall within the above ratios.

The size of the molecules of the copolymers of the invention can be measured using known analytical techniques such as size-exclusion chromatography size, using as standards of proteins with known size of the molecule (the following examples 15 and 16 offer examples of the application of such techniques). Using exclusion chromatography size the size of the molecule of the copolymer obtained as its Stokes radius. In the present invention on the size of the molecules of the copolymers special limitation is imposed. 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 is in the range from 3.1 to 6.2 nm or in the range from 1.5 to 4.7 nm.

Copolymers and their pharmacologically acceptable salts of the present invention can be obtained by carrying out a suitable reaction copolymerization known specialistov the corresponding region [see, for example, "Comprehensive Polymer Science" published by Pergamon Press (Oxford) in 1989], using the original monomers described by the following formulas (IV) and (V):

(where m, Alk and R1, R2and R3are 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 subjected to fractionation using gel-filtration chromatography.

The monomers state of the art are known, or they can be obtained in accordance with the method, a well-known specialist in the field [see, e.g., J. M. Harris, "Laboratory synthesis of polyethylene glycol derivatives", Rev. Macromol. Chem. Phys. C25, 326-373 (1985) and Japanese patent No. 2621308]. The monomer described by formula (V)can be easily obtained by conducting to maleic anhydride of one or more reactions selected from the group consisting of (a) hydrolysis, (b) ammonolysis, (C) aminolysis and (d) of the United States.

In an alternative embodiment, the copolymer or its pharmacologically acceptable salt can be obtained as a result of one or more parts of the anhydride of the carboxylic acids described by formula (III), in the copolymer, which contains as a form of elementary links

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

where:

m, Alk, R1and R2are defined above, and

(b) the specified structural elementary link that contains a link anhydride carboxylic acids described by formula (III):

one or more reactions selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) the alcoholysis. Source copolymers containing basic units described by formulas (I) and (III), or a well-known state of the art (for example, copolymers such as S-K and AM CH, can be purchased in the company NOF Corporation), or they can be obtained in accordance with the method, a well-known specialist in the relevant field [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, the publication of Japanese patent application No. 2003-105040 and the publication of Japanese patent application No. 2003-105003].

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

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

In the present invention "hydrolysis" refers to the reaction to the disclosure of the loop for link anhydride carboxylic acids described by formula (III), under the action of water with obtaining structural elementary level, described by formula (II), where R3represents a hydroxy group. On the nature of the actual hydrolysis particular limitation is imposed up until it comes to the method commonly used by specialists in the respective field.

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

Used reagent in a suitable case is water. If water is used as solvent, then adding an additional amount of water is not necessary. In addition, for the purpose of accelerating the reaction, you can add the basis of the definition. Examples of such bases include: inorganic bases, such as carbonates of alkali metals (e.g. sodium carbonate, potassium carbonate and lithium carbonate), bicarbonates of alkali metals (e.g. sodium bicarbonate, potassium bicarbonate and lithium bicarbonate), hydrides of alkali metals (e.g. lithium hydride, sodium hydride and potassium hydride), hydroxides of alkali metals (e.g. sodium hydroxide, potassium hydroxide, barium hydroxide and lithium hydroxide), alkali metal fluorides (for example, sodium fluoride and potassium fluoride; and organic bases such as alkoxides of alkali metals (for example, sodium methoxide, ethoxide sodium, potassium methoxide, ethoxide potassium tert-piperonyl potassium and lithium methoxide), N-methylmorpholine, triethylamine, Tripropylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinedione, 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 preferred bases are organic bases, and most preferred is pyridine. In this regard it should be noted that when the hydrolysis reaction proceeds satisfactorily in the absence of the basis of the Oia, in addition the Foundation is not necessary.

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

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

After completion of the reaction, the desired compound hydrolysis reaction can be isolated from the reaction mixture using conventional method known to the expert in the relevant field. For example, the resulting reaction mixture can be condensed using ultrafiltration membrane, and then subjected to freeze drying to obtain the target compound reactions. Alternatively, the desired compound can be in the form of a solution, without highlighting it when it needs to be used to modify proteins.

Thus, the desired compound can optionally be subjected to additional purification using conventional techniques, such as gel-filtration chromatography. To obtain a connection across which the student specific desired average degree of polymerization or desirable molecular weight, subjected to cleaning compound can be further fractionate using gel-filtration chromatography.

In the present invention the ammonolysis" refers to the reaction to the disclosure of the loop for link anhydride carboxylic acids described by formula (III), under the action of ammonia with getting the link described by the formula (II), where R3represents an amino group. On the nature of the actual reaction of ammonolysis particular limitation is imposed up until it comes to the method commonly used by specialists in the respective field.

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

Examples suitable for use reagents include gaseous ammonia and aqueous ammonia solution, and the preferred ammonia.

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

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

After completion of the reaction, the desired compound ammonolysis reaction can be isolated from the reaction mixture using conventional method known to the expert in the relevant field. For selection of the target compounds resulting reaction mixture can, for example, be treated as follows: (1) dialysis through a semipermeable membrane with an acid, such as aqueous solution of acetic acid to remove excess ammonia (dialysis is carried out in such conditions under which the solution of the reaction mixture does not become acidic, adding water as needed) followed by condensation with the use of the ultrafiltration membrane, and then freeze-drying the thus obtained condensate; or (2) the addition of an aqueous sodium hydroxide solution and an immiscible organic solvent, such as diethyl ether, shaking the resulting mixture (shaking can be two or more than two times as n is donosti), and after that selected the aqueous layer containing the target compound is subjected to freeze drying to obtain the target compounds. Alternatively, the desired compound can be in the form of a solution, without highlighting it when it needs to be used to modify proteins.

Thus, the desired compound can optionally be subjected to additional purification using conventional techniques, such as gel-filtration chromatography. For connection, characterized by a particular desired average degree of polymerization or desirable molecular weight, subjected to cleaning compound can be further fractionate using gel-filtration chromatography.

It should be noted that the target connection ammonolysis reaction may contain elementary structural units described by formula (II), where R3represents a hydroxy group by hydrolysis of some elementary units described by formula (III), in the initial compound under the action of water present in the solvent or reagent. It should also be noted that to improve storage to the target compound can be added to the base.

In the present invention "aminals" refers to a reaction R is hiding cycle for link anhydride carboxylic acid, described by formula (III), under the action of an amine with getting the link described by the formula (II), where R3represents a group described by formula-NR4R5where R4and R5are defined above. On the nature of the actual reaction aminolysis particular limitation is imposed up until it comes to the method commonly used by specialists in the respective field.

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

Used reagent obviously depends on the nature of the target groups described by the formula-NR4R5. 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-hydroxyisopropyl and their aqueous solutions. In number Yes the different reagents are preferred reagents include an aqueous solution of dimethylamine and 1-amino-2-propanol; the most preferred aqueous solution of dimethylamine.

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

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

After completion of the reaction, the desired compound reaction aminolysis can be isolated from the reaction mixture using conventional method known to the expert in the relevant field. For selection of the target compounds resulting reaction mixture can, for example, be treated as follows: (1) dialysis through a semipermeable membrane with an acid, such as aqueous solution of acetic acid to remove excess ammonia (dialysis is carried out in such conditions under which the solution of the reaction mixture does not become acidic, adding water as needed) followed by condensation with the use of the ultrafiltration membrane, and then freeze-drying the thus obtained condensate; or (2) adding an aqueous solution of hydroxide NAT the Oia and the immiscible organic solvent, such as diethyl ether, shaking the resulting mixture (shaking can be two or more than two times as needed), and then selected the aqueous layer containing the target compound is subjected to freeze drying to obtain the target compounds. Alternatively, the desired compound can be in the form of a solution, without highlighting it when it needs to be used to modify proteins.

Thus, the desired compound can optionally be subjected to additional purification using conventional techniques, such as gel-filtration chromatography. For connection, characterized by a particular desired average degree of polymerization or desirable molecular weight, subjected to cleaning compound can be further fractionate using gel-filtration chromatography.

It should be noted that the target connection response aminolysis may contain elementary structural units described by formula (II), where R3represents a hydroxy group by hydrolysis of some elementary units described by formula (III), in the initial compound under the action of water present in the solvent or reagent. You tacheometry, what to improve storage to the target compound can be added to the base.

In the present invention "alcoholism" refers to a reaction to the disclosure of the loop for link anhydride carboxylic acids described by formula (III), under the influence of alcohol or kilowog alcohol with getting the link described by the formula (II), where R3represents alkoxy or aryloxy group defined above. On the nature of the actual reaction of alcoholysis particular limitation is imposed up until it comes to the method commonly used by specialists in the respective 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 hexamethylphosphorotriamide. Among these solvents, preferred is water. It should be noted that the reagent can also be used as a solvent.

Used reagent obviously depends on the nature of the target alkoxy - or aryloxy group. Typical examples of the alcohol used to obtain the desired alkoxy group include methanol, ethanol, ethylene glycol, n-propanol, propylene glycol, 2-hydroxy-n-propanol and isopropanol and aqueous solutions, while typical examples of the alcohol used DL is getting desirable aryloxy group, include phenol and p-NITROPHENOL. Among these reagents are preferred is ethanol.

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

The reaction time varies depending on the reaction temperature, the starting compound, reagent and type of solvent used, but it usually is in the range from 10 minutes to 3 days, and preferably in the range of from 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 conventional method known to the expert in the relevant field. For example, for removal of excess alcohol could to carry out condensation reaction mixture by using ultrafiltration membranes, addition of water, and after that additional condensation of a mixture of two or more than two times using ultrafiltration membrane, and the resulting condensate is subjected to freeze drying to obtain the target compound. Alternatively, the desired compound can be in the form of a solution, without highlighting it when it needs to be used to modify proteins.

Thus, the received desired connection can optionally be subjected to additional purification using conventional techniques, such as, for example, the use of gel-filtration column. For connection, characterized by a particular desired average degree of polymerization or desirable molecular weight, subjected to cleaning compound can be further fractionate using gel-filtration chromatography.

It should be noted that the target connection reaction of alcoholysis may contain elementary structural units described by formula (II), where R3represents a hydroxy group by hydrolysis of some elementary units described by formula (III), in the initial compound under the action of water present in the solvent or reagent. It should also be noted that to improve storage to the target compound can be added to the base.

Then, when the desired copolymer of the present invention is pharmacologically acceptable salt, a method of obtaining this special salt is no limit to until it comes to the method commonly used by specialists in the relevant field. For example, a pharmacologically acceptable salt can be obtained by dissolving the copolymer of the invention in an organic solvent, adding to the thus obtained solution of the base and then collecting the resulting in achiev is Tata salt, which precipitates.

To regulate the average molecular weight, the share configuration with striping and parity of specified composition, for copolymers of the present invention may use a wide set of techniques.

It is well known that the monomer is maleic anhydride has a tendency to polymerization interleaved with comonomers, including polyoxyethylenesorbitan simple fluids [e.g., 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 configuration of the polymer specialist in the relevant field can be adjusted (for example, see T. Ohtsu and M. Kinoshita, Koubunshi Gousei no Jikkenhou pp. 125-154, Called-Dojin 1972). In addition, despite the fact that maleic anhydride has a tendency to polymerization interleaved with comonomers, specialists in the relevant field is well known for the possibility that the value specified composition, proportion, formed in the copolymer elementary parts of maleic anhydride, will exceed 50% (mol.) (Japanese patent№ 2621308, № 2701295, № 2803265, № 3271265, № 3035675 and no 3106265 and publication of Japanese patent applications No. 2003-105003 and No. 2003-105040).

In the General case for polymers with high molecular weight and a high degree of configuration che what adowanie polymerization should be carried out in mild conditions, such as low temperature and low concentration of initiator. Increased monomer concentration and decreased concentration of the solvent, which reduces the relative concentration of the initiator, as a result result in increased molecular weight and increased share configuration interleaved.

Alternatively, to obtain polymers with low molecular weight and low degree of configuration with alternating favorable high temperature, high concentration of initiator, low monomer concentration and high concentration of solvent. In addition, when these relatively harsh conditions of polymerization and/or in the presence of maleic anhydride in excess of 50% (mol.) from the total number of monomers in the mixture of monomers in the ratio specified composition, the resulting copolymer, the proportion of elementary parts of maleic anhydride may exceed 50% (mol.).

Some examples of the polymerization conditions for polyoxyethyleneglycol simple diapir and maleic anhydride are described in T. Yoshimoto et al., Biochemical Biophysical Research Communication, Vol. 148, 876-882 (1987). In accordance with this article was stirred polyoxyethyleneglycol simple fluids, maleic anhydride, toluene and benzoyl peroxide (initiator) and conducted the floor is merisalu boiling under reflux at 80° With over 7 hours. The resulting polymer was characterized by a molecular weight equal to 13 kDa, and had 8 of the PEG chains and 8 elementary parts of maleic anhydride. Configuration with alternating for copolymer was proposed based on the fact that the copolymer was characterized by the ratio specified by the composition of 1:1, and that the monomer is maleic anhydride tends to be copolymerized with comonomers interleaved. If you will require a copolymer characterized by a low molecular weight and low degree of configuration with alternating, then you will need to use the increased concentration of the initiator, a lower monomer concentration and/or increased concentration of the solvent. For example, if the solvent in the polymerization using toluene, and polymerization is carried out at 1 atmosphere, then the polymerization can be conducted at temperatures up to its boiling point equal to 110°C. in Addition, in the case of another solvent, the boiling temperature which exceeds the boiling point of toluene is preferred elevated temperature. In such cases appropriately you will need to select the type of initiator, since each initiator is characterized by its own specific rate constant of decomposition, and when the data increases the R temperatures will decompose certain initiators. State of the art already known to many initiators with different speeds decomposition [e.g., see Polymer Handbook, Third Edition, pp. II/1-II/65, Ed. by J. Brandrup and E. Immergut, John Wiley & Sons (1989)].

In other documents of the prior art, such as the publication of Japanese patent application No. 2003-105003 also describes alternative solvents, initiators and reaction conditions, leading to changes in the balance of defined composition, molecular weight and share the configuration with the alternation in the copolymerization reactions polyoxyethyleneglycol simple diapir and maleic anhydride. Xylene, for example, has a higher boiling point at 1 atmosphere (140° (C) in comparison with toluene. It is also known that in some cases, the copolymerization reaction can be conducted without using any solvent that removes the restriction on the polymerization temperature defined by the boiling point of the solvent. Also describes and various alternative initiators, such as benzoyl peroxide, di-tert-butylperoxide and tert-butylperoxyisopropyl and azo-initiators, such as azobisisobutyronitrile. To reduce the average molecular weight of the copolymer can also result from the use of reagents transfer chain.

If the ratio defined by the composition, the copolymer is required, the proportion of elementary parts of maleic anhydride in the total number of Monomeric elementary units, largest exceeding 50%, then in the mixture of monomers will need to use a high percentage of monomer of maleic anhydride with or without the use of such relatively hard conditions of polymerization.

When you get a pharmaceutically active ingredient with the use of the copolymer of the present invention or its pharmacologically acceptable salt and the protein or its analogue or variant, the ratio between the copolymer and protein or an analogue or variant of the special limitation is imposed up until the resulting complex will be characterized desired pharmaceutical activity. Typically, the mass ratio of the copolymer of the present invention or its pharmacologically acceptable salt and a protein or an 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 the practical application of pharmaceutically active ingredient containing copolymer of the present invention or its pharmacologically acceptable salt and a protein or an analogue or variant, usually obtained in the form of a solution of protein-containing copolymer, or p is dergoth lyophilized solution, containing copolymer of the present invention or its pharmacologically acceptable salt and a protein or an analogue or variant. In the latter case, subjected to a lyophilized form of the active ingredient is dissolved just before it will need to be used. Alternatively, the pharmaceutically active ingredient can be obtained in the form of a kit. In this case, the copolymer of the present invention or its pharmacologically acceptable salt and a protein or an analogue or variant stored in different containers and then mixed with obtaining the desired pharmaceutically active ingredient immediately before use. Among the various forms the most preferred form is subjected to freeze drying.

When the copolymer of the present invention or its pharmacologically acceptable salt is used as a modifier protein, the ratio between the copolymer or its pharmacologically acceptable salt and a protein or an analogue or variant of no particular restrictions on the form of any specific values are not imposed until such time as they achieve the desired modification of the protein. Typically, the mass ratio of the copolymer of the present invention or its pharmacologically acceptable salt and a protein or as is the dialogue or the option 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.

When the copolymer of the present invention is used as a modifier protein, the method of inoculation protein especial limitation is imposed up until it comes to the method that is normally used for protein modification. For example, a protein or an analogue or variant can be modified as follows.

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

The modifier protein is dissolved in the above solvent, and then the thus obtained solution is added to aqueous solution of the specified protein or analogue or variant. If necessary, the resulting solution can be shaken to facilitate the reaction. At desire it is possible to carry out fine-tuning of the pH of the resulting mixture by the addition of acids and/or bases. On the relationship between the modifier protein of the present invention and the above-mentioned protein or an analogue or variant of the restrictions in any specific values is not restricted as long as it leads to the attainment of the desired modification of the protein. Usually the ratio is in the range from 0.01 to 100 (wt.); preferably in the range of from 0.1 to 50; more preferably in the range from 1 to 10; and most preferably it is in the range from 1 to 1.5:1.

