The complex containing inhibitory osteoclastogenic factor (ocif) and polysaccharide

 

The invention relates to the field of medical drugs. The essence of the invention is a new complex containing at least one substance selected from the group: enzyme inhibition osteoclastogenic factor (OCIF), its analogs and its variants, which is associated with a substance selected from the group consisting of polysaccharides and derivatives thereof. Complex detects the property of prolonged retention in the bloodstream after administration, making it applicable in the treatment and prevention of bone metabolic diseases. The technical result is an expansion of the means for normalization of bone metabolism. 7 N. and 27 C.p. f-crystals, 8 PL.

The technical field,

This invention relates to a complex containing at least one inhibitory osteoclastogenic factor (hereinafter referred to as OCIF or analogue or variant and at least one polysaccharide or its derivative, to a method for the specified complex, a drug for treating or preventing metabolic bone diseases containing this complex as an active ingredient, and to the use of this complex in the treatment or prevention of bone IU is existing in a living organism, and, therefore, play an important role not only in maintaining the body, but also functioning as the largest stored calcium organ in the body. Bones play an important role in the maintenance of calcium homeostasis. It is known that activation of osteoclasts, which play an important role in bone resorption, causing excessive influx of calcium into the blood from the bones with disturbance of calcium homeostasis in the blood inducyruya hypercalcemia. This induction of hypercalcemia through activation of osteoclasts and stimulation of bone resorption can be induced by cytokines that are secreted abnormally due to the spread of cancer to the bone [for example, see Jean-Jacques Body, Current and Future Directions in Medical Therapy: Hypercalcemia, CANCER Supplement, 88(12), 3054-3058 (2000)]. The prognosis for patients suffering from cancer hypercalcemia is usually poor, and, therefore, highly desirable is to find effective treatment for this condition.

It is known that in rheumatoid arthritis, such as rheumatoid arthritis, etc., or osteoarthritis, abnormal formation or abnormal activation of osteoclasts is one of the main causes of various symptoms, which are present in the bones and joints of patients suffering from these conditions [see, id Arthritis, Bone, 30(2), 340-346 (2002)]. Pain in the joints and bones caused by rheumatoid arthritis, such as rheumatoid arthritis and osteoarthritis, is extremely intense and is extremely detrimental to the quality of life for patients suffering from these conditions. Again, therefore, is highly desirable finding effective treatment for such conditions.

It is also known that osteoclasts play a role in osteoporosis. The balance of bone resorption stimulated by osteoclasts and bone formation stimulated by osteoblasts, is gradually inclined in the direction of bone resorption due to decreased secretion of female hormones after menopause or due to aging, resulting in bone density decreases and occurs osteoporosis, if this decrease in bone density is quite heavy. When aging patients with high risk of osteoporosis suffer a fracture, the possibility that they will become bedridden, is very high, and it became a social problem in the growing aging population in all parts of the world [for example, see Bruno Fautrel and Francis Guillemin, Cost of illness studies in rheumatic diseases, Current Opinion in Rheumatology, 14, 121-126 (2002)]. Thus is a vigorous search for an effective way of lecinena hormones, such as estrogen, and the use of agents that inhibit the activity of osteoclasts, such as bisphosphonates or calcitonin [see, for example, Mohammed M. Iqbal and Tanveer Sobhan, Osteoporosis: A Review, Missouri Medicine, 99(1), 19-23 (2002).]. However, hormones can have unwanted side effects, such as increased risk of carcinogenesis induction of endometriosis and abnormal bleeding from the genital organs [e.g., see Joyce Penrose White and Judith S. Schilling, Postmenopausal Hormone Replacement: Historical Perspectives and Current Concerns, Clinical Excellence for Nurse Practitioners, 4(5), 277-285 (2000)]. Although it is known that bisphosphonates can easily bind excess calcium in the blood and accumulate in the bones, some researchers doubt the extent to which it can be improved bone strength with their use. In addition, it was also reported that there is a danger of deterioration of kidney function associated with their use [for example, see Jonathan R. Green, Yves Seltenmeyer, Knut A. Jaeggi and Leo Wildler, Renal Tolerability Profile of Novel, Potent Bisphosphonates in Two Short-Term Rat Model, Pharmacology and Toxicology, 80, 225-230 (1997)]. As for calcitonin, increase bone density, obtained with its use, is unfortunately temporary. It was also reported that the interruption of the injection of calcitonin may cause regression of the condition to be treated, while casinogo treatment due to the appearance of circulating antibodies to this calcitonin in humans [S. L. Porcel, J. A. Cumplido, B. dela Hoz, M. Cuevas and E. Losada, Anaphylaxis to calcitonin, Allergologia et Immunopathologia, 28(4), 243-245 (2000)].

As explained above, osteoclasts play a major role in stimulation of bone resorption, which is an important factor contributing to the increase in the concentration of calcium in the blood. However, it is believed that none of the above drugs is not active in the suppression of the formation of osteoclasts. Thus, none of these conventional medicines is not suitable for a fundamental treatment of bone metabolic diseases, as these drugs are able to deal only with symptoms, not causes.

Not so long ago it was shown that OCIF is an endogenous protein which inhibits the differentiation of precursor cells of osteoclasts in osteoclasts and/or bone-resorption activity of Mature osteoclasts (see WO-A-96/26217 and EP-A-0816380). In view of the fact that the above-mentioned metabolic bone diseases, such as hypercalcemia, osteoporosis and rheumatoid arthritis are the result of at least some degree of bone resorption, it was hoped that these diseases could be treated successfully using OCIF field, have been confirmed due to osteoclast. However, OCIF is the main protein that has an isoelectric point of about 9, and it disappears very quickly from the bloodstream after administration. An attempt to solve the problem described in WO-A-2000/24416 and EP-A-1127578, where the length of time during which OCIF remains in the blood after administration (and therefore OCIF), was extended to some extent by the joint introduction of OCIF with a polysaccharide, such as heparin, or textresult. However, the prolonged retention time achieved as a result, may be insufficient to obtain so prolonged retention OCIF in the blood, to make it a true candidate for use in the treatment of bone metabolic diseases, such as hypercalcemia, osteoporosis and rheumatism. Thus, there is a need for an improved method of prolongirovanie the length of time during which OCIF remains in the bloodstream after administration.

The invention

Thus, the purpose of this invention is the provision of a preparation containing OCIF, which allows you to prolong the time during which OCIF is retained in the bloodstream after administration, ensuring thus the agent (the funds), which actually as hypercalcemia, osteoporosis and rheumatism, is enhanced and prolonged.

Other objectives and advantages of this invention will become apparent as the description.

Thus, this invention provides a complex containing at least one substance selected from the group consisting of OCIF, its analogs and its variants, which is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof.

This invention also provides a method of prolonging the time during which OCIF or analogue or variant is retained in the bloodstream after administration to the patient through education complex at least one specified OCIF specified analog or specified variant with at least one polysaccharide or its variant.

This invention also provides a pharmaceutical composition comprising an effective amount of pharmacologically active agent (means) together with a carrier or diluent, where the aforementioned pharmacologically active agent is a compound containing at least one substance selected from the group consisting of OCIF, its analogs and its variants, which spasenoski, it provides such pharmaceutical composition for the treatment or prevention of bone metabolic diseases.

This invention also provides a method of treatment or prevention of bone metabolic diseases, providing for the introduction to the patient an effective amount of a complex containing at least one substance selected from the group consisting of OCIF, its analogs and its variants, which is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof.

This invention also provides the use of a complex containing at least one substance selected from the group consisting of OCIF, its analogs and its variants, which is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof, in the preparation of medicines for the prevention or treatment of bone metabolic diseases.

Detailed description of the invention

The authors found that by incubating at least one substance selected from the OCIF, its analogues and variants, with at least one substance selected from polysaccharides and derivatives thereof, under conditions that are economical and options associated with the specified at least one substance selected from polysaccharides or derivatives thereof, is formed by this agent, in which the effect of said OCIF or analogue or variant in the prevention and treatment of bone metabolic diseases, mediated by osteoclasts, such as hypercalcemia, osteoporosis and rheumatism, is enhanced or prolonged. This is due to the fact that the length of time during which the said OCIF or analogue or variant is retained in the bloodstream after injection, prolonged in comparison with combinations of OCIF and polysaccharides previous prior art, described in WO-A-2000/24416 and EP-A-112578.

As noted above, the complexes of this invention contain at least one substance selected from the OCIF, its analogues and variants that are associated with at least one substance selected from polysaccharides and derivatives thereof. In the specified complex of OCIF and the polysaccharide are linked together by a chemical bond such as a covalent bond (for example, with the formation of Schiff bases), an ionic bond or a coordinate bond or a non-chemical communications, such as hydrophobic interaction, hydrogen bond, electrostatic maintains the t to be the natural type or it can be a recombinant type, and its origin is not particularly limited. OCIF natural type indicates OCIF, which is obtained in the form of natural protein produced by the extraction, purification and/or separation from the body, body fluids, cell cultures derived from human or animal, not a person. Recombinant OCIF type, its analogue or variant is a recombinant protein obtained by extraction, purification and/or allocation of the specified protein from the host, usually used in such ways, such as a prokaryotic cell, the master (for example, Escherichia coli) or eukaryotic cell, such as cell line human or not human that was transformed by 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].

The origin of OCIF, its analogs and its variants used in this invention is not particularly limited, and they can be obtained from a person or animal (not human). Preferably, they can be obtained from a mammal, such as human, rat, mouse, rabbit, dog, cat, cow, pig, sheep or goat; or birds, such as poultry, GU is ucaut of man.

OCIF or analogue as used in this invention may be OCIF monomer type (for example, in people with a monomer having a molecular weight, measured by electrophoresis in LTO-SDS page under non conditions of about 60000 or dimeric type (for example, in people dimer having a molecular weight of about 120000 as measured by electrophoresis in LTO-SDS page under non conditions) [see EP-A-0816380 (WO-A-96/26217)].

It is known that OCIF is transmitted in cells in the form of a polypeptide containing a signal peptide at its aminocore, and that then he Matures with use of the processing, including the removal of the specified signal peptide [for example, see the recombinant methods described in EP-A-0816380 (WO-A-96/26217)]. OCIF, an analogue or variant used in this invention includes as a polypeptide containing a signal peptide and the Mature form. Preferred examples include OCIF with signal peptide, which amino acids -21 to +380 of SEQ ID NO:1 list of sequences, and Mature OCIF without the signal peptide, which amino acids +1 to +380 of SEQ ID NO: list of sequences. Of them, particularly preferred is a Mature OCIF.

It is also known that the methionine maranantha protein in the cell-master, in particular prokaryotic cell host, such as Escherichia coli. This is achieved by adding a triplet of nucleotides having the sequence ATG (AUG) to the 5’-end of polynucleotide encoding the Mature form of OCIF, its analogue or variant, and the embedding obtained polynucleotide expressing in a suitable vector. Then this desired Mature protein having a methionine at its aminocore, can be expressed a suitable cell host, which was transformed specified expressing recombinant vector. In addition, one or more amino acids can be added to the specified protein at the position adjacent to the triplet of nucleotides ATG, added at the 5’end of polynucleotide encoding the Mature form of OCIF, its analogue or variant.

