Method of treating interleukin-6-dependent diseases

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a therapeutic agent for treating rheumatoid arthritis administered in a dose of 4 mg/kg for 4 weeks and more that provides an equivalent concentration of the interleukin-6 receptor antibody (IL-6R antibody), containing the interleukin-6 receptor antibody (IL-6R antibody).

EFFECT: invention provides the simultaneous intensification of the therapeutic action on rheumatoid arthritis and reduced hypersensitivity.

46 cl, 4 ex

 

The technical field to which the invention relates.

The present invention relates to methods for treating interleukin-6(IL-6)-dependent diseases using a combination of an antagonist of interleukin-6 (antagonist IL-6), in particular antibodies to receptor of interleukin-6 (IL-6R) (antibody to IL-6R), and immunosuppressants, and by administering antibodies to IL-6R in high dosage.

The level of technology.

IL-6 is a cytokine, which is also called factor-2, stimulating b cells (BSF2, cell stimulating factor-2)or interferon-β2. IL-6 was opened as a differentiation factor involved in the activation of cells derived from b-lymphocytes (T. Harada et al., Nature, 324:73-76, 1986), and subsequently it was found that IL-6 is a multifunctional cytokine that affects the function of various cells (Akira, S. et al., Adv. in Immunology, 54:1-78, 1993). It was shown that IL-6 induces the maturation of cells derived from T-lymphocytes (M. Lotz et al., J. Exp. Med., 167:1253-1258, 1998).

IL-6 informs the cells of their biological activity through two types of proteins. One of them is the receptor for IL-6 (IL-6R), which is a ligand-binding protein with a molecular mass of about 80 kDa, which is joined by IL-6 (Taga T. et al., J. Exp. Med., 166:967-981, 1987; K. Yamasaki et al., Science 241:825-828, 1987). In addition to the membrane-associated type, which penetrates and is expressed in the cell membrane, the receptor for IL-6 was also obnarugen the form of a soluble receptor of IL-6, consisting mainly of the extracellular part of the receptor.

International publication WO 92/19759 describes the different types of antibodies to IL-6R, such as humanized antibodies to IL-6R and chimeric antibodies to IL-6R. WO 96/11020 describes a therapeutic tool in rheumatoid arthritis and the inhibitor of the growth of synovial cells (cells of the inner lining of the joints), the primary ingredient of which is the antagonist of IL-6, such as an antibody to IL-6R. WO 96/12503 describes the treatment of diseases, for which the characteristic of the production of IL-6, such as plasmocytes, hyperimmunoglobulinemia, anemia, nephritis, cachexia, rheumatoid arthritis, disease Castleman (Castleman''s disease) and mesangial proliferative nephritis. WO 98/42377 describes protective/therapeutic agent for diseases related to sensitized T-cells, such as multiple sclerosis, uveitis, thyroiditis, delayed hypersensitivity, contact dermatitis and atopic dermatitis, the active ingredient of which is an antibody to IL-6R.

WO 98/42377 describes a therapeutic agent in systemic erythematous, an active ingredient which is an antibody to IL-6R. WO 99/47170 describes therapeutic agent for Crohn's disease, the active ingredient of which is an antibody to IL-6R. WO 00/10607 describes therapeutic agent for pancreatitis, the active ingredient of which is an antibody to IL-6R. WO 02/3492 describes a therapeutic agent for psoriasis, the active ingredient of which is an antibody to IL-6R. In addition, WO 02/080969 describes a therapeutic agent for juvenile idiopathic arthritis, the active ingredient of which is an antibody to IL-6R.

Disclosure of the invention.

As described above, have been known various preventive and therapeutic agent, the active ingredient of which is an antibody to IL-6R. However, it was not known that in the treatment of IL-6-dependent diseases by a combination of antibodies to IL-6R and immunosuppressants, such as methotrexate (MTX), it is possible to obtain synergistic effects that immunosuppressant, such as methotrexate (MTX) may reduce or prevent allergic reactions in the treatment of rheumatoid arthritis using antibodies to IL-6R, and that antibody to IL-6R in high dosage can reduce or prevent allergic reactions in the treatment of rheumatoid arthritis with methotrexate.

Therefore, the present invention provides a pharmaceutical composition for the treatment of IL-6-dependent diseases, comprising the antagonist of interleukin-6 (antagonist IL-6) and immunosuppressants.

The invention also provides a pharmaceutical composition comprising an immunosuppressant, to enhance the effect when using the antagonist of IL-6R in the treatment of IL-6-C is independent diseases.

The invention also provides a pharmaceutical composition comprising an immunosuppressant for the prevention or attenuation of allergic reactions in the treatment of IL-6-dependent diseases with the help of antagonist IL-6.

The invention also provides a therapeutic agent comprising an antagonist of IL-6 for the treatment of IL-6-dependent diseases by administering high doses of the drug.

The invention also provides a pharmaceutical composition comprising an antagonist of IL-6 in the high dose for the prevention or attenuation of allergic reactions in the treatment of IL-6-dependent diseases.

The antagonist of IL-6 preferably is an antibody to IL-6R. Antibody to IL-6R is preferably a monoclonal antibody to IL-6R. Preferably, the antibody to IL-6R was a monoclonal antibody to IL-6R person. Or preferably, an antibody to IL-6R was a monoclonal antibody to IL-6R mouse. Preferably, the antibody to IL-6R antibody was recombinant type. Preferably, a monoclonal antibody to IL-6R person was, for example, antibody RM-1. Preferably, a monoclonal antibody to IL-6R mouse was, for example, antibody MR16-1. In addition, the antibody may be a chimeric antibody, humanized antibody or human antibody to IL-6R. Particularly preferred antibody to IL-6R is, n is the sample, humanitariannet antibody RM-1.

When sharing antagonist IL-6, in particular, antibodies to IL-6R, and immunosuppressant dosage of antagonist IL-6, in particular, antibodies to IL-6R is, for example, intravenously from 0.02 to 150 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R, preferably from 0.5 to 30 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R, and most preferably using from 2 to 8 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

With the introduction of antagonist IL-6, in particular, antibodies to IL-6R in the high dose, the dosage of antagonist IL-6, in particular, antibodies to IL-6R is, for example, when administered intravenously in not less than 4 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R, preferably from 6 to 16 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R, or most preferably from 6 to 10 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

When used as immunogenes the Anta MTX dosage MTX is for example, from 1 to 100 mg/person/week or the dosage at which the blood is equivalent to the concentration of MTX, preferably from 4 to 50 mg/person/week or dose at which the blood is equivalent to the concentration of MTX, or especially preferably from 10 to 25 mg/person/week or dose at which the blood is equivalent to the concentration of MTX.

The dosage at which the blood find drug (e.g., antibody to IL-6R or MTX), means dosages giving equivalent therapeutic effect, and even when reaching a certain concentration in the blood is different because of the way the introduction of means, for example, by intravenous injection or subcutaneous injection, it is regarded as the dosage at which the blood find such a concentration of a drug (e.g., antibodies to IL-6R or MTX), which is achieved equivalent therapeutic effect.

Examples of IL-6-dependent diseases include

acute and chronic inflammatory diseases and autoimmune diseases: jade, mesangial proliferative nephritis, Crohn's disease, ulcerative colitis, pancreatitis, juvenile idiopathic arthritis or systemic juvenile idiopathic arthritis, vasculitis, Kawasaki disease, rheumatoid arthritis, systemic erythematous, psoriasis, Sjogren syndrome, a disease of Steele in adults;

neoplastic diseases: multiple myeloma, a disease of Castleman, malignant lymphoma, cancer of the kidney;

infectious diseases: HIV, the virus infection Epstein-Barr (EBV);

cachexia: cachexia;

other diseases: plasmocytes, hyperimmunoglobulinemia, anemia, etc. and is preferably rheumatoid arthritis, plasmocytes, hyperimmunoglobulinemia, anemia, nephritis, cachexia, multiple myeloma, disease Castleman, mesangial proliferative nephritis, systemic erythematous, Crohn's disease, pancreatitis, psoriasis, juvenile idiopathic arthritis or systemic juvenile idiopathic arthritis.

The pharmaceutical composition of the present invention can be administered orally or parenterally and regularly or when necessary. For example, you can choose intravenous injection, such as the introduction using the dropper, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, injection into the rectum, oral drugs are covered with enteric-soluble shell, and the like, and suitable route of administration of medicines, you can choose the right way depending on the age and condition of the patient. The upper and lower limits of the effective dose depends on the frequency of the input is the means, for example, the dosage of funds for one time increase during the long interval between meals, while the dosage means reduce in the short interval between meals.

Preferred effective dose and method of administration of the antibody to the receptor of IL-6, for example, is such a number, in which blood find free antibody. As typical examples, there are ways of introducing medicines with split doses in several doses, for example, via intravenous injection such as drip, subcutaneous injection, which is carried out in accordance with the schedule twice a week, once a week, once in two weeks once in 4 weeks once every 6 weeks, once every 8 weeks or so the Schedule can be adjusted by increasing the interval between meals two times a week or once a week to once in 2 weeks, one times in 3 weeks once in 4 weeks once in 6 weeks and once per 8 weeks during the monitoring of the disease and changes in results of laboratory tests blood.

When co-administration with MTX dosage antibodies to IL-6R is common, for example, in the treatment of rheumatoid arthritis this dosage more than 0.5 mg/kg per week or the dosage at which the blood is equivalent to the or higher Antirheumatic effect. For example, intravenous, held once in four weeks, the dosage ranges from 0.02 to 150 mg/kg, preferably from 0.5 to 30 mg/kg and more preferably from 2 to 8 mg/kg

Antibody to IL-6R and the immunosuppressant is administered simultaneously or with a time interval.

Immunosuppressants include Antirheumatic means adrenocorticoids hormonal drugs and TPI include, for example, the following medicines.

Immunosuppressants, Antirheumatic means, means including adrenocorticoid hormones

Immunosuppressants

Alkylating tools

Cyclophosphamide

Metabolic antagonists

Azathioprine, methotrexate, mizoribine

Inhibitors of the activity of T-cells

Cyclosporine, tacrolimus

Antirheumatic means:

Hydroxychloroquin, sulfasalazin, Leflunomide, etanercept, infliximab, adalimumab, D-penicillamine, oral gold preparations, injectable preparations of gold (for intramuscular injection), minocycline, gold sodium thiomalate, auranofin, lobenzarit, bucillamine, actarit;

Means, including adrenocorticoid hormones:

Cortisone, hydrocortisone

Cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, forgotson acetate

Prednisolone, prednisolone

Predni the'olonne, prednisolone sodium succinate, prednisolone sodium phosphate, halopedia acetate

Methylprednisolone

Methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate

Triamcinolone

Triamcinolone, triamcinolone acetate triamcinolone actinide

Dexamethasone

Dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, dexamethasone palmitate

Betamethasone

Betamethasone, betamethasone sodium phosphate

Paramethasone

Paramethasone acetate

The dosage of immunosuppressant, for example, with co-administration of MTX for the treatment of rheumatoid arthritis, for example, in oral administration, is from 1 to 100 mg/person per week, preferably from 4 to 50 mg, and most preferably from 7.5 to 25 mg/person.

High dosage of antibodies to IL-6R includes dosage, can prevent or reduce allergic reaction, this dosage is equivalent to or exceeds the minimum dosage that is effective for the treatment of IL-6-dependent diseases. For example, in the treatment of rheumatoid arthritis intravenous drip infusion produced every four weeks, the dosage of 4 mg/kg or more, preferably from 6 to 16 mg/kg and most preferably from 6 to 10 mg/kg

The above-described method, the interval between doses and dosage are particularly the illustration of the preferred examples, and appropriately, you can choose the way of the reception interval and the dose at which achieved similar therapeutic effect. For example, measuring the concentrations of various drugs in the blood, you can choose the way of the reception interval and the dose at which effects are achieved, similar to those described above as examples. The invention includes a method of reception interval and the dose at which blood is achieved concentrations equivalent to those described in the examples above.