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

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

Sometimes in the course of the method of modification of the protein under the conditions used are such that some of the elementary structural units described by formula (II)in the copolymer are converted into elementary units described by formula (II)with another entity. For example, when R3in elementary structural units described by formula (II)represents a group other than hydroxy groups, the conditions used for the method of modification, such that some of these groups of R3subjected to hydrolysis to obtain elementary structural units described by formula (II), where R3represents a hydroxy group.

As noted above, the complexes of the present invention contain at least one protein or an analogue or variant that bind at least one copolymer of the present invention or its pharmacologically acceptable salt. In the specified complex protein or an analogue or variant and a copolymer or a pharmacologically acceptable salt connected with each other via chemical bonding such as covalent bond, (e.g., the formation of Schiff bases, the formation of the amide bond and the formation of ester bonds), the ionic bond Il the coordination bond, or using non-chemical communications, such as hydrophobic interaction, hydrogen bond, electrostatic interaction, or affinity binding.

Preferably when conducting exclusion chromatography size or SDS-PAGE in non conditions of the complexes of the present invention in dissociated form to any significant extent not find. More preferred complexes of the present invention in dissociated form to any significant extent not find when conducting exclusion chromatography size and SDS-PAGE in non conditions. In addition, even more preferably the rate of destruction, capable of detecting the protein complex ELISA method (enzyme-linked immunosorbent assay) for the complexes of the present invention obtained by modifying the protein structure of the copolymers and their pharmacologically acceptable salts, are very small (preferably the rate of destruction is 20% or less, more preferably 15% or less, and most preferably 10% or less).

In the present invention similar protein is defined as the protein encoded by the cDNA molecule cloned from a cDNA library derived from animal cells, body fluids or tissues as a result of hybridization with the use the of the cDNA of a protein in severe conditions (60 to 70° With a 6×SSC).

In the present invention the variant protein is defined as a protein that is a result of substitution, deletion, addition or insertion of one or several amino acids in the protein source, and which is still detects at least part of the activity of the original protein on detektiruya level.

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

OCIF, an analogue or variant used in the present invention may relate to the natural type, or they can relate to recombinant type, and their origin special limitation is imposed. OCIF natural type indicates OCIF, which is obtained in the form of protein, formed under natural conditions, resulting in the extraction, cleaning and/or discharge from the body, body fluids, cell cultures or cell environment, derived from a human or animal, not a person. Recombinant OCIF type, its analogue or variant represent a recombinant protein obtained by extraction, purification and/or selection of the specified protein from the host body, usually used in such methods, such as a prokaryotic cell, the master (for example,Escherichia coli) or eukaryotic cell, such as cell line from the human body or the body, not that change under the action of a vector containing polynucleotide that encodes OCIF, an analogue or a variant of it [for example, see the recombinant methods described in EP-A-0816380 (WO-A-96/26217) and WO-A-97/23614].

On the origin of OCIF, its analogs and its variants used in the present invention, particular limitation is imposed, and you can get them from the human body or an animal, not a person. Preferably you can get them from the body of a mammal, such as human, rat, mouse, rabbit, dog, cat, cow, pig, sheep or goat, or poultry, such as chicken, goose, chicken or Turkey. More preferably, they are derived from mammals, and most preferably, they are derived from the human body.

OCIF or analogue, IP is alzhemier in the present invention, may refer to OCIF monomer type (for example, the monomer is characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions) or dimeric type (for example, the dimer is characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions) [see EP-A-0816380 (WO-A-96/26217)]. Preferably a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 as measured by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions, and more preferably it is a dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions.

It is known that OCIF translates into cells in the form of a polypeptide containing a signal peptide at its amine end, and that after he undergoes maturation in the processing, including the removal of specified signal peptide, [for example, see the recombinant methods described in EP-A-0816380 (WO-A-96/26217) and WO-A-97/23614]. OCIF, an analogue or variant used in the present invention, VK is ucaut as a polypeptide, containing the signal peptide and the Mature form. Preferred examples include human OCIF, containing a signal peptide comprising amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences, and the Mature form of 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, particularly preferred is human OCIF in Mature form.

It is also known that such a Mature form of OCIF, its analogue or variant can be added methionine, if this form is expressed as recombinant protein in the cell of the host, particularly in prokaryotic cell host, such asEscherichia coli. This is done by adding a nucleotide triplet, including the sequence ATG (Aug), to the 5' end of polynucleotide encoding the Mature form of OCIF, its analogue or variant, and insert the resulting polynucleotide expressing in a suitable vector. Desirable Mature protein having a methionine at its amino end, then can be expressed using a suitable host cell that has been modified because specified expressing recombinant vector. In addition, one or more amino acids can be in order to add to the specified squirrel in position, adjacent to the methionine at the amino end, the result of adding the additional nucleotide triplets next door to the triplet ATG added to the 5' end of polynucleotide encoding the Mature form of OCIF, its analogue or variant. Target Mature form of OCIF, its analogue or variant containing a methionine at the amino end, you can clean and separate from the culture of a modified host cell in accordance with a commonly used method. In addition, one or more amino acids can be inserted into the Mature form OCIF containing methionine at the amino end, its analogue or variant in position adjacent to the methionine and closer to the carboxy-end compared with methionine.

In the present invention similar OCIF refers to a protein encoded by polynucleotides that exists in vivo in cells, body fluids and/or organs of a human or animal, not a person, such as those in the examples above. Specific preferred examples of such analogues include OCIF2, OCIF3, OCIF4 and OCIF5 [see EP-A-0816380 (WO96/26217)]. Such analogues OCIF or their active fragments can be obtained in a manner such as the following: RNA extracted from a cell, organ, tissue or body fluids of a person or animal, not a person; using inverse TRANS is riptease synthesize the first chain cDNA which complementary specified RNA, and then using the first circuit as the template using a DNA polymerase synthesize the second circuit indicated cDNA; the thus obtained double strand cDNA is inserted into a suitable commonly used expressing vector; suitable commonly used cell-host after this change under the action of the thus obtained vector; host, generating the desired peptide, then select to use the techniques of hybridization, such as hybridization of platelet or hybridization of phage, using OCIF cDNA or its fragment as a probe under stringent conditions [see EP-A-0816380 (WO-A-96/26217)]; and then, finally, the desired analog OCIF is expressed by a commonly used method using the thus obtained host cell.

In the present invention variant OCIF refers to a protein that contains the amino acid sequence, where the amino acid sequence of OCIF or analogue replace, delete, add or insert one or more amino acid residues, 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 a single nucleotide, or several nucleotides in the nucleotide sequence that is tidey sequence, encoding OCIF or analogue, using the method of polymerase chain reaction (hereafter in this document referred to as PCR), methods of genetic recombination or the way nucleases splitting using ectonucleoside or endonuclease, such as restriction enzyme; transforming a eukaryotic host cell, such as an animal cell, or a prokaryotic host cell, such asEscherichia coliunder the action of expressing vector, which produces the insertion of the obtained nucleotide encoding the desired variant OCIF; and after this extraction, purification and/or selection of the desired peptide from the protein-containing fractions obtained using cell culture transformed specified owner in accordance with the method, a well-known specialist in the respective field.

Versions of the form OCIF, the carboxy-end of the polypeptide OCIF remove a substantial portion of the amino acid sequence, also known for the fact that they retain at least some degree of OCIF activity [see, for example, EP-A-0816380 (WO-A-96/26217) and WO-A-97/23614]. Such versions of the types of OCIF, preserving at least some activity of the full polypeptide OCIF, are also included in the OCIF variants of the present invention.

In addition, OCIF or process is automated form, which are subjected to a fusion with immunoglobulin domain, such as an Fc domain (e.g., fused polypeptide, in which the Fc domain of human IgG attached to the carboxy-end of the OCIF), and which retain at least some activity of the full polypeptide OCIF known (see WO-A-97/23614), and such fused proteins are also included in the OCIF variants of the present invention.

Any educated in natural conditions OCIF or analogue or recombinant OCIF or analogue or variant can contain a sugar chain, which is attached to OCIF or analogue or variant phase posttranslate. Educated in natural conditions OCIF or analogue containing the sugar chain can be obtained from cell cultures, tissues, organs, body fluids or cell lines derived from human or animal body, not a person, using conventional techniques. Recombinant OCIF or analogue or variant containing a sugar chain can be obtained from the culture eukaryotic host cell transformed with the use of a vector containing the nucleotide sequence encoding any OCIF or analogue or variant, such as those described and are given as examples above. Examples of suitable host cells that can be used and which have the ability posttranslate the traditional modification OCIF or analogue or variant attach chain of sugar, include cells of Chinese hamster ovary cells and COS [Yasuda, H. et al., Endocrinology, 139, 1329-1337 (1998)]. OCIF or analogue or variant containing such a chain of sugar, suitable for use in obtaining complexes of the present invention. Chain sugar OCIF or analogue or variant containing a sugar chain, it is possible to artificially modify (in particular, under the action of 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 equivalent, may be a solution obtained in accordance with the method described above, or a composition obtained by freeze drying the solution. In an alternative embodiment, such pharmaceutical composition may be prepared in accordance with the alternative method, or it may take the form of a kit. In the latter case, the copolymer of the present invention or its pharmacologically acceptable salt and a protein or an analogue or variant stored in different containers and then mixed with obtaining the desired pharmaceutical composition immediately prior to use.

The pharmaceutical compositions of the present invention is not necessarily moroccopolisario to contain the base. On the basis of any restrictions in any specific reason not imposed until such time as it is the base is usually used in pharmaceutical compositions. Examples of such bases include: inorganic bases, such as carbonates of alkali metals (e.g. sodium carbonate, potassium carbonate and lithium carbonate), bicarbonates of alkali metals (e.g. sodium bicarbonate, potassium bicarbonate and lithium bicarbonate), hydrides of alkali metals (e.g. lithium hydride, sodium hydride and potassium hydride), hydroxides of alkali metals (e.g. sodium hydroxide, potassium hydroxide, barium hydroxide and lithium hydroxide), alkali metal fluorides (for example, sodium fluoride and potassium fluoride; and organic bases such as alkoxides of alkali metals (for example, sodium methoxide, ethoxide sodium, potassium methoxide, ethoxide potassium tert-piperonyl potassium and lithium methoxide), mercaptans alkali metals (for example, methyl mercaptan sodium and ethyl mercaptan sodium), N-methylmorpholine, triethylamine, Tripropylamine, tributylamine, diisopropylethylamine, dicyclohexylamine, N-methylpiperidine, pyridine, 4-pyrrolidinedione, 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-dia is bicyclo[5.4.0]undec-7-ene (DBU). Among these preferred bases are the hydroxides of alkali metals, and particularly preferred is sodium hydroxide. In this regard it should be noted that part or all of a number of bases can form salts with the copolymer of the present invention.

Examples of pharmaceutical compositions according to the present invention include a complex between at least one substance selected from the copolymer of the present invention or its pharmacologically acceptable salt, and protein or an analogue or variant selected from bFGF (used for treatment or prevention of ischemic arteriopathies disease and narutowicza skin ulcers), EGF (used for the treatment or prophylaxis of peptic ulcer disease, such as ulcerative colitis and long-term dysfunction of the epithelium of the cornea), PDGF (used for the treatment of wounds), BDNF and NGF (used for treatment or prevention of diseases of the Central nervous system, such as Parkinson's disease and Alzheimer's disease), HGH (used for treatment or prevention of the growth hormone deficiency syndrome low secretion of growth hormone, Turner syndrome and cartilage degeneration), HGF (used for the treatment or prevention of diabetes, atherosclerosis, such as heart attack, brain, and liver fibrosis) and VEGF (semices the e arteriopathies disease and occlusive peripheral arteries).

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

The metabolism in the bones, the treatment or prevention of which can be accomplished with the use of pharmaceutical compositions of the present invention include: primary osteoporosis (senile osteoporosis, postmenopausal osteoporosis and idiopathic juvenile osteoporosis); endocrine osteoporosis (gipertireoidizmom, hyperparathyreoidism, hyperadrenocorticism and acromegaly); osteoporosis associated with hypogonadism (hypopituitarism, syndrome Kleinfelter syndrome and Those who Nera); hereditary and congenital osteoporosis (imperfect osteogenesis, homocystinuria syndrome Menkes and family autonomic dysfunction); osteopenia caused by the reduction of the load of its own weight or by fixation and immobilization of the limb; deforming the fibrosa; osteomyelitis; infectious lesion caused by ratifikaciey bone, hypercalcemia, arising as consequences of solid cancer (breast cancer, lung cancer, kidney cancer and prostate cancer); hematological malignancy (multiple myeloma, lymphoma and leukemia); idiopathic hypercalcemia; hypercalcemia associated gipertireoidizmom or kidney dysfunction; osteopenia arising as consequences of steroid treatment drugs; osteopenia arising as consequences of receiving other drugs (e.g., immunosuppressive drugs such as methotrexate and cyclosporine A, heparin, and anti-epileptics); osteopenia associated with impaired kidney function; osteopenia associated with surgery or diseases of the digestive organs (for example, obstruction of the small intestine, obstruction of the large intestine, chronic hepatitis, gastric resection, primary biliary cirrhosis of the liver and qi is the rose of the liver); osteopenia caused by different types of arthritis, such as rheumatoid arthritis; osteoclasia and destruction of the joint, caused by various types of arthritis, such as rheumatoid arthritis; rheumatoid arthritis, related to the type of myelodysplastic Tripathi; osteoarthritis; rarefication periodontal bone; cancer metastasis in bone (osteolytic metastases); bone necrosis or necrosis of bone cells associated with traumatic injury, Gaucher's disease, sickle cell anemia, systemic lupus erythematous or nontraumatic rupture; the fibrosa, such as nephrogenic osteodystrophy; osteopenia associated with hypophosphatasia or diabetes; osteopenia associated with malnutrition or malnutrition; and other osteopenia. In addition, in the present invention in metabolic bone also included cachexia due to the above solid cancer, cancer metastasis in bone (osteolytic metastases) or hematological malignancies (see Japanese patent application (Kokai) No. 2000-178200).

The pharmaceutical composition of the present invention described above, people or animals, other than humans, can safely be taken orally or neironalna. Dosage form pharmaceutical HDMI is the proper way, you can choose depending on the type of disease, the extent of disease and condition, age, sex and weight of the patient. For example, the pharmaceutical composition can be administered orally, using the form of tablets, capsules, powders, granules or syrup; enter using the form of intravenous injection, individually or in combination with a conventional adjuvant, such as glucose, amino acids, and the like, or using a separate form for intramuscular, subcutaneous, intradermal or intraperitoneal injection; enter transdermal, using the form of poultices; enter transnasal, using the form of nasal drops; to enter transmucosal or in the oral cavity using the form transmucosal drug; or enter intrarectal, using the form of suppository. The composition of these drugs can be a conventional manner using well-known adjuvants that are commonly used in the medical field, such as fillers, binders, disintegrant, lubricants, flavorings, soljubilizatory, suspendida, dyes, pH regulators, preservatives, gelling agents, surfactants and other substances to cover.

When the pharmaceutical composition was prepared in the form of tablets, you can use a variety of media known state of 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, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate and polyvinylpyrrolidone; disintegrant, such as dry starch, sodium alginate, powdered agar, powdered laminaran, sodium bicarbonate, calcium carbonate, esters derived from polyoxyethylenesorbitan and fatty acids, sodium lauryl sulfate, monoglyceride of stearic acid, starch and lactose; disintegration inhibitors such as sucrose, stearin, cacao butter and gidrirovannoe oil; absorption accelerators, such as Quaternary ammonium bases and sodium lauryl sulfate; humectants, such as glycerin and starch; binders 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, if necessary on a tablet can be coated in the form commonly used shell. Examples of such film-coated tablets, include the pill with sugar coating the pill with a gelatin coating the tablet with enterolab is determined as being coated, the tablet film-coated, two-layer and multilayer tablet tablet.

When the pharmaceutical composition was prepared in the form of pills, you can use a variety of media known state of the art. Examples of such carriers include excipients such as glucose, lactose, cocoa butter, starch, gidrirovannoe vegetable oil, kaolin and talc; binders such as powdered Arabian gum, powdered tragakant, gelatin and ethanol; and disintegrant, such as agar-based laminaran.

When the pharmaceutical composition was prepared in the form of a suppository, you can use a variety of media known state of the art. Examples of such carriers include polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin and semi-synthetic glycerides.

When the pharmaceutical composition was prepared in the form of injection, it is preferable that the composition is in the form of a solution or suspension was subjected to sterilization and made isotonic with respect to blood. When this composition was prepared in the form of a solution, emulsion, suspension or essentially homogeneous aqueous solution, possibly using different diluents known state of the art. Examples of such diluents include in the u, ethanol, propylene glycol, ethoxylated isostearoyl alcohol, polioksidony isostearoyl alcohol and esters derived from polyoxyethylenesorbitan and fatty acids. In this case, the pharmaceutical composition may further contain common salt, glucose or glycerin in an amount sufficient to maintain isotonicity with respect to blood. In addition, the pharmaceutical composition may also contain commonly used soljubilizatory, buffer substances, softeners, pH regulators, stabilizers or solubilizing components. Such an injection can be lyophilized.