The present invention similar OCIF refers to the protein encoded by polynucleotides that exists in natural cells, bodily fluids and/or organs of humans or animals (not humans), such as shown in the examples above. Specific preferred examples of such analogues include OCIF2, OCIF3, OCIF4 and OCIF5 [see EP-A-0816380 (WO-A-96/26217)]. Such analogues OCIF or their active fragments can be obtained using the following method: RNA akatora complementary specified RNA, synthesized using reverse transcriptase, and then the second circuit indicated synthesize cDNA using the first circuit as the template using a DNA polymerase; the thus obtained double-stranded cDNA is inserted into suitable, commonly used expressing vector; then a suitable, commonly used cell host transform thus obtained vector; then the cells of the host, producing the desired peptide is subjected to screening using the method of hybridization, such as hybridization of phage plaques or hybridization, using OCIF cDNA or its fragment as probe under stringent hybridization conditions [see EP-A-0816380 (WO-A-96/26217)]; and then, finally, the desired analog OCIF is an accepted way thus received to the host-cell.

In this variant of the invention OCIF refers to a protein that has the amino acid sequence in which one or more amino acid residues have been replaced, delegated, added or insertion in the amino acid sequence of OCIF or analogue, and still has at least a portion of OCIF activity. Such OCIF variants can be obtained, for example, by using the nucleotide sequence, encoding OCIF or analogue, using the method of polymerase chain reaction (hereinafter referred to as PCR), methods of genetic recombination or way of splitting the nucleases using ectonucleoside or endonuclease, such as restriction enzyme; transforming a eukaryotic host cell, such as an animal cell, or a prokaryotic host cell, such as Escherichia coli expressing vector, in which was embedded the obtained nucleotide encoding the desired variant OCIF; and then the extraction, purification and/or highlight the desired peptide from the protein fraction produced by culture of cells transformed specified host in accordance with the method, a well-known specialist in this field.

It is known that the shortened form OCIF, in which a significant part of the amino acid sequence has been delegated from carboxylic polypeptide OCIF, also retain at least part of the OCIF activity [for example, see EP-A-0816380 (WO-A-96/26217) and WO-A-97/23614]. These shortened types of OCIF, preserving at least part of the activity of the full polypeptide OCIF, also included in the OCIF variants of the present invention.

Also known OCIF or its shortened form, which chrysoidine to carboxylic OCIF), and which retain at least some portion of the full activity of the polypeptide OCIF (see WO-A-97/23614), and such fused proteins are also included in the OCIF variants of the present invention.

It was also shown that OCIF or analogue or variant can be chemically modified and still retain the applicable activity and, in some cases, there may be benefits, such as increased stability or reduced immunogenicity. Such chemical modification can include the derivatization of only a single site in the molecule OCIF or analogue or variant or in more than one site. For example, it was shown that OCIF and its variants (derivatives), such as a shortened form, can be chemically modified by one or more water-soluble polymers such as polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose and polyvinyl alcohol, and can be found in the enhanced biological activity (for example, see WO-A-97/23614). Such chemically modified types of OCIF or analogue or variant is also included in OCIF variants of the present invention.

Examples of known OCIF variants that are suitable for use in P3, OCIF-DCR4, OCIF-DDD1, OCIF-DDD2, OCIF-CL, OCIF-CC, OCIF-CDD2, OCIF-CDD1, OCIF-CCR4, OCIF-CCR3, OCIF-CBst, OCIF-CSph, OCIF-CBsp, OCIF-CPst [see EP-A-0816380 (WO-A-96/26217)], muOPG[22-401]-Fc, muOPG[22-194]-Fc, muOPG[22-185]-Fc, muOPG[22-180]-Fc, muOPG[22-401], muOPG[22-401]C195, muOPG[22-401]C202, muOPG[22-401]C277, muOPG[22-401]C319, muOPG[22-401]C400, muOPG[22-185], muOPG[22-194], muOPG[22-200], muOPG[22-212], muOPG[22-293], muOPG[22-355], huOPG[22-401]-Fc, huOPG[22-201]-Fc, huOPG[22-401]-Fc P26A, huOPG[22-401]-Fc Y28F, huOPG[22-401], huOPG[27-401]-Fc, huOPG[29-401]-Fc, huOPG[32-401]-Fc, muOPG met[22-194], muOPG met[22-194] 5k PEG, muOPG met[22-194] 20k PEG, huOPG met[22-194]P25A, huOPG met[22-194]P25A 5k PEG, huOPG met[22-194]P25A 20k PEG, huOPG met[22-194]P25A 31k PEG, huOPG met[22-194]P25A 57k PEG, huOPG met[22-194]RA 12k PEG, huOPG met[22-194]P25A 20k branched PEG, huOPG met[22-194]P25A 8k PEG dimer, huOPG met[22-194]P25A with disulfide cross-link (WO-A-97/23614), OPG[22-194]-Fc, OPG[22-201]-Fc, OPG[22-194]-FcC, OPG [22-201]-FcC, OPG [22-194]-FcG10, metFcC-OPG[22-194] (WO-A-2001/17543), OPG[22-194]-FcC, OPG[22-194]-FcG10, FcC-OPG[22-194], metFcC-OPG[22-194], metFcC-22-194, OPG[22-194]-Fc, OPG[22-194]-FcC, metOPG[22-194], metOPG[22-201], OPG[22-293], OPG[22-401] and metFcC-22-194 (WO-A-2001/18203).

Of them, preferable examples include: OCIF-C19S, OCIF-C20S, OCIF-C21S, OCIF-C22S, OCIF-C23S, OCIF-DCR1, OCIF-DCR2, OCIF-DCR3, OCIF-DCR4, OCIF-DDD1, OCIF-DDD2, OCIF-CL, OCIF-CC, OCIF-CDD2, OCIF-CDD1, OCIF-CCR4, OCIF-CCR3, OCIF-CBst, OCIF-CSph, OCIF-CBsp, OCIF-CPst, muOPG[22-401]-Fc, muOPG[22-194]-Fc, muOPG[22-185]-Fc, m22-401]-Fc Y28F, huOPG[22-401], huOPG[27-401]-Fc, huOPG[29-401]-Fc, huOPG[32-401]-Fc, muOPG met[22-194]5k PEG, muOPG met[22-194]20k PEG, huOPG met[22-194]P25A 5k PEG, huOPG met[22-194]P25A 20k PEG, huOPG met[22-194]P25A 31k PEG, huOPG met[22-194]P25A 57k PEG, huOPG met[22-194]P25A 12k PEG, huOPG met[22-194]P25A 20k branched PEG, huOPG met[22-194]P25A 8k PEG dimer, huOPG met[22-194]P25A with disulfide cross-link, OPG[22-194]-Fc, OPG[22-201]-Fc, OPG [22-194 ]-FcWith, OPG[22-201]-FcC, OPG [22-194]-FcG10, metFcC-OPG[22-194], OPG[22-194]-FcC, OPG [22-194]-FcG10, FcC-OPG[22-194], metFcC-OPG[22-194], metFcC-22-194, OPG[22-194]-Fc, OPG[22-194]-FcC, metOPG[22-194], metOPG[22-201], OPG[22-293], OPG[22-401] and metFcC-22-194.

OCIF or analogue or variant of the present invention may contain a sugar chain as part of the molecule. Any natural produced OCIF or analogue or recombinant OCIF or analogue or variant can contain a sugar chain that is attached to OCIF or analogue or variant excision. Natural produced OCIF or analogue containing sugar chain can be obtained from cell cultures, tissues, organs, body fluids or cell lines derived from human or animal (not human) using conventional sea eukaryotic host cell, transformed by a vector containing a nucleotide sequence encoding OCIF or analogue or variant, such as analogs and variants described and shown as examples above. Examples of suitable host cells, which can be used that is capable of post-translational modifications of OCIF or analogue or variant thus, to attach the sugar chain include cells of Chinese hamster ovary cells and COS [Yasuda, H. et al., Endocrinology, 139, 1329-1337 (1998)]. OCIF or analogue or variant containing a sugar chain, suitable for use in the formation of complexes of the present invention.

If, on the other hand, it is desirable to obtain recombinant OCIF or analogue or variant that has no sugar chain, which was attached as post-translational modifications, the preferred cells of the host are prokaryotic cells such as Escherichia coli.

The polysaccharide used in the formation of complexes of the present invention is a polymer (Picanol) produced by glycosidic bonds between two or more monosaccharides, and preferably is heteropolysaccharides (heteroglycans), consisting of at least doget to be used in the complex of the present invention.

In this invention a derivative of the polysaccharide is a polysaccharide in which at least a portion of the molecule specified polysaccharide substituted by one or more molecules and/or residues other than saccharide or sugar. Preferred derivatives include acid esters of polysaccharides and especially preferred are sulfate esters of polysaccharides.

Examples of natural polysaccharides suitable for use in the formation of complexes of this invention include hyaluronic acid, chondroitinase acid, dermatan acid, heparan acid, keratan-acid, carrageenan, pectin and heparin. Examples of synthetic polysaccharides suitable for use in the formation of complexes of this invention include dextran, whereas examples of suitable synthetic derivatives of polysaccharides include textresult. From polysaccharides and derivatives thereof are most preferred for use in the formation of complexes of the present invention is textresult.

In this invention polysaccharides and their derivatives, such as extrasolar include and their salts. The most preferred salt doctranslate is its hydroxide(hereinafter called DS5: produced by Meito Sangyo Co., Ltd.) and sodium salt of dextransucrase 5000 and sodium salt of dextransucrase 10000 (both are manufactured by Waco Pure Chemical Industries, Ltd.).

The molecular weight of dextransucrase calculated as follows.

1) Measurement of molecular weight dextran

Molecular weight dextran may be calculated according to the recipe Sato below [e.g., see Manual for Pharmacopoeia of Japan, the thirteenth revision, published by Hirokawashoten (1998), article concerning dextran 40], is based on measuring the limiting viscosity of the specified dextran.

The limiting viscosity = 9,0010-4molecular weight050

2) Measurement of sulfur content

The sulphur content of interest doctranslate can be measured in wt.% using any conventional method known in this field, such as a method described in the article concerning the sodium salt of dextransucrase sulfur 5 in the Pharmacopoeia of Japan [14threvision, published by Jihou (2001)].