The implementation of the invention.

Antagonists of IL-6 used in the present invention can be used regardless of their origin, type and shape as long as they demonstrate a preventive or therapeutic effect in IL-6-dependent diseases.

Antagonists of IL-6 are substances that inhibit the biological activity of IL-6. Antagonists of IL-6 are preferably substances having inhibitory activity of binding to any protein of IL-6, IL-6R or gp130. Antagonists of IL-6 include antibody to IL-6, antibody to IL-6R, gp130 antibody to modified IL-6, a modified soluble IL-6R or partial peptides of IL-6 or IL-6R, as well as substances with a low molecular weight, having similar activity.

Antibody to IL-6, used in this image is the situation, can be obtained as polyclonal or monoclonal antibodies, using means known in this technical field. As antibodies to IL-6 used in the present invention, preferred are monoclonal antibodies derived from mammals. Monoclonal antibodies mammals include antibodies produced by hybridomas and antibodies produced by cells of the host, which were transformed with expression vector comprising genetically-engineered gene antibodies. This antibody binding to IL-6, inhibits the binding of IL-6 receptor, IL-6 and, thus, blocks the transmission of biologically active signal of IL-6 in cells.

Examples of such antibodies include antibody MN (Matsuda T. et al., Eur. J. Immunol., 18:951-956, 1988) and SK2 antibody (Sato, K. et al., The 21 st Proceedings of the Japanese Society for Immunology, 21:166, 1991).

Hybridoma producing antibodies to IL-6, can basically be constructed using a known procedure described below. That is, hybridoma may be obtained by immunization using as a sensitizing antigen, IL-6, in accordance with the traditional method of immunization; merging the obtained immune cells with known technology parent cells in the conventional method of merging cells; and selecting cells producing a monoclonal antibody, using traditionnal the screening method.

In particular, antibody to IL-6 can be obtained as follows. For example, human IL-6 that is used as the sensitizing antigen for obtaining antibodies can be obtained using the gene/amino acid sequence of IL-6, described in detail in Eur. J. Biochem., 168:543-550, 1987; J. Immunol., 140:1534-1541, 1987; or in Agr. Biol. Chem., 54:2685-2688, 1990.

The sequence of the gene IL-6 is inserted into a known expression vector, which transform appropriate cells of the host, then use the known method of interest protein IL-6 purified from the cells or the supernatant of the cell culture and purified protein of IL-6 can be used as a sensitizing antigen. Also, as a sensitizing antigen can be used fused protein formed by the combination of protein, IL-6 and another protein.

Antibody to the receptor of IL-6 used in the present invention, can be obtained as polyclonal or monoclonal antibodies using the method known in this technical field. As antibodies to the receptor of IL-6 used in the present invention, preferred is a monoclonal antibody, particularly an antibody, derived from mammals. Monoclonal antibodies mammals include antibodies produced by hybridomas and antibodies produced by cells of the host, which biotransformation using the expression vector, including genetically engineered antibody gene. This antibody binds with the receptor of IL-6, inhibits the binding of IL-6 receptor, IL-6 and, thus, blocks the transmission of biologically active signal of IL-6 in cells.

Examples of such antibodies include antibody MR16-1 (T. Tamura et al., Proc. Natl. Acad. Sci. USA, 90:11924-11928, 1993), antibody PM-1 (Hirata Y. et al., J. Immunol., 143:2900-2906, 1989), antibody AUK12-20 antibody AUK64-7 or antibody AUK146-15 (International Patent Application Publication No. WO 92-19759). Among them, most preferred is an antibody RM-1.

Cell line hybridoma RM-1, which produces the antibody RM-1, was deposited for the international recognition of 12 July 1989 under the terms of the Budapest Treaty as FERM BP-2998 International Depositary for patentable microorganisms (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Pref.). Cell line rat-mouse hybridoma MR16-1, which produces the antibody MR16-1 was deposited for the international recognition of March 13, 1997 under the terms of the Budapest Treaty as FERM BP-5875 International Depositary for patentable microorganisms (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Pref.).

Hybridoma producing monoclonal antibody to the receptor of IL-6, can be obtained using known procedures described below. That is, hybridoma may be obtained by immunization using as sensitizing the Academy of Sciences of the Egan receptor IL-6, in accordance with the traditional method of immunization; merging the obtained immune cells with known technology parent cells in the conventional method of merging cells; and selecting cells producing a monoclonal antibody, using traditional screening method.

In particular, the antibody to the receptor of IL-6 can be obtained as follows. For example, human receptors IL-6, is used as the sensitizing antigen for obtaining antibodies can be obtained using the gene or the amino acid sequence of the receptor for IL-6, described in detail in European Patent Application Publication No. EP 325474 and JP-A-3-155795, respectively.

There are two types of receptor proteins IL-6, one of them expressed on the cells, and the other is separated from the cell membrane (soluble receptor IL-6) (Yasukawa K. et al., J. Biochem., 108:673-676, 1990). Soluble receptor of IL-6 is formed largely from the extracellular part of the receptor of IL-6 and differs from the membrane-associated receptor of IL-6 that does not contain a transmembrane site or transmembrane plot together with intracellular site. Both protein receptor of IL-6 can be used as a sensitizing antigen for production of antibodies to receptor of IL-6 used in the present invention.

The sequence of the gene for the receptor of IL-6 include the well-known expression century the PRS, which transform appropriate cells of the host, and then of interest to a protein receptor of IL-6 purified from the cells or culture supernatant using a known method, and purified protein receptor of IL-6 can be used as a sensitizing antigen. Also, as a sensitizing antigen can be used cells expressing the receptor of IL-6, or a protein formed by the combination of the protein receptor of IL-6 and another protein.

The strain E. coli HB101-pIBIBSF2R, cells which contain the plasmid pIBIBSF2R, including cDNA, which encodes the receptor for IL-6 person, was deposited for the international recognition of 9 January 1989 under the terms of the Budapest Treaty as FERM BP-2232 International Depositary for patentable microorganisms (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Pref.).

Antibody to gp130 used in the present invention, can be obtained as polyclonal or monoclonal antibodies using a known method. As antibodies to gp130 used in the present invention, preferably used is a monoclonal antibody derived from mammals. Monoclonal antibodies mammals include antibodies produced by hybridomas and antibodies produced by cells of the host, which were transformed using the expression vector, including GE is eticheski constructed gene antibodies. This antibody binding to gp130, inhibits binding of IL-6/receptor IL-6 gp130, blocking the transfer of biologically active signal of IL-6 in cells.

Examples of such antibodies include antibody AM (JP-A-3-219894), antibody B and antibody N (US 5571513), antibody (B-S12 and antibody-R8 (JP-A-8-291199).

Hybridoma producing monoclonal antibody to gp130, can be obtained using known procedures described below. That is, hybridoma may be obtained by immunization using as a sensitizing antigen gp130, in accordance with the traditional method of immunization; merging the obtained immune cells with known technology parent cells in the conventional method of merging cells; and selecting cells producing a monoclonal antibody, using traditional screening method.

In particular, the monoclonal antibody can be obtained as follows. For example, gp130, is used as the sensitizing antigen for obtaining antibodies can be obtained using the gene/amino acid sequence of gp130, is described in detail in European Patent Application Publication No. EP 411946.

The sequence of the gene gp130 include in a known expression vector system, which transform a suitable cell host, then interest gp130 protein purified from the cells or cell culture supernatant using and the known method, and purified protein gp130 can be used as a sensitizing antigen. Also as a sensitizing antigen can be used cells expressing gp130, or a fused protein formed by the combination of gp130 and another protein.

Animals immunized with sensitizing antigen is not limited, but they are preferably selected for their compatibility with the parent cells used to merge cells. Usually they include rodents, such as mouse, rat and hamster.

Immunization of the animal sensitizing antigen is carried out using a known method. As the main ways, for example, is used intraperitoneal or subcutaneous injection of an animal sensitizing antigen. In particular, in the preferred embodiment, sensitizing antigen, which is appropriately diluted and suspended in PBS (phosphate saline buffer or saline solution, mixed to emulsify with a suitable number of conventional adjuvant, for example, complete adjuvant's adjuvant, and then introduce him to a mammal in several techniques range from 4 to 21 days. Also during immunization sensitizing antigen may be used in suitable media.

Animal subjected to immunization in this way, and immunization under versaut fact, what in the serum increases the level of specific antibodies. Then immunized with cells extracted from a mammal and is used to merge cells. Preferred cells to merge cells, in particular cells of the spleen.

As the myeloma cells mammals used as parent cells-partners to merge with the above immunized cells, preferably using known cell lines such as P3X63Ag8.653 (Kearney J.F. et al., J. Immunol., 123:1548-1550, 1979), P3X63Ag8U.1 (Current Topics in Microbiology and Immunology, 81:1-7, 1978), NS-1 (Kohler G. and Milstein C., Eur. J. Immunol., 6:511-519, 1976), MOC-11 (D.H. Margulies et al., Cell, 8:405-415, 1976), SP2/0 (Shulman, M. et al., Nature 276:269-270, 1978), FO (de St. Groth S.F. et al., J. Immunol. Methods 35:1-21, 1980), S194 (Trowbridge, I.S., J. Exp. Med., 148:313-323, 1978) and R210 (Galfre, G. et al., Nature, 277:131-133, 1979).

Merging the above immune cells with myeloma cells essentially can be carried out in accordance with the known method such as the method described by Milstein et al. (Kohler G. and Milstein, C., Methods Enzymol., 73:3-46, 1981).

More specifically, the specified cell fusion is carried out, for example, in conventional nutrient culture medium in the presence of a catalyst merge cells. As a catalyst for the fusion of cells, for example, it is possible to use polyethylene glycol (PEG), Sendai virus (HVJ) or something similar, and, in addition, to increase the effectiveness of the merger, then the request can be in order to add/use a helper tool such as dimethyl sulfoxide.

The preferred ratio of immune cells and the myeloma cells is, for example, from 1 - to 10-fold excess of immune cells against myeloma cells. As the medium used to merge cells, you can use medium RPMI 1640, MEM medium suitable for growth of the cell lines, myeloma, and other standard environment used for this type of cell culture, and, in addition, you can enter serum supplements such as fetal calf serum (FCS).

To merge target cells merged cells (hybridoma) is obtained by thoroughly mixing a certain amount described above immunized cells with myeloma cells in the above environment, adding a solution of PEG, for example, a solution of PEG with a molecular weight of from 1000 to 6000, pre-warmed to 37°C, in standard concentrations from 30% to 60% (weight/volume), followed by stirring. Then, using the procedure repeated, successive addition of a suitable culture medium and centrifugation to remove supernatant, agents, merge cells, and so on, the presence of which is undesirable for the growth of hybridoma.

Hybridoma can be selected by culturing in conventional breeding environment, for example, in the culture medium HAT (medium containing gipoksantin, aminopterin and timid is). Cultivation in the environment HAT continue usually from several days to several weeks, during a time sufficient for cell death (not fused cells), which differs from the desired hybrid. Then apply the usual method of limiting dilution and conduct screening and cloning of hybridomas producing the desired antibody.

In addition to obtaining the above hybridoma, immunosera antigen animal (but not person) with sensibilizirovannoy of human lymphocytes by protein antigen or cells expressing the antigen in vitro, and fusing the sensitized lymphocytes with myeloma cells of a person, for example U266 cells (see JP-B-1-59878), it is possible to obtain the desired human antibody having the activity of binding to the desired antigen or cells expressing the antigen. In addition, the antigen or cells expressing the antigen, you can enter a transgenic animal having a set of genes of human antibodies, in order to obtain the desired human antibody in accordance with the method described above (see International Patent Application Publication Nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096, WO 96/33735).