In addition, the pharmaceutical composition according to the invention optionally can also contain dyes, preservatives, fragrances, flavorings, sweeteners or other drug.

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

In the present invention, the dosage of the complex of OCIF or analogue or variant with a copolymer of Riego pharmacologically acceptable salt, contained in the pharmaceutical compositions of the present invention depends on various factors, including the condition and age of the patient, method of application and form of the drug. In the General case, the number entered an adult for once, is in the range of the upper limit in the range of from 1 to 50 mg/kg up to the lower limit in the range of from 0.001 to 0.1 mg/kg, where the preferred range is from 0.01 to 1 mg/kg, and more preferred is the range from 0.1 to 1 mg/kg

The pharmaceutical composition corresponding to the present invention, it is necessary to take one every few months once a month once every few days, once a day or several times a day depending on the type of ingredient contained in the pharmaceutical composition, method of application and form of the drug. When the composition comprises a complex of OCIF or analogue or variant with a copolymer or a pharmacologically acceptable salt intended for use as a tool in the treatment or prevention of metabolic bone in accordance with the present invention, it is necessary to take one every few months once a month once every few days, once a day or several times a day depending on the type of active Ingram the rate, contained in the tool, intended for the treatment or prevention of metabolic disorders in the bones, method of application and form of the drug.

The copolymer of the present invention and its pharmacologically acceptable salts are suitable as polymer modifier, can result in complex with homogeneous properties, in particular low education disordered cross-linked structures with protein, the best preservation of the protein activity and excellent retention of protein in the blood after administration of specified property. This makes it particularly suitable as a modifier in the modification of the pharmaceutical properties of proteins that possess useful pharmaceutical activity.

Examples

The following examples, reference examples and examples of the tests are intended to further illustrate the present invention, and they in no way imply a limitation of the scope of the invention. In the following examples, m and R3represent the same as defined above for formulas (I) and (II), "aspect]. comp." is a value defined by the composition [i.e., the ratio between the elementary structural units (I) and (II)], "cf. step. floor." represents the average degree of polymerization for the obtained copolymer, and "aspect]. the andetc.for the obtained copolymer represents the ratio of asked by hydrolysis [i.e., the average ratio between the elementary structural units, where R3is a HE, and elementary structural units, where R3differs from IT].

Example 1

Obtaining hydrolyzed poly(PEG500-MA) [poly(PEG500-MA)g], where m=6-16, R3=OH, aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 10:0 (compound No. 1)

As the starting compound used is a copolymer polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by an average molecular weight of approximately 500, and the average degree of polymerization of the main chain is in the range from 30 to 40 [AM-0530K, produced by NOF Corporation (hereafter in this document referred to as "poly(PEG500-MA)")]. To 3 g of poly(PEG500-MA) for dissolution specified source compounds were added 50 ml of distilled water and the thus obtained solution was stirred at 40°C for 15 hours. The resulting solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (nature is soumeya limit molecular weight, 10,000, produced by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 1.3 g of the specified compounds poly(PEG500-MA)g (compound No. 1) as an oily compound.

Poly(PEG500-MA)g (compound No. 1) was purified using gel filtration as follows. 100 mg of poly(PEG500-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, produced by Amersham-Pharmacia). The first 1 ml of eluent was discarded. Then each column was applied 1.5 ml of 0.001 n sodium hydroxide solution and the elution from the column was received and rejected another 1.5 ml Then for each column put 2.5 ml of 0.001 n sodium hydroxide solution and the elution from the column was given 2.5 ml, and that this fraction contained the purified compound poly(PEG500-MA)g (connection # 1). Purified fractions of the four columns were combined and received 10 ml of purified solution of the target compound. Exit after cleanup phase (determined spectrophotometrically at measuring the optical density poly(PEG500-MA)g in region 210 nm before and after cleaning) recorded equal to 80% (80 mg polymer), and the concentration was subjected to atom cleaning solution determined is equal to 8 mg/ml

The content of carboxyl groups in the target compound was identified by means of conductometric titration as follows. 7.5 ml solution of purified target compound obtained above (containing 60 mg of target compound), was diluted to a volume of 50 ml using distilled water and the pH of the thus obtained solution was brought to 12 with 1 M aqueous sodium hydroxide solution. To the solution in portions of 0.1 ml was added 0.1 M hydrochloric acid, after each addition of hydrochloric acid were measuring the pH and conductivity of the solution. Then the content of carboxyl groups in the copolymer was calculated from the amount of 0.1 M hydrochloric acid is added to the buffer region of conduction (i.e., the area on the graphic dependence of conductivity on the number of added hydrochloric acid in which the carboxyl group of the copolymer of the target compounds act as a buffer, with the specified region corresponds to the range of pH from about 10.5 to 3); the molar amount of hydrochloric acid used in the buffer region conductivity is equal to the molar amount of carboxylic groups in the copolymer of the target compounds. As a result, the content of carboxyl groups in 1 g of the target compound is predelli equal 3,22 mmol. Hence the ratio of the specified composition, calculated equal to 1:1,07. This numerical value was used in the following examples, where used the same original connection.

Example 2

Obtaining hydrolyzed poly(PEG1500-MA) [poly(PEG1500-MA)g], where m=28-38, R3=OH, aspect]. comp.=approximately 1:1, cf. step. floor.=10-15, and aspect]. HYDR.=approximately 10:0 (compound No. 2)

As the starting compound used is a copolymer polyoxyethyleneglycol simple diapir (m=28-38, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which the polyoxyethylene is characterized by an average molecular weight of approximately 1500 and the average degree of polymerization of the main chain is in the range of 10-15 (AM-1510K, produced by NOF Corporation) [hereafter in this document referred to as "poly(PEG1500-MA)"]. To 1.5 g of poly(PEG1500-MA) for dissolution specified source compounds were added 25 ml of distilled water and the thus obtained solution was stirred at room temperature for 20 hours. At the end of this period the solution are condensed in the same manner as that described in above example 1, using ultrafiltration membranes made from the floor is eferalgan on the basis of simple ether (characterized by the limiting molecular weight, 10,000, produced by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 0.8 g of target compound poly(PEG1500-MA)g (compound No. 2) as an oily compound.

The content of carboxyl groups in the target connection poly(PEG1500-MA)g (the compound No. 2) was measured in the same manner as that described in above example 1, and the content of carboxyl groups in 1 g of target compound was determined equal to 1.63 mmol. Hence the ratio of the specified composition, calculated equal to 1:1,4. This numerical value was used in the following examples, where used the same original connection.

Example 3

The product of ammonolysis of the poly(PEG500-MA) [poly(PEG500-MA)], where m=6-16, R3=NH2that aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 0:10 (connection # 3)

To 1 g of poly(PEG500-MA) (AM-C, produced by NOF Corporation) to dissolve the specified source compounds were added 9.5 g of the water-ammonia solution (ammonia: 28% (wt.)) and the thus obtained solution was stirred at room temperature for 16 hours. At the end of this period of time still the way the resulting solution was subjected to dialysis through a membrane of regenerated cellulose (characterized limit molecular weight in the range of 12000 to 14000, produced by Sanko Junyaku Co., Ltd., model number UC36-32-100) using one day to 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 for 1 day to 1 liter of water. After update 1 liter of water dialysis was performed additionally for another day. As a result of such dialysis of the product was removed excess amount of ammonia. The thus obtained solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 0,99 g of target compound poly(PEG500-MA)a (compound No. 3) as an oily compound.

The content of carboxyl groups in 1 g of poly(PEG500-MA)a (compound No. 3) was determined by the method conductometric titration in the same manner as that described in above example 1, and the content of carboxyl groups in 1 g of target compound was determined equal to 1.55 mmol.

In the used reaction conditions may be that the remainder of the maleic anhydride is the way the n to undergo reaction with the disclosure of the loop in the flow not only of ammonolysis, but also hydrolysis due to the presence in the reaction system of water. The relation between residues of maleic anhydride subjected to ammonolysis, and residues of maleic anhydride subjected to hydrolysis, was calculated as follows.

In example 1, was determined by the level of the content of carboxyl groups in 1 g of poly(PEG500-MA)g (compound No. 1). Hence, the received numerical value for the level of the content of carboxylic groups for the case of complete hydrolysis of all residues of maleic acid (3,22 mmol per 1 g of polymer). This results in the calculations was determined by the weight of the poly(PEG500-MA)g (compound No. 1) one mol of carboxyl group (1/3,22×10-3g) and the weight of the poly(PEG500-MA)g (compound No. 1) one mol of maleic acid residue, obtained by solving cycle [2 × weight of poly(PEG500-MA)g (compound No. 1) one mol of maleic acid residue, obtained during the opening cycle, since there are 2 carboxyl groups on one fully subjected to hydrolysis residue of maleic acid], resulting in 311 g 621 g, respectively. These values were determined by the weight of the poly(PEG500-MA) (i.e., the weight of the copolymer before hydrolysis) per gram functional group and a weight of poly(PEG500-MA)a (compound No. 3), obtained by adding to the poly(PEG500-MA)ammonia, one gram of functional groups. Specifically, the weight of the poly(PEG500-MA) (i.e., the weight of the copolymer before hydrolysis) to one mole of the residue of maleic anhydride was obtained by subtracting the molecular weight of a molecule of water (18 g) of weight fully hydrolyzed copolymer, resulting in a given numerical value 603, the Weight of the poly(PEG500-MA)a (compound No. 3) on one mole of the carboxyl group was obtained by adding the molecular weight of a molecule of ammonia (17 g) to the weight of the poly(PEG500-MA), resulting in a given numerical value equal to 620 g Of this value in the calculation (1 g/620) determined theoretical level of the content of carboxyl groups in 1 g of poly(PEG500-MA)a (compound No. 3), in which all residues of maleic anhydride were subjected to ammonolysis of (i.e., in the absence of hydrolysis), and it was obtained equal to 1.61 mmol. From the content of carboxyl groups in 1 g of poly(PEG500-MA)g (compound No. 1), in which all residues of maleic anhydride were subjected to hydrolysis, theoretical content of carboxyl groups in 1 g of poly(PEG500-MA)a (compound No. 3), in which all residues of maleic anhydride were subjected to ammonolysis, and the actual content of carboxyl groups in 1 g of poly(PEG500-MA)a (compound No. 3), the measurement is tion above (i.e., 1.55 mmol), was calculated the ratio between residues of maleic anhydride subjected to ammonolysis in the target compound, and the total number of residues of maleic anhydride in the source connection and it was determined to be 1.0. Thus, it was confirmed that essentially all traces of maleic anhydride were subjected to ammonolysis, and that virtually no hydrolysis was not leaking.

Example 4

The product of the reaction between dimethylamine and poly(PEG500-MA) [poly(PEG500-MA)DMA], where m=6-16, R3=NMe2that aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 0:10 (compound No. 4)

To 10 g of poly(PEG500-MA) (AM-C, produced by NOF Corporation) to its dissolution was added 71 g of an aqueous solution of dimethylamine (concentration of dimethylamine: 50% (wt.)) and the thus obtained solution was stirred at room temperature for 20 hours. At the end of this period of time thus obtained solution was subjected to dialysis through a membrane of regenerated cellulose (characterized limit molecular weight in the range of 12000 to 14000 and produced by Sanko Junyaku Co., Ltd., model number UC36-32-100) using in one day 10 liters of an aqueous solution of acetic acid the concentration of 0.1% (wt.), and then it was further subjected to dialysis using within 1 day 10 litres of water. After update 10 liters of water dialysis was performed additionally for another day. As a result of such dialysis of the product was removed excess dimethylamine. The thus obtained solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610), and the resulting condensate was subjected to freeze drying to obtain 6.3 g of target compound poly(PEG500-MA)DMA (compound No. 4) in the form of oily compounds.

The content of carboxyl groups in 1 g of poly(PEG500-MA)DMA (compound No. 4) was determined in the same manner as that described in above example 1, and he was determined equal to 1.53 mmol. Using a calculation similar to that described in example 3 for the product of ammonolysis, the calculation was set theoretical level the content of carboxyl groups in 1 g of poly(PEG500-MA)DMA (compound No. 4), in which all residues of maleic anhydride were subjected to aminals, and it was determined equal to 1.54 mmol. From results data calculated the ratio between the action of maleic anhydride, subjected to aminals in the target compound, and the total number of residues of maleic anhydride in the source connection and it was determined to be 1.0. Thus, it was confirmed that essentially all traces of maleic anhydride were subjected to aminals under the action of dimethylamine, and that virtually no hydrolysis was not leaking.

Example 5

The product of the reaction between 1-amino-2-propanol and poly(PEG500-MA) [poly(PEG500-MA)IPA], where m=6-16, R3=NH(CH2CH(OH)CH3), aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 0:10 (compound No. 5)

To 1.5 g of poly(PEG500-MA) (AM-C, produced by NOF Corporation) to its dissolution was added 14 g of 1-amino-2-propanol and the resulting solution was stirred at room temperature for 16 hours. At the end of this period the solution was added 300 ml of distilled water and to neutralize the solution was additionally added glacial acetic acid. The thus obtained solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporaion, model number PBGC07610), receiving 50 ml of condensate. The condensate was added 300 ml of distilled water and the resulting solution again are condensed in the same manner. This cycle dilution condensate using distilled water, and then re-condensation was repeated five times to remove excess 1-amino-2-propanol. The resulting condensate was subjected to freeze drying to obtain 1.3 g of target compound poly(PEG500-MA)IPA (compound No. 5) in the form of oily compounds.

The content of carboxyl groups in 1 g of poly(PEG500-MA)IPA (compound No. 5) was determined in the same manner as that described in above example 1, and he was determined equal to 1.55 mmol. Using a calculation similar to that described in example 3 for the product of ammonolysis, the calculation was set theoretical level the content of carboxyl groups in 1 g of poly(PEG500-MA)IPA (compound No. 5), in which all residues of maleic anhydride were subjected to aminals, and it was determined equal to 1.47 mmol. From results data calculated the ratio between residues of maleic anhydride subjected to aminals in the target compound, and the total number of residues of maleic anhydride in the source connection and it was determined to be 1.0. Thus, it was p who tveretina, that essentially all traces of maleic anhydride in the initial compound were subjected to aminals under the action of 1-amino-2-propanol, and that virtually no hydrolysis was not leaking.

Example 6

The product of the alcoholysis reaction between ethanol and poly(PEG500-MA) [poly(PEG500-MA)EA], where m=6-16, R3=OCH2CH3that aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 4:6 (compound No. 6)

To 1.5 g of poly(PEG500-MA) (AM-C, produced by NOF Corporation) to its dissolution was added 25 g of absolute ethanol, and the thus obtained solution was stirred at room temperature for 16 hours. At the end of this period of time was added 300 ml of water and the resulting solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610), receiving 50 ml of condensate. The condensate was added 300 ml of distilled water and the resulting solution again are condensed in the same manner. This cycle dilution condensate using distilled water and then repeat the Noah condensation was repeated five times to remove excess ethanol. The resulting condensate was subjected to freeze drying to obtain 0.8 g of target compound poly(PEG500-MA)EA (compound No. 6) in the form of oily compounds.

The content of carboxyl groups in 1 g of poly(PEG500-MA)EA (compound No. 6) was determined in the same manner as that described in above example 1, and he was determined equal of 2.16 mmol. Using a calculation similar to that described in example 3 for the product of ammonolysis, the calculation was set theoretical level the content of carboxyl groups in 1 g of poly(PEG500-MA)EA (compound No. 6), in which all residues of maleic anhydride were subjected to alcoholysis, and it was determined equal to 1.47 mmol. From this result, the expected correlation between residues of maleic anhydride subjected to alcoholysis in the target compound, and the total number of residues of maleic anhydride in the source connection and it was determined equal to 0.6. Thus, it can be seen that 60% of residues of maleic anhydride in the initial compound were subjected to alcoholysis, and the remaining 40% of residues of maleic anhydride were subjected to hydrolysis.

Example 7

The product of ammonolysis of the poly(PEG1500-MA) [poly(PEG1500-MA)], where m=28-38, R3=NH2that aspect]. comp.=p is blithedale 1:1, cf. step. floor.=10-15, and aspect]. HYDR.=approximately 4:6 (compound No. 7)

To 1.5 g of poly(PEG1500-MA) (AM-C, produced by NOF Corporation) to its dissolution was added 14.5 g of the water-ammonia solution (ammonia: 28% (wt.)) and the thus obtained solution was stirred at room temperature for 20 hours. At the end of this period the solution was added 300 ml of distilled water and to neutralize the specified solution was additionally added glacial acetic acid. The resulting solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610), receiving 50 ml of condensate. The condensate was added 300 ml of distilled water and the resulting solution again are condensed in the same manner. This cycle dilution condensate using distilled water, and then re-condensation was repeated five times to remove excess ammonia. The resulting condensate was subjected to freeze drying to obtain 0.7 g of target compound poly(PEG1500-MA)a (compound No. 7) in the form of oily compounds.