Although the molecular mass of glucose, which is an elementary link of dextran equal to 180, the actual molecular weight of a glucose unit in a molecule of dextran equal to 162, and this value is calculated by subtracting the molecular weight of the water molecule dextran. The hydrogen atom is replaced by a sodium-sulfate groups (SO3Na: one gram-equivalent = 103) in each glucose level of dextransucrase, which is substituted therefore. Using this information, the degree of substitution of the molecule doctranslate (hereinafter referred to as “degree of substitution”) can be determined from the following formula:

Sulfur content (wt.%)=[32the degree of substitution/(162+102the degree of substitution)]100

3) Calculate the molecular weight of dextransucrase

Since, as noted above, the actual molecular weight of the glucose level in doctranslate equal to 162, the molecular mass of dextransucrase can be calculated on the basis of this information and the degree of substitution determined as described in (2) above, using the following formula:

The molecular mass of dextransucrase = molecular weight dextran(162+102the degree of substitution)/162

It is known that polysaccharides exhibit a molecular weight distribution, for example, each different type of dextransucrase manifests a certain molecular weight distribution. The molecular weight of any polysacharide molecular weight polysaccharides, used in this invention is in no way limited. The range of the average molecular weight is most preferred polysaccharide derivative of the present invention, dextransucrase, usually equal 1500-12000 and more preferably equal 1800-6000. Molecular mass (mean±standard deviation) DS5 approximately 1950±70 (n=7). The degree of substitution of sulfur (mean±standard deviation) DS5, calculated as described above, is approximately 0,32±0,01 (n=7). The average molecular weight of the sodium salt of dextransucrase 5000 and sodium salt of dextransucrase approximately 10000 5000 and approximately 10,000 respectively. The polysaccharides used for obtaining complexes of this invention can be used without any additional purification and/or fractionation or purification and/or fractionation them before applying. In this invention, the polysaccharides or their derivatives do not include any sugar chain that is attached to recombinant OCIF or its analogs or variants or natural produced OCIF or its analogs or variants of excision and/or endogenous cells or tissues or the body of man or animals, into, the substance selected from the group consisting of polysaccharides and derivatives thereof, complexes of this invention will vary depending on various factors, including the identity of the components of the specified complex and the conditions under which receive this complex. There are no special restrictions molar ratio of a substance selected from the group consisting of OCIF, its analogues and variants, the substance selected from the group consisting of polysaccharides and derivatives thereof, complexes of the present invention. In the preferred complexes of the present invention containing a substance selected from the group consisting of OCIF, its analogs and its variants, and textresult, the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to doctranslate is from 1:1 to 1:10; more preferably, this molar ratio is from 1:1 to 1:8; more preferably, this molar ratio is from 1:1 to 1:5 and most preferably this molar ratio is from 1:1 to 1:4,5.

As mentioned above, OCIF or analogue or variant can exist as a monomer or can form dimers, so OCIF or analogue or variant, present the Ohm, heterooligomeric, homopolymers or heteropolymers containing more than two Monomeric units OCIF, its analogue or variant (for example, see U.S. patent US 6369027). The molar ratio of a substance selected from the group consisting of OCIF, its analogues and variants, the substance selected from the group consisting of polysaccharides and their derivatives, the complex containing OCIF or analogue or variant and polysaccharides or their derivative, calculated as the number of molecules of the polysaccharide or its derivative, a monomer OCIF, option or similar.

The number of molecules of the polysaccharide or its derivative in the complex of the present invention preferably can be defined as follows. The content of neutral sugars tested complex [designated as (x)] and the same content of the reference sample, which contains not located in the complex, free OCIF or analogue or variant [designated as (y)], quantitatively determined by using method using phenol and sulfuric acid (which is described in detail elsewhere in this application). Then the amount of the polysaccharide or its derivative, which is associated with OCIF or analogue or variant in the test complex, determine Vici the tion, associated with OCIF or analogue or variant, calculated according to (I) or (II) as shown below:

(I) the resulting number to the number of the polysaccharide or its derivative, which is associated with OCIF or analogue or variant, is divided by the average molecular weight of the specified polysaccharide or its derivative. The resulting number represents the total number of molecules of the polysaccharide or its derivative in the test complex.

(II) the resulting number to the number of the polysaccharide or its derivative, which is associated with OCIF or analogue or variant, is divided by the amount (mg) of the specified OCIF, its analogue or variant specified in the complex. Then the resulting number, which is the amount of the polysaccharide or its derivative at 1 mg OCIF, its analogue or variant in this complex, is used to calculate the number of molecules of the polysaccharide or its derivative on one molecule OCIF, its analogue or variant based on the molecular weight of the specified OCIF, its analogue or variant [for example, in accordance with example 4(d), below].

The number of molecules of OCIF or analogue or variant in the complex of the present invention preferably can be determined using the method of immunological analysis, such as the JV is I, which can be used for their characteristics is their affinity towards heparin. Heparin is a polysaccharide containing D-glucosamine, D-glucuronic acid and D-Euronova acid, which partially or completely modified sulfate and acetyl groups. A preferred characteristic of the complexes of the present invention is that the strength of adsorption of the specified complex of OCIF or analogue or variant of heparin is lower than the strength of adsorption of the free, not in the complex of OCIF or analogue or variant. The degree of adsorption can be determined using a column Packed with beads of crosslinked to a high degree of agarose, which was immobilized heparin (for example, heparin is derived from mucosal bovine intestine). The appropriate columns of this type include column HiTrap heparin HP, HiPrep 16/10 Heparin and Heparin Sepharose (all can be obtained from Amersham Pharmacia). The strength of adsorption (affinity) of the complex can be determined according to any suitable method well known to a qualified specialist in this field, to determine the affinity of proteins against polysaccharides. Preferably the degree adseg low ionic strength, but which eluted from the specified column in conditions of high ionic strength, with a number of complex, which is not associated with the column of heparin under conditions of low ionic strength (ionic strength can be installed with the use of a salt of a strong acid, such as sodium chloride). Thus, the degree of adsorption of the complex on the heparin can be defined as follows:

(a) a Column Packed with media, such as granules of cross-linked agarose, which was immobilized heparin, balance buffer with low ionic strength (e.g., sodium phosphate buffer, containing 0.1-0.8 M sodium chloride).

(b) the Complex of the present invention, which is tested, dissolved in the same buffer of low ionic strength, which is used in (a), and applied on the column and then collect the first eluate (fraction A).

(c) Then the column is washed additionally with the same buffer of low ionic strength, which is used in stage (a), and collect the second eluate (fraction B).

(d) Then the column is washed with a buffer having a relatively high ionic strength (e.g., sodium phosphate buffer containing 1.0 to 2.0 M sodium chloride) and then collect the third eluate (fraction C).

(e) Then determine the number of comodi immunoassay).

(f) Then determine the degree of adsorption of the complex on the heparin according to the following formula:

The greater the strength of binding of the complex with the column, the greater the value (s) (as it may be removed from the column using eluents having a relatively high ionic strength) and, consequently, the greater the degree of adsorption. The degree of adsorption complexes of the present invention, measured by the above formula, will vary to some extent depending on the type of heparin columns and conditions, providing a definition. However, the degree of adsorption of the free, not in the complex of OCIF will always be equal to approximately 1.0, whereas the degree of adsorption complexes OCIF of the present invention is equal to less than 1.0, which demonstrates that the strength of binding complexes containing OCIF or analogue or variant according to this invention, heparin is weaker than the strength of binding free, not in the complex of OCIF or analogue or variant (e.g., using porcine heparin immobilized on agarose pellets, such as column HiTrap heparin HP, the first and second ALSI 10 mm sodium phosphate, Butera what I the degree of adsorption complexes of this invention containing OCIF or analogue or variant, is not more than 0.7, preferably not more than 0.6, and particularly preferably not more than 0.5).

Another preferred characteristic of the complexes of the present invention, which can be used for their characteristics, is the ratio of the number of molecules of OCIF or analogue or variant present in the specified complex, measured by the method of immunological analysis (e.g., ELISA), to the number of molecules of OCIF or analogue or variant present in the specified complex, as measured by the method of measuring total protein present in the specified complex [for example, according to the method of Lowry: Lowry, O. H. et al., J. Biol. Chem., 193, 263-275 (1951), by absorption280) when staining with silver or BSA-method].

The number of molecules of OCIF or analogue or variant present in the specified complex, can be measured by the method of immunological analysis using, for example, ELISA. Antibodies for use in binding to the immobilized phase or for marking reporter enzyme, such as peroxidase, ELISA is any antibody of interest OCIF or analogue or variant, which I what I OI-26, isolated from the culture of hybridoma producing antibody OI-26 (FERM BP-6421), and OI-19 isolated from the culture of hybridoma producing antibody OI-19 (FERM BP-6420), while suitable antibodies for use as antibodies labeled reporter enzyme in the mobile phase, include monoclonal antibody OI-4, isolated from the culture of hybridoma producing antibody OI-4 (FERM BP-6419) labeled with peroxidase. A typical procedure for measuring the number of molecules of OCIF or analogue or variant in complex procedure is as follows:

(a) a Known concentration of free, not in the complex of OCIF is used to produce the calibration curve.

(b) Conduct ELISA on interest complex and then the calibration curve is used to ascertain the concentration of OCIF.

(c) using the information obtained in (b), and the molecular weight of the monomer OCIF calculate the number of molecules of OCIF in the test complex.

The number of molecules of OCIF or analogue or variant present in the specified complex, measured according to the method of measuring the total amount of protein present in the specified complex can be determined using, for example, the Lowry method. Typical is the following full-time curve.

(b) Then use the method of Lowry to determine the total protein concentration in the test complex, and the calibration curve used to determine the concentration of OCIF.

(c) using the information obtained in (b), and the molecular weight of the monomer OCIF calculate the number of molecules of OCIF in the test complex.

The actual ratio varies depending on the type of immunoassay method and/or method used to measure total protein. A preferred variant of the present invention includes a complex derived from human OCIF or analogue or variant with doctranslate, in which the ratio of the number of molecules of a specified OCIF or analogue or variant present in the specified complex, specific enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies against human OCIF (FERM BP-6420) as the antibodies bound to the solid phase, and monoclonal antibody OI-4 anti-human OCIF, purified from culture hybridoma producing antibody OI-4 (FERM BP-6419), peroxidase labeled in the mobile phase, to the number of molecules of OCIF or analogue or variant present in the specified complex, defined by the dimension containing the compared is at least 0.6, but not more than 1.1, and most preferably this ratio is at least 0.7 to, but not more than 1.1.

Preferred complexes of this invention include:

(a) complex in which the specified substance selected from the group consisting of OCIF, its analogs and its variants, is a Monomeric human OCIF having a molecular weight, measured by electrophoresis in LTO-page in non conditions, approximately 60000, or dimeric human OCIF having a molecular weight of about 120000 as measured by electrophoresis in LTO-page in non conditions, and these polysaccharides and their derivatives selected from the group consisting of hyaluronic acid, chondroitinase acid, dermatan acid, heparan-acid, keratan-acid, carrageenan, pectin, heparin, dextran and derivatives thereof, and the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the specified substance selected from the group consisting of polysaccharides and derivatives thereof is from 1:1 to 1:10;

(b) complex in which the specified substance selected from the group consisting of OCIF, its analogs and its variants, is monomials conditions, approximately 60000, or dimeric human OCIF having a molecular weight of about 120000 as measured by electrophoresis in LTO-page in non conditions, and these polysaccharides and their derivatives selected from the group consisting of dextransucrase and its salts, and the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the specified substance selected from the group consisting of polysaccharides and derivatives thereof is from 1:1 to 1:10;

(c) complex in which the specified substance selected from the group consisting of OCIF, its analogs and its variants, is a Monomeric or dimeric human OCIF, wherein said monomer or one of the links specified dimer OCIF contains amino acids +1 to +380 of SEQ ID NO:1 list of sequences, as specified in the derived polysaccharide is a sodium salt of dextransucrase having an average molecular weight of from 1500 to 12000, and the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to this sodium salt of doctranslate is from 1:1 to 1:10;

(d) complex (C), where the molar ratio of the specified substances, selected and who:1 to 1:8;

(e) complex (C), where the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the said sodium salt of doctranslate is from 1:1 to 1:5; and

(f) the complex according to any one of (C)-(e), where the specified derived polysaccharide is a sodium salt of dextransucrase having an average molecular weight of from 1800 to 6000.