Designed hybridoma producing monoclonal antibody, can be grown in standard culture medium or can be stored for a long time in the frozen state at a temperature of Idaho nitrogen.

To obtain monoclonal antibodies from hybridomas, you can use the way in which hybridoma cultivated in the usual way and get monoclonal antibody in the supernatant of the cell culture, or by the manner in which hybridoma enter and grow in animals that are compatible with the specified hybridomas, and the antibodies obtained as ascites. The first method is suitable for obtaining highly purified antibodies, whereas the latter method is suitable for production of large quantities of antibodies.

For example, getting hybridoma producing antibody to the receptor of IL-6, can be performed using the method disclosed in JP-A-3-139293. You can apply the way in which hybridoma producing antibody PM-1 deposited for the international recognition of 12 July 1989 under the terms of the Budapest Treaty as FERM BP-2998 International Depositary for patentable microorganisms (AIST Tsukuba Central 6, 1-1, Higashi 1-chome, Tsukuba-shi, Ibaraki Pref.), administered intraperitoneally to BALB/c mice to obtain ascites, and antibodies RM-1 purified from these ascites. Also, you can apply the way in which these hybridoma cultivated in a suitable culture medium, for example, in the medium RPMI 1640, in hybridoma medium SFM (produced by GIBCO-BRL), in the environment PFHM-II (produced by GIBCO-BRL)containing 10%fetal calf serum and 5%MB-Condimed H1 (manufacturing firms is s Boehringer Mannheim), and antibody PM-1 purified from the supernatant of cell culture.

In the present invention as monoclonal antibodies can be used recombinant antibody type produced by cloning the gene of the antibody from hybridoma, the inclusion of this gene in a suitable vector, its introduction into the cells of the host, and the use of genetic engineering techniques (see, for example, C.A.K. Borrebaeck, and Larrick J.W. Therapeutic Monoclonal Antibodies, published in the United Kingdom by Macmillan Publishers Ltd. 1990).

Specifically, mRNA encoding the variable (V) region of the desired antibodies, isolated from cells producing the desired antibody, such as hybridoma. For isolation of mRNA receive a total RNA preparation using a known method, such as method of ultracentrifugation in the presence of guanidine (J.M. Chirgwin et al., Biochemistry, 18:5294-5299, 1979), AGPC method (Chomczynski, P. et al., Anal. Biochem., 162:156-159, 1987), and mRNA receive, using a set of firm Pharmacia "mRNA Purification Kit". Also mRNA can be distinguished directly using the set of firm Pharmacia "QuickPrep mRNA Purification Kit".

cDNA V region antibodies are synthesized from the obtained mRNA using reverse transcriptase. The cDNA synthesis is performed using a set of "AMV Reverse Transcriptase First-strand cDNA Synthesis Kit" and similar sets. Also, for synthesis and amplification of cDNA can use a set of "5'-Ampli FINDER RACE kit (produced by Clontech), and the method of 5'-RACE, in which the om is used polymerase chain reaction (PCR) (Frohman M.A. et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002, 1988; A. Belyavsky et al., Nucleic Acids Res., 17:2919-2932, 1989). The desired DNA fragment is purified from the PCR product and are ligated into a DNA vector. Then the obtained recombinant vector is introduced into E. coli, and selected colonies to highlight the desired recombinant vector. The sequence of bases of the desired DNA is confirmed by using a known method, such as dideoxy-sequencing method.

If you get a DNA encoding the V region of the desired antibodies, it are ligated to the DNA molecule encoding the constant region (C-region) of the desired antibodies, and then it is included in the expression vector. Or DNA encoding the V region of the antibody, can be included in expression vector containing the C-region of the antibody.

To produce the antibody used in the present invention, the gene of the antibody include in the expression vector for its expression was under the control of expression regulatory areas, for example, enhancer and promoter, as described below. Then, using this expression vector can be transformed cell hosts.

In the present invention in order to reduce allogenic antigenicity for a person can be applied recombinant antibodies artificially modified gene, for example, chimeric antibody, humanitariannet antibody and a human antibody. These modified the s antibodies can be obtained using known methods.

A chimeric antibody can be obtained by ligating obtained as described above, DNA encoding the V region of the antibody, DNA encoding the C-region of a human antibody, and subsequent to its inclusion in the expression vector is introduced into cells of the hosts for production (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. WO 92/19759). Using these known methods it is possible to obtain chimeric antibody useful in the present invention.

For example, plasmids which contain DNA encoding the V region of the L - and H-chain chimeric antibody RM-1, were named the ppm-k3 and ppm-h1, respectively, and the strains of E. coli with these plasmids have been deposited for the international recognition of 12 February 19991 year under the terms of the Budapest Treaty as NCIMB 40366 and NCIMB 40362, respectively, of the National collections of industrial, food and marine bacteria (23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom).

Humanized antibodies are human antibodies with a modified form, are antibodies in which the hypervariable segment (CDR, complementarity determining region) of the mammal, not the person, for example, mouse antibody transplanted in the CDR section of human antibodies using known, conventional recombinant DNA (see European Patent Application Publication No. EP 125023, International Patent Application Publication No. O 92/19759).

In particular, the DNA sequence, which was designed for ligating the CDR of mouse antibodies in karisny plot (FR, framework region of human antibodies synthesized by the method of PCR using oligonucleotides having at the ends of the overlapping area. The resulting DNA are ligated to DNA encoding the C-region of a human antibody, and then include in the expression vector is introduced into cells of the hosts for the production of gumanitarnogo antibody (see European Patent Application Publication No. EP 239400, International Patent Application Publication No. WO 92/19759).

As FR antibodies person, very through CDR, choose those in which hypervariable area forms a good range of binding antigen. If desired, you can replace amino acids in the frame area of the variable segment of antibodies to the hypervariable area of the human antibodies with a modified form could form the corresponding region of the binding of antigen (Sato, K. et al., Cancer Res., 53:851-856, 1993).

For chimeric antibodies and gumanitarnogo antibodies used-region of a human antibody. The C-region of a human antibody includes γ, and, for example, you can use Cγ1, γ2, γ3 and γ4. The C-region of a human antibody can be modified to improve the stability of the antibody or its products.

Chimeric antibody includes variabel the second region of the antibody, originating from a mammal other than human, and C-region derived from human antibody. Humanitariannet antibody comprises CDR plot antibody derived from a mammal other than human, and the frame section and the C-region derived from human antibody. In line with this, they have low antigenicity in the human body and, thus, useful as antibodies used in the present invention.

Preferred typical examples gumanitarnogo antibodies used in the present invention include humanitariannet antibody PM-1 (see International Patent Application Publication No. WO 92/19759).

Also, in addition to the methods described above for obtaining human antibodies known technology for producing human antibodies by panning using the library of human antibodies. For example, the variable region of a human antibody can Express on the surface of the phage in the form of single-chain antibodies (scFv) using the method of phage display (phage display), and from the library you can select phage that bind to the antigen. Genetic analysis of selected phage can determine the sequence of DNA encoding the variable region of a human antibody that binds to the antigen. If the sequence of Dnsapi scFv, which binds the antigen was detected using a suitable expression vector can be constructed sequence to obtain human antibodies. These methods are already well known, and they can be found in WO 92/01047, WO 92/20791, WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438 and WO 95/15388.

Gene antibodies, constructed as described above, it is possible to Express and receive using a known method. In the case of mammalian cells gene antibodies can be Express using DNA, which is effectively connected commonly used promoter, expressed antibody gene and the signal sequence of the poly-A, located on the 3'end of the DNA, or by using a vector that includes them. For example, as the promoter/enhancer can be used immediate early promoter/enhancer of the human cytomegalovirus.

In addition, as other promoters/enhancers that can be used for expression of the antibodies used in the present invention, it is possible to use viral promoters/enhancers retrovirus, virus polyoma, adenovirus and virus 40 monkeys (SV40), and the promoter/enhancer derived from the gene for elongation factor human 1α (HEF1α) of mammalian cells.

For example, the expression can be performed using the method of Mulligan et al. (R. Mulligan et al., Nature, 277:p.108-114, 1979) using a promoter/anchan the EPA SV40 or by the method of Mizushima et al. (Mizushima, S. and Nagata, S., Nucleic Acids Res., 18:5322, 1990) using the promoter/enhancer of the gene HEF1α.

In the case of E. coli gene can Express through effective compounds commonly used promoter, a signal sequence for secretion of antibodies and expressed the gene of the antibody. For example, promoters may include promoters of genes lacZ and araB. When using the promoters of genes lacZ and araB, you can use the method of Ward et al. (E.S. Ward et al., Nature, 341:544-546, 1989; Ward E.S. et al., FASEB J., 6:2422-2427, 1992) and the method of Better et al. (Better, M. et al., Science, 240:1041-1043, 1988), respectively.

As a signal sequence for secretion of antibodies can be used signal sequence pe1B (Lei S.P. et al., J. Bacteriol., 169:4379-4383, 1987) in the case of the production of protein in periplasm E. coli. The antibody produced in periplasm, isolated and then used after a suitable change of the spatial structure of antibodies (see, for example, WO 96/30394).

As of the start points of replication (origin), you can use viral origin derived from SV40, a virus polyoma, adenovirus, virus, bovine papillomavirus (BPV). In addition, to increase the number of gene copies in the system host cells, the expression vector may include as a selective marker gene aminoglycosidetherapy (ARN), gene timedancing (TS), gene xanthine-guanine-phosphoribosyltransferase E. coli (Ecogpt)gene dihydro who ultratotty (dhfr), etc.

For producing antibodies used in the present invention, it is possible to use any producing system. To obtain antibodies are producing in vitro and in vivo. The system of production in vitro includes producing a system in which the use of eukaryotic cells, and producing a system in which the use of prokaryotic cells.

When using eukaryotic cells are producing systems that use animal cells, plant cells or cells of fungi. The animal cells include (1) mammalian cells such as Cho, COS, myeloma cells, KSS (cells from the kidneys of baby hamster), HeLa, Vero, etc. (2) cells of amphibians such as Xenopus oocytes or (3) insect cells such as sf9, sf21, Tn5, etc.. Among plant cells are known, for example, cells that originate from Nicotiana tabacum, and which can be grown as callus (undifferentiated tissue of plants that can be grown in vitro). The cells of fungi include yeasts such as the genus Saccharomyces, in particular Saccharomyces cerevisae, or filamentous fungi such as the genus Aspergillus, in particular Aspergillus niger.

If using prokaryotic cells, there are producing systems that use bacterial cells. Bacterial cells include E. coli and Bacillus subtilis.

The antibody can be obtained by centuries the tion of the gene of the desired antibodies in these cells through their transformation and culturing the transformed cells in vitro. The cultivation is carried out using known methods. For example, the culture medium can be used DMEM, MEM, RPMI 1640 and IMDM, and together with them you can use serum supplements such as fetal calf serum (FCS). Antibodies can be obtained in vivo by transferring the cells into which has been introduced a gene antibodies, into the abdominal cavity of the animal.

On the other hand, systems that produce in vivo include systems in which animals are used, and those that use the plant cells. When using animal cells are producing systems that apply mammals and insects.

As mammals can be used goats, pigs, sheep, mice, cattle, etc. (Vicki Glaser, Spectrum Biotechnology applications, 1993). Also, as insects can be used silkworms. Plants can be applied, for example, tobacco.