The level of sod is Rania carboxyl groups in 1 g of poly(PEG 1500-MA)and was determined in the same manner as described in example 1 and it was determined equal to 1.12 mmol. As in the case of ammonolysis of the poly(PEG500-MA)and from example 3, it may be that the residues of maleic anhydride in the source connection of poly(PEG1500-MA) will be able to be as ammonolysis and hydrolysis. Therefore, the fraction of residues of maleic anhydride subjected to ammonolysis, and the fraction of residues of maleic anhydride subjected to hydrolysis, was calculated as follows.

In example 2, was determined by the level of the content of carboxyl groups in 1 g of poly(PEG1500-MA)g (compound No. 2). Hence, the received numerical value for the level of the content of carboxylic groups for the case of complete hydrolysis of all residues of maleic acid (1,63 mmol per 1 g of polymer). From this result, the expected mass of the poly(PEG1500-MA)g (compound No. 2) on one mole of the carboxyl group (1/1,63×10-3g) and the weight of the poly(PEG1500-MA)g (compound No. 2) on one mol of maleic acid residue, obtained by solving cycle [2 × weight of poly(PEG1500-MA)g (compound No. 2) on one mol of maleic acid residue, obtained during the opening cycle, since there are 2 carboxyl groups on the one subjected to complete hydrolysis residue of maleic acid], which resulted in 613 g and 1227 g, respectively. From receiving the Noah thus the mass of the poly(PEG 1500-MA)g and using the approach similar to the above example 3, was calculated theoretical level the content of carboxyl groups in 1 g of poly(PEG1500-MA)a (compound No. 7), in which all residues of maleic anhydride were subjected to ammonolysis, and it was received is 0.82 mmol. From these results and the actual content of carboxyl groups in the poly(PEG1500-MA)a (compound No. 7), measured above (1.12 mmol), the calculations have established the relation between residues of maleic anhydride subjected to ammonolysis in the target compound, and the total number of residues of maleic anhydride in the source connection and it was determined equal to 0.6. Thus, it was confirmed that 60% of residues of maleic anhydride in the source connection of poly(PEG1500-MA) were subjected to ammonolysis, and the remaining 40% of residues of maleic anhydride were subjected to hydrolysis.

Example 8

The product of the reaction between dimethylamine and poly(PEG1500-MA) [poly(PEG1500-MA)DMA], where m=28-38, R3=NMe2that aspect]. comp.=approximately 1:1, cf. step. floor.=10-15, and aspect]. HYDR.=approximately 0:10 (compound No. 8)

To 1 g of poly(PEG1500-MA) (AM-C, produced by NOF Corporation) to its dissolution was added 11 wodnego solution of dimethylamine (characterized by a concentration of 50% (wt.)) and the thus obtained solution was stirred at room temperature for 20 hours. At the end of this period the solution was added 300 ml of distilled water and to neutralize the solution was additionally added glacial acetic acid. The resulting solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610), receiving 50 ml of condensate. The condensate was added 300 ml of distilled water and the resulting solution again are condensed in the same manner. This cycle dilution condensate using distilled water, and then re-condensation was repeated five times to remove excess amine. The resulting condensate was subjected to freeze drying to obtain the target compounds poly(PEG1500-MA)DMA (compound No. 8) in the form of oily compounds.

The content of carboxyl groups in 1 g of poly(PEG1500-MA)DMA (compound No. 8) was determined in the same manner as described in example 1, and it has been calculated equal to 0.82 mmol. Using the approach similar to what was used in example 3, theoretical level of the content of carboxyl groups in 1 g of poly(PEG1500-MA)DMA, in which all traces of maleinovogo anhydride were subjected to aminals, in the course of calculations determined equal to 0.80 mmol. From these results during calculations have established the relation between residues of maleic anhydride subjected to aminals in the target compound, and the total number of residues of maleic anhydride in the source connection and it was determined to be 1.0. Thus, it was confirmed that essentially all traces of maleic anhydride were subjected to aminals under the action of dimethylamine.

Example 9

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

Each of the polymeric modifiers obtained in examples 1 to 8, was dissolved in physiological solution 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 of sodium dihydrophosphate and 150 mm sodium chloride, at a suitable ratio with the receiving buffer, characterized by the value of pH of 6.0) and obtained solutions are characterized by the concentration of modifier in the range from 1 to 20 mg/ml For each solution of each modifier, turn mixed of 0.625 ml of the resulting solution of polymer modifier and of 0.625 ml of a solution containing purified Mature human OCIF (OCIF, polucen is in accordance with the description in WO 96/26217 and EP 816380) [protein concentration: 2 mg/ml, medium: PBS (pH 6.0)], and the thus obtained mixture was allowed to stand at a temperature in the range from 4°to 37°C for at least one hour to obtain a series of solutions [environment: PBS (pH 6.0)], containing OCIF, modified with polymeric modifiers of examples 1 to 8, in which the ratio between the modifier and OCIF in each solution was determined by the concentration of the added solution of modifier and reaction conditions. Mass ratio modifier/OCIF for some of the obtained complexes (and the conditions in which they were received) shown in table 6 in the following test example 2. The size of the molecule for each of the resulting complexes was measured as explained in the following test example 11. The detection rate of OCIF in the complexes was measured using ELISA method as described in the following test example 3.

In addition, for each of the polymer modifiers in exactly the same way received the OCIF solution modified with polymeric modifier, except that the medium used with PBS pH 7.4.

Example 10

Obtaining the sodium salt of the product of ammonolysis of the poly(PEG500-MA) [poly(PEG500-MA)a-Na], where m=6-16, R3=NH 2that aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 3.1:6,9 (compound No. 9)

As the starting compound used is a copolymer polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by an average molecular weight of approximately 500, and the average degree of polymerization of the main chain is in the range from 30 to 40 (AM-0530K, produced by NOF Corporation, with the batch number M) [hereafter in this document referred to as "poly(PEG500-MA)"]. To 10.1 g of the specified parent compound poly(PEG500-MA) was added 61 ml of 0.5 M solution of ammonia/1,4-dioxane and the resulting solution was stirred at 25°C for 20 hours. At the end of this period the solution was added 200 ml of diethyl ether and 100 ml of 0.2 M aqueous solution of sodium hydroxide and the mixture was intensively shaken for approximately 3 minutes. After the occurrence of phase separation of organic and aqueous layers, the lower layer was collected. In the selected layer was added 200 ml of diethyl ether and 60 ml of 1,4-dioxane and the resulting solution again was intensively shaken. After phase separation of the resulting bottom is th layer was collected, and then subjected to freeze drying and received a 10.1 g of target compound sodium salt of poly(PEG500-MA)a (compound No. 9) in the form of a solid phase is yellow.

The content 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

The product of ammonolysis of the poly(PEG500-MA) [poly(PEG500-MA)], where m=6-16, R3=NH2that aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 1.4:8,6 (compound No. 10)

To 1 g of poly(PEG500-MA) (AM-C, produced by NOF Corporation, with the batch number M) for its dissolution was added 9.5 g of the water-ammonia solution with a concentration of 28% (wt./wt.) and the thus obtained solution was stirred at 25°C for 4 hours. At the end of this period of time was added 250 ml of water and ammonia solution with a concentration of 0.28% and the resulting solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610) and the thus obtained condensate was subjected to is whether lyophilized to obtain 0.8 g of target compound poly(PEG 500-MA)a (compound No. 10) in the form of oily compounds.

The content 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

The product of the reaction between dimethylamine and poly(PEG500-MA) [salt of poly(PEG500-MA)DMA-Na], where m=6-16, R3=NMe2that aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=around 2.9:7,1 (compound No. 11)

To 5 g of poly(PEG500-MA) (AM-C, lot number: M received in the company NOF Corporation) for its dissolution was added 35 g of an aqueous solution of dimethylamine with a concentration of 50% (wt./wt.) and the resulting solution was stirred at 25°C for 3 hours, then additionally stirred at 4°C for 16 hours. At the end of this period of time was added 100 ml of 0.1 M aqueous sodium hydroxide solution, and the thus obtained solution was subjected to freeze drying to obtain 5.4 g of target compound salts of poly(PEG500-MA)DMA-Na (compound No. 11) in the form of a solid phase is yellow.

The content of carboxyl groups and the ratio specified by hydrolysis, for the target polymer was determined as described in the following examples the E. testing 1.

Example 13

Obtaining hydrolyzed poly(PEG500-MA) [poly(PEG500-MA)g], where m=6-16, R3=HE, aspect]. comp.=approximately 1:1, cf. step. floor.=30-40, and aspect]. HYDR.=approximately 10:0 (compound No. 12)

To 1 g of poly(PEG500-MA) (AM-C, produced by NOF Corporation, with the batch number M) for its dissolution was added 17 ml of distilled water and the resulting solution was stirred at 40°C for 4 hours. At the end of this period of time there was added 250 ml of water and ammonia solution with a concentration of 0.28% (wt./wt.) and the thus obtained solution are condensed using ultrafiltration membranes made from polyethersulfone on the basis of simple ether (characterized by the limiting molecular weight of 10,000, and produced by Millipore Corporation, model number PBGC07610). The thus obtained condensate was subjected to freeze drying to obtain 0.7 g of target compound poly(PEG500-MA)g (compound No. 12) in the form of oily compounds.

The content 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 modi is tatrami of examples 10 to 13 and OCIF

Each of the polymeric modifiers obtained in examples 10 to 13, was dissolved in physiological solution 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 of sodium dihydrophosphate and 150 mm sodium chloride, at a suitable ratio with the receiving buffer, characterized by a pH equal to 7.0) and received solutions, characterized by a range of concentrations of the modifier in the range from 1.25 to 105 mg/ml For each polymer modifier, turn, with a volumetric ratio of 1:1 was stirred thus obtained solutions of polymer modifiers and the solution containing the purified Mature human OCIF (OCIF obtained in accordance with the description in WO 96/26217 and EP 816380) [protein concentration from 0.25 to 14 mg/ml, medium: PBS (pH 6.0)], and obtained the solutions characterized by different mass ratios between the modifier and OCIF. The thus obtained solution pH was brought to 5.0, and 5.5, and 6.0, about 6.5, a 7.0 or 7.4 using 1 M hydrochloric acid or 1 M aqueous solution of sodium hydroxide. Each of the thus obtained solution was left to stand at 25°With over a period of time ranging from 12 hours to one time and received the solution of the complex between the polymer modifier of the present invention and OCIF. The resulting solutions were stored at 4°C. the Size of the molecule of the resulting complexes was measured as explained in the following examples, tests 6 and 11. The detection rate of OCIF in the complexes was measured using ELISA method as described in the following test example 8.

Example 15

Obtaining the sodium salt of the product of ammonolysis of the poly(PEG500-MA) [salts of poly(PEG500-MA)and-Na], characterized by an adjustable size of the molecule, where m=6-16, R3=NH2that aspect]. comp.=approximately 1:1, cf. step. floor. shown below, and aspect]. HYDR.=approximately 1.4:8,6 (compound No. 13-19)

100 mg of sodium salt of poly(PEG500-MA)and(compound No. 9)obtained in example 10, was dissolved in 1 ml of saline phosphate buffer (PBS, characterized by the value of pH of 7.4, obtained by mixing an aqueous solution containing 10 mm sodium hydrogen phosphate and 150 mm sodium chloride, and an aqueous solution containing 10 mm of sodium dihydrophosphate and 150 mm sodium chloride, at a suitable ratio). The thus obtained solution was fractionally way gel-filtration chromatography. Used two different following set of conditions gel filtration:

(1) the Method faction who scan using Superose 6 (hereafter in this document referred to by way SRF)

Column: Superose 6 HR 10/30 Amersham Bioscience

The temperature of the column: 8°

Mobile phase: PBS (pH 7,4)

The wavelength of detection: 280 nm

Flow rate: 0.3 ml/min

The number entered in column: 100 µl

(2) Method of fractionation using Superdex 200 (hereafter in this document referred to by way SDF)

Column: Superdex 200 HR 16/60 Amersham Biosciences

The column temperature: room temperature

Mobile phase: PBS (pH 7,4)

The wavelength of detection: 280 nm

Flow rate: 2 ml/min

The number entered in column 5 ál

Polymer modifiers, elution which was performed using these two methods of fractionation during elution in the range from x to y minutes, was identified as poly(PEG500-MA)a-Na(SRFx-y) and poly(PEG500-MA)a-Na(SDFx-y), respectively.

The concentration of polymer modifier in each fraction (aqueous solution) was determined by the method of high performance liquid chromatography. The conditions used in high-performance liquid chromatography, was the following:

Column: Shodex OHpak SB-806M HQ (purchased in the company SHOWA DENKO K. K.)

Protective column: Shodex OHpak SB-G (acquired in the company SHOWA DENKO K. K.)

The column temperature: 40°

Mobile phase: aqueous solution of 50 mm sodium hydrogen phosphate with bringing the pH to,0 using 1 M hydrochloric acid

The wavelength of detection: 210 nm

Flow rate: 0.5 ml/min

The number entered in column: 50 ál

For poly(PEG500-MA)a-Na(SRFx-y) and poly(PEG500-MA)a-Na(SDFx-y), obtained respectively as a result of fractionation in accordance with the method SRF and SDF way, spent more gel-filtration chromatography (method SRF) using the same elution conditions as described above for the first fractionation in accordance with the method SRF, to estimate the size of molecules for each of the samples after fractionation. As standard samples used proteins, characterized by the known size of the molecules (the acquired company Amersham Bioscience). In this respect, molecular weight and size of the molecules for each of the proteins shown in the appropriate directory under which they were acquired.

The calculated size of the molecules to polymer modifiers obtained by fractionation shown in the following table 1.

td align="left">  
Table 1
Molecular weightThe Stokes radius (nm)Retention time (min)
Standard protein
Thyroglobulin6690008,5038,48
Ferritin4400006,1044,85
Catalase2320005,2248,77
Aldolase1580004,8150,11
Albumin670003,5551,85
Ovalbumin430003,0553,95
Chymotrypsinogen And250002,0959,26
Ribonuclease And137001,6460,23
The modifier of example 15No. of connections
poly(PEG500-MA)and-Na (nericcio-failed)9,3 or less **35-65 *9
poly(PEG500-MA)and-Na (SRF50-55output is 23%3,1-6,2 **45-55 *13
poly(PEG500-MA)a/sub> -Na (SRF55-60output is 29%1,5-4,7 **50-60 *14
poly(PEG500-MA)and-Na (SRF60-65output 12%of 3.1 or less **55-65 *15
poly(PEG500-MA)and-Na (SDF46-52output is 22%7.8 or less **40-65 *16
poly(PEG500-MA)and-Na (SDF52-58output is 22%6,2 or less **45-65 *17
poly(PEG500-MA)and-Na (SDF58-64output is 13%of 3.1 or less **55-65 *18
poly(PEG500-MA)and-Na (SDF60-70output is 13%of 3.1 or less **55-65 *19
* The range for retention time indicates that the sample is characterized by a 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 those cases, when the lower limit is not shown, that is, when the radius of the Stokes shown as "XX or less", we can say that the sample with the contains low molecular weight component, which cannot be assessed using gel-filtration chromatography, carried out under the conditions of this example.

Using this approach has received various polymers poly(PEG500-MA)and-Na, characterized by different sizes of molecules (compound No. 13-19). The average degree of polymerization of the compounds of the№№ 13, 14, 15, 16, 17, 18 and 19 was calculated as in the preceding examples, and, as was shown, it was <30, <<30, <<<30, <30, <<30, <<<30 <<<30, respectively.

Example 16

The product of ammonolysis of the poly(PEG1500-MA) [poly(PEG1500-MA)and], characterized by an adjustable size of the molecule, where m=28-38, R3=NH2that aspect]. comp.=approximately 1:1, cf. step. floor. below (compound No. 20-22)

On the basis of [poly(PEG1500-MA)and] (compound No. 7)obtained in the above example 7, is used as the starting compound, and using the same approach as used in the above example 15, in the same way as in example 15, was obtained poly(PEG1500-MA)and(SRFx-y). The elution conditions in the case of elution SRF were the same as in example 15. The outputs of polymer modifiers, polucen the x as a result of fractionation, shown in the following table 2.

Table 2
The modifier of example 16No. of connections
poly(PEG1500-MA)and, (refractionary)7
poly(PEG1500-MA)and, (SRF50-55), and 12.2%20
poly(PEG1500-MA)and, (SRF55-60), to 13.1%21
poly(PEG1500-MA)and, (SRF60-65), and 16.4%22

Using this approach, received various polymers poly(PEG1500-MA)andcharacterized by different sizes of molecules (compound No. 20-22). The average degree of polymerization of the compounds of No. 20, 21 and 22 was calculated as in the preceding examples, and, as was shown, it was <10, <<10 <<<10, respectively.