The complexes of this invention can be obtained using any suitable method, which promotes the binding of the polysaccharide or its variant with OCIF or analogue or variant. In the following embodiment, the present invention is provided a method of producing complex containing at least one substance selected from the group consisting of OCIF, its analogs and its variants, which is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof, and specified the method comprises the stage of incubation of the specified at least one substance selected from the group consisting of OCIF, its analogs and its variants, with the specified at least one substance selected from the group consisting of polysaccharides and derivatives thereof, under conditions favorable ovariotomy, and then remove any free polysaccharides or their variants that are not associated with the specified OCIF, analogues or variants.

Incubation of the specified at least one substance selected from the group consisting of OCIF, its analogs and its variants, with the specified at least one substance selected from the group consisting of polysaccharides and their derivatives, performed in any suitable conditions, but usually the incubation takes place in aqueous conditions. Preferably the incubation is performed in alkaline conditions. More preferably, the incubation is carried out by a pH of from 9.5 to 12. Most preferably the incubation performed at pH 10 to 11.

During incubation the concentration range of the specified OCIF, its analogue or variant in the aqueous solution is not limited particularly, as long as it is suitable for the formation of the desired complex. Usually the maximum concentration specified OCIF, its analogue or variant in the aqueous solution is from 0.1 to 0.5 mm, and the minimum concentration is from 0.001 to 0.05 mm. Preferably the concentration of the specified OCIF, its analogue or variant in the aqueous solution is from 0.01 to 0.2 mm and most preferably is from 0.05 to 0.1 mm. In the case of OCIF maximalpower concentration of OCIF in aqueous solution is from 1 to 20 mg/ml and more preferably it is 5 to 10 mg/ml

During incubation the concentration range of the specified polysaccharide or its variant in the aqueous solution is not limited particularly, as long as it is suitable for the formation of the desired complex. Usually the maximum concentration specified polysaccharide or its derivative in an aqueous solution is from 0.1 to 0.5 M, and the minimum concentration is from 0,00005 to 0.05 M. Preferably, the concentration of the specified polysaccharide or its derivative in an aqueous solution is equal to 0.005 to 0.25 M, and most preferably is from 0.05 to 0.1 M In the case of sodium salt of dextransucrase sulfur 5 maximum concentration specified polysaccharide or its variant in the aqueous solution is from 200 to 1000 mg/ml, and the minimum concentration is from 0.1 to 100 mg/ml, Preferably the concentration of the specified polysaccharide or its variant in the aqueous solution is from 10 to 500 mg/ml and most preferably is from 100 to 200 mg/ml

During the incubation temperature is not specifically limited, as long as it is suitable for the formation of the desired complex. Usually the upper limit of the temperature for incubation is equal to from 10 to 50C, and the lower limit of its equal from 0 to 4C. Prepact temperature is from 4 to 10C.

As noted above, the complex of the present invention does not contain free polysaccharides or their variants that are not associated with OCIF or analogue or variant. The method used for deleting free polysaccharides and their variants, is not limited to, yet he is the way, which is conveniently used in processes such as purification, separation and/or fractionation. Examples of suitable methods include ion exchange chromatography, adsorption chromatography, the distribution chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, crystallization, salting out and ultrafiltration. Of them are preferred gel-filtration chromatography (hereinafter referred to as “gel-filtration and ultrafiltration, and gel filtration is the most preferred.

There are no special restrictions gel used for gel-filtration in the removal of free polysaccharides or their variants of the desired complex after incubation, as it can be used to separate the fractions containing the desired complex from the free polysaccharide or its variants that are not associated with OCIF. Suitable Ave is etenia, containing at least one substance selected from the group consisting of OCIF, its analogues and variants that is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof, can be distinguished from the free, not in the complex of OCIF or analogue or variant per se using a variety of techniques, including isoelectric point, the sugar content and by immunological detection.

Isoelectric point can be measured by any conventional method isoelectric electrophoresis, well known to specialists in this field. OCIF is the main protein, and its isoelectric point is approximately equal to PI 9. This is significantly higher than the isoelectric point of the complexes of this invention containing OCIF and polysaccharides and their variants, such as extrasolar, typical values of PI are in the area of 5-7. Thus, using this method can easily be distinguished in the complex and is not located in the complex of OCIF.

The sugar content of the complexes of this invention and free, not in the complex of OCIF or analogue or variant can be measured using m typical examples include a method with phenol and sulfuric acid [M Dibois et al., Anal. Chem., 28, 350 (1956)]. Because the content of total sugar complex of the present invention containing OCIF or analogue or variant, and the polysaccharide or its version is higher than the content of the OCIF, they can be distinguished from each other.

Another alternative way to distinguish not located in the complex of OCIF or analogue or variant from the complexes of the present invention containing the specified OCIF or analogue or variant that is associated with the polysaccharide or its variant is the identification number of the polysaccharide or its variants in each of them, using antibodies that specifically binds to the specified polysaccharide or option.

To measure the amount of protein in OCIF or analogue or variant or complex of the present invention containing OCIF or analogue or variant and the polysaccharide or its variant can be used with any method commonly used to measure total protein content. Suitable examples include a method of Lowry [Lowry, O. H. et al., J. Biol. Chem., 193, 263-275 (1951)], absorption (280 nm) with silver staining and BSA-way.

Free, not in the complex of OCIF or analogue or variant, or OCIF or it is the use of the method, which applies at least one monoclonal antibody against OCIF. Examples of suitable monoclonal antibodies against OCIF, preferably used for the immunological measurement of human OCIF include antibody produced by hybridomas OI-19 (FERM BP-6420), the antibody produced by hybridomas OI-4 (FERM BP-6419), and the antibody produced by hybridomas OI-26 (FERM BP-6421) (for example, see WO-A-99/15691). These antibodies are referred to as “antibody OI-19”, “antibody OI-4 and antibody OI-26”, respectively, in this invention. Antibody OI-19 antibody OI-4 bind as a monomer OCIF and dimer OCIF with equivalent affinity, whereas OI-26 specifically binds a dimer OCIF. Immunological measurement can be performed using the antibodies of this type in accordance with any method known to the person skilled in the art (see for example WO-A-99/15691). Examples of suitable methods include enzyme-linked immunosorbent assay (indicated as “EIA”), radioimmunoassay analysis, solid-phase enzyme-linked immunosorbent assay (ELISA) and sandwich enzyme-linked immunosorbent assay (sandwich-EIA). One of them is preferred ELISA. If OCIF is of human origin, ELISA can be preferably used using antibody OI-19 or antibody OI-inim enzyme used for labeling antibodies, is peroxidase (referred to as “POD”).

Hybridoma producing antibody OI-4, was deposited as “OI-4” in National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology at 1-3, Higashi 1 chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan (which has since become an International Depositary patenting organisms, National Institute of Advanced Industrial Science and Technology at AIST Tsukuba Central 6, 1-1, Higashi 1 chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan) on October 16, 1997 (Heisei-9), and it was given the number FERM P-16473. She was transferred to the international Depository number Deposit FERM BP-6419 13 July 1998 (Heisei-10).

Hybridoma producing antibody OI-19, was deposited as “OI-19” in National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology at 1-3, Higashi 1 chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan (which has since become an International Depositary patenting organisms, National Institute of Advanced Industrial Science and Technology at AIST Tsukuba Central 6, 1-1, Higashi 1 chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan) on October 16, 1997 (Heisei-9), and it was given the number FERM P-16474. She was transferred to the international Depository number Deposit FERM BP-6420 13 July 1998 (Heisei-10).

Hybridoma producing antibody OI-26 was deposited as “OI-26” in National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology at 1-3, Higashi 1 chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan (which has since become the international who Ibaraki-ken 305-8566 Japan) on October 16, 1997 (Heisei-9), and it was given the number FERM P-16475. She was transferred to the international Depository number Deposit FERM BP-6421 13 July 1998 (Heisei-10) (see WO-A-99/15691).

The concentration in the blood or serum of the complex of the present invention containing OCIF or analogue or variant and the polysaccharide or its variant, can be measured as follows. First, this complex is administered to a human or animal (not human). Then after a certain length of time from it take blood or serum. Then the concentration of the specified complex in the blood or serum was measured using ELISA using at least one monoclonal antibody against OCIF, as described elsewhere in this application (see WO-A-99/15691).

The complex of this invention containing at least one substance selected from the group consisting of OCIF, its analogues and variants that is associated with at least one substance selected from the group consisting of polysaccharides and their derivatives, applicable in the treatment or prevention of bone metabolic diseases. In this invention metabolic bone disease is any disease that is characterized by low net amount of bone in a patient, Stradale for the treatment or prevention of a specified disease. Bone metabolic diseases that can be treated or prevented by the complex of the present invention, include: primary osteoporosis (senile osteoporosis, postmenopausal osteoporosis and idiopathic juvenile osteoporosis); endocrine osteoporosis (hyperthyroidism, hyperparathyroidism, Cushing's syndrome and acromegaly); osteoporosis accompanying hypogonadism (hypopituitarism, syndrome Kleinfelter and Turner syndrome); hereditary and congenital osteoporosis (imperfect osteogenesis, homocystinuria syndrome Menkes syndrome Riley-Dey); osteopenia caused by weakening of the gravitational load or fixation and immobilization of the limb; Paget's disease; osteomyelitis; infectious lesion, due to bone loss; hypercalcemia resulting from solid cancer (e.g. breast cancer, lung cancer, kidney cancer and prostate cancer); hematological malignant disease (multiple myeloma, lymphoma and leukemia); idiopathic hypercalcemia; hypercalcemia associated with hyperthyroidism or kidney dysfunction; osteopenia resulting from the introduction of other drugs (for example, immunosupression, such as the methods of the AI kidney; osteopenia resulting from surgery or disease of an organ of digestion (e.g., disorders of the small intestine, disorders of the colon, chronic hepatitis, gastrectomy, biliary cirrhosis and cirrhosis); osteopenia caused by different types of arthritis, such as rheumatoid arthritis, osteoclasia; the destruction of the joints, caused by different types of arthritis, such as rheumatoid arthritis, fibrositis; osteoarthritis; vacuum periodontal bone; cancer metastasis bone (softening and destruction of bone tissue); avascular necrosis or death of osteocytes that accompany traumatic injury, Gaucher disease, sickle cell anemia, systemic lupus erythematosus or nontraumatic injury; the fibrosa, such as renal osteodystrophy; osteopenia accompanying hypomelanus phosphatasebuy or diabetes; osteopenia accompanying malnutrition or eating disorders; and other osteopenia. Bone metabolic diseases also include cachexia caused by solid carcinoma or cancer bone metastasis or malignant hematological disease (see publication of the patent application Anisa least one substance, selected from the group consisting of OCIF, its analogues and variants that is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof, together with pharmaceutically acceptable carrier or diluent, can be safely administered orally or neironalna a person or an animal (not human). Dosage form may be appropriately selected and it will vary depending on various factors such as the type of the subject to treatment of the disease, the degree of the specified diseases, and age, sex and weight of the patient. For example, the complex may be administered orally in the form of tablets, capsules, powders, granules or syrup, inetservices intravenously alone or in combination with conventional auxiliary agents such as glucose, amino acids, or similar, to inetservices intramuscularly, subcutaneously, intradermally or intraperitoneally separately, be administered transdermally in the form of a poultice, injected transnasal in the form of nasal drops, to enter through the mucous membranes or into the oral cavity in the form of the agent for applying to mucosa or entered intrarectal in the form of a suppository. These preparations can be prepared obseryations, binding agents, disintegrant, lubricating agents, flavouring agents, solubilization, suspendisse agents, dyes, pH regulators, antiseptic agents, gelling agents, surfactants and the means for covering.