Genes of antibodies injected into the cells of these animals or plants, and such animals or plants are produced antibodies, which then emit. For example, the gene of the antibody receive in the form of a fused gene by inserting into the middle of the gene encoding a protein that is produced by hereditary in milk, for example, such as a gene β-casein goats. The DNA fragment containing the fused gene, which was inserted gene of the antibody is administered with the aid of the completion of the injection into the embryo goats, and the embryo is introduced into a female goat. The desired antibody is obtained from milk produced by the transgenic goat born from the goats that has been implantable embryo, or milk from the offspring of such transgenic goats. To increase the amount of milk containing the desired antibody transgenic goat can give appropriate hormones (Ebert K.M. et al., Bio/Technology, 12:699-702, 1994).

If using silkworms, it infecting baculovirus in which the inserted gene of the desired antibodies, and the desired antibody is isolated from fluid obtained from the body of the silkworm (Maeda, S. et al., Nature, 315:592-594, 1985). In addition, if you use tobacco, the gene of the desired antibody are inserted into the plant expression vector, for example, pMON 530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. Tobacco, for example, Nicotiana tabacum, infect these bacteria, and leaves the tobacco receive the desired antibody (Julian K.S., Ma, et al., Eur. J. Immunol., 24:131-138, 1994).

If the antibody is obtained by producing systems in vitro or in vivo, as described above, DNA encoding a heavy chain (H chain) or light chain (L chain) of antibody can be individually integrated into expression vectors, which can simultaneously transform the owner, or DNA encoding the H and L chains can be included in the same expression vector, which can be transformed host (see International Patent Appliction Publication No. WO 94-11523).

The antibody used in the present invention, can be fragments of antibodies or their modified versions as long as the antibody can be adequately used. For example, fragments of antibodies may include Fab, F(ab')2, Fv or single-chain Fv (scFv)in which Fv fragments from H-chain and L-chain linked by a suitable linker.

In particular, in order to obtain fragments of antibodies, the antibody is treated with an enzyme such as papain, pepsin, or construct the gene encoding the antibody fragment and insert it in the expression vector, which Express in a suitable cell host (see, for example, With .S. et al., J. Immunol., 152:2968-2976, 1994; Better, M. and Horwitz A.N., Methods in Enzymology, 178:476-496, 1989; Plueckthun, A. and Scerra A., Methods in Enzymology, 178:497-515, 1989; Lamoyi, E., Methods in Enzymology, 121:652-663, 1989; Rousseaux, J. et al., Methods in Enzymology, 121, 663-666, 1989; Bird, R. E. et al., TIBTECH, 9:132-137, 1991).

Fragment scFv can be obtained by legirovaniem V-region of the H-chain V-region L-chain antibodies. In this scFv V region of H chain and the V region of the L-chain is preferably connected via a linker, preferably is a peptide linker (Huston J.S., et al, Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988). V region of H chain and the V region of the L-chain scFv can be from any of the aforementioned antibodies. As a peptide linker to connect the V-regions can be used, for example, a single-chain peptide comprising 12-19 aminokislotnykh residues.

DNA encoding scFv is obtained by PCR using the amplification of the DNA encoding the desired amino acid sequence, DNA encoding the H chain or the V region of H chain of the above antibodies, which are used as matrices, using a pair of primers that define both their end; then at the next stage amplified DNA encoding the plot peptide linker, and both ends of the specified DNA combine with a pair of primers that define their connection with the H-chain and L-chain, respectively.

In addition, after constructing DNA encoding scFv, using conventional methods can be obtained expression vector containing the DNA, and the owner, who transformed expression vector, and in accordance with conventional methods, scFv can be obtained using the obtained transformed organism-owner.

For these fragments, antibodies can also get their genes to Express and produce them in the body of the host, as noted above. Used in the present invention the term "antibody" also includes those fragments of antibodies.

As modified antibodies can be used antibody associated with various molecules such as polyethylene glycol (PEG). Used in the present invention the term "antibody" also includes these modifications EN is the body. To obtain such modifications antibodies derived antibody chemically modify. These methods are already established in this field of technology.

Produced and expressed, as described above, the antibody can be isolated from external and internal compartments host cells and purified to a homogeneous state. Separation and purification of the antibodies used in the present invention, can be performed using affinity chromatography. The column used for affinity chromatography include column with protein a and protein G. the Media used in the column with the protein And include Hyper D, POROS, Sepharose F, etc. Without any restrictions can be used any suitable conventional methods of separation/purification of normal proteins.

For example, the antibody used in the present invention, can be selected by appropriate selection and combination of chromatography that is different from the above-mentioned affinity chromatography, using filtration, ultra filtration, desalting, dialysis, etc. Examples of chromatography include Ion exchange chromatography, hydrophobic chromatography, gel filtration, etc. These kinds of chromatography can be applied in the format of HPLC (high performance liquid chromatography). You can also use reverse-phase HPLC.

Dimension to the concentrations of the antibodies, obtained as described above can be performed by measuring the optical absorption or by using enzyme-linked immunosorbent assay (ELISA). That is, in the measurement of optical absorption, antibody diluted appropriately with PBS (-) and then measure the optical absorption at 280 nm and calculate the concentration, assuming that 1 mg/ml gives an absorption equal to 1.35 OD. When using the ELISA measurement is carried out as follows. Thus, 100 μl of goat antibodies to immunoglobulins IgG-man (produced by TAG), diluted to a concentration of 1 μg/ml in 0.1 M bicarbonate buffer (pH 9,6), bring in a 96-well plate (produced by NUNC) and incubated overnight at 4°C for immobilization of antibodies. After blocking, make 100 ál of each diluted appropriately antibodies used in the present invention, or a sample comprising the antibody, or human IgG (produced by Cappel) as standard and incubated at room temperature for 1 hour.

After washing make 100 μl of the diluted 5000 times of antibodies to immunoglobulins human IgG labeled with alkaline phosphatase (produced by Bio Source), and incubated at room temperature for 1 hour. After rinsing, add and incubated substrate solution, and then to calculate the concentration of the desired antibody, measure the absorption of PR is 405 nm by using a spectrophotometer Microplate Reader Model 3550 (produced by Bio-Rad).

Modified IL-6 used in the present invention, is a substance that possess the activity of the receptor binding of IL-6 and which do not provide the biologically active signal IL-6. Modified IL-6 does not pass biologically active signal IL-6 as it is competitive in relation to IL-6 binds to the receptor of IL-6 blocking, thus, signaling IL-6.

Modified IL-6 design by obtaining variants of IL-6 by replacing amino acid residues in the amino acid sequence of IL-6. IL-6, which is the source of the modified IL-6 may be of any origin, but if we take into account the antigenicity, preferably it should be a human IL-6.

In particular, the modification of the IL-6 hold, predicting the secondary structure of the amino acid sequence of IL-6 using known in this technical field program molecular modeling amino acid sequence, for example, the program WHATIF (Vriend et al. J. Mol. Graphics, 8:52-56, 1990), then evaluating the impact of the replacement of individual amino acid residues in the whole protein. After the choice was made of suitable amino acid residues for amino acid replacement, get the gene encoding the modified IL-6, receiving its variants using commonly used for this method, the polymerase is epnoe reaction (PCR), in which amino acids are substituted, using as matrix vector comprising a sequence of bases, the human gene encoding IL-6. Modified IL-6 can be obtained, if necessary, inserting it into a suitable expression vector in accordance with the methods of expression, production and purification of the above-mentioned recombinant antibodies.

Typical examples of the modified IL-6 revealed in the work Brakenhoff et al., J. Biol. Chem., 269:86-93, 1994 and Savino et al., EMBO J. 13:1357-1367, 1994, WO 96/18648 and WO 96/17869.

Partial peptides of IL-6 or a partial peptide of the receptor of IL-6 used in the present invention have the activity of the receptor binding of IL-6 or IL-6, respectively, and are substances which do not transmit the biological activity of IL-6. That is, partial peptides of IL-6 or a partial peptide of the receptor of IL-6 specifically inhibit the binding of IL-6 receptor, IL-6, joining the receptor of IL-6 or IL-6, respectively, and locking them. As a result, as they do not provide the biologically active signal IL-6, they block the signaling of IL-6.

A partial peptide of IL-6 or a partial peptide of the receptor of IL-6 are peptides, including some or the complete amino acid sequence involved in the binding of IL-6 receptor, IL-6, in the amino acid sequence of IL-6 or receptor of IL-6. Such a peptide is usually on the et from 10 to 80, preferably from 20 to 50, even more preferably from 20 to 40 amino acid residues. A partial peptide of IL-6 or a partial peptide of the receptor of IL-6 can be constructed by defining a region involved in binding to IL-6 or its receptor IL-6 in the amino acid sequence of IL-6 or receptor of IL-6, and through the synthesis of some part or the entire amino acid sequence of conventional methods, such as genetic engineering or method of peptide synthesis.

To obtain a partial peptide of IL-6 or a partial peptide of the receptor of IL-6 using genetic engineering, the DNA sequence encoding the desired peptide is introduced into the expression vector using the methods of expression, production and purification mentioned above for recombinant antibodies.

To obtain a partial peptide of IL-6 or a partial peptide of the receptor of IL-6 using peptide synthesis, you can use the methods commonly used in peptide synthesis, for example, solid-phase synthesis method or liquid-phase synthesis method.

In particular, it is possible to carry out the synthesis in accordance with the method described in Zoku Iyakuhin, Kaihatsu, Vol. 14, Peptido Gousei, (Ed. Yajima, H., Hirokawa Shoten, 1991). As solid-phase synthesis method is applied, for example, a method in which a peptide chain are increasing by attaching the amino acid corresponding to the C-end of the synthesized peptide is IDA, to the substrate, which is insoluble in organic solvents, and alternately repeating the reaction in which a specific amino acid sequentially condense in the direction from C - to N-ends in the amino acid whose α-amino group and the functional group of the side chain protected with suitable protective groups, and the reaction in which the specified protective group of an α-amino groups of amino acids are removed from the amino acid or peptide attached to the substrate. Methods of solid-phase peptide synthesis in General are classified as the BOC-and Fmoc method-method, depending on the type of protective groups.

After synthesis of the desired peptide is carried out in such a way, conduct the reaction unprotect and the reaction of cleavage of the peptide chain from the substrate. In the reaction of cleavage of the peptide chain from the media in Eastern method is usually used hydrogen fluoride or triftorbyenzola, and Fmoc-method - triperoxonane acid (TFA). In Eastern method of the above-mentioned substrate is protected peptide is treated with hydrogen fluoride in the presence of anisole. Then, to obtain the peptide spend the removal of the protective group and cleavage of the peptide from the substrate. To obtain the crude peptide drug it lyophilizer. On the other hand, in the Fmoc method, for example, TFA, the reaction unprotect and the reaction of cleavage of the peptide from the substrate mo is but performed using the procedures similar to that described above.

The crude peptide can be extracted and purified by using HPLC method. The elution can be performed in optimal conditions, using a solvent system of water-acetonitrile, which is usually used for protein purification. Fractions corresponding to the peaks of the profile of the resulting chromatography, collecting and lyophilizers. Treated thus peptide fractions identify, by analyzing the molecular weight using mass spectroscopic analysis, analysis of amino acid composition or analysis of amino acid sequence.

Typical examples of the partial peptides of IL-6 and receptor of IL-6 are disclosed in JP-A-2-188600, 7-324097 and 8-311098, as well as in US patent 5210075.

The pharmaceutical compositions of the present invention can include pharmaceutically acceptable carriers or additives depending on the route of administration tools. Examples of such carriers or additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethyl cellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium salt carboxymethylamino starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum Arabic, casein, gelatin, agarose, diglycerin, the PCC is lepicol, the polyethylene glycol, vaseline, paraffin, stearic alcohol, stearic acid, human serum albumin (HSA), mannitol, sorbitol, lactose, pharmaceutically acceptable surfactants, etc. depending on the composition of the used additives are selected from the above compounds or their combinations, but not limited to.