Example 17

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

Complexes of polymer-OCIF of the present invention was obtained in the form of aqueous solutions using essentially the same preparative approach, as in the above example 14, using as initial components aqueous solutions of compounds No. 13 to 22 obtained in the above PR is measures 15 and 16 (medium: PBS, characterized by the value of pH of 7.4), and an aqueous solution of purified Mature human OCIF (OCIF obtained in accordance with the description in WO 96/26217 and EP 816380) (medium: PBS, characterized by the value of pH of 6.0). More specifically, in the case of each of the modifiers was stirred his solution in PBS (pH 7.4) with a concentration of 0.5 mg/ml (obtained as described in the above example 9) and OCIF solution in PBS (pH 6.0) with a concentration of 0.5 mg/ml at a volume ratio of 1:1. The resulting reaction mixture was left to stand at 25°C for 7 days. The size of the molecules in the resulting complexes were measured as explained in the following test example 11.

Example 18

The product of ammonolysis of the poly(PEG500-MA) [salts of poly(PEG500-MA)and-Na], characterized by an adjustable size of the molecule: (a) m=6-16, R3=NH2that aspect]. comp.=approximately 1:2, and cf. step. floor.=20-30, (compound No. 27), and (b) m=6-16, R3=NH2that aspect]. comp.=approximately 1:1, and cf. step. floor.=approximately 15 (compound No. 28)

Two polymer of the present invention poly(PEG500-MA)and(compound No. 27) and poly(PEG500-MA)and(compound No. 28) was prepared as follows. The original link is m for the first one was poly(PEG 500-MA) (AM-K made using methods similar to that described in Japanese patent No. 2621308 and publications of Japanese patent applications No. 2003-105040 and 2003-104003) - copolymer polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by an average molecular weight of approximately 500, and the average degree of polymerization of the main chain is in the range from 20 to 30, the ratio between elementary units polyoxyethyleneglycol simple diapir and elementary parts of maleic anhydride is 1:2, and the average molecular weight of approximately 6000 [srednekislye molecular weight is about 6000 and the molecular mass distribution (Mw/Mn) is approximately equal to 1.25]. The reference compound for the second of them was poly(PEG500-MA) (AM-K made using methods similar to that described in Japanese patent No. 2621308 and publications of Japanese patent applications No. 2003-105040 and 2003-104003) - copolymer polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by a medium is nd molecular weight, approximately equal to 500, the average degree of polymerization of the main chain is approximately 15, and the average molecular weight of approximately 10,000. Both the parent compound was subjected to ammonolysis using conditions essentially identical to the conditions used in example 10, and the received target compound salt of poly(PEG500-MA)and-Na (compound No. 27) and salt of poly(PEG500-MA)and-Na (compound No. 28). The content of carboxyl groups in the target polymer was determined as described in the following test example 1, and how it was discovered, it is by 2.73 mmol/g for connection No. 27 and 2.05 mmol/g for connection No. 28.

Example 19

Obtaining complexes between the polymer modifiers from example 18 and OCIF

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

Example 20

Assessment of the composition of poly(PEG500-MA) and receipt and evaluation of poly(PEG500-MA)a (compounds No. 29-53) under various conditions

20(1) determining the ratio of specified composition for poly(PEG500-MA)

Have tested for several copolymers polyoxyethyleneglycol simple diapir and maleic anhydride poly(PEG500-MA) with different composition in order to determine the ratio between the elementary parts of PEG-allylmethylamine simple diapir and maleic anhydride (ratio of specified composition) in them (different party AM C made using methods similar to that described in Japanese patent No. 2621308 and publications of Japanese patent applications No. 2003-105040 and 2003-104003 - statistical copolymers polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by an average molecular weight of approximately 500, and the average degree of polymerization of the main chain is in the range from 20 to 30. Srednekislye mole is alarna mass (M n) and index of molecular weight distribution (Mw/Mnwhere Mwrepresents a mass-average molecular mass) for each batch tested copolymers shown below. The molecular mass (MM) for each batch of poly(PEG500-MA) was determined by the method of gel-filtration chromatography using poly(ethylene glycol) with a predefined MM as standard. Therefore, MM poly(PEG500-MA), shown below, is not absolute MM, and is a relative MM, measured using PEG as standard.

MO, Mn=6431; Mw/Mn=1,27

3N549, Mn=6360; Mw/Mn=1,23

3N550, Mn=5891; Mw/Mn=1,28

3N569, Mn=5897; Mw/Mn=1,25

In order to determine the ratio of the specified composition, for each of the copolymers, it was necessary to transform them into the corresponding sodium salt of hydrolyzed copolymers, i.e., sodium salt of poly(PEG500-MA)was Used conditions of hydrolysis consisted of the following.

To 1 g of poly(PEG500-MA) (party MO) was added 25 ml of 1,4-dioxane, 100 ml of simple ether and 42 ml of 0.1 G. of an aqueous solution of NaOH. The resulting mixture then was intensively shaken, and after she gave enabled the industry to settle, collected thus obtained water layer. The thus obtained aqueous layer was stirred at 40°within 2 hours after filtration through filter paper (704×40 m/m, obtained in the company Nihon Rikagaku-kiki Corporation). At the end of this period the resulting solution was subjected to freeze drying. Sodium salt of hydrolyzed product obtained from the starting compound, namely sodium salt of poly(PEG500-MA)g (the party MO), received in the form of a solid phase yellow (yield 100%).

To 1 g of poly(PEG500-MA) (party 3N549) was added 12 ml of 1,4-dioxane and 9 ml of 1 N. aqueous solution of NaOH. The resulting mixture was then stirred at room temperature for 24 hours after the end of this period of time was added 1 ml of 1 N. aqueous NaOH solution and the resulting solution was subjected to freeze drying. Sodium salt of hydrolyzed product obtained from the starting compound, namely sodium salt of poly(PEG500-MA)g (the party 3N549), received in the form of a solid phase yellow (yield 100%).

To 2 g of poly(PEG500-MA) (party 3N550) was added 5 ml of 1,4-dioxane and 9 ml of 1 N. aqueous solution of NaOH. The resulting mixture is then stirred at 40°C for 23 h At the end of this period the reaction mixture was subjected to life Inoi drying. Sodium salt of hydrolyzed product obtained from the starting compound, namely sodium salt of poly(PEG500-MA)g (the party 3N550), received in the form of a solid phase yellow (yield 100%).

To 2 g of poly(PEG500-MA) (party 3N569) was added 5 ml of 1,4-dioxane and 9 ml of 1 N. aqueous solution of NaOH. The resulting mixture is then stirred at 40°C for 23 h At the end of this period the reaction mixture was subjected to freeze drying. Sodium salt of hydrolyzed product obtained from the starting compound, namely sodium salt of poly(PEG500-MA)g, was obtained as a solid phase yellow (yield 100%).

The content of carboxyl groups for each of the sodium salts of poly(PEG500-MA)g, thus obtained, was measured by the method conductometric titration as described above. The results shown in the table below 3.

Sodium salt of poly(PEG500-MA)g contains the following two Monomeric elementary level:

If the number of Monomeric elementary parts of sodium salt of maleic acid in 1 elementary link of PEG-allylmethylamine simple diapir denoted as "a", and theoretical minimum repeating the elementary level will include one elementary frame link guides the PEG-allylmethylamine simple diapir and "and" elementary parts of sodium maleate, then the formula mass (FM) minimum repeating the elementary level will be obtained in accordance with equation 1:

FM[(PEG-allylmethylamine simple W)1+ (sodium salt of maleic acid)and] = FM(PEG-allylmethylamine simple W) + [FM(sodium salt of maleic acid)] = 541 + 160A (equation 1)

The number of carboxyl groups in the minimum repeating an elementary link of the sodium salt of poly(PEG500-MA)g is equal to 2A. Therefore, the content of carboxyl groups (mmol/g) in the minimum repeating an elementary link of the sodium salt of poly(PEG500-MA)g is obtained in accordance with equation 2:

C=2A/(541+160A)·1000 (equation 2)

The ratio between the number of elementary parts of sodium salt of maleic acid and the number of elementary units PEG-allylmethylamine simple diapir in actual sodium salts of poly(PEG500-MA)g is the same as the value in the minimum repeating elementary units. Therefore, the value is the same in the minimum repeating the elementary level and in the actual sodium salts of poly(PEG500-MA)g, the content of carboxyl groups which were identified by means of conductometric titration (see above). Thus, the level measurement is the contents of carboxyl groups and using equation 2 we can obtain the value of the ratio, asked composition, "a", namely the number of elementary parts of sodium salt of maleic acid in 1 elementary link of PEG-allylmethylamine simple diapir [which corresponds to the composition of the monomer compositions for poly(PEG500-MA)]. The results shown in the table below 3.

Table 3
Party poly(PEG-MA)The number of carboxyl groups (mmol/g)MA/PEG-allylmethylamine simple fluids
Sodium salt of poly (PEG-MA)gNo. 1MOof 4.662,01
No. 2M3N549to 5.212,42
No. 3M3N5505,983,10
No. 4M3N5696,143,26

The ratio of the specified composition for the sodium salts of poly(PEG500-MA)d was in the range of from 1:2 to 1:3,3. It is obvious that the ratio of the specified composition is the same in the cases of poly(PEG500-MA) and sodium salt of poly(PEG500-MA)g, so that the ratio defined by the composition of the monomer mixture, for different source copolymers of poly(PEG500-MA), as was podtverjdeno, was in the range of from 1:2 to 1:3:3.

20(2) Ammonolysis of the poly(PEG500-MA) in the different conditions obtaining poly(PEG500-MA)a (compound No. 29-53) and the determination of the ratios set by hydrolysis, these compounds

The use of the ratio defined by the composition (a) and content of carboxyl groups to sodium salts of poly(PEG500-MA)g ratio set by hydrolysis (i.e., the ratio between the elementary parts of maleic anhydride in the original polymer, subjected to ammonolysis, and data elementary parts subjected to hydrolysis) can be calculated as follows. The formula of the sodium salt of elementary link amidinophenoxy maleic acid (sodium salt polyamide maleic acid) shown below:

Similarly, as in the case of the above sodium salts of poly(PEG500-MA)g, we can assume that the minimum elementary link of the sodium salt of poly(PEG500-MA)and includes one elementary link of PEG-allylmethylamine simple diapir, Oh elementary parts of sodium salt polyamide maleic acid and(1-x) of the elementary parts of sodium salt of maleic acid. Formula weight minimum basic level of sodium salt of poly(PEG500

FM[(PEG-allylmethylamine simple W)1+ (sodium salt polyamide maleic acid)Oh+ (sodium salt of maleic acid)and(1-x)] = FM(PEG-allylmethylamine simple W) + Ah[FM(sodium salt polyamide maleic acid)] + a(1-x)[FM(sodium salt of maleic acid)]=541+Ah+160A(1-x)=541+160A-Ah (equation 3)

Because the level of the content of carboxyl groups in a minimum basic level of sodium salt of poly(PEG500-MA)and is a Ah+2A(1-x)=2A-Ah, the content of carboxyl groups (mmol/g) in the minimum basic level of sodium salt of poly(PEG500-MA)and can be obtained from equation 4:

C=10000×(2A-s)/(541+160A-Ah) (equation 4)

Thus, the value specified by hydrolysis, (1-x):x can be determined from equation 4 using the content of carboxyl groups (mmol/g), which can be obtained by the method conductometric titration of sodium salt of poly(PEG500-MA), and the values "a", which was obtained above.

Some examples of the preparation of poly(PEG500-MA)and the calculated ratio set by the hydrolysis product of the poly(PEG500-MA)as shown below.

(a) 100 mg of poly(PEG500-MA) was added a 0.5 M solution of ammonia/1,4-dioxane (amounts are specified in the following table 4) and the resulting solution p is remedial at temperatures and times, presented in the following table 4. At the end of the reaction time to the reaction mixture was added approximately 0.4 ml (range, 0.3 to 0.5 ml) 1 N. aqueous solution of NaOH. The resulting solution was subjected to freeze drying and obtained the sodium salt of poly(PEG500-MA)and in the form of a solid phase.

"The degree of completion of reaction of amine component" for each of the sodium salts of poly(PEG500-MA)and [i.e., the percentage of elementary parts of maleic anhydride in the original polymer, subjected to ammonolysis of (100·*x)]received data in different reaction conditions shown in the following table 4.

No. 8-19, 25, 27-37 represent the target polymers poly(PEG500-MA)a (compound No. 29-52). Each of these polymers is statistical, and m=6-16, R3=NH2the ratio defined by composition=1:2,4, and average degree of polymerization=20-30.

As shown in table 4, all the reaction products of poly(PEG500-MA)a (compound No. 29-52) are characterized by a very high degree of completion of reaction of amine component, which indicates the achievement of the extent of reaction for the amino group, very close to 100%, using different reaction conditions described above.

A few extra the examples of alternative methods for obtaining poly(PEG 500-MA)as described below:

(b) of 0.44 g of poly(PEG500-MA) was dissolved in 9 ml of dimethylformamide. Then in the solution at 50°was barbotirovany gaseous ammonia. The final amount of ammonia in the reaction mixture amounted to 0.67, the Thus obtained reaction mixture was sealed in a glass ampoule under nitrogen atmosphere, and then stirred at room temperature for 24 hours after the end of this period of time ammonia gas evaporated under reduced pressure after the glass ampoule was opened. After this reaction mixture was added dropwise to 90 ml of diethyl ether. The thus obtained precipitate was collected and dried under reduced pressure. In the form of a yellow powder was obtained 0.28 g of ammonium salt of poly(PEG500-MA)a (compound No. 53) (marked as No. 41 in the above table 4). The polymer is statistical, and m=6-16, R3=NH2the ratio defined by composition=1:3,10, and the average degree of polymerization=20-30.

(C) Ammonium salt of poly(PEG500-MA)and received in the same way as (b), using toluene instead of dimethylformamide.

(4) Ammonium salt of poly(PEG500-MA)and received in the same way as (b), using 1,4-dioxane instead of dimethylformamide.

Example 21

Obtaining the reaction product al is agelisa between ethanol and poly(PEG 500-MA) [poly(PEG500-MA)EA-Na], where m=6-16, R3=OCH2CH3that aspect]. comp.=approximately 1:3, cf. step. floor.=20-30, aspect]. HYDR.=approximately 3.1:6,9 (compound No. 54)

As the starting compound used poly(PEG500-MA) (AM-C, party M3N550 made using methods similar to that described in Japanese patent No. 2621308 and publications of Japanese patent applications No. 2003-105040 and 2003-104003) - copolymer polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by an average molecular weight of approximately 500, the average degree of polymerization of the main chain is in the range from 20 to 30, the ratio between elementary units polyoxyethyleneglycol simple diapir and elementary parts of maleic anhydride is about 1:3, srednekislye molecular mass (Mnequal 5891 and the molecular mass distribution (Mn/Mw) is 1,28 (see the above example 20). To 100 mg of the specified parent compound was added 1 ml of ethanol and the resulting reaction mixture was left to stand for 16 hours at 40°C. In the eye of the Chania this period of time was added 52 μl of 2.5 N. the sodium hydroxide solution in ethanol. Thus the resulting mixture was concentrated at 35°With the result of evaporation, and then dried under vacuum to obtain the sodium salt of poly(PEG500-MA)EA (compound No. 54) in the form of an oily substance. The value specified by hydrolysis, was calculated using the method described in the above examples 3 and 6. Namely, by determining the content of carboxyl groups in the sodium salt of poly(PEG500-MA)EA (compound No. 54) and the corresponding sodium salts of poly(PEG500-MA)g calculated the ratio between residues of maleic anhydride in the target connection is subjected to alcoholysis, and the total number of residues of maleic anhydride in the original connection, and it was determined to be equal to 0.69. Thus, it can be seen that 69% of residues of maleic anhydride in the initial compound was subjected to alcoholysis, and the remaining 31% of residues of maleic anhydride was subjected to hydrolysis.

Example 22

Obtaining complexes between the polymer modifier of example 21 and OCIF

Complexes of polymer-OCIF of the present invention was obtained in the form of aqueous solutions using essentially the same preparative approach, as in the above example 14, using an aqueous solution of compounds is 54, obtained in the above example 21 [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 of sodium dihydrophosphate and 150 mm sodium chloride, at a suitable ratio with the receiving buffer, characterized by the value of pH of 7.4)], and an aqueous solution of purified Mature human OCIF (OCIF was obtained 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 specified OCIF solution with a concentration of 4 mg/ml was added 0,472 ml solution of compound No. 54 with a concentration of 17.2 mg/ml and receive a solution 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°to obtain aqueous solutions of the desired complexes of the present invention. The sizes of the complexes were measured in the following test example 13.

Example 23

The product of the alcoholysis reaction between ethanol and poly(PEG500-MA) [poly(PEG500-MA)EA], where m=6-16, R3=OCH2CH3that aspect]. comp.=PR is approximately 1:3, cf. step. floor.=20-30 (compound No. 55)

As the starting compound used poly(PEG500-MA) (AM-C, party M3N550 made using methods similar to that described in Japanese patent No. 2621308 and publications of Japanese patent applications No. 2003-105040 and 2003-104003) statistical copolymer polyoxyethyleneglycol simple diapir (m=6-16, Alk=ethylene, R1=hydrogen, and R2=methyl) and maleic anhydride, in which polyoxyethylene side chain is characterized by an average molecular weight of approximately 500, the average degree of polymerization of the main chain is in the range from 20 to 30, the ratio between elementary units polyoxyethyleneglycol simple diapir and elementary parts of maleic anhydride is about 1:3, srednekislye molecular mass (Mnequal 5891 and the molecular mass distribution (Mn/Mw) is 1,28 (see the above example 20). To 50 mg of the specified parent compound was added 0.5 ml of ethanol and the resulting reaction mixture was left to stand at 37°C for 24 hours. The target connection poly(PEG500-MA)EA (compound No. 55) was obtained in the form of a solution in ethanol [the concentration of the poly(PEG500-MA)EA: 100 mg/ml].