When preparing the complexes of this invention in the form of tablets can be used in any media known in the field. These carriers include, for example, fillers such as lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicate or etc.; binding agents such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, or etc.; disintegrators such as dry starch, sodium alginate, agar powder, powder of laminaran, sodium bicarbonate, acid, calcium carbonate, esters of fatty acids and polyoxyethylenesorbitan, sodium lauryl sulfate, monoglyceride of stearic acid, starch, lactose or similar; inhibitors of destruction, such as white sugar, stearin, cacao butter, hydrogenated oil, or etc.; absorption accelerators, such as hmal or similar; absorbents such as starch, lactose, kaolin, bentonite, colloidal silica, or etc.; and lubricants such as purified talc, stearic acid, metal salts of stearic acid such as calcium stearate and magnesium stearate, talc, boric acid powder, polyethylene glycol, etc., in Addition, if desired, tablets may have a coating, for example, for education covered sugar tablets, coated gelatin tablets, tablets with intersolubility coating, film-coated tablets, tablets with dual coating or tablet with multi-layer coating.

When preparing the complexes of this invention in the form of pills, the drug may contain carriers, known in the field, for example fillers such as glucose, lactose, cocoa butter, powder starch, utverjdenie vegetable oil, kaolin, talc, or etc.; binding agents, such as powder Arabian gum, powder tragakant, gelatin, ethanol and so on; and disintegrators such as laminaran, agar, or etc.

When preparing the complexes of this invention in the form of a suppository preparation may contain conventional carriers, such as polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohol is njection solutions preferably, the drug in the form of a solution or suspension are sterilized and do is isotonic with respect to blood. If the drugs are in the form of a solution, emulsion or suspension, may be used any solvent known and commonly used in this field, examples of which include water, ethanol, propylene glycol, ethoxylated isostearoyl alcohol, polioksidony isostearoyl alcohol and ethers of fatty acids and polyoxyethylenesorbitan. In addition, in such injectable compositions of these preparations may also contain salts, glucose, glycerol, or, etc., in sufficient quantity to maintain isotonicity with respect to blood. They may also contain additional agents, including soljubilizatory, buffer agents, anesthetic agents, pH regulators, stabilizers, and solubilizing agents. Injectable solutions can be dried after cooking.

The preparations of this invention may also contain additional additives such as coloring agents, preservatives, perfumes, improves the taste and odor agents, sweeteners or other ingredients.

There are no special restrictions regarding the amount of the complex of the present invention containing at least one substance selected from the group consisting of OCIF, its analogues and variants, and at Marsili for introduction to to prevent or treat metabolic bone disease, but it is usually in an amount of 0.1-70% by weight and preferably 1-30% by weight of the entire composition.

The dose of the complex in accordance with this invention will vary depending on various factors, including subject to treatment status, age, sex and body weight of the patient and the route of administration. However, the number entered an adult, is usually in the range having an upper limit of -30 to 1000 mg and the lower limit of 0.001 to 0.03 mg per day. The preferred range is from 0.03 to 30 mg per day. Enter the amount is usually in the range having an upper limit of 1 to 20 mg/kg / day and lower limit of 0.01 to 0.5 μg/kg / day. The preferred range is from 0.5 μg/kg to 1 mg/kg / day. The complex of this invention can be injected once a day or more than once a day, depending on such factors as the form of administration and the condition of the patient.

The following examples, reference examples and test examples are intended to further illustrate this invention and are not intended to limit in any way the present invention.

Example 1

Obtaining complexes, with the th dimeric human OCIF, having a molecular weight of 120,000 was received in accordance with the procedure described in EP-A-0816380 (WO-A-96/26217) in the examples. Namely, pBKOCIF, plasmid vector containing a nucleotide sequence that encodes human OCIF, containing a signal peptide derived from pBK/01F10 strain-transformant E. coli [deposited as FERM BP-5267 according to the Budapest Treaty at the National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology at 1-3, Higashi 1 chome, Tsukuba-shi Ibaraki-ken 305-8566 Japan (which has since become an International Depositary patenting organisms, National Institute of Advanced Industrial Science and Technology), obtained according to example 11 of EP-A-0816380, were digested with restrictase SalI and EcoRI. Nucleotide which encodes human OCIF, containing a signal peptide, which is equivalent to the OCIF cDNA man, was extracted according to the procedure described in example 14 of EP-A-0816380. After isolation and purification of the specified nucleotide was built in expressing vector pcDL-SR296 (Molecular and Cellular Biology, vol.8, p.466, 1988) and then strain E. coli DH5(Gibco BRL) was transformed them (see the procedure described in example 14 of EP-A-0816380). Thus obtained recombinant vector, named pSROCIF, were extracted from specified is th recombinant Mature human OCIF. Namely, the cells are Cho dhFr (ATCC CRL 9096) were transfusional recombinant plasmid pSROCIF, obtained as described above, and selected plasmid expressing dihydrotetrazolo (DHFR) (plasmid pBAdDSV described in WO-A-92/01053), and then expressing the DHFR transfectant. Transformants that were extracted large quantities of OCIF, cloned. Clones, air-conditioned environment which contained OCIF in high concentrations, were selected and received the clone expressing the highest amount of OCIF, namely - 5561. The culture thus obtained clone was 5561 kondicionirovanie and was filtered and then applied to a column of Heparin Sepharose-FF (2,610 cm, Pharmacia Co.) and subjected to column chromatography using a linear gradient of sodium chloride as eluent. Then the fraction with the OCIF activity, loireau from approximately 0.6 to 1.2 M sodium chloride, was applied to the affinity column (blue-5PW, 0,55.0 cm, Tosoh Co.) and subjected to affinity chromatography using a linear gradient of sodium chloride as eluent. Erwerbende fractions were subjected to electrophoresis in LTO-polyacrylamide gel under reducing and non conditions and the fractions containing the protein bands rOCIF with average molecular masses 60000 and 120000, what was obtained in example 14 of EP-A-0816380. Amino acid sequence of the Monomeric peptide shown in SEQ ID NO:1 list of sequences, which is identical to the full sequence of SEQ ID NO:4 or amino acids No. 1 to No. 380 of SEQ ID NO:5 of WO-A-96/26217 and EP-A-0816380.

Then to combined fractions containing the received OCIF person, adding 1/100 volume of 25% triperoxonane acid and the resulting mixture was applied to a reversed-phase column (PROTEIN-RP, 2.0 mm250 mm, supplied by YMC Co.), which was pre-equilibrated to 30% acetonitrile containing 0.1% triperoxonane acid. Then the column was suirable linear gradient from 30 to 55% acetonitrile at a speed of current of 0.2 ml/min for 50 minutes Faction two peaks were collected separately and then liofilizirovanny. Then fraction, which found the band, having an average molecular weight of 120,000 during electrophoresis on LTO-page in reducing conditions, used in the following examples as a dimeric human OCIF (see examples 17 and 18 WO-A-96/26217 and EP-A-0816380).

1(b) Obtaining complexes containing OCIF and textresult

Purified dimeric human OCIF, obtained as described in example 1 (a) above, was dissolved in 10 mm buffer solution of sodium phosphate (pH 6 is translate sulfur 5 (produced by Meito Sangyo Co., Ltd., hereafter referred to as “DS5”) was dissolved in the thus obtained aqueous solution to a final concentration of 40, 100, 130, 150, 200, 400, 500, 510 or 1000 mg/ml, and then thereto was added 1 n sodium hydroxide to a final pH of 10, 10.5 or 11. The resulting aqueous solutions were incubated at 4, 7, 25, or 37With over 1, 3, 6, 18, 24, 48, 72, 96, 144, 168 or 288 hours.

At the end of this period, 4 ml of each resulting solution was applied to a gel filtration column Superdex 200 prep grade (internal column diameter: 16 mm; length: 60 cm; limiting the displacement molecular weight: 1300000; produced by Amersham Pharmacia Biotech), previously equilibrated with 10 mm sodium phosphate buffer (pH 6) containing 0.3 M sodium chloride, and then was suirable the same buffer at a speed of current of 2 ml/min. and the Absorbance at a wavelength of 280 nm was monitored using a measuring absorption in the UV spectrophotometer, and the eluate at retention time of about 28 to 36 min was collected. Free DS5, which was not associated with OCIF was suirable at retention time of about 50-70 minutes All stages of this procedure, the gel filtration was performed at room temperature. The obtained preparations, which contained the desired complexes of dimeric human OCIF and DS5, froze and hranicena natural human OCIF

Natural produced OCIF person received in accordance with the procedure described in the examples of WO-A-96/26217 and EP-A-0816380 from cell cultures of lung fibroblasts human embryo IMR-90 (ATCC-CCL186).

Example 2

Obtaining complexes containing OCIF and nextresult (II)

Purified dimeric human OCIF, obtained as described in example 1 (a) above, was dissolved in 10 mm buffer solution of sodium phosphate (pH 6.0) containing 0.15 M sodium chloride, to obtain a solution having a concentration of OCIF 5 mg/ml Sodium salt of dextransucrase having a molecular weight of 5000 (produced Waco Pure Chemical Industries, Ltd., hereafter referred to as “DS 5000”), was dissolved in the thus obtained aqueous solution to obtain a final concentration of DS 5000 150 mg/ml and then was added 1 n sodium hydroxide to a final pH of 10.5. Thus obtained aqueous solution was incubated at 4With in 24 hours.

At the end of this time period, 4 ml of the resulting solution was applied to a gel filtration column Superdex 200 prep grade for chromatography as described in example 1(b) above. The absorbance at a wavelength of 280 nm was monitored using a measuring absorption in the ultraviolet spectrophotometer, and the eluate with time derivan 40-65 minutes

The obtained preparations, which contained the desired complexes of dimeric human OCIF and DS5000, was frozen and stored at -60C. Conditions obtain for this complex are summarized in table 2.

Example 3

Measurement of isoelectric point

Purified recombinant dimeric human OCIF, obtained as described in example 1(a) above, and the complex of OCIF and dextransucrase, obtained as described in example 1(b) above, and which was designated as the Drug is No. 22 in table 1, was applied separately for gel electrophoresis with isoelectric focusing IEF PAGE (pH range 3-10 manufactured by Iwaki Glass) using a set of buffers of pH 3-7 for IEF (isoelectric focusing) (Technical Frontier Co.) and applied voltage to this gel in accordance with the following regime: 100 V for 1 h, then 200 V for 1 h and finally 500 within 30 minutes After completion of the electrophoresis, the gel obtained in each case were stained with Kumasi blue.