Examples

The present invention will now be described in detail below using examples and typical examples, but the scope of the present invention is not limited to these examples.

Example 1

MRA is a recombinant humanitariannet monoclonal antibody (IgG1 subtype) receptor interleukin-6 person, which inhibits the function of the cytokine interleukin-6 (IL-6). In early studies in Japan and Europe MRA demonstrated perspectives in the treatment of rheumatoid arthritis and was well tolerated by the patients.

It was a large-scale clinical trial MRA stage II to determine the optimal dose of MRA, which was given alone or in combination with methotrexate for treatment of rheumatoid arthritis. The potential efficacy of multiple intravenous MRA techniques both in monotherapy and in combination with methotrexate, was evaluated in patients with active rheumatoid arthritis despite treatment with methotrexate for the particular prom is a terrible time, and it was compared with methotrexate monotherapy. Evaluated the efficacy, safety and tolerability MR A.

Methods

The entities. Registered patients with rheumatoid arthritis, which was diagnosed on the basis of the classification of diseases of the American College of rheumatology (ACR) 1987, and which lasted at least 6 months. Patients had to be the active form of the disease and inadequate response or disease outbreak after taking only methotrexate (MTX), which was given to patients at least 6 months at a dosage of at least 12.5 mg every week or 10 mg every week in the case of lactose intolerance.

Model study. Double-blind, randomized study with parallel groups using the center randomized method.

The dosage and the reception means. Seven groups: 0 mg/kg MRA (placebo dosage form containing a neutral substance) + MTX, 2 mg/kg MRA + MTX, 4 mg/kg MRA + MTX, 8 mg/kg MRA + MTX, 2 mg/kg MRA + MTX placebo, 4 mg/kg MRA + MTX placebo and 8 mg/kg MRA + MTX placebo. Welcome MRA or placebo performed using intravenous infusion every 4 weeks. Receiving MTX or MTX placebo exercise orally, once per week at a dose of 10-25 mg/week.

The way of learning. The prescribed dose was administered via intravenous infusion over four times the 4-week intervals, and the efficacy and safety were evaluated with a 2-week intervals for 16 weeks, and the final observation was performed at 20 weeks. The main efficacy criterion was the level of ACR 20 at 16 week (4 weeks after last dose). Additional criteria included the levels of ACR 50 and ACR 70, 16 week (4 weeks after last dose).

Criteria of the ACR improvement. Cases in the following 7 points by the number of swollen joints, number of painful joints is improved by 20% or more, and improvement of 20% or more is observed in three of the remaining five points, defined as 20%or higher percentage of improvement in ACR criteria. In addition, the cases of 50%and 70%improvement mean the cases when the condition more than 20% of the body parts of a patient with improved performance improved by 50% and 70%, respectively:

(1) the number of swollen joints;

(2) the number of painful joints;

(3) assessment of pain by the patient;

(4) overall assessment of disease activity by the patient;

(5) assessment of disease activity physician;

(6) assessment of physical function of the patient;

(7) CRP or ESR.

Statistically significant compared with the control group the percentage of improvement is higher than the ACR 20, was observed in all groups, except the group receiving only the MRA in the amount of 2 mg/kg group (MRA 8 mg/kg + MTX), the percentage improvement in ACR 50 and 70 with Talal 53,1% and 36.7%, respectively, which was statistically more significant in comparison with the control group, in which these figures were 28,6% and 16.3%, respectively (table 1).

Table 1
2 mg/kg MRA4 mg/kg MRA8 mg/kg MRAMTX
ACR 2030,8%61,1%62,7%40,8%
ACR 505,8%27,8%41,2%26,6%
ACR 701,9%5,6%15,7%16,3%
2 mg/kg MRA + MTX4 mg/kg MRA + MTX8 mg/kg MRA + MTX
ACR 2064,0%63,3%73,5%
ACR 5032,0% 36,7%53,1%
ACR 7014,0%12,2%36,7%

In groups, taking only the MRA, a statistically significant dependence on dose was observed for ACR 20. In addition, for ACR 50 and ACR 70, a statistically significant dose-dependent response was observed in the treated group, only the MRA and MTX-combination group.

The decrease in the number of swollen joints (table 2).

The average number of swollen joints at baseline was the same among all groups subjected to the treatment.

Was accumulating a reduction in the average number of swollen joints by increasing exposure time in all seven groups, subjected to treatment. The average decrease in the number of swollen joints in the "MRA 8 mg/kg group was statistically significant compared to the reduction in the MTH group (p=0,010). At the 16th week, the mean difference (95% confidence interval (CI)) between "MRA 8 mg/kg group and the MTH group amounted 2,31 (-4,07, be 0,55). Found a statistically significant linear dependence on dose between groups exposed to monotherapy MRA (p<0,001). The average decrease in the number of swollen joints in the "MRA 8 mg/kg + MTX group was statistically significant. what to reduction in the MTH group (p< 0,001). The mean difference (95% CI) between MRA 8 mg/kg group and the MTH group amounted 3,62 (-5,39, -1,84). Found a statistically significant linear dependence on dose between groups exposed to monotherapy MRA (p=0.004).

Table 2
2 mg/kg MRA4 mg/kg MRA8 mg/kg MRAMTX
Baseline: N
Mean ± STD. off. (SD)
52545149
11,6±4,611,1±4,412,2±5,212,7±4,2
Change from baseline to 16 weeks N
Mean ± SD
42434339
to-4.5±5,7-5,8±4,1-8,4±4,6-5,7±6,1
2 mg/kg MRA + MTX4 mg/kg MRA + MTX8 mg/kg MRA + MTX
Baseline: N
Mean ± SD
504949
11,9±4,311,9±3,911,8±3,9
Change from baseline to 16 weeks N
Mean ± SD
464244
is 6.2±4,6-6,8±5,4-9,4±4,0

Among 359 registered patients of group safety assessment, a complete analysis and PPS (set Protocol) amounted to 359, 354 and 307, respectively. There were 359 patients, 299 completed the full study and 60 patients were excluded. Among the excluded patients, 33 were adverse effects, one appeared complication of another disease, 7 people abandoned the study because of the drugs that are prohibited for use, five - because of the reluctance to bring the study to the end and 22 due to the lack of effectiveness (including multiple reasons).

Among the serious adverse events, causal relationships have not been Viasna is s, it was reported five cases of infection. That is, it was reported on one patient with an abscess of the foot and osteomyelitis in 2 mg/kg MRA group, one patient with a chest infection and pleurisy, one patient with sepsis in 8 mg/kg MRA + MTX group and one patient with infection of the joint in "8 mg/kg MRA + MTX group. In addition, it was reported five cases of hypersensitivity as a serious complication, a causal relationship which has not been, this four patients with hypersensitivity in 2 mg/kg MRA group and one patient with hypersensitivity 4 mg/kg MRA group. All these cases of hypersensitivity was observed in the treatment without MTX after the 3rd and 4th reception.

With regard to laboratory data of the study of the liver, although the application of the MRA and observed increased levels of ALT and AST, this increase was equivalent to that seen in other patients with rheumatoid arthritis. In the "MRA" groups saw improvements in the results of laboratory tests related to lipids (total cholesterol, HDL-cholesterol and triglycerides). However, net changes in atherogenic index was not found.

Some patients have observed a small temporary reduction in the number of neutrophils. Observed clinically significant change settings that are typical for active disease, for example, the reduction of CRP and ESR and the increase of hemoglobin, was a dose-dependent manner.

Reaction to the infusion

The reaction to the infusion was defined as an adverse effect that have taken place over 24 hours study after insertion. The number of patients who experienced a reaction to the infusion, in each group, treated, allowed to assume the existence of a possible inverse relationship to the dose for MRA.

Antibody to MRA

Investigated the appearance of antibodies to the MRA. In groups of 8 mg/kg (monotherapy or combination therapy with MTX), such antibodies were not found. In groups of 2 or 4 mg/kg number of cases was lower in the group with combination therapy with MTX compared to groups exposed to monotherapy MRA.

Results

When alone MRA and combination therapy MRA and methotrexate observed a clear dose-dependent response. The effectiveness of the MRA for the treatment of patients with rheumatoid arthritis was confirmed as monotherapy MRA, and for combination therapy MRA and methotrexate. The safe use of MRA was confirmed as monotherapy MRA, and for combination therapy MRA and methotrexate.

Typical example 1. Obtaining soluble recetor IL-6 person

Soluble receptor of IL-6 were obtained using the method of PCR using the plasmid pBSF2R.236, sod is Rasa cDNA, which encodes the receptor for IL-6, and was obtained according to Yamasaki et al. (K. Yamasaki et al., Science 241:825-828, 1988). Plasmid pBSF2R.236 were digested using restrictase SphI to obtain cDNA for the receptor of IL-6, which is then inserted into the vector SR (produced by Amersham). Using synthetic oligonucleotide primers designed to enter the stop codon in the cDNA of the receptor for IL-6 using PCR method with the use of "in vitro Mutagenesis System" (produced by Amersham) was implemented variations in the cDNA of the receptor for IL-6. The result of this procedure occurred enter the stop codon 345-th position of the amino acid chain that led to a cDNA that encodes a soluble receptor of IL-6.

In order to Express the cDNA in cells SNO, cDNA soluble receptor of IL-6 ligated into a plasmid pSV (produced by Pharmacia)to obtain plasmid pSVL344. cDNA soluble receptor of IL-6, split with restricted Hindlll and Sail, was inserted into plasmid pECEdhfr containing dhfr cDNA to obtain the expression plasmid pECEdhfr344 for cells SNO.

Using the method of deposition of calcium phosphate (Chen, S. et al., Mol. Cell. Biol., 7:2745-2751, 1987), 10 μg of plasmid pECEdhfr344 was transferrable in dhfr-CHO-cell line DXB-11 (G. Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216-4220, 1980). Transfected Cho cells were grown for 3 weeks in selection medium α-MEM, not containing nucleosides, containing 1 mm gluta the ina, 10%cialisovernight FCS, 100 U/ml penicillin and 100 μg/ml streptomycin.

Selection of Cho cells was performed using the method of limiting dilution to obtain a clone from a single Cho cells. Cell clone Cho was propagated in a medium containing from 20 nm to 200 nm methotrexate (MTX), to obtain a line of Cho cells E, which produces human soluble receptor of IL-6. Line Cho-cells E cultivated in an environment of Dulbecco modification Iscove (IMDM, manufactured by Gibco)containing 5%FBS. The supernatant of the cell culture was collected and opredelyali using ELISA method the concentration of the soluble receptor of IL-6 in the supernatant of cell culture. The results confirmed that the supernatant of the cell culture is a soluble receptor of IL-6.

Typical example 2. Obtaining antibodies to human IL-6

Mouse BALB/c mice were immunized with 10 μg of recombinant IL-6 (Harada et al., Immunol. Lett., 17:41, 1988) together with complete adjuvant's adjuvant and immunization was repeated every week until until in serum were not detected antibodies to IL-6. Immune cells extracted from the local lymph nodes and then they were merged with the line of myeloma cells P3U1, using polyethylene glycol 1500. Hybridoma were selected according to the method Oi et al. (Selective Methods in Cellular Immunology, W.H. Freeman and Co., San Francisco, 351, 1980), which use the environment HAT, and ubeida is of n in obtaining hybridoma, which produces antibodies to IL-6 persons.