Example 24

aluchemie complexes between the polymer modifier of example 23 and OCIF

Complexes of polymer-OCIF of the present invention was obtained in the form of aqueous solutions using essentially the same preparative approach, as in the above example 14, using a solution of compound No. 55 in ethanol, obtained in the above example 23, and an aqueous solution of purified Mature human OCIF (OCIF was obtained 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, 0.5 ml of the specified OCIF solution with a concentration of 5 mg/ml was added to 37.5 μl of a solution of the compound No. 55 in ethanol and the resulting reaction mixture was allowed to stand for 3 days at 25°to obtain desirable solutions of the complexes of the present invention. The sizes of the complexes were measured in the following test example 13.

Comparative example 1

Getting copolymer monometoksipolietilenglikolya-metilfenidato ether-maleic acid (PEG-PMMA)

Grafted copolymer monometoksipolietilenglikolya-metilfenidato ether-maleic acid (PEG-PMMA) received in accordance with the method described in example 2 of Japanese patent application (Kokai) No. Hei 11-302199.

Comparative example 2

Obtaining complex containing copolymer monomethoxy the ethylene glycol-metilfenidato ether-maleic acid (PEG-PMMA) and OCIF protein

Treated human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380), modified using PEG-PMA, obtained as described in the above comparative example 1 was obtained in the form of a solution [environment: PBS (pH 6,0)] essentially the same way as in example 9. More specifically, stirred 1 ml OCIF [OCIF concentration: 2 mg/ml; medium: PBS (pH 6.0)] and 1 ml PEG-PMA [concentration of PEG-PMA: 2 or 20 mg/ml; medium: PBS (pH 6.0)] and to retrieve the target complex, and the mixture was left to stand for 24 hours at 25°C.

Comparative example 3

Obtaining complex containing polymer modifier and OCIF protein using poly(PEG500-MA) as a polymer modifier

To 28,4 µl of the aqueous solution subjected to the purification of human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380) (polymer concentration: 3.5 mg/ml, medium: 0.5 M aqueous solution of NaH2PO4, the pH of which was brought to 7.6 using a 5 M aqueous solution of NaOH) was added 2,2 μl solution in dimethyl sulfoxide containing poly(PEG500-MA) [AM-C, produced by NOF Corporation (hereafter in this document referred to as "poly(PEG500MA)"] (the concentration of the polymer: from 35 to 350 mg/ml) and thus receive the hydrated solution (concentration of OCIF: 3.2 mg/ml, the concentration of the poly(PEG500-MA): 2.5 mg/ml or 6.3 mg/ml) was shaken at 25°C for 40 hours. At the end of this period, the solution was diluted using PBS (pH 7.0) and received the OCIF solution modified with polymer modifier, with a concentration of OCIF, equal to 0.25 mg/ml of the Thus obtained solution was stored at 4°C.

The test example 1

The measurement of the content of carboxyl groups of the polymers of examples 10 to 13 method conductometric titration

The content of carboxyl groups for each of the polymers [poly(PEG500-MA)a (compounds No. 9 and 10), poly(PEG500-MA)DMA (compound No. 11) and poly(PEG500-MA)g (compound No. 12)]obtained in examples 10 to 13, were identified by means of conductometric titration as follows.

First, each of the polymers was subjected to purification using gel filtration as follows. For each polymer 100 mg of 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, produced by Amersham-Pharmacia). The first 1 ml of eluent was discarded. Then each column was applied 1.5 ml of 0.001 n sodium hydroxide solution and the elution from the column poluchenii threw another 1.5 ml Then each column was applied to 2.5 ml of 0.001 n sodium hydroxide solution and the elution from the column was given 2.5 ml, and that this fraction contained the purified compound. Purified fractions of the four columns were combined and received a purified solution of the target compounds. Exit after cleanup phase (determined spectrophotometrically at measuring the optical density poly(PEG500-MA)g in region 210 nm before and after cleaning) recorded equal to 80%, and the concentration in the treated solution was determined equal to 8 mg/ml

For each of the solutions treated polymer aliquot (2.5 to 7.5 ml) was brought to 50 ml using distilled water or 0.001 M aqueous solution of sodium hydroxide, and then to the resulting solution was added 1 M aqueous sodium hydroxide solution to bring the pH to 12. Then to the solution or portions of 0.1 ml, or continuously at a speed of 0.1 ml/min was added 0.1 M hydrochloric acid. In the first case, the pH and conductivity were measured after each addition, and in the second case, the measurement was made every 15 seconds. After that, the content of carboxyl groups in the polymer was calculated from the amount of 0.1 M hydrochloric acid is added to the buffer conductivity regions (corresponding to the range of pH in the pre is Elah approximately from 10 to 5.5). The results shown in the table below 5.

Table 5
Number exampleThe type of polymer (and no connection)The content of carboxyl groups (mmol/g polymer)The degree of transmission of ammonolysis/ aminolysis (%)The value specified by hydrolysis
10poly(PEG500-MA)and (9)2,10693,1:6,9
11poly(PEG500-MA)and (10)1,83861,4:8,6
12poly(PEG500-MA)DMA (11)2,02712,9:7,1
13poly(PEG500-MA)g (12)3,21-10:0

In the case of the polymers of examples 10 to 12 residues of maleic anhydride in the initial compound, subjected to ammonolysis or aminals, and residues of maleic anhydride subjected to hydrolysis, in each case determined in the result of the calculation as follows. As shown in the above table 5, the content of carboxyl groups in 1 g of poly(PEG500-MA)g (compound No. 12) is is 3.21 mmol. Proceeding and the data values were determined by the weight of the poly(PEG 500-MA) (i.e., the weight of the copolymer before hydrolysis) per gram functional group and a weight of poly(PEG500-MA)a (compounds No. 9 and 10)obtained by adding ammonia to the poly(PEG500-MA), one gram of functional groups. Specifically, the weight of the poly(PEG500-MA) (i.e., the weight of the copolymer before hydrolysis) to one mole of the residue of maleic anhydride was obtained by subtracting the molecular weight of a molecule of water (18 g) of weight fully hydrolyzed copolymer, which resulted in a numerical value equal to 605, the Weight of the poly(PEG500-MA)a (compounds No. 9 and 10) on one mole of the carboxyl group was obtained by adding the molecular weight of a molecule of ammonia (17 g) to the weight of the poly(PEG500-MA), which resulted in a numerical value equal to 622, on the Basis of this value to the result of the calculation (1 g/622) determined theoretical level of the content of carboxyl groups in 1 g of poly(PEG500-MA)a (compounds No. 9 and 10), in which all residues of maleic anhydride were subjected to ammonolysis of (i.e., in the absence of hydrolysis), and, as it was found, he makes 1.61 mmol. In the same way by adding the molecular weight of the molecule of the amine to the weight of the poly(PEG500-MA) was determined by the weight of the poly(PEG500-MA)DMA (connection who is 11), obtained by adding the amine to poly(PEG500-MA), one mole of the carboxyl group, which resulted in a numerical value equal to 650, on the Basis of this value in the calculations was determined by theoretical level of the content of carboxyl groups in 1 g of poly(PEG500-MA)DMA, in which all residues of maleic anhydride were subjected to aminals under the action of dimethylamine, and it is found that he is 1.54 mmol.

For each of the polymers of examples 10 to 12, the ratio set by the hydrolysis (the percentage of parent compound, subjected to hydrolysis, in comparison with the ammonolysis or aminolysis), and the degree of completion of reaction ammonolysis or aminolysis (%) was determined from the content of carboxyl groups in 1 g of poly(PEG500-MA)g (compound No. 11), in which all residues of maleic anhydride were subjected to hydrolysis, theoretical content of carboxyl groups in 1 g of poly(PEG500-MA)a (compounds No. 9 and 10), in which all residues of maleic anhydride were subjected to ammonolysis, theoretical content of carboxyl groups in 1 g of poly(PEG500-MA)DMA (compound No. 11), in which all residues of maleic anhydride were subjected to aminals under the action of dimethylamine, and the actual measured levels of the content of the carboxyl groups in 1 g of each of the polymers of examples 10 to 12 in accordance with the measurement method described above conductometric titration. The results shown in the above table 5.

The test example 2

Evaluation of the retention of complexes OCIF-modifier in the blood for example rats

Each of the samples described in example 9, comparative example 2, and unmodified treated human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380) appropriately diluted using PBS (pH 6.0) so that the concentration of OCIF was 0.25 mg/ml Each of the diluted samples, thus obtained, was injected through a vein in the tail female Wistar rats, aged five weeks (having a body weight of approximately 100 g, and which would not give them food in one day) so to dose OCIF would be 0.5 mg/kg (2 ml/kg volume). Later, 6 hours after injection of rat heart were selected with 200 µl of blood, and then using ELISA method was measured concentration of OCIF in the serum, while conditions were those described in the following test example 3.

The concentration of OCIF in the serum when measured after injection of each sample shown in the following table 6.

Modifier (and no connection)
Table 6< / br>
The OCIF concentration in serum after intravenous injection of each sample OCIF
Modifier/ OCIF (mass ratio)The OCIF concentration in the serum after 6 hours after administration (ng/ml)Conditions under stirring*
Unmodified OCIF-25±19-
PEG-PMMA1101±26-
10361±33-
poly(PEG1500-MA)g (2)10502±70-
poly(PEG1500-MA)and (7)101029±30-
poly(PEG1500-MA)DMA (8)12145±721-
poly(PEG500-MA)g (1)10750±80-
2,5434±92-
poly(PEG500-MA)and (3)103416±440-
102445±19537°
2,53428±27-
2,5484±921 hour
13004±158-
12275±130pH 7.4
13786±46140 hours
1777±153
0,52951±512-
poly(PEG500-MA)DMA (4)11014±331-
*Basic conditions under stirring was OCIF concentration of 1 mg/ml, pH 6.0, 16 hours and 25°C. In table 6 are the only conditions that differ from the data.

From the above table 6 one can see 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 retention of protein when introduced him alone. From the comparison of the complex prior art between PEG-PMMA and protein (described in Japanese laid patent No. Hei 11-302199 and obtained in the above comparative example 2) and of the complexes of the present invention, which is characterized by the same mass ratio between the modifier and protein (1 or 10), you can also see 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 prior art between the EG-PMMA and protein.

The test example 3

Assessment of detection OCIF by ELISA method

One of the problems faced in the case of protein modifiers of the prior art, is that the binding of the modifier protein is the reason of the modification of protein structure and/or shielding of the protein due to the formation of bulk systems. In order to test the complexes OCIF-modifier of the present invention, a method ELISA was performed 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 the unmodified, subjected to cleaning, OCIF person (OCIF was obtained as described in WO 96/26217 and EP 816380). Method ELISA was implemented as follows.

Monoclonal antibody against human OCIF OI-19 (obtained in accordance with the method described in AR) was dissolved in 0.1 M solution of acid sodium carbonate (pH 9,6), and received the solution characterized by the concentration OI-19 equal to 10 ág/ml 100 μl of the thus obtained solution OI-19 was placed in each well of 96-well immunoplate (produced by Nunc), and the tablets were left to Mature at 4°With during the night. At the end of this time period in each well for blocking was added 50%Block Ace (acquiring the emy company Snow Brand Milk Products Co., Ltd.) and then the tablets three times washed using PBS (wash buffer)containing 0.1% Tween 20. Treated human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380) was dissolved in a primary reaction buffer (i.e., 0.2 M Tris-buffer solution, containing 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 concentrations of OCIF. 100 µl of each of the thus obtained solutions with different concentrations of OCIF was added to each well, the tablets were shaken at room temperature for 2 hours, and then each well was washed six times with wash buffer. Then spent 10000-fold dilution POD-OI-4 (i.e., antibody, labeled with peroxidase and recognize OCIF, obtained as described in EP 0974671) using a secondary reaction buffer (i.e., 0.1 M Tris-buffer solution, containing 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 tablets were shaken at room temperature for 2 hours, and then each well was washed six times. Once this was accomplished, to each well was added 100 μl of substrate solution (p the agent, soluble TMB available in the company Scytek) and tablets were shaken at room temperature over a period of time lasting from 10 to 15 minutes. Then to each well was added 100 ál stopping the reaction (containing TMB stop buffer available in the company Scytek), and the tablets were subjected to careful shaking. At the end of this time period for each well using a microplate reader (MEML 001, produced by Molecular Device corporation) was performed measuring the optical density at the wavelength region of 450 nm. Based on these results, obtained calibration curve in the form of the dependence of optical density on the concentration of OCIF. After receiving this calibration curve method was repeated for each of the tested complexes between OCIF and the modifier in each well was added 100 μl each of solutions containing the test complex, reaction with POD-OI-4 was performed according to the same method as above, and then for each well using a microplate reader was measured by optical density at the wavelength region of 450 nm. Comparison with optical densities obtained using a calibration curve made it possible to obtain the concentration of OCIF, detected using ELISA method for each of the complexes, which conducted the ü measurement.

For each of the samples used to calculate the degree of failure to identify OCIF by ELISA method, and the results are shown in the following table 7. The degree of failure to identify OCIF by ELISA method was determined using the following equation:

Extent=[1-(OCIF concentration measured by ELISA method)/(concentration of OCIF, measured by the method of Lowry)]×100

In the above equation Lowry method for measuring the concentration of OCIF in the complexes was determined as described in Japanese patent application No. 2002-190407. This allowed us to obtain a measure of total concentration of OCIF in the complexes. The degree of failure to identify OCIF complex method ELISA represents a measure of the conformation changes of OCIF caused by complexation with the modifier. The low degree of failure to identify is the evidence that OCIF complex can easily be contacted with antibodies against OCIF as OI-19, and OI-4, which thus shows the structure of OCIF complex minor modification or lack of it.

Table 7< / br>
The pistons due to sample OCIF by ELISA method
Modifier (and no connection)Modifier/OCIF (mass ratio)The degree of failure to identify OCIF by ELISA method (%) (based on the Nemo is aficionado OCIF)
OCIF--
PEG-PMMA125
1039
poly(PEG1500-MA)g (2)1013
poly(PEG1500-MA)and (7)1015
poly(PEG1500-MA)DMA (8)100
poly(PEG500-MA)g (1)1017
poly(PEG500-MA)and (3)100
2,50
poly(PEG500-MA)DMA (4)112

From the results shown in the above table 7, it can be seen that in the case of complexes between the polymer modifier and the protein of the present invention, the degree of failure to identify OCIF by ELISA method in the modification of the structure of the protein was significantly decreased in comparison with a high degree, which he received in the case of the complex prior art between PEG-PMMA and protein obtained in the above test example 2.

Thus, on the basis of results examples tests 2 and 3, it was confirmed that the decrease in sensitivity in the detection of protein caused by excessive modification b is the left main coronary artery in the case of complexes between the polymer modifier and the protein of the present invention, very small, and, in addition, the retention in the blood for protein in these complexes is much better compared to achievable in the case of complexes of the prior art.

The test example 4

Measurement of the concentration of OCIF in the blood

Each of the samples obtained as described in example 9 and subjected to the purification of human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380) appropriately diluted using PBS (characterized by a pH in the range from 6.0 to 7.4) so that the concentration of OCIF would be in the range from 0.1 to 1 mg/ml of Each of the thus obtained diluted samples was then introduced through the saphenous vein of the leg or subcutaneously into the back of the female monkeys of the genus Cynomolgus the age of six or seven years (with mass of a body in the range from 2 to 4 kg, and which would not give them food in one day) so to get a dose of OCIF in the range from 0.1 to 1 mg/kg (1 ml/kg volume). After a pre-specified period of time after administration in the range from five minutes to one month of its blood vessel hips were selected with 500 µl of blood and the concentration of OCIF in the serum were measured using ELISA method described in the above test example 3.

The test example 5

Bone density measurement

Each of the exemplary is, obtained as described in example 9 and subjected to the purification of human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380) appropriately diluted using PBS (characterized by a pH in the range from 6.0 to 7.4) so that the concentration of OCIF would be in the range from 0.7 to 3.5 mg/ml then received adjuvant using dead cellsMycobacterium butyricumand liquid paraffin, and it was given as an injection into the skin of the root of the tail female Lewis rats aged 5 to 10 weeks to stimulate the development of the indicated rat arthritis. Two weeks after the injection of adjuvant rat via the tail vein or subcutaneously in the back was administered to each of the samples so that the dose of OCIF would be in the range from 1.4 to 7 mg/kg (2 ml/kg volume). Three weeks after injection of the adjuvant rat analyzed to extract both thighs and measured the density of her bones.