From the obtained, as described above, gel electrophoresis has determined that the isoelectric point of dimeric human OCIF was about PI 9 and isoelectric point of the complex of OCIF and doctranslate designated as the Drug No. 22, was about a pI of 6.5, by comparing pictranslator in the complex, containing OCIF and textresult

4(a) Obtaining an initial solution of the monoclonal antibody against human OCIF OI-4, peroxidase labeled

In this stage monoclonal antibody against human OCIF was labelled with peroxidase using the kit for activated maleimido horseradish peroxidase (EZ-Link Maleimide Activated Horseradish Buffer Kit manufactured by Pierce) according to the Protocol II described in the booklet of instructions attached to the kit. Details of this procedure are as follows.

Monoclonal antibody against human OCIF OI-4 was isolated from the culture hybridoma producing antibody OI-4 (FERM BP-6419), according to the method described in example 4 of EP-A-0974671 (WO-A-99/156991), and then diluted to a final protein concentration of 1 mg/ml 10 mm phosphate buffer (pH 7,6).

N-Succinimidyl-3-acetylthiourea (provided in this kit EZ-Link Maleimide Activated Horseradish Buffer Kit) was dissolved in dimethylformamide to obtain a solution having a concentration of 10 mg/ml immediately before use. An aliquot of 4 μl was added to 1 ml of diluted OI-4-containing solution obtained as described above, and then the resulting solution was incubated at room temperature for 30 minutes. At the end of this time period, 20 µl of a solution, poluokrugli of maleimide (provided in this kit EZ-Link Maleimide Activated Horseradish Buffer Kit) was added thereto and the resulting solution was incubated at room temperature for 2 hours. At the end of this period of time that the reaction mixture was applied to a polyacrylamide desalting column (10 ml contained in the specified collection EZ-Link Maleimide Activated Horseradish Buffer Kit), pre-equilibrated with 30 ml of buffer for conjugation of maleimide (also represented in the specified set), and then buffer for conjugation of maleimide inflicted on the specified column. The eluate was collected in fractions of 0.5 ml Fractions 7-10, containing the antibody were pooled. Then 100 μl of a solution obtained by dissolving 5 mg of activated maleimido horseradish peroxidase (EZ-Link Maleimide Activated Horseradish Buffer) (contained in the specified collection EZ-Link Maleimide Activated Horseradish Buffer Kit) in 500 ál of distilled water immediately before use, was added to the combined fractions of the eluate and the mixture was incubated at room temperature for one hour. After incubation were added to a mixture of equal volume of glycerol and thus obtained solution was stored at -20C.

The solution obtained by the above method, was used as the initial solution of the monoclonal antibody against human OCIF OI-4,the definition of the amount of OCIF

The amount of OCIF present in any of the complexes obtained in examples 1 and 2 above, and in combination, prepared in reference example 1 below, was measured by enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies against OCIF, and the details of this procedure were as follows.

Monoclonal antibody against human OCIF OI-26 was isolated from the culture hybridoma producing antibody OI-26 (FERM BP-6421), according to the method described in example 4 of EP-A-0974671 (WO-A-99/15691), and then diluted with 0.1 M sodium bicarbonate to obtain a solution having a final protein concentration of 5 µg/ml. Aliquot of his volume of 100 µl was transferred into each well of 96-well microtiter tablet (Maxisorp manufactured by NUNC), and then the tablet was hermetically and incubated at 4With during the night. At the end of this period, each well was washed three times with 250 μl of phosphate buffered saline (SFR) (pH 7.4) containing 0.1% Polysorbate 20. 20 μl of solution dilution buffer [containing 0.2 M Tris-HCl, 40% Block ACE (Dainippon Pharmaceutical Co., Ltd.), and 0.1% Polysorbate 20; pH 7.4] was added to each well and the tablet then kept at room temperature for 20 minutes to BC what about the above, preferably diluted solution dilution buffer used above for blocking holes. To construct the calibration curve solution dilution buffer containing human OCIF in known concentrations were used as standards. Solution dilution buffer was used as control. 50 μl of each sample was transferred into each well.

After adding the samples to the wells, 50 μl of a solution obtained by diluting the original solution POD-OI-4 [prepared as described in example 4(a) above] 1500-fold volume of solution dilution buffer [0.2 M Tris-HCl, 40% Block ACE (Dainippon Pharmaceutical Co., Ltd.), 0.1% Polysorbate 20; pH 7.4] was added to each well and the tablet then incubated at room temperature for 2 hours. At the end of this period, each well was washed four times with 250 μl of phosphate buffer containing 0.1% Polysorbate 20 (hereinafter referred to as “PB”, pH 7.4).

0.1 M citric acid and 0.2 M intrigejosa were mixed and used as the substrate solution (pH 4.5). Its an aliquot of 32.5 ml was transferred into a test tube and to it was added a 6.5 μl of hydrogen peroxide. Then 13 mg tablets o-phenylenediamine dihydrochloride (OPD) (manufactured by Waco Pure Chemical Industries, Ltd.) rasti then incubated at room temperature for 15 minutes. At the end of this period, 50 μl of a solution to stop reaction containing purified water and concentrated sulfuric acid in a ratio of 250:50 by volume, was added to each well. After careful mixing of the solutions in the wells using a shaker (Titer mixer MB-1; manufactured Japan Trika) absorption of each well was measured at a wavelength of 490 nm using a microplate reader (SPECTRAFLUOR: made TECAN).

On the basis of the calibration curve obtained, as explained above, absorption standard solutions of human OCIF at known concentrations, calculated the amount of OCIF person in each sample.

4(C) determining the amount of dextransucrase

The number of dextransucrase in each complex, obtained as described in examples 1 and 2, measured in the form of neutral sugars by the method with phenol and sulfuric acid, the details of which are as follows:

The solution, having a concentration in the range of 10-60 µg/ml DS5 (produced by Meito Sangyo Co., Ltd.) or DS5000 (produced by Waco Pure Chemical Industries, Ltd.), prepared using diluent (0.01 M citric acid, 0.3 M sodium chloride, 0.01% of an aqueous solution of Polysorbate 80: pH 6.0) and was used as a standard solution. 0.2 ml of each standard is Yali and quickly mixed. After incubating the resulting mixture at 60C for 20 seconds in a water bath and to it was added 1.0 ml of concentrated sulfuric acid. After careful but rapid mixing, the tube was incubated for 10 minutes at room temperature, again quickly mixed and then incubated for 20 minutes at room temperature. At the end of this period, the absorption solution in the tube was measured at a wavelength of 490 nm using a spectrophotometer (UV-240: produced by Shimadzu Seisakusho K. K.).

This absorption and the calibration curve was determined by the content of neutral sugars. OCIF person contains sugar chain. Thus, the number of dextransucrase associated with human OCIF in the analyzed medication, is calculated by subtracting a value of the content of neutral sugars of the OCIF person of value, which was measured for any of the analyzed drug.

4 (d) Calculation of molar ratio of OCIF and doctranslate in complex containing OCIF and textresult

The number of dextransucrase present in the analyzed drug, as defined, as described in example 4 (C) above, divided by the number of OCIF person present in the analyzed the existing 1 mg OCIF person in the analyzed drug.

Thus obtained is then used to calculate the molar ratio of OCIF in the form of monomer and dextransucrase in the analyzed drug by calculating the number of molecules of dextransucrase per molecule of monomer OCIF, based on the assumption that the molecular mass of the monomer OCIF equal to 60000 molecular weight DS5 equal 1950, molecular weight DS5000 equal to 5000.

The results obtained are shown in table 3.

Example 5

The stability of the binding between OCIF and doctranslate in complexes OCIF/textresult

Gel-filtration of the complex containing OCIF and textresult, repeated twice as described in example 4(C) above, and the number of dextransucrase present in the complex, was measured after each of the above gel filtration. The details were as follows.

5(a) Incubation of OCIF and doctranslate

Used the procedure described above in example 1(b). Recombinant dimeric human OCIF, obtained as described in example 1(a), was dissolved in 10 mm sodium phosphate buffer (pH 6.0) containing 0.15 M sodium chloride, to obtain a solution having a concentration of OCIF 5 mg/ml DS5 was dissolved in the thus obtained solution with a final concentration DS5 150 mg/ml and the PRS were incubated at 4With within 7 days from the receipt of a solution containing a complex of dimeric human OCIF and DS5.

5(b) gel-filtration

The solution containing the complex of dimeric human OCIF and DS5, obtained at the end of incubation in example 5(a) above was subjected to gel filtration in accordance with the method described in example 1(b) above. The fraction at retention time of about 28 to 36 minutes were collected, whereas free textresult, which was not associated with OCIF, elyuirovaniya at retention time of about 50-70 minutes.

5(C) Measurement of protein content

The amount of protein present in the complex, was measured by the method of Lowry [Lowry, O. H. et al., J. Biol. Chem., 193, 263-275 (1951)], as follows.

0.2 g of the pentahydrate of copper sulfate (II) (Waco Pure Chemical) was dissolved in water to a final volume of 50 ml 0.4 g dihydrate sodium tartrate (Waco Pure Chemical) was dissolved in water to a final volume of 50 ml 20 g of sodium carbonate was dissolved in water to a final volume of 100 ml Three aqueous solution, thus obtained, was mixed in a ratio of 1:1:2 by volume immediately prior to use (the resulting solution was named “solution A”). 10 g of sodium dodecyl sulfate (Nacalai Tesque Inc.) was dissolved in water to a final volume of 200 ml (solution was named “solution” “solution”). Solution A, solution b and solution C were mixed in the ratio 1:2:1 by volume immediately prior to use.

Separately reagent Polina-Ciocalteu (Waco Pure Chemical) and water were mixed in a ratio of 1:5 by volume immediately prior to use. 2.76 g of dihydrate trinational salt of citric acid (Wacp Pure Chemical), of 0.13 g of citric acid monohydrate (Waco Pure Chemical) and 17.5 g of sodium chloride and 0.1 g of Polysorbate 80 was dissolved in water to a final volume of 1 liter (pH 6.9) to obtain the solution, called the “diluent”.

to 9.5 ml of the diluent was added to 500 ál of standard bovine serum albumin (Pierce Co. Ltd.), containing 2 mg/ml bovine serum albumin (“BSA”) in 0.9% aqueous sodium chloride containing sodium azide at a concentration of less than 0.1%, to obtain the solution, called “solution of BSA 100 μg/ml”. 3.5 ml, 3 ml, 2.5 ml or 2 ml of diluent was added to 1.5 ml, 2 ml, 2.5 ml or 3 ml BSA 100 μg/ml with obtaining, respectively, solutions, called “solution of BSA 30 μg/ml, BSA solution 40 mg/ml, solution of BSA and 50 μg/ml and a solution of BSA 60 µg/ml respectively. 3 ml of the diluent was added to 1.5 ml BSA 60 μg/ml to obtain a solution called “restorane with obtaining a solution with a final concentration of about 40 µg of protein per 1 ml 1 ml solution of BSA 20 mg/ml solution of BSA 30 μg/ml solution of BSA 40 μg/ml solution of BSA and 50 μg/ml solution of BSA 60 μg/ml, dilute the sample diluent (n=3) was transferred into a test tube, was added to 1 ml of alkaline copper reagent and the resulting solution was mixed and incubated at room temperature for 10 minutes. Then thereto was added 0.5 ml of diluted reagent Polina-Ciocalteu and the resulting solution was mixed and incubated at room temperature for 30 minutes. At the end of this period, the absorption of each mixture at a wavelength of 750 nm was measured using a cell made of quartz, which width was 10 mm, using UV spectrophotometer (Lambda 20: Perkin Elmer Co. Ltd.). Then the amount of protein contained in the sample was calculated on the basis of the calibration curve, obtained using the acquisitions of standard solutions of BSA (values given to the amount of BSA).