For hybridoma producing antibodies to IL-6 person, a measurement was performed binding of IL-6, as described below. So, 96-well microtiter plate made of flexible polyvinyl (produced by Dynatech Laboratories, Inc., Alexandria, VA), was senzibilizirani 100 μl of goat antibodies to mouse IgG immunoglobulins (10 µl/ml, produced by Cooper Biomedical, Inc., Malvern, PA) in 0.1 M carbonate-bicarbonate buffer (pH 9,6) at 4°C over night. Then the tablet was treated with 100 μl of PBS containing 1%bovine serum albumin (BSA) for 2 hours at room temperature.

The tablet were washed in PBS-buffer, each well was brought to 100 μl of hybridoma supernatant culture, and incubated at 4°C over night. The tablet was washed, in each well was made125I-labeled recombinant IL-6 concentrations up to 2000 imp. in min/0.5 ng/well, the tablet was washed and then using a gamma counter (Beckman Gamma 9000, Beckman Instruments, Fullerton, CA) measured the radioactivity in each well. The result of 216 clones of hybridoma 32 clones were positive for measuring the binding of IL-6. Of these clones eventually was obtained a stable clone MH166.BSF2. Antibodies to IL-6 MN produced by hybridomas, belonged to subtype IgG1κ.

Then we studied the activity of antibodies MN to neutralize the growth of cells hybridoma using IL-6-dependent KLO is MH166.BSF2 mouse hybridoma. Cells MH166.BSF2 made in quantities of 1×104/200 μl/well, there was submitted a sample containing antibodies MIN, then incubated for 48 hours and added with 0.5 µci/well H-thymidine (New England Nuclear, Boston, MA). After additional growth over the next 6 hours the cells were placed on a glass fiber filter and processed it using the automatic device "Harvester" (Labo Mash Science Co., Tokyo, Japan). As control was used rabbit antibody to IL-6.

Resulting antibodies MN inhibited dose-dependent way the inclusion of3H-thymidine into cells MH60.BSF2 induced IL-6. It showed that antibodies MN neutralizes the activity of IL-6.

A typical example 3. Obtaining antibodies to the human receptor, IL-6

For cleaning specific receptor IL-6 antibodies MT obtained by the method of Hirata et al. (Y. Hirata et al. J. Immunol., 143:2900-2906, 1989), it was attached to CNBr-activation of Sepharose 4B (produced by Pharmacia Fine Chemicals, Piskataway, NJ) in accordance with the attached Protocol (Yamasaki K. et al., Science 241:825-828, 1988). Cell line human myeloma U266 was solubilization in 1 mm solution of para-aminobenzenesulfonamide (produced by Wako Chemicals)dissolved in 1%detienne, 10 mm ethanolamine (7,8) and 1.5 M NaCl (digitalonly buffer)and mixed with the antibody MT attached to the beads Sepharose 4B. Then Shari and washed six times digitanium buffer and provide them as a partially purified preparation of the receptor of IL-6 for immunization.

Mouse BALB/c mice were immunized four times every ten days the above partially purified receptor, IL-6, obtained from 3×109cells U266, and then using the standard method received hybridoma. Supernatant hybridoma culture from the wells with positive growth analyzed for the activity of the receptor binding of IL-6 in accordance with the method described below. 5×107of U266 cells were labeled35S-methionine (2.5 MCI) and were solubilization the above digitanium buffer. Solubilization U266 cells were mixed with 0.04 ml volume antibodies MT attached to the beads sepharose 4B, and were then washed six times digitanium buffer, then35S-methionine-labeled receptor of IL-6 was suirable 0.25 ml digitalanalog buffer (pH 3,4) and neutralized 0,025 ml of 1 M Tris (pH 7,4).

The supernatant of the cell culture hybridoma (0.05 ml) was mixed with 0.01 ml Protein G-Sepharose (produced by Pharmacia). After washing Sepharose incubated with 0,005 ml35S-methionine-labeled receptor, IL-6, obtained as described above. Immunoprecipitate analyzed using SDS page electrophoresis in the presence of sodium dodecyl sulfate (SDS), and investigated supernatant cell culture hybridoma, which reacted with the receptor of IL-6. The result was revealed reaction-positive clone hybridoma RM-1 (FERM THE P-2998). The antibody produced by hybridomas RM-1, belongs to the subtype IgG1κ.

Using a cell line of human myeloma U266 studied the inhibitory activity of antibodies produced by hybridomas RM-1, aimed at the process of binding IL-6 receptor IL-6 person. Human recombinant IL-6 was obtained from E. coli (Harada et al., Immunol. Lett., 17:41-45, 1988) and tagged125I (Taga T. et al., J. Exp. Med., 166:967-981, 1987) using the reagent of the Bolton-hunter (New England Nuclear, Boston, MA).

Cells U266 in the number of 4×105were grown in 70% (v/v) supernatant cell culture hybridoma RM-1 and in the presence of IL-6, labeled125I (14000 imp. in min). The sample (70 μl) was layered on 300 ál of FCS in polyethylene microcentrifuge tube with a volume of 400 μl and centrifuged, followed by measurement of the radioactivity of the cells.

In the result, it was shown that the antibody produced by hybridomas RM-1, inhibits the binding of IL-6 receptor IL-6.

A typical example 4. Getting mouse antibodies to the receptor of IL-6

Monoclonal antibody directed against murine receptor of IL-6 was obtained according to the method described by Saito et al., J. Immunol., 147:168-173, 1991.

The CHO cells that produce soluble murine receptor, IL-6, were grown in culture medium IMDM containing 10%FCS, and soluble murine receptor for IL-6 was purified from the culture supernatant of cells using affinity is alongi, in which antibodies RS12 (see Saito et al., J. Immunol., 147:168-173, 1991) to the murine receptor of IL-6 were sewn to the gel Affigel 10 (produced by Biorad).

Thus obtained soluble murine receptor for IL-6 (50 μg) was mixed with complete adjuvant's adjuvant, was administered by injection into the abdominal cavity of Wistar rats. Two weeks later the animals were additionally immunization incomplete adjuvant's adjuvant. 45 day was selected cells of the spleen in rats, and 2×108cells were merged with 1×107cells of mouse myeloma RSS in accordance with the traditional method, using 50%PEG 1500 (produced by Boehringer Mannheim) followed by screening hybridomas in culture medium HAT.

Supernatant cell culture hybridoma made in wells coated with rabbit antibody to immunoglobulin IgG rat (produced by Cappel), where mouse soluble receptor of IL-6 reacts. Then, using rabbit antibodies to mouse receptor of IL-6 and sheep antibodies to rabbit immunoglobulins IgG labeled with alkaline phosphatase using the ELISA method was selected hybridoma producing antibodies to soluble murine receptor of IL-6. Clone hybridoma that has been shown the ability to produce such an antibody, and was twice subclinically and received a single clone of hybridoma. This clone was named MR16-1.

Neutralizing activity of the antibodies, produced this hybridomas aimed at the process of transmission of signals from IL-6 mouse was studied by including3H-thymidine using cells MH60.BSF2 (Matsuda T. et al., J. Immunol., 18:951-956, 1988). Cells MH60.BSF2 were placed in a 96-well plate at the rate of 1×104cells/200 μl/well. The tablet was made 10 IG/ml murine IL-6 and antibody MR16-1, or antibodies RS12 from 12,3 to 1000 ng/ml, and then the cells were grown at 37°C for 44 hours under 5%CO2and then thereto was added 1 µci/well3H-thymidine. After 4 hours was measured by the inclusion of3H-thymidine. In the result, it was found that the antibody MR16-1 was suppressed by the inclusion of3H-thymidine into cells MH60.BSF2.

Thus, it was shown that the antibody produced by hybridomas MR16-1 (FERM BP-5875), inhibits the binding of IL-6 receptor IL-6.

1. Therapeutic agent for the treatment of rheumatoid arthritis for reception in a dose of 4 mg/kg/4 weeks or more or the dose at which the blood is equivalent to the concentration of antibodies to receptor of interleukin-6 (antibodies to IL-6R), comprising the antibody to the receptor of interleukin-6 (antibody to IL-6R).

2. A therapeutic agent of claim 1, wherein the antibody to IL-6R is a monoclonal antibody to IL-6R.

3. Therapeutic agent according to claim 2, in which the antibody to IL-6R is a monoclonal antibody to IL-6R man is ka.

4. Therapeutic agent according to claim 2, in which the antibody to IL-6R is a monoclonal antibody to IL-6R mouse.

5. Therapeutic agent according to any one of claim 2 to 4, in which the antibody to IL-6R is a recombinant antibody.

6. Therapeutic agent according to claim 3, in which the monoclonal antibody to IL-6R person is an antibody RM-1.

7. Therapeutic agent according to claim 4, in which the monoclonal antibody to IL-6R mouse is an antibody MR16-1.

8. Therapeutic agent according to any one of claims 1 to 4, 6, or 7, in which the antibody to IL-6R is a chimeric antibody, humanized antibody or the antibody to IL-6R human type.

9. A therapeutic agent of claim 8, in which humanitariannet antibody to IL-6R is a humanized antibody RM-1.

10. Therapeutic agent according to claim 1, in which the dosage of the antibodies to IL-6R is from 6 to 16 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

11. A therapeutic agent of claim 10, in which the dosage of the antibodies to IL-6R is from 6 to 10 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

12. Pharmaceutical composition comprising the antibody to the receptor of interleukin-6 (antibody to IL-6R) in the dose of 4 mg/kg/4 weeks and up or the dosage at which the blood has reached the W is equivalent to the concentration of antibodies to receptor of interleukin-6 (antibody to IL-6R) to reduce or prevent allergic reactions in the treatment of rheumatoid arthritis.

13. The pharmaceutical composition according to item 12, in which the antibody IL-6R is a monoclonal antibody to IL-6R.

14. The pharmaceutical composition according to item 13, in which the antibody to IL-6R is a monoclonal antibody to IL-6R person.

15. The pharmaceutical composition according to item 13, in which the antibody to IL-6R is a monoclonal antibody to IL-6R mouse.

16. The pharmaceutical composition according to any one of p-15, in which the antibody to IL-6R is a recombinant antibody.

17. The pharmaceutical composition according to 14, in which the monoclonal antibody to IL-6R person is an antibody RM-1.

18. The pharmaceutical composition according to item 15, in which the monoclonal antibody to IL-6R mouse is an antibody MR16-1.

19. The pharmaceutical composition according to any one of p-15, 17 or 18, in which the antibody to IL-6R is a chimeric antibody, humanized antibody or the antibody to IL-6R human type.

20. The pharmaceutical composition according to claim 19, in which humanitariannet antibody to IL-6R is a humanized antibody RM-1.

21. The pharmaceutical composition according to item 12, in which the dosage of the antibodies to IL-6R is from 6 to 16 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

22. The pharmaceutical composition according to item 21, in which the dosage of the antibodies to IL-6R is from 6 to 10 mg/kg/4 weeks or doses is roku, when the blood is equivalent to the concentration of antibodies to IL-6R.

23. The use of antibodies to receptor of interleukin-6 (antibodies to IL-6R) for the production of therapeutic agent for rheumatoid arthritis for reception in a dose of 4 mg/kg/4 weeks or more or the dose at which the blood is equivalent to the concentration of antibodies to receptor of interleukin-6 (antibodies to IL-6R), comprising the antibody to the receptor of interleukin-6 (antibody to IL-6R).

24. The use of antibodies to receptor of interleukin-6 (antibodies to IL-6R) for the production of pharmaceutical compositions comprising the antibody to the receptor of interleukin-6 (antibody to IL-6R) in the dose of 4 mg/kg/4 weeks or more or the dose at which the blood is equivalent to the concentration of antibodies to receptor of interleukin-6 (antibodies to IL-6R), to reduce or prevent allergic reactions in the treatment of rheumatoid arthritis.