Example test 6

Estimating the size of a molecule by the method of polyacrylamide gel electrophoresis in the presence of SDS in non conditions

The size of the molecule for each of the complexes between the polymer modifier and OCIF, obtained as described in example 14 and comparative example 3, and subjected to cleaning OCIF person (OCIF was received as described WO 96/26217 and EP 816380) was estimated using SDS-PAGE in non conditions as follows.

To 10 μl each of the test samples [which is optionally diluted using saline 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 of sodium dihydrophosphate and 150 mm sodium chloride, at a suitable volume ratio of the receiving buffer, characterized by a pH equal to 7.0) so that the protein concentration was least 250 μg/ml] was added 5 μl containing LDS (lithium dodecyl sulphate) the sample buffer (4X) NuPAGE (trade mark) (obtained in the company Invitrogen Life Technology) and 5 μl of purified water and each of the resulting solutions were heated at 95°C for 7 minutes. At the end of this period of time to a polyacrylamide gel for electrophoresis in the presence of SDS (3-8%increase Tris-acetate gel, having a thickness of 1 mm and manufactured in the company NOVEX) was added the full amount of the reaction mixture and the gel applied voltage of 150 V using a power supply unit (PhoreStar Pro, produced by Anatech). After electrophoresis the protein on the gel was stained using Kumasi blue in accordance with the method, well known to experts in soo which corresponds to the region.

As can be seen from figures 1 and 2, OCIF, modified with polymeric modifier of the present invention, stably detected as substances, characterized by a molecular weight greater than the molecular weight of the unmodified OCIF (120 kDa) with the ratio of components in the mixture (primary band in the area of from 130 to 150 kDa and a secondary band in the region from 180 to 200 kDa were detected based on labels, markers of molecular weight), and, in addition, neither found any complex, characterized by a molecular weight in excess of 210 kDa. Similar results (not shown) were also obtained for other polymeric modifiers of the present invention, which represented the products of aminolysis and alcoholysis. On the other hand, as can be seen from figure 3, in the case of OCIF modified using conventional polymer modifier is poly(PEG500-MA)obtained in comparative example 3, the number of received raw complex significantly increases the proportion of modifier.

This result demonstrated that in comparison with structurally quite similar polymeric modifier is poly(PEG500-MA) prior art the use of a polymeric modifier of the present invention can provide value is to suppress the formation of raw complexes, which are not preferable from the viewpoint of pharmacy, and receive complex between the polymer modifier and protein, which has stable properties regardless of the ratio of components in the mixture.

The test example 7

Evaluation of the activity of poly(PEG500-MA)and the formation of the covalent bond

Determined reactivity for each of the polymeric modifiers poly(PEG500-MA)a (compound No. 9) and poly(PEG500-MA)g (compound No. 12) in relation to tetramethylpropylenediamine, which is a fluorescent substance characterized by the presence of amino groups and a molecular weight equal to 514,62, (available in the company Molecular Probes, Inc.) (hereafter in this document referred to as "Rho-NH2") and the activity of the modifiers in the formation of covalent bonds has been evaluated as a result of comparison of the reactivity of two polymeric modifiers as follows.

To 18,9 μl of PBS (pH 6.0)containing 1.08 mg/ml Rho-NH2added to 3.8 μl of solutions of each of the poly(PEG500-MA)g (compound No. 12) (obtained as described in the above example 13) and poly(PEG500-MA)a (compound No. 9) [concentration of polymer: 21 mg/ml, medium: PBS (pH brought to 9.5 with 1 M NaOH)] (obtained as described privedennom above example 10). The thus obtained solution was left to stand at 25°C for 3 days. At the end of this period the reaction mixture was fractionally using gel filtration chromatography (column: PD-10, produced by Amersham Biotech, mobile phase: purified water), as follows. On gel-filtration column was applied 0.5 ml of the reaction mixture and discard 0.5 ml of the effluent from the column. In a column was added 2 ml of distilled water and the elution was received and rejected a further 2 ml column was added 2 ml of distilled water and the elution was given 2 ml, and this fraction contained the fraction of the polymer. Data on the number of Rho-NH2contained in the fractions of the polymer was obtained by the method of fluorescence spectrometry (wavelength of excitation: 544 nm, the wavelength fluorescence: 571 nm, medium: purified water, bringing the pH to 3), making the calculation of the ratio between the amount of Rho-NH2contained in each complex between the polymer and Rho-NH2and the full amount of Rho-NH2in the reaction mixture, i.e., the degree of binding between Rho-NH2and polymer.

The results shown in the following table 8.

Table 8< / br>
Reactivity floor is measured modifier
The degree of binding between Rho-NH2and polymer* (%)
Rho-NH2+ poly(PEG500-MA)11,5, 12,6
Rho-NH2+ poly(PEG500-MA)gof 0.8, and 1.6
Only one Rho-NH20, 0
*This test was performed twice.

From the above table 8 one can see that the reaction mixture between the polymer modifier is poly(PEG500-MA)a (compound No. 9) and Rho-NH2there is a fairly high level of Rho-NH2, detected in a fraction of the polymer. This case is different from the reaction mixture between the polymer modifier is poly(PEG500-MA)g (compound No. 12) and Rho-NH2which is only a low level of Rho-NH2, detected in a fraction of the polymer. If a column has introduced only one Rho-NH2then in the "fraction of polymer" no elution of Rho-NH2not happened. These results indicate that the polymer modifier is poly(PEG500-MA)a (compound No. 9) of the present invention is firmly bound to the amino group Rho-NH2. On the other hand, the degree of binding to the polymer modifier is poly(PEG500-MA)g (compound No. 12) of the present invention is small, the site is allegedly testifying this polymer modifier with the amino group Rho-NH2firmly bound.

The test example 8

Assessment of detection OCIF by ELISA method

Method ELISA was performed to determine the degree of detection of each of the complexes between OCIF and polymer modifier obtained in accordance with the description in the above example 14 and comparative example 3, on the basis of the unmodified treated human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380). Method ELISA was implemented as follows.

Monoclonal antibody against human OCIF OI-19 (obtained in accordance with the method described in AR) was dissolved in 0.1 M solution of acid sodium carbonate (pH 9,6), and received the solution characterized by the concentration OI-19 equal to 10 ág/ml 100 μl of the thus obtained solution OI-19 was placed in each well of 96-well immunoplate (produced by Nunc), and the tablets were left to Mature at 4°With during the night. At the end of this time period in each well for blocking was added 50%Block Ace (acquired company Snow Brand Milk Products Co., Ltd.) and then the tablets three times washed using PBS (wash buffer)containing 0.1% Tween 20. Treated human OCIF (OCIF was obtained as described in WO 96/26217 and EP 816380) solution is whether in the primary reaction buffer (i.e., 0.2 M Tris-buffer solution, containing 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 concentrations of OCIF. 100 µl of each of the thus obtained solutions with different concentrations of OCIF was added to each well, the tablets were shaken at room temperature for 2 hours, and then each well was washed six times with wash buffer. Then spent 10000-fold dilution POD-OI-4 (i.e., antibody, labeled with peroxidase and recognize OCIF, obtained as described in EP 0974671) using a secondary reaction buffer (i.e., 0.1 M Tris-buffer solution, containing 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 tablets were shaken at room temperature for 2 hours, and after each well was washed six times. Once this was accomplished, to each well was added 100 μl of substrate solution (reagent, TMB soluble in available in the company Scytek) and tablets were shaken at room temperature over a period of time lasting from 10 to 15 minutes. Then to each well was added 100 ál stopping the reaction (with whom containing a series of TMB stop buffer, available in the company Scytek), and the tablets were subjected to careful shaking. At the end of this time period for each well using a microplate reader (MEML 001, produced by Molecular Device corporation) was performed measuring the optical density at the wavelength region of 450 nm. The concentration of OCIF in each standard sample OCIF was calculated based on a calibration curve obtained using solutions of OCIF having a known concentration.

For each of the samples tested of the complex was measured by optical density and in accordance with the explanations in the above test example 3 were calculated OCIF concentration calculated from the calibration curve, and the degree of failure to identify OCIF by ELISA method. The results obtained are shown in the following table 9.

Table 9< / br>
The pistons due to sample OCIF by ELISA method
Modifier (and no connection)Modifier/ OCIF (mass ratio)The degree of failure to identify OCIF by ELISA method (%) (based on the unmodified OCIF)
OCIF individually--
poly(PEG500-MA)g (12)18
With the eh poly(PEG 500-MA)a-Na (9)10
0,750
0,50
0,250
poly(PEG500-MA)7,898
1,989
0,7860

From the above table 9 it is easy to see that for each of the complexes between the polymer modifier and the protein of the present invention obtained in example 14, the degree of failure to identify OCIF by ELISA method, due to the modification of the protein, was significantly reduced compared with the complex between the polymer and protein prior art, obtained in comparative example 3, for which the degree of failure to identify OCIF by ELISA method 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 small decrease in the sensitivity of detection of the ELISA method, which can be caused by excessive protein modification and/or education surround the complex.

The test example 9

Evaluation of the retention in the blood for example rats

Each of the samples described in example 14, and unmodified treated human OCIF (CIF received so as described in WO 96/26217 and EP 816380) appropriately diluted using PBS (pH 7.0) so that the concentration of OCIF was 0.25 or 0.025 mg/ml Each of the diluted samples, thus obtained, was injected through the femoral vein to the female Wistar rats, aged five weeks (having a body weight of approximately 100 g, and which would not give them food in one day) so that the dose of OCIF would be 0.5 or 0.05 mg/kg (2 ml/kg volume). Later, 6 hours after administration of the jugular vein of rats were selected with 200 µl of blood, and then by the above method was measured by ELISA the concentration of OCIF in the serum.

The concentration of OCIF in the serum when measured after injection of each sample shown in the following table 10.

Table 10< / br>
The OCIF concentration in serum after intravenous injection of each sample OCIF
Modifier (and no connection)Modifier/ OCIF (mass ratio)Dosage (mg/kg)The OCIF concentration in the serum after 6 hours after administration (ng/ml)
OCIF individually-0,518
poly(PEG500-MA)g (12)10,05 127
Salt of poly(PEG500-MA)a-Na (9)10,05603
10,55549
0,750,54770
0,50,54292
0,250,53020

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

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

The test example 10

Evaluation of the retention in the blood for example rats

For each of the samples obtained in example 17 and example 19, evaluated by the same method as in test example 9. The concentration of OCIF in the serum when measured after administration of each sample thus is antiroman the following table 11.

Table 11< / br>
The OCIF concentration in serum after intravenous injection of each sample OCIF
Number exampleModifier (and no connection)FactionModifier/OCIF (mass ratio)Dosage (mg/kg)The OCIF concentration in the serum after 6 hours after administration (ng/ml)
17poly(PEG500-MA)and-Na
14SRF55-6010,05437
142,50,05460
15SRF60-6510,05478
19SDF60-7010,54255
17poly(PEG1500-MA)and
21SRF55-6010,5256
21 2,50,05334
19poly(PEG500-MA)and
27Refractionary10,56,138 for
28Refractionary10,56713

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

The test example 11

Estimating the size of a molecule by the method of exclusion chromatography size

The size of the molecule for each of the complexes between the polymer modifier and OCIF obtained in example 9, example 14, example 17 and example 19, was estimated by the method of exclusion chromatography size. The test conditions shown in the following table 12.

Table 12< / br>
Conditions when conducting exclusion chromatography size
Apparatus for chromium is ographie: Explorer 10S (Amersham Biotech)

Column: Superdex 200 HR10/30 (Amersham Biotech)

Mobile phase: a physiological solution with phosphate buffer (8 mm Na2HPO4, 15 mm KN2RHO4, 145 mm NaCl, 0.5 g/l NaN3)

Temperature analysis: 4°

The wavelength of detection: 280 nm

The flow rate of mobile phase: 0.6 ml/min

Retention time the following standard proteins in the above conditions shown in the following table 13.

Table 13< / br>
The retention time of each standard protein
The type of proteinMolecular mass (kDa)The Stokes radius (nm)Retention time (min)
Ferritin4406,1018,41
Aldose1584,8122,48
Ovalbumin433,0525,19
Ribonuclease13,71,6429,56

Based on the results exclusion chromatography size, the Stokes radius for uncomplexed OCIF determined equal 5,63 nm and the Stokes radius for each set of polymer modifier-OCIF of this invention to define is whether, as follows:

The complexes obtained in example 9:

Complexes of poly(PEG500-MA)g (connection # 1)-OCIF (Stokes radii in the range from 6,13 nm and 7,32 nm),

Complexes of poly(PEG500-MA)a (compound No. 3)-OCIF (6,12 nm and 6,54 nm),

The complex of poly(PEG500-MA)DMA (compound No. 4)-OCIF (6,39 nm),

The complex of poly(PEG500-MA)IPA (compound No. 5)-OCIF (6,26 nm),

The complex of poly(PEG500-MA)EA (compound No. 6)-OCIF (6,44 nm),

The complex of poly(PEG1500-MA)g (connection # 2)-OCIF (6,44 of 6.71 nm to nm),

The complex of poly(PEG1500-MA)a (compound No. 7)-OCIF (6,40 nm to 6.47 nm),

The complex of poly(PEG1500-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 the Stokes radius, which is approximately 1 nm exceeds the relevant value for uncomplexed OCIF in physiological solution with phosphate buffer was used as the mobile phase. In addition, each of the samples, the peak arising from the presence of unmodified OCIF, not recorded, which indicates the stability of the complexes.

The Stokes radii for other complexes was determined in conditions similar to those described above. The results consisted of the following:

The complexes obtained in example 14:

The complexes obtained in the conditions: OCIF concentration of 5 mg/ml; the concentration of the polymer modifier is in the range of from 1.25 mg/ml to 5 mg/ml; pH 7.4; 25°S; 36 hours; the environment in the form of a saline solution with phosphate buffer (concentration of phosphate, 10 mm; NaCl concentration of 150 mm), as shown, are characterized by the Stokes radii in the range from 6.2 nm to 6.5 nm. In addition, each of the samples, the peak arising from the presence of unmodified OCIF, not recorded, which indicates the stability of the complexes.

The complexes obtained in example 17:

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

The complexes obtained in example 19:

The complexes obtained in the conditions of incubation: OCIF concentration of 5 mg/ml; the concentration of polymer modifier 5 mg/ml; pH 5.5; 25°; 168 h; medium in the form of saline phosphate buffer (concentration of phosphate, 10 mm; NaCl concentration of 150 mm), as shown, are characterized for themselves Stokes in the range of 6.3 nm to 6.8 nm. In addition, each of the samples, the peak arising from the presence of unmodified OCIF, not recorded, which indicates the stability of the complexes.

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

The test example 12

Evaluation of the retention in the blood for example, rats and assessment of detection OCIF by ELISA method

Tests to determine the retention in the blood and the degree of detection OCIF by ELISA method was performed for the complex obtained in example 22 (complex between compound No. 54 and OCIF) (incubation temperature: 25° (C) in the same way as described in the above examples, tests 2 and 3. Equivalent dose for OCIF was set at 0.1 mg/kg Concentration of OCIF in serum were found, amounted to 189 ng/ml after 6 hours after intravenous injection of the complex. Thus, it was confirmed that the complexes obtained using the sodium salt of poly(PEG500-MA)EA (compound No. 54) in the above example 22, demonstrated an excellent level of retention in the blood. In addition, the decrease in sensitivity in the detection of OCIF by ELISA method, determined from utochnim by modification of the protein in the complex under the action of the modifier, as it was found, very small.

The example tests 13

Estimating the size of the complex

The size of the molecules in the complexes obtained in examples 22 (a complex between the compound No. 54 and OCIF) and 24 (a complex between the compound No. 55 and OCIF), was estimated by gel-filtration chromatography as described in the above test example 11. It was found that the size of the molecules in these complexes demonstrates a high level of uniformity. In the case of complexes between the connection 54 and OCIF, educated at 4°, 10°and 25°S, the Stokes radii, as shown, are equal to 6.0 nm to 6.0 nm and 6.0 nm. In the case of complexes between compound No. 55 and OCIF the Stokes radii were equal to 6.4 nm. Thus, each of the complexes obtained in example 22 and 24, were characterized by a large Stokes radius in comparison with unmodified treated human OCIF (Stokes radius: 5,63 nm).

Sample preparation

The solution containing the complex obtained in sterile conditions in the same way as described in the above example 14, was subjected to freeze drying and received lyophilized drug.

1. The copolymer or its pharmacologically PR is acceptable salt, which contain as forming their elementary parts

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

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

Alk represents alkylenes group containing from 1 to 6 carbon atoms, and

R1and R2are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms, and

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

where R3represents a

hydroxyl group,

alkoxy group containing from 1 to 6 carbon atoms, which optionally can be substituted by an aryl group containing from 6 to 14 carbon atoms,

aryloxy-group containing from 6 to 14 carbon atoms, which optionally may be substituted by a nitro-group, or

the group has the formula-NR4R5where R4and R5are identical or different from each other, and each of them before the hat is a hydrogen atom or alkyl group, containing from 1 to 6 carbon atoms, which optionally may be substituted by a hydroxy-group,

moreover, the arrangement of structural units represented by the formula (I)and structural units represented by the formula (II)is selected from the following (i)to(iv):

(i) the location of said structural units of the formula (I) and formula (II) as a sequence of alternating "head to head"

(ii) arrangement of structural units of the formula (I) and formula (II) as a sequence of alternating "head to tail"

(iii) the location of said structural units of the 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 said structural units of the formula (I) and formula (II) in the form of arbitrary sequence; and

where the ratio between the structural units represented by the formula (I)and structural units represented by the formula (II)in the 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 those who envoy or trimethylene group.