5(d) determining the amount of dextransucrase

The number of dextransucrase associated with human OCIF in the complex, which was obtained after the first gel filtration in example 5 (b) above, was measured using the procedure described in example 4(C) above.

5(e) a Second gel filtration

United is and MW 30000, Millipore Amicon Co., Ltd. and they were centrifuged at 2000 rpm for 20 minutes using a centrifuge (himacCT60, Hitachi Seisakusho Co., Ltd.). Unfiltered concentrated solutions obtained from the two filter elements Centriprep, collected and combined. The resulting solution was subjected to gel filtration as described in example 1(b) above, and the fraction at retention time of about 28 to 36 minutes was collected and combined. Then the protein and sugar in the complex present in the combined fractions was measured as described in examples 5(C) and 5(d) above.

5(f) Third gel filtration

The combined collected fractions obtained in example 5(e) above, was transferred to a two filter elements Centriprep (UM-30, cut-off MW 30000, Millipore Amicon Co., Ltd. and they were centrifuged at 2000 rpm for 20 minutes using a centrifuge (himacCT60, Hitachi Seisakusho Co., Ltd.). Unfiltered concentrated solutions obtained from the two filter elements Centriprep, collected and combined. The resulting solution was subjected to gel filtration as described in example 1(b) above, and the fraction at retention time of about 28 to 36 minutes was collected and combined. Then the protein and sugar in the complex present in the combined fractions was measured as described in examples 5(C) and 5(d) videosurface, present in the complex obtained in the fractions after the first gel filtration in example 5(b) above, the second gel filtration in example 5(e) above and the third gel filtration in example 5(f) above, calculated in accordance with example 4(d) described above. The results obtained are summarized in table 4.

From the above will be immediately obvious that the molar ratio of OCIF to doctranslate in the complex of the present invention is a constant for three gel filtration, indicating a high degree of stability of the binding between OCIF and doctranslate in the complexes of the present invention.

Example 6

The degree of adsorption complex of OCIF and doctranslate on crosslinked heparin column

6(a) Chromatography on a heparin column

All procedures column chromatography in this example were performed at the speed of the current 4 ml per minute.

Heparin sewn column (HiTrap Heparin HP column, Lot. 289212, Amersham Pharmacia Biotech) pre-balanced 5 ml of 10 mm sodium phosphate buffer containing 0.7 M sodium chloride. The preparation of table 1 of example 1 was taken and diluted to a final protein concentration of 0.1 mg/ml 10 mm sodium phosphate buffer containing 0.7 M sodium chloride. 1 ml of the thus obtained diluted with the aqueous buffer, containing 0.7 M sodium chloride, was applied to the specified column and collected 5 ml of the second eluate (fraction). Finally, 4 ml of 10 mm sodium phosphate buffer containing 2 M sodium chloride, was applied to the specified column and collected 4 ml of the eluate (fraction C).

6(b) measuring the amount of OCIF in the eluate

100 ál of 0.1 M sodium bicarbonate (pH 9,6), which was dissolved monoclonal antibody against human OCIF 01-19 (FERM BP-6420) at a concentration of 10 µg per ml, was transferred into each well of 96-well microtiter tablet (Maxisorp: NUNC Co. Ltd.). The tablet was hermetically and then incubated at 4With during the night. At the end of this period, the solution in each well was removed by decantation, to each well was added 300 μl of 50% Block ACE (produced by Dainippon Pharmaceutical Co., Ltd.), and the tablet then incubated at room temperature for 2 hours. After removal of the solution from each well, each well was washed three times with 300 ál SFR (pH 7.4) containing 0.1% Polysorbate 20, using SERA WASHER MW-96R (Bio-Tec Co. Ltd.).

After making the holes, as described above, 20 μl of each of the three eluates (fractions a, b and C) obtained in example 6 (a) above was diluted to a final volume of 120 μl of 0.2 M Tris-Hcl (pH 7.4) containing 40% Block ACE, 10 μg/ml m the time a known amount of human OCIF was dissolved in 120 μl of 0.2 M Tris-Hcl (pH 7.4) containing 40% Block ACE, 10 μg/ml mouse immunoglobulin G and 0.1% Polysorbate 20, and then diluted with the same volume of pure water. Thus obtained solution was used as standard.

100 μl each of the diluted eluates and standard was added in one hole (each) pre-prepared microtiter tablet, as described above, and then this tablet incubated at room temperature for 2 hours with careful stirring using a microplate shaker (NS-P: Ichi Seiei-Do Co. Ltd.). At the end of this time the solution was removed from each well and then each well was washed six times with 300 μl SFR (pH 7.4) containing 0.1% Polysorbate 20, with SERA WASHER MW-96R (Bio-Tec Co. Ltd.). Then 100 μl of 0.1 M Tris-HCl (pH 7.4) containing 25% Block ACE, 10 μg/ml mouse immunoglobulin G and 0.1% Polysorbate 20, to which was added the original solution POD-OI-4, prepared as described in example 4(a) above, getting a 0.01% solution (about./vol.), was added to each well and the plate incubated at room temperature for 2 hours with careful mixing using the same microplate shaker. After UD is Itanium SERA WASHER MW-96R (Bio-Tec Co. Ltd.).

After washing the wells with 100 μl of 3,3’,5,5’-tetramethylbenzidine (TMB) - soluble reagent (Scytec Co., Ltd. was added to each well and the tablet then incubated at room temperature for 10-15 minutes with careful mixing using the same microplate shaker, which is described above. At the end of this time period in each well was added 100 μl of TMB stop buffer (Scytec Co Ltd.). After careful mixing of the tablet using a microplate shaker for about 1 minute absorption of each well at a wavelength of 450 nm was measured using a microplate reader (THERMO SPECTRA: TECAN Co. Ltd.). Then the amount of OCIF contained in each of fractions a, b and C [designated as (a), (b) and (C)], was calculated by using the calibration curve obtained by plotting the absorbance of each standard described above, depending on the concentration. Then the degree of adsorption of the tested complex of OCIF and doctranslate on crosslinked heparin column was calculated in accordance with the following formula:

The results are summarized below in table 5 for 7 from the complexes obtained in example 1 above. Also provides the corresponding result for not finding is paranavai column, than the complexes of the present invention. It was also found that the complexes of this invention can be further characterized according to their degree of adsorption on crosslinked heparin column.

Example 7

Immunological detection of a complex of OCIF-textresult

7(a) measuring the amount of protein

The amount of protein contained in the drug complex of example 1 described above was determined according to the method described in example 5(C) above.

7(b) Immunological measurement of the amount of OCIF

The amount of OCIF contained in the drug complex of example 1, described above, was determined immunologically by using ELISA method described in example 6 above.

7(C) Calculate the coefficient of immunological detection

The value obtained in example 7(b) above, divided by the corresponding value obtained in example 7(a) above, and the thus obtained value was called the “coefficient of immunological detection”.

These results are summarized in table 6. Given the result for is not located in the complex of OCIF. It was also found that the complexes of this invention can be further characterized by their coefficient immunological detection.

is translate (molecular weight of 5000 or 10000) was prepared in the following manner using the procedures described in example 1 of EP-A-1127578 (WO-A-2000/24416).

Purified dimeric human OCIF having a molecular weight of about 120000, obtained as described in example 1(a) above, was dissolved in 10 mm solution of sodium phosphate buffer (pH 6.0) containing 0.15 M sodium chloride and 0.01% Polysorbate 80, to obtain a solution having a concentration of OCIF 0.25 mg/ml DS 5000 (manufactured Waco Pure Chemical Industries, Ltd.), described above in example 2, or the sodium salt of dextransucrase having a molecular weight of 10000 (produced Waco Pure Chemical Industries, Ltd., hereafter referred to as “DS10000”) was dissolved in the obtained aqueous solution to obtain a solution having a final concentration of sodium salt of tetrasulfate 1 or 4 mg/ml, and then thereto was added sodium hydroxide to a final pH of 7. Thus obtained aqueous solutions were incubated at 4With in 24 hours of obtaining the desired preparations containing OCIF and DS5000 or DS10000, which are then used for comparative purposes in test example 1.

Terms of preparation for each combination are summarized in table 7.

Test example 1

The measurement of the concentration of complexes containing OCIF and textresult, serum

1(a) Injecting and blood collection

Five weeks females the Chennai, as described in any of examples 1, 2 or reference example 1, which were subject to the test, was diluted to a concentration of 0.25 mg/ml using SFR (pH 7.4) containing 0.01% Polysorbate 80, for receiving the injection solution, which is then injected in the tail of one of the tested rats through the tail vein in a disposable dose rate of 2 ml/kg body weight. Later, 6 hours after administration, the blood was taken from the rat heart.

1(b) Fractionation of serum

After keeping blood collected as described in 1(a) above, to coagulate at room temperature for 30 minutes, it was given the serum in the form of a supernatant by centrifugation of the blood at 14000 rpm for 3 minutes using a rotor with a diameter of 10 cm

1(C) quantification of OCIF in serum

100 µl of a solution, in which the monoclonal antibody OI-19 against human OCIF (see EP-A-0974671 (WO-A-99/15691)] was dissolved in a 0.1 solution of sodium bicarbonate to a final concentration of OCIF 10 kg/ml, was added into each well of 96-well microtiter tablet (Maxisorp: machines are manufactured by NUNC), and then the tablet was hermetically and gave him to stand overnight at 4C. Then a solution of antibody was removed by decanting and to each well dabwali to stand at room temperature for 2 hours. At the end of this period, each well was washed three times with 300 ál SFR (pH 7.4) containing 0.1% Polysorbate 20.

100 μl of purified water and 120 μl of buffer solution for dilution [composition: 0.2 M Tris-chloride-hydrogen acid, 40% Block ACE (produced by Dainippon Pharmaceutical Co., Ltd.), 10 μg/ml mouse immunoglobulin G and 0.1% Polysorbate 20; pH 7.4] was added to 20 μl of the test serum, which was collected as described in 1 (b) above, and stirred. As a control, 100 μl of purified water and 120 μl of dilution buffer containing dimer OCIF man in a known concentration, was added to 20 μl of distilled water and stirred.