25. The use according to any one of p and 24, wherein the antibody to IL-6R is a monoclonal antibody to IL-6R.

26. Use A.25, wherein the antibody to IL-6R is a monoclonal antibody to human IL-6R.

27. Use A.25, wherein the antibody to IL-6R is a monoclonal antibody to mouse IL-6R.

28. The use according to any one of p and 24, wherein the antibody to IL-6R is a recombinant antibody.

29. Use p, the ri which monoclonal antibody to human IL-6R antibody is RM-1.

30. The application of item 27, wherein the monoclonal antibody to the murine receptor IL-6 is an antibody MR16-1.

31. The use according to any one of p and 24, wherein the antibody to IL-6R is a chimeric antibody, humanized antibody or the antibody to IL-6R human type.

32. Use p where humanitariannet antibody to IL-6R is a humanized antibody RM-1.

33. The use according to any one of p and 24, in which the dosage of the antibodies to IL-6R is from 6 to 16 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

34. Use p, in which the dosage of the antibodies to IL-6R is from 6 to 10 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.

35. The method including the introduction of antibodies to receptor of interleukin-6 (antibodies to IL-6R) in the dose of 4 mg/kg/4 weeks or more or the dose at which the blood is equivalent to the concentration of antibodies to receptor of interleukin-6 (antibodies to IL-6R) to a patient in need of such treatment, as a method of treatment of rheumatoid arthritis by injecting antibodies to receptor of interleukin-6 (antibodies to IL-6R) in the dose of 4 mg/kg/4 weeks or more or in a dose, at which the blood achieved the equivalent concentration of antibodies to receptor of interleukin-6 (antibodies to I-6R).

36. The way to reduce or prevent allergic reactions in the treatment of rheumatoid arthritis, including the dose of 4 mg/kg/4 weeks or more or the dose at which the blood is equivalent to the concentration of antibodies to receptor of interleukin-6 (antibodies to IL-6R), antibodies to the receptor of interleukin-6 (antibodies to IL-6R) to a patient in need of such treatment.

37. The method according to any of PP and 36, in which the antibody to IL-6R is a monoclonal antibody to IL-6R.

38. The method according to clause 37, in which the antibody to IL-6R is a monoclonal antibody to human IL-6R.

39. The method according to clause 37, in which the antibody to IL-6R is a monoclonal antibody to mouse IL-6R.

40. The method according to any of PP and 36, in which the antibody to IL-6R is a recombinant antibody.

41. The method according to § 38, in which the monoclonal antibody to human IL-6R antibody is RM-1.

42. The method according to § 39, in which the monoclonal antibody to mouse IL-6R antibody is MR 16-1.

43. The method according to any of PP and 36, in which the antibody to IL-6R is a chimeric antibody, humanized antibody or the antibody to IL-6R human type.

44. The method according to item 43, in which humanitariannet antibody to IL-6R is a humanized antibody RM-1.

45. The method according to any of PP and 36, in which the dosage of the antibodies to IL-6R is from 6 to 16 mg/kg/4 is not the eating or dosage, when the blood is equivalent to the concentration of antibodies to IL-6R.

46. The method according to item 45, in which the dosage of the antibodies to IL-6R is from 6 to 10 mg/kg/4 weeks or the dosage at which the blood is equivalent to the concentration of antibodies to IL-6R.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention refers to a compound of formula (I):

,

where R1 represents NR7C(O)R8 or NR9R10; R2 represents hydrogen; R3 represents halogen; R4 represents hydrogen, halogen, cyano, hydroxy, C1-4alkyl, C1-4alkoxy, CF3, OCF3, C1-4alkylthio, S(O)(C1-4alkyl), S(O)2(C1-4alkyl), CO2H or CO2(C1-4alkyl); R5 represents C1-6alkyl (replaced with NR11R12 or heterocyclyl that represents nonaromatic 5-7-membered ring containing 1 or 2 heteroatoms independently chosen from a group containing nitrogen, oxygen or sulphur); R6 represents hydrogen, halogen, hydroxy, C1-4alkoxy, CO2H or C1-6alkyl (possibly replaced with NR15R16 group, morpholinyl or thiomorpholinyl); R7 represents hydrogen; R8 represents C3-6cycloalkyl (possibly replaced with NR24R25 group), phenyl or heteroaryl, which represents aromatic 5- or 6-membered ring containing 1 to 3 heteroatoms independently chosen from the group containing nitrogen, oxygen and sulphur, and which is probably condensed with one 6-membered aromatic or nonaromatic carbocyclic ring or with one 6-membered aromatic heterocyclic ring, where the above 6-membered aromatic heterocyclic ring includes 1 to 3 heteroatoms independently chosen from a group containing nitrogen, oxygen and sulphur; R9 represents hydrogen or C1-6alkyl (possibly replaced with pyrazolyl); R10 represents C1-6alkyl (possibly replaced with phenyl or heteroaryl group, which represents aromatic 5- or 6-membered ring containing 1 or 2 heteroatoms independently chosen from the group containing nitrogen, oxygen or sulphur, and which is possibly condensed with one 6-membered heterocyclic ring, where the above 6-membered aromatic heterocyclic ring contains 1 or 2 heteroatoms independently chosen from the group containing nitrogen, oxygen or sulphur; where the above phenyl and heteroaryl groups in R8, R9 and R10 are possibly independently replaced with the following group: halogen, hydroxy, C(O)R42, C1-6alkyl, C1-6hydroxyalkyl, C1-6halogenoalkyl, C1-6alkoxy(C1-6)alkyl or C3-10cycloalkyl; unless otherwise stated, heterocyclyl is possibly replaced with group of C1-6alkyl, (C1-6alkyl)OH, (C1-6alkyl)C(O)NR51R52 or pyrrolidinyl; R42 represents C1-6alkyl; R12, R15 and R25 independently represent C1-6alkyl (possibly replaced with hydroxy or NR55R56 group); R11, R16, R24, R51, R52, R55 and R56 independently represent hydrogen or C1-6alkyl; or to its pharmaceutically acceptable salts.

EFFECT: new compounds are obtained, which can be used in medicine for treatment of PDE4-mediated disease state.

10 cl, 2 tbl, 202 ex

FIELD: biotechnologies.

SUBSTANCE: peptide of DGSVVVNKVSELPAGHGLNVNTLSYGDLAAD structure is used for suppression of allergic inflammation of respiratory passages, for prophylaxis and treatment of arthritis, as well as for pain relief. A peptide is effective as an adjuvant and for stimulation of IL-12 products in a cell.

EFFECT: peptide allows increasing IL-12 products by 10 times relative to normal levels of IL-12 cellular production.

19 cl, 25 dwg, 10 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to organic chemistry and specifically to 5-phenyl-1H-pyrazin-2-one derivatives of general formula II or pharmaceutically acceptable salts thereof, where R denotes -R1 or - R1-R2-R3; R1 denotes aryl or heteroaryl, and is optionally substituted with one or two R1'; where each R1' independently denotes C1-6alkyl, halogen or C1-6halogenalkyl; R2 denotes -C(=O), -CH2-; R3 denotes R4; where R4 denotes an amino group or heterocycloalkyl, and is optionally substituted with one or two substitutes selected from C1-6alkyl, hydroxy group, oxo group, C1-6hydroxyalkyl, C1-6alkoxy group; Q denotes CH2; Y1 denotes C1-6alkyl; Y2 denotes Y2b; where Y2b denotes C1-6alkyl, optionally substituted with one Y2b'; where Y2b' denotes a hydroxy group, n and m are equal to 0; Y4 denotes Y4c or Y4d; where Y4c denotes lower cycloalkyl, optionally substituted with halogen; and Y4d denotes an amino group, optionally substituted with one or more C1-6alkyl; where "aryl" denotes phenyl or naphthyl, "heteroaryl" denotes a monocyclic or bicyclic radical containing 5 to 9 atoms in the ring, which contains at least one aromatic ring containing 5 to 6 atoms in the ring, with one or two N or O heteroatoms, wherein the remaining atoms in the ring are carbon atoms, under the condition that the binding point of the heteroaryl radical is in the aromatic ring, "heterocycloalkyl" denotes a monovalent saturated cyclic radical consisting of one ring containing 5 to 6 atoms in the ring, with one or two ring heteroatoms selected from N, O or SO2. The invention also relates to use of the compound of formula II or a pharmaceutical composition based on the compound of formula II.

EFFECT: obtaining novel compounds that are useful for modulating Btk activity and treating diseases associated with excessive activity of Btk.

7 cl, 2 tbl, 53 ex

FIELD: chemistry.

SUBSTANCE: present invention relates to novel compounds of formula I: or salts thereof, where: A1 denotes hydrogen, CN, CI, F, Br, OMe, (1-4C alkyl) or cyclopropyl; A2 denotes hydrogen, Cl, Br, F, (1-4C alkyl) or cyclopropyl; W denotes -C(=O)NR1- or -NR2C(=O)-; each of R1 and R2 denotes hydrogen or methyl; L denotes a chemical bond, -(CR3R4)n-(CRaRb)m-(CR5R6)-*, (2-4C)alkenylene, -O(1-4C alkyl)-*, -(1-4C alkyl)-O-*, -(1-4C alkyl)-S-*, (3-6C)cycloalkylene or hetCyc1, where the symbol "*" indicates the binding position of G, under the condition that if W denotes -C(=O)NR2-, then L is not -(CH=CH)-; m equals 0, 1 or 2; n equals 0 or 1; Ra and Rb are independently selected from hydrogen and (1-4C alkyl); R3 denotes hydrogen, (1-4C alkyl) or CH2OH; R4 denotes hydrogen or methyl; R5 denotes hydrogen, (1-4C alkyl), OH, -O(1-4C alkyl) or F; R6 denotes hydrogen, F or methyl; or R5 and R6 together with the carbon atom with which they are bonded form a cyclopropyl ring, hetCyc1 denotes a group of formula where t equals 1 or 2 and p equals 0 or 1, and the symbol "*" indicates the position of binding with G; G denotes Ar1, Ar2, naphthyl, benzo-condensed (5-6C)cycloalkyl ring, optionally substituted with one or more substitutes independently selected from Cl and OMe, benzo-condensed 5-6-member heterocyclic ring with 1-2 heteroatoms independently selected from O and N, (3-6C)cycloalkyl ring, optionally substituted with one or more substitutes independently selected from (1-4C)alkyl, oxaspirononanyl ring or t-butyl; Ar1 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, Cl, Br, CF3, (1-4C)alkyl, OH, -O(1-4C alkyl), -S(1-3C alkyl), -SCF3, cyclopropyl, -CH2N(1-3C alkyl)2, -O-(2-3C)fluoroalkyl, -O-(1-3C)difluoroalkyl-O-(1-3C)trifluoroalkyl, -OCH2(cyclopropyl) and (3-4C)alkynyl; Ar2 denotes phenyl, substituted with Ar3, -O-Ar4, hetAr1 or -O-hetAr2, where Ar2 is further optionally substituted with one or more substitutes independently selected from F, O or CF3; Ar3 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, CI, Br and (1-4C alkyl); Ar4 denotes phenyl, optionally substituted with one or more substitutes independently selected from F, CI, Br and (1-4C alkyl); hetAr1 denotes a 6-member heteroaryl with 1-2 nitrogen atoms, optionally substituted with one or more substitutes independently selected from (1-4C alkyl); hetAr2 denotes a 6-member heteroaryl with 1-2 nitrogen atoms, optionally substituted with one or more substitutes independently selected from (1-4C alkyl) and CF3; R7a, R7 and R8 each independently denotes hydrogen or methyl; R9 denotes hydrogen, methyl, fluorine or NO2; and R10 denotes hydrogen, methyl or fluorine; where A1, A2, W, L, G, R7a, R7b, R8, R9 and R10 assume values given in the description, which are DP2 receptor modulators which are effective in treating immunological diseases.