3. The copolymer or its pharmacologically acceptable salt according to claim 2, where Alk represents 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 R1represents a hydrogen atom.

9. The copolymer or its pharmacologically acceptable salt according to claim 1, where R2represents a methyl group.

10. The copolymer or its pharmacologically acceptable salt according to claim 1, where R3represents a hydroxyl group or alkoxygroup containing from 1 to 6 carbon atoms.

11. The copolymer or its pharmacologically acceptable salt according to claim 10, containing at least one structural unit described by formula (II)in which R3is alkoxygroup containing from 1 to 6 carbon atoms, and optionally at least one structural unit described the data by the formula (II), in which R3represents a hydroxyl group, where the ratio between the structural units described by formula (II)in which R represents a hydroxy group, and structural units described by formula (II)in which R3is alkoxygroup 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 R3is alkoxygroup containing from 1 to 6 carbon atoms.

13. The copolymer or its pharmacologically acceptable salt of claim 10, where the specified alkoxygroup containing from 1 to 6 carbon atoms, represents ethoxypropan.

14. The copolymer or its pharmacologically acceptable salt of claim 10, where R3represents a hydroxyl group or a group described by formula-NR4R5where R4and R5are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms.

15. The copolymer or its pharmacologically acceptable salt 14 containing at least one structural unit described by formula (II)in which R3represents a group described by formula-NR4R5where R4and R5are identical or different, and each of them presented yet a hydrogen atom or alkyl group, containing from 1 to 6 carbon atoms, and optionally at least one structural unit described by formula (II)in which R3represents a hydroxyl group, where the ratio between the structural units described by formula (II)in which R3represents a hydroxy group, and structural units described by formula (II)in which R3represents a group described by formula-NR4R5is 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 R3represents a hydroxy group, and structural units described by formula (II)in which R3represents a group described by formula-NR4R5is in the range from 4:6 to 0:10.

17. The copolymer or its pharmacologically acceptable salt according to any one of p-16, where R3represents a group described by formula-NR4R5where R4and R5are identical or different and each represents a hydrogen atom or an alkyl group containing from 1 to 6 carbon atoms.

18. The copolymer or its pharmacologically acceptable salt according to any one of p-16, where the group has the formula-NR4R5is an amino group, m is filamentgroup or dimethylaminopropyl.

19. The copolymer or its pharmacologically acceptable salt according to any one of p-16, where the group has the formula-NR4R5is an amino group.

20. The copolymer or its pharmacologically acceptable salt according to any one of p-16, where the group has the formula-NR4R5is dimethylaminopropyl.

21. The copolymer or its pharmacologically acceptable salt according to claim 1, where R3represents a hydroxyl group.

22. The copolymer or its pharmacologically acceptable salt according to claim 1, where R3represents 1-amino-2-propanolol 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 a 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 a 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 a 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 structural SV is but of the formula (II) are arranged in a random sequence, and the ratio between the structural unit described by formula (I)and a structural unit described by formula (II)is 1:1.

27. The copolymer or its pharmacologically acceptable salt according to claim 1, where the structural unit represented by formula (I)and a structural unit represented by formula (II), are arranged in a random order, and the ratio between the structural unit described by formula (I)and a 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 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 p, where the average degree of polymerization is in the range from 20 to 30.

32. The copolymer or its pharmacologically acceptable salt 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 the Stokes radius is 9.3 nm or less.

34. The copolymer or its pharmacologically acceptable salt according to claim 3, where the Stokes radius is 7.3 nm or less.

35. The copolymer or its pharmacologically acceptable salt according to clause 34, where the Stokes radius is 6.2 nm or less.

36. The copolymer or its pharmacologically acceptable salt p where their Stokes radius is 4.7 or less.

37. The copolymer or its pharmacologically acceptable salt p where their Stokes radius is 3.1 nm or less.

38. The copolymer or its pharmacologically acceptable salt p where their Stokes radius is in the range from 1.5 to 4.7 nm.

39. The copolymer or its pharmacologically acceptable salt p 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 ethylene group, R1represents a hydrogen atom, R2represents a methyl group, a m, R, the ratio between the structural units described by formulas (I) and (II), (ratio of specified composition) and, where appropriate, the relationship between the units described by formula (II), where R3represents a hydroxy-group, and the basic units described by formula (II), where R3represents a group other than hydroxyl (ratio set by hydrolysis), choose from the following:

(i) m is in the range from 6 to 16, R3depict is to place a hydroxy-group, the ratio of the specified 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, R3represents a hydroxy-group, the ratio of the specified 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, R3represents the amino group, the ratio of the specified 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 represents dimethylaminopropyl, the ratio of the specified composition is 1:1, and the average degree of polymerization is in the range from 30 to 40;

(v) m is in the range from 6 to 16, R3represents 1-amino-2-propanolol group, the ratio of the specified composition is 1:1, and the average degree of polymerization is in the range from 30 to 40;

(vi) m is in the range from 6 to 16, R3choose from ethoxy - and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, and the ratio specified by hydrolysis, 4:6;

(vii) m is in the range from 28 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is away in the range from 10 to 15, and the ratio specified by hydrolysis, 4:6;

(viii) m is in the range from 28 to 38, R3is dimethylaminopropyl, the ratio of the specified 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, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, the value specified by hydrolysis, as well 3,1:6,9, and the copolymer is a sodium salt;

(x) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, and the ratio specified by hydrolysis, is equal to 1.4:8,6;

(xi) m is in the range from 6 to 16, R3selected from dimethylamino - and hydroxy groups, the ratio of the specified composition is 1:1, the average degree of polymerization is in the range from 30 to 40, the value specified by hydrolysis, equal to 2.9:7,1, and the copolymer is a sodium salt;

(xii) m is in the range from 6 to 16, R3represents the amino group, the ratio of the specified composition is 1:2,4, and the average degree of polymerization is in the range from 20 to 30;

(xiii) m is in the range is from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 0.4:9,6;

(xiv) m is in the range from 6 to 16, and R are selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 2.9:7,1;

(xv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 0.9:9,1;

(xvi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.5 to 9.5;

(xvii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.3:8,7;

(xviii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, who Rudnaya degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 1.9:8,1;

(xix) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 1.0:9,0;

(XX) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.8:9,2;

(xxi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, as well 4,6:5,4;

(xxii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio set by the hydrolysis is 1.2:8,8;

(xxiii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio set by the hydrolysis is 2.0:8,0;

(xxiv) m is a dia is the azone from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.1:8,9;

(xxv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 2.4:7,6;

(xxvi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 0.9:9,1;

(xxvii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 1.5:8,5;

(xxviii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.7:9,3;

(xxix) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, the average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, as well 4,5:5,5;

(xxx) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.4:8,6;

(xxxi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.7:9,3;

(xxxii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is 0.8:9,2;

(xxxiii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 1.4:8,6;

(xxxiv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition, is equal to 1:3,1, average degree of polymerization is in the range from 20 to 30, and sootnoshenie is, asked by hydrolysis, is 0.7:9,3;

(xxxv) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, is equal to 0.9:9,1;

(xxxvi) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:2,4, average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, equal to 1.9:8,1;

(xxxvii) m is in the range from 6 to 16, R3choose from ethoxy - and hydroxy groups, the ratio of the specified composition is approximately equal to 1:3, the average degree of polymerization is in the range from 20 to 30, and the ratio specified by hydrolysis, as well 3,1:6,9;

(xxxviii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 9.3 nm or less;

(xxxix) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 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, R3vybirayuthij amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 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, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 3.1 nm or less;

(xlii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 1.4:8,6, and the Stokes radius is 7.8 nm or less;

(xliii) m is in the range from 6 to 16, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 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, R3selected from amino and hydroxy groups, the ratio of the specified composition is 1:1, the ratio specified by hydrolysis, is equal to 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 parts of the anhydride of the carboxylic acids described by formula (III), in the copolymer, which contains as forming its elementary parts (a) one or several flats which are structural links which may be identical or different from each other, and which are described in the following formula (I)

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

Alk represents alkylenes group containing from 1 to 6 carbon atoms, and

R1and R2are identical or different and each represents a hydrogen atom or alkyl group, and

(b) the specified structural elementary link that contains a link anhydride carboxylic acids described by formula (III)

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

42. The copolymer or its pharmacologically acceptable salt according to paragraph 41, where the structural unit described by formula (I)and a structural unit described by formula (III)in the copolymer is oriented in a sequence of alternating "head to head", the sequence of alternating "head to tail" or 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 a structural unit described by formula (III)in the copolymer guiding the Ana in the form of statistical sequence.

44. The copolymer or its pharmacologically acceptable salt according to any one of p-43, where Alk represents ethylene or trimethylene group.

45. The copolymer or its pharmacologically acceptable salt according to item 44, where Alk represents ethylene group.

46. The copolymer or its pharmacologically acceptable salt according to any one of p-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 p, 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 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 p-43, where R1represents a hydrogen atom.

51. The copolymer or its pharmacologically acceptable salt according to any one of p-43, where R2represents a methyl group.

52. The copolymer or its pharmacologically acceptable salt according to any one of p-43, where the ratio between the structural unit described by formula (I)and a structural unit obtained by introducing one or more structural units described by formula (III), in one or n is how many reactions, selected from the group consisting of (i) hydrolysis, (ii) ammonolysis, (iii) aminolysis and (iv) the United States, 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 a 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) the United States, 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 a 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) the United States, 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 § 42, where the ratio between the structural unit described by formula (I)and a 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) ammonal is for, (iii) aminolysis and (iv) the United States, 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 a 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) the United States, is in the range from 1:2 to 1:4.

57. The copolymer or its pharmacologically acceptable salt according to any one of p-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 § 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 § 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 § 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 p-43, which is obtained by ammonolysis of a fragment of a carboxylic acid anhydride of the formula (III) copolymer.

63. The copolymer of Riego pharmacologically acceptable salt according to item 62, upon receipt of which the ammonolysis of the used aqueous solution of ammonia.

64. The copolymer or its pharmacologically acceptable salt according to any one of p-43, which is obtained by aminolysis fragment anhydride of carboxylic acid of the formula (III) copolymer.

65. The copolymer or its pharmacologically acceptable salt p, upon receipt of which aminolysis used aqueous solution of dimethylamine.

66. The copolymer or its pharmacologically acceptable salt according to any one of p-43, which is obtained by alcoholysis of a fragment of a carboxylic acid anhydride of the formula (III) copolymer.

67. The copolymer or its pharmacologically acceptable salt p, upon receipt of which the United States used ethanol.

68. Pharmaceutical composition for prevention or treatment of metabolic bone containing a pharmaceutically acceptable diluent or carrier, at least one protein or an analogue or variant and at least one copolymer or its pharmacologically acceptable salt according to any one of claims 1 to 67, and this protein, or an analogue or variant, is a factor inhibiting osteoclastogenesis (OCIF), or its analogue or variant.

69. The pharmaceutical composition according p where specified OCIF or analogue or variant, is a natural or recombinant type OCIF.

70. Pharmaceutical to notice on p, where specified OCIF or analogue or variant, is a monomer or dimer.

71. The pharmaceutical composition according p where specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 according to measurement by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 as measured by the method of SDS-PAGE in non conditions.

72. The pharmaceutical composition according p where specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

73. The pharmaceutical composition according p where specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

74. Modifier capable of modifying a protein or an analogue or variant, where the specified modifier contains a copolymer or its pharmacologically acceptable salt according to any one of claims 1 to 67.

75. Modifier capable of modifying a protein or an analogue or variant, p, where protein is the main protein.

76. Modifier capable of modifying a protein or an analogue or variant according to item 75, where the main protein is a basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), factor inhibiting osteoclast is enesa, (OCIF), platelet-derived growth factor (PDGF), isolated from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepaticotomy growth factor (HGF) or growth factor vascular endothelial (VEGF), or an analogue or variant.

77. Modifier capable of modifying a protein or an analogue or variant according to item 75, where the main protein is a factor inhibiting osteoclastogenesis (OCIF), or its analogue or variant.

78. Modifier capable of modifying a protein or an analogue or variant, p where specified OCIF or analogue or variant relate to OCIF natural type or recombinant type.

79. Modifier capable of modifying a protein or an analogue or variant according to item 75, where specified OCIF or analogue or variant, represent a monomer or dimer.

80. Modifier capable of modifying a protein or an analogue or variant, p where specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 according to measurement by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 according to measurement by the method of SDS-PAGE in non conditions.

81. Modifier capable of modifying a protein or an analogue or variant, p, g is e specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

82. Modifier capable of modifying a protein or an analogue or variant, p where specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

83. The complex containing at least one protein, or an analogue or variant, which is associated at least one copolymer or its pharmacologically acceptable salt according to any one of claims 1 to 67.

84. Complex p, where protein is the main protein.

85. Complex p, where the main protein is a basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), factor inhibiting osteoclastogenesis, (OCIF), platelet-derived growth factor (PDGF), isolated from brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), human growth hormone (HGH), hepaticotomy growth factor (HGF) or growth factor vascular endothelial (VEGF) or an analogue or variant.

86. Complex p, where the main protein is a factor inhibiting osteoclastogenesis (OCIF), or its analogue or variant.

87. Complex p where specified OCIF or analogue or variant, refer to OCIF natural type or recombinant type.

88. Complex p where specified OCIF or analogue or variant, represent a monomer or dimer.

89. Complex p where specified OCIF, not only is em a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 according to measurement by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 according to measurement by the method of SDS-PAGE in non conditions.

90. Complex p where specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

91. Complex p where specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

92. Pharmaceutical composition for prevention or treatment of metabolic substance in the bones, containing an effective amount of pharmacologically active substances together with a carrier or diluent for him, where the aforementioned pharmacologically active substance is present in the form of a complex according to any one of p-91.

93. How to extend the time within which the factor inhibiting osteoclastogenesis (OCIF), or its analogue or variant, is retained in the bloodstream after ingestion by the patient as the result of complex formation between the specified protein, or an analogue or variant, and at least one copolymer, or its pharmacologically acceptable salt according to any one of claims 1 to 67.

94. The method according to p where specified OCIF or analogue or variant include what I OCIF natural type or recombinant type.

95. The method according to p where specified OCIF or analogue or variant, represent a monomer or dimer.

96. The method according to p where specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 according to measurement by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 according to measurement by the method of SDS-PAGE in non conditions.

97. The method according to p where specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

98. The method according to p where specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID No.№1 list of sequences.

99. Method for the treatment or prevention of metabolic disorders in the bones, including the patient an effective amount of a complex containing the complex, including the factor of inhibition of osteoclastogenesis (OCIF), or its analogue or variant, which is associated at least one copolymer, or its pharmacologically acceptable salt according to any one of claims 1 to 67.

100. The method of prevention or treatment of metabolic bone on p where specified OCIF or analogue or variant, OCIF is a natural type or recombinant type.

101. A method of preventing or cured the I of metabolic bone on p, where specified OCIF or analogue or variant, is a monomer or dimer.

102. The method of prevention or treatment of metabolic bone on p where specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 according to measurement by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 according to measurement by the method of SDS-PAGE in non conditions.

103. The method of prevention or treatment of metabolic bone on p where specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

104. The method of prevention or treatment of metabolic bone on p where specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

105. The use of complex, containing a protein or an analogue or variant that is associated with at least one copolymer, or its pharmacologically acceptable salt according to any one of claims 1 to 67, for manufacturing a medicinal product intended for the prevention or treatment of metabolic bone susceptible to the effect of said protein, or its analogue or variant, where this protein is with the combat factor inhibiting osteoclastogenesis (OCIF), or option or similar.

106. Use p where specified OCIF or analogue or variant, refers to OCIF natural type or recombinant type.

107. Use p where specified OCIF or analogue or variant, is a monomer or dimer.

108. Use p where specified OCIF is a monomer OCIF person, characterized by a molecular weight approximately equal to 60000 according to measurement by the method of SDS-PAGE in non conditions, or dimer OCIF person, characterized by a molecular weight of approximately 120000 according to measurement by the method of SDS-PAGE in non conditions.

109. Use p where specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

110. Use p where specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID No. No. 1 list of sequences.

Priority items:

24.03.2003 according to claims 1-10, 12-14, 17-22, 26, 28, 30, 41-51, 57, 62-67;

16.10.2003 under § § 11, 16, 55, 59;

24.03.2004 on PP, 23-25, 27, 29, 31-40, 52-54, 56, 58, 60-61, 68-110.



 

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20 cl, 10 tbl, 5 ex

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4 cl, 1 tbl

FIELD: organic chemistry, polymers.

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4 cl, 6 ex, 1 tbl

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4 cl, 8 ex, 1 tbl

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