100 µl of each of the preparations of serum, thus obtained, was added to each well and then the plate was allowed to stand at room temperature for 2 hours. Each well was washed six times after completion of the reaction, 300 μl of a solution containing 0.1% Polysorbate 20 (pH 7.4). Then 100 μl of the solution obtained 1000-fold dilution of the original solution POD-OI-4 obtained in example 4(a), as described above, the reconstitution solution [composition: 0.1 M Tris-chloride-hydrogen acid, 20% Block ACE (acquired by Dainippon Pharmaceutical Co., Ltd.), 10 μg/ml mouse immunoglobulin G and 0.1% Polysorbate 20: pH 7.4], debaveye time each well was washed six times with 300 μl SFR (pH 7.4), containing 0.1% Polysorbate 20. Then 100 μl of substrate solution (TMB soluble reagent: manufactured Scytec) was added to each well and the plate was allowed to stand at room temperature for 10-15 minutes. Then 100 μl of a solution to stop reaction (TMB stop buffer produced Scytec) was added to each well.

After mixing by using the apparatus for shaking (microplate shaker NS-P; produced by Iuchi Seiei-Do, Ltd.) the absorption of each well at a wavelength of 450 nm was measured using a microplate reader (THERMO SPECTRA produced by TECAN). Then the concentration of OCIF in the test serum was calculated from a calibration curve using a standard solution of OCIF. The dose was calculated as dose OCIF per kg of body weight (mg/kg) by measuring the concentration of OCIF for each injection solution, prepared as described in 1(a), similarly as it did in the case of serum.

1(d) Concentration in serum

OCIF in serum, obtained as described in 1(b) above were quantified for each sample according to the method described above in 1(C). The results are shown in table 8.

As shown in table 8, the concentration of serum preparations of the present invention, administered at a dose of 0.5 mg/kg of body weight, Psylocke 1 drug at the same dose.

The benefits of this invention

As demonstrated above, the complexes of this invention containing at least one OCIF, an analogue or variant and at least one polysaccharide or its variant, are retained in the blood after administration in significantly higher concentrations in comparison with the known combinations containing OCIF and polysaccharides, such as described in WO-A-2000/24416. The complexes of this invention are applicable for the prevention or treatment of various metabolic bone diseases such as osteoporosis, hypercalcemia, lytic bone metastasis, bone loss due to rheumatoid arthritis, osteopenia due to steroid drugs, multiple myeloma, osteopenia or hypercalcemia caused by renal dysfunction, renal osteodystrophy, osteoarthritis and so on

Claims

1. Compleo, selected from the group consisting of enzyme inhibition osteoclastogenic factor (OCIF), its analogs and its variants, which is associated with at least one substance selected from the group consisting of polysaccharides and derivatives thereof.

2. Complex p. 1, where the specified substance selected from the group consisting of OCIF, its analogs and its variants natural type or recombinant type.

3. Complex under item 1 or 2, where the specified substance selected from the group consisting of OCIF, its analogs and its variants, is a monomer or dimer.

4. Complex on p. 3, where the specified substance selected from the group consisting of OCIF, its analogs and its variants, is Monomeric human OCIF having a molecular weight, measured by electrophoresis in LTO-page in non conditions, approximately 60000, or dimeric human OCIF having a molecular weight of about 120000 as measured by electrophoresis in LTO-page in non conditions.

5. The complex according to any one of paragraphs.1-4, where the specified OCIF contains amino acids -21 to +380 of the sequence SEQ ID NO:1 list of sequences.

6. The complex according to any one of paragraphs.1-4, where the specified OCIF contains amino acids +1 to +380 of the sequence SEQ ID NO:1 list C group, composed of hyaluronic acid, chondroitinase acid, dermatan (acid), heparana (acid), ceratina (acid), carrageenan, pectin, heparin, dextran and derivatives thereof.

8. Complex under item 7, where the specified derived polysaccharide selected from dextransucrase and its salts.

9. Complex p. 8, where the specified derived polysaccharide is sodium salt doctranslate.

10. Complex p. 9, where the average molecular weight of the specified doctranslate equal from 1500 to 12000.

11. Complex p. 9, where the average molecular weight of the specified doctranslate equal from 1800 to 6000.

12. The complex according to any one of paragraphs.1-11, where the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the specified substance selected from the group consisting of polysaccharides and derivatives thereof is from 1:1 to 1:10.

13. Complex p. 12, where the specified molar ratio is from 1:1 to 1:8.

14. The complex according to any one of paragraphs.1-13, where the strength of adsorption of the specified complex containing OCIF or analogue or variant on the heparin is lower than the strength of adsorption of the free, not in the complex of OCIF or analogue or variant.

15. Complex under item 14, the DG is,7:

(a) a column Packed with media, such as granules of cross-linked agarose, which was immobilized heparin, balance buffer with low ionic strength, containing 0.1-0.8 M sodium chloride;

(b) complex, which is being tested, dissolved in the same buffer of low ionic strength, which is used in (a), and applied on the column and then collect the first eluate (fraction A);

(c) then the column is washed additionally with the same buffer of low ionic strength, which is used in stage (a), and collect the second eluate (fraction C);

(d) then the column is washed with buffer with high ionic strength, containing 1.0 to 2.0 M sodium chloride, and then collect the third eluate (fraction C);

(e) then determine the amount of complex present in each of fractions a, b and C, denoted as (a), (b) and (C) respectively, using immunoassay and

(f) then determine the degree of adsorption of the complex on the heparin in accordance with the following formula:

16. The complex according to any one of paragraphs.1-15 containing OCIF or analogue or variant and extrasolar or its salt, where the ratio of the number of molecules of a specified OCIF or analogue or variant present in the specified complex, opredelennoye, isolated from the culture of hybridoma producing antibody OI-19 (FERM BP-6420), as the antibodies bound to the solid phase, and monoclonal antibody OI-4 anti-human OCIF isolated from the culture of hybridoma producing antibody OI-4 (FERM BP-6419), peroxidase labeled in the mobile phase, to the number of molecules of OCIF or analogue or variant present in the specified complex determined by measuring the total protein content by the Lowry method, is from 0.5 to 1.2.

17. Complex p. 16, where the specified ratio is from 0.6 to 1.1.

18. Complex p. 16, where the specified ratio is from 0.7 to 1.1.

19. Complex p. 1, where the specified substance selected from the group consisting of OCIF, its analogs and its variants, is Monomeric human OCIF having a molecular weight, measured by electrophoresis in LTO-page in non conditions, approximately 60000, or dimeric human OCIF having a molecular weight of about 120000 as measured by electrophoresis in LTO-page in non conditions, and these polysaccharides and their derivatives selected from the group consisting of hyaluronic acid, chondroitinase acid, dermatan (acid), heparana (acid), ceratina (Kislov, selected from the group consisting of OCIF, its analogs and its variants, the substance selected from the group consisting of polysaccharides and its derivatives, is from 1:1 to 1:10.

20. Complex p. 1, where the specified substance selected from the group consisting of OCIF, its analogs and its variants, is Monomeric human OCIF having a molecular weight, measured by electrophoresis in LTO-page in non conditions, approximately 60000, or dimeric human OCIF having a molecular weight of about 120000 as measured by electrophoresis in LTO-page in non conditions, and these polysaccharides and their derivatives selected from the group consisting of dextransucrase and its salts, and the molar ratio of the specified substances, selected from the group consisting of OCIF, its analogs and its variants, the substance selected from the group consisting of polysaccharides and its derivatives, is from 1:1 to 1:10.

21. Complex p. 1, where the specified substance selected from the group consisting of OCIF, its analogs and its variants, is Monomeric or dimeric human OCIF, wherein said monomer or one of the links specified dimer OCIF contains amino acids +1 to +380 of the sequence SEQ ID NO:1 list item is Yuyu molecular weight of from 1500 to 12000, moreover, the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the sodium salt of doctranslate is from 1:1 to 1:10.

22. Complex p. 21, where the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the said sodium salt of doctranslate is from 1:1 to 1:8.

23. Complex p. 21, where the molar ratio of the specified substance selected from the group consisting of OCIF, its analogs and its variants, to the said sodium salt of doctranslate is from 1:1 to 1:5.

24. The complex according to any one of paragraphs.21-23, where the specified derived polysaccharide is sodium salt doctranslate having an average molecular weight of from 1800 to 6000.

25. Method of prolonging the time during which OCIF or analogue or variant is retained in the bloodstream after administration to the patient through the education system before the introduction of at least one specified OCIF, its specified equivalent or specified variant as defined in any of paragraphs.1-6, with at least one polysaccharide or its derivative defined in any of paragraphs.1 and 7-11.

26. Pharmaceutical composition for treatment or prevention of bone with a carrier or diluent, where specified pharmacologically active agent is a complex according to any one of paragraphs.1-24.

27. Agent containing a therapeutically effective amount of a complex according to any one of paragraphs.1-24, for use for the prevention or treatment of bone metabolic diseases.

28. Agent p. 27, where these bone metabolic disease selected from the group consisting of osteoporosis, osteopenia, Paget's disease, osteomyelitis, infectious lesion, caused by the rarefaction of bone, hypercalcemia, osteoclasia, employed or osteopenia caused by rheumatism, osteoarthritis, depression periodontal bone cancer metastasis bone, osteonecrosis or death of osteocytes that accompany traumatic injury, Gaucher disease, sickle cell anemia, systemic lupus erythematosus or nontraumatic injury, osteodystrophies and cachexia caused by solid carcinoma, or cancer bone metastasis or malignant hematological disease.

29. The method of prevention or treatment of bone metabolic diseases in a patient suffering from them, introducing a specified patient an effective amount of complexi of osteoporosis, osteopenia, Paget's disease, osteomyelitis, infectious lesion, caused by the rarefaction of bone, hypercalcemia, osteoclasia, employed or osteopenia caused by rheumatism, osteoarthritis, depression periodontal bone cancer metastasis bone, osteonecrosis or death of osteocytes that accompany traumatic injury, Gaucher disease, sickle cell anemia, systemic lupus erythematosus or nontraumatic injury, osteodystrophies and cachexia caused by solid carcinoma, or cancer bone metastasis or malignant hematological disease.

31. A method of producing a complex according to any one of paragraphs.1-24, where the specified method comprises the stage of incubation of at least one substance selected from the group consisting of OCIF, its analogs and its variants, as defined in any of paragraphs.1-6, with at least one substance selected from the group consisting of polysaccharides and derivatives thereof as defined in any of paragraphs.1 and 7-11, at a pH of from 9.5 to 12, and then remove any free polysaccharides or their derivatives, which are not associated with the specified OCIF, analogues or variants.

32. The method according to p. 31, where incubation of the specified item is owned at least one substance, selected from the group consisting of polysaccharides and their derivatives, performed at pH 10 to 11.

33. The method according to p. 31 or 32, where any free polysaccharide or its derivatives that are not associated with the specified OCIF, analogues or variants after incubation remove the gel-filtration chromatography.

34. The complex according to any one of paragraphs.1-24, where specified complex get method in accordance with any of paragraphs.31-33.

 

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The invention relates to biotechnology and can be used to create a functioning tyrosine-specific chimeras proteinkinase

The invention relates to the production of endostatin mouse and man

The invention relates to medicine and concerns transducers cytochrome P450 retroviral vectors

The invention relates to the field of medical genetics
The invention relates to medicine, gynecology, and can be used for the treatment of uterine cervix using locoregionally citicentre
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