EFFECT: inventions relate to a method of producing compounds of formula 1, a pharmaceutical composition based on said compounds and a method of treatment.

30 cl, 1 tbl, 239 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. A method for preparing the vaccine provides crossing of two different species of the four: T.spiralis, T.nativa, T.pseudospiralis and T.britovi, and selecting the line having the lower pathogenic and higher immunogenic properties by infecting with murine Trichinella larvae and controlling the embryogenesis; 1.5-2 months later, infected mouse's muscular tissue is separated, and the invasion larvae are recovered to be used for re-infection in a dose of 200-300 larvae; a degree of infection is determined, while the vaccine is prepared of the Trichinella line that leads to a mildest disease, while the re-infection causes no disease. Milk of the animals immunised with the vaccine prepared as described above is used for the purpose of prevention and treatment of the immune disorders. The vaccine described above is also used to prepare the serum. The vaccine is orally administered in a dose of 5000 to 20000 living larvae starting from the 10-14th day from the administration of the preparation; a usual therapeutic correction is required as that for trichinellosis, while the blood is sampled 30 days after the administration of the preparation. The prepared serum is administered into an individual according to the following schedule: either orally starting from 0.05 ml per one intake 3 times a day and increased to 1.2 ml by adding 0.05 ml to each intake, and then decreased in reverse order, or in the form of injections parenterally in a dose of 1-2 ml every 2 days, and in a dose of 10-15 ml into an animal three times to prevent and treat the immune disorders.

EFFECT: using the inventions described above enables more effective prevention and treatment of the immune disorders.

4 cl, 9 ex

FIELD: medicine.

SUBSTANCE: present invention generally refers to immunology, particularly to bolstering the immune system in the elderly age and represents a composition. The composition for treating and preventing the diseases related to the altered immune system specified in a group consisting of infections, particularly bacterial, viral, mycotic and/or parasitic infections; the autoimmune diseases; the inborn immunity diseases, e.g. NK cell deficiency, phagocyte deficiency, such as leukocyte adhesion deficiency, cyclic neutropenia; and combinations thereof containing a protein fraction comprising acetylneuraminic acid related to a threonine-rich peptide/protein frame.

EFFECT: invention provides bolstering the immune system more effectively.

13 cl, 2 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biopharmacology and concerns drug preparations for treating various autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, systemic scleroderma, insulin dependent diabetes mellitus and others, due to its immunoregulatory properties. The drug preparation contains a mixture of four ingredients taken in the following weight relation: trophoblastic β-1-glycoprotein 2.0 to 5.0%; immunoglobulin A 10.0 to 30.0%; immunoglobulin M 15 to 35.0%; immunoglobulin G - the rest up to 100%.

EFFECT: invention provides the higher clinical effectiveness in both remitted, and aggravated autoimmune diseases, with the substantial improvement of the immunoregulatory and immunomodulatory properties of the drug preparation.

1 cl, 5 dwg

FIELD: biotechnologies.

SUBSTANCE: strain is deposited in CNCM and has number 1-3689. Strain has ability to inhibit the growth of pathogenic microorganisms in culture, and namely Escherichia coli, Salmonella enteritidis and Listeria monocytogenes. Strain has anti-inflammatory properties at the ratio of IL-10/IL-12 equal to 11.8.

EFFECT: food product containing the above living strain has antimicrobial or immunomodulating properties.

6 cl, 3 dwg, 4 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.

EFFECT: invention can be further used in therapy of diseases associated with IgE.

46 cl, 19 dwg, 5 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes humanised anti-NKG2A antibody obtained from murine antibody Z270, which is characterised through amino-acid sequences of variable domains, and method of its obtainment. Besides, a pharmaceutical composition is described, which contains an antibody according to the invention, a treatment method and application of the antibody in production of a medicine to be injected into a patient who is a human being suffering the disorder chosen from cancer, virus disease, inflammatory disorder and autoimmune disorder.

EFFECT: invention can be further used in therapy.

14 cl, 20 dwg, 2 tbl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely neurology, and concerns treating multiple sclerosis. That is ensured by administering a pharmaceutical composition containing an activated-potentiated form of human gamma-interferon (IFN-γ) antibodies and an activated-potentiated form of brain-specific protein S-100 antibodies.

EFFECT: drug provides the effective treatment of multiple sclerosis ensured by the synergetic action of the ingredients of the composition.

11 cl, 2 ex, 1 tbl, 4 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present group of inventions refers to medicine, namely to endocrinology, and concerns treating diabetes. That is ensured by administering a pharmaceutical composition containing an activated potentiated form of insulin receptor antibodies to and an activated potentiated form of endothelial NO-synthase antibodies.

EFFECT: method provides the effective treatment of diabetes ensured by a synergetic effect of the ingredients of the composition on reducing insulin resistance and hyperglycemia.

23 cl, 6 dwg, 5 ex

Anti-axl antibodies // 2506276

FIELD: chemistry.

SUBSTANCE: present invention relates to immunology. Disclosed are monoclonal antibodies which bind to the extracellular domain of receptor tyrosine kinase AXL and which at least partially inhibit AXL activity, as well as antigen-binding fragments. Also provided is an isolated nucleic acid molecule, a host cell and a method of producing a monoclonal antibody and an antigen-binding fragment thereof, as well as use of the monoclonal antibody or antigen-binding fragment thereof to produce a drug, pharmaceutical compositions, a method of diagnosing and a method of preventing or treating a condition associated with expression, overexpression and/or hyperactivity of AXL.

EFFECT: invention can be used in therapy and diagnosis of diseases associated with AXL.

23 cl, 20 dwg, 24 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: group of inventions relates to biotechnology. A method for preparing the vaccine provides crossing of two different species of the four: T.spiralis, T.nativa, T.pseudospiralis and T.britovi, and selecting the line having the lower pathogenic and higher immunogenic properties by infecting with murine Trichinella larvae and controlling the embryogenesis; 1.5-2 months later, infected mouse's muscular tissue is separated, and the invasion larvae are recovered to be used for re-infection in a dose of 200-300 larvae; a degree of infection is determined, while the vaccine is prepared of the Trichinella line that leads to a mildest disease, while the re-infection causes no disease. Milk of the animals immunised with the vaccine prepared as described above is used for the purpose of prevention and treatment of the immune disorders. The vaccine described above is also used to prepare the serum. The vaccine is orally administered in a dose of 5000 to 20000 living larvae starting from the 10-14th day from the administration of the preparation; a usual therapeutic correction is required as that for trichinellosis, while the blood is sampled 30 days after the administration of the preparation. The prepared serum is administered into an individual according to the following schedule: either orally starting from 0.05 ml per one intake 3 times a day and increased to 1.2 ml by adding 0.05 ml to each intake, and then decreased in reverse order, or in the form of injections parenterally in a dose of 1-2 ml every 2 days, and in a dose of 10-15 ml into an animal three times to prevent and treat the immune disorders.

EFFECT: using the inventions described above enables more effective prevention and treatment of the immune disorders.

4 cl, 9 ex

FIELD: medicine.

SUBSTANCE: invention refers to diagnostic techniques for hepatic fibrosis in an individual involving urokinase plasminogen, matrix metalloproteinase 9 and β-2-microglobulin expression tests to derive a score and to diagnose. Also, the present invention refers to a diagnostic kit for hepatic fibrosis comprising a first antibody specifically bound to an urokinase plasminogen activator (uPA), a second antibody specifically bound to matrix metalloproteinase 9 (MMP9) and a third antibody specifically bound to β-2-microglobulin (β-2-MG).

EFFECT: higher diagnostic accuracy.

17 cl, 33 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: invention pertains to the versions of antibodies against IL-6 receptor, variable regions of light and heavy chains of which are modified by introduction of amino-acid replacement. There revealed is a pharmaceutical composition for treating the diseases associated with IL-6 and containing the said version of antibody.

EFFECT: invention allows efficient treatment of the diseases associated with IL-6.

8 cl, 48 dwg, 16 tbl, 20 ex

FIELD: biotechnologies.

SUBSTANCE: there proposed is an antibody specific to TENB2 containing light and heavy chains. Heavy chain contains substitution for free (reaction capable) cysteine A121C that corresponds to A114C (Kabat numbering) or A118C (Eu numbering). Conjugate versions are proposed for prostate cancer treatment containing antibody covalently bound to auristatin, also by means of linker. The following is described: pharmaceutical composition for prostate cancer treatment that uses as active beginning the antibody or its conjugate; product for prostate cancer treatment on the basis of such composition. The invention proposes: method for defining protein TENB2 in the sample - on the basis of antibody as well as analysis for revealing prostate cancer cells at mammal and method for cell proliferation inhibiting on the base of antibody conjugate and auristatin. There described is the method for obtaining antibody conjugate (Ab) and auristatin (D) with expression Ab-(L-D)p, where p is equal from 1 to 4, and L is linker.

EFFECT: invention application provides conjugates with increased stability in serum in comparison with the same conjugates without A121C substitution in antibody that can be used in medicine.

33 cl, 18 dwg, 2 tbl, 4 ex

FIELD: veterinary.

SUBSTANCE: method involves the subcutaneous injections of donor animals' hyperimmune blood serum containing antihemagglutinin in titres to herpes virus-3 1:1280, to rednose virus - min. 1:256, to viral diarrhea -1:1024 and to adenovirus -1:128; in a dose of 2.0 ml/kg every 24 hours until recovered completely; besides, 20-30 minutes before feeding, a pytopreparation of 70% alcohol tincture of purple echinacea (Echinacea purpurea L) herb and blossom, common pine (Pinus sylvestris) buttons, horseheal (Inula helenium) roots and rhizomes, common licorice (Glycyrrhiza glabra L.) roots and harmala shrub (Peganum harmala) herb taken in relation 1:1:1:1:0.5 in the amount of 7-8% aqueous solution is administered in a dose of 3.0-3.5 ml/kg of body weight every 12 hours until recovered completely.

EFFECT: higher clinical effectiveness and natural body resistance.

4 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: methods, compositions and sets for diagnostics of osteoarthritis at cat family are disclosed. According to the invention, the methods involve detection of differential expression at least of one marker in a sample of an organism, preferably in blood sample, where biomarker is differentially expressed at osteoarthritis.

EFFECT: improving diagnostics accuracy.

23 cl, 4 dwg, 7 tbl, 3 ex

Antibodies to her // 2504553

FIELD: biotechnologies.

SUBSTANCE: invention describes versions of bispecific antibodies specifically bound to EGFR and HER3, which contain amino-acid sequences of variable regions of heavy and light chains respectively, SEQ ID NO: 30 and 29; or SEQ ID NO: 28 and 27; or SEQ ID NO: 28 and 29; or contain complementary regions CDR of heavy and light chains of the above sequences of variable regions. The invention describes nucleic acid coding a versions antibody, and a host cell containing the above nucleic acid and used for expression of the anitbody. Immunoconjugate containing antibody versions and cytotoxic agent used for treatment of cancer containing cells that express EGFR and HER3 are presented. A method for obtaining a bispecific antibody, which involves cultivation of a host cell so that an antibody is generated, is described. The invention describes a pharmaceutical composition for treatment of cancer containing EGFR- and HER3-expressing cells, which contains effective amount of bispecific antibody and pharmaceutically acceptable carrier. The invention proposes a treatment method of cancer containing EGFR- and HER3-expressing cells and an inhibition method of biological activity of EGFR and/or HER3 of a specimen, which involve introduction of effective amount of a bispecific antibody. Use of the above antibody in production of a remedy for treatment of cancer, the cells of which express EGFR and HER3, is described.

EFFECT: invention allows obtaining bispecific antibodies binding EGFR and HER3, which are not conjugates of two antibodies.

22 cl, 33 dwg, 4 tbl, 19 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

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