HK1237650A1 - Composition for treating il-6-related diseases - Google Patents

Description

Compositions for treating IL-6 related diseases

Technical Field

The present invention relates to pharmaceutical compositions or dosing regimens for the treatment of IL-6 related diseases.

Background

Interleukin-6 (IL-6) is a cytokine, also known as B cell stimulating factor 2(BSF2) or interferon beta 2. IL-6 was found to be a differentiation factor involved in B-lymphoid cell activation (non-patent document 1), and was later found to be a multifunctional cytokine affecting the functions of various cells (non-patent document 2). It has been reported that IL-6 causes maturation of T lymphoid cells (non-patent document 3).

IL-6 transmits its biological activity via two classes of proteins on the cell. One of them is the IL-6 receptor, the receptor is combined with IL-6 ligand binding protein with molecular weight of about 80kD (non-patent documents 4 and 5). In addition to the membrane-bound form expressed on the cell membrane, the IL-6 receptor exists as a soluble IL-6 receptor, which is composed primarily of its extracellular region and penetrates through the cell membrane.

The other is the membrane protein gp130, which has a molecular weight of about 130kDa and is involved in signal transduction with non-ligand binding. IL-6 and IL-6 receptor form IL-6/IL-6 receptor complex, and then the complex and gp130 binding, thereby IL-6 biological activity is delivered to the cell (non-patent literature 6).

IL-6 inhibitors are substances that inhibit the transmission of IL-6 biological activity. At present, antibodies against IL-6 (anti-IL-6 antibodies), antibodies against IL-6 receptor (anti-IL-6 receptor antibodies), antibodies against gp130 (anti-gp 130 antibodies), IL-6 variants, IL-6 or IL-6 receptor partial peptides and the like are known.

There are several reports on anti-IL-6 receptor antibodies (non-patent documents 7 and 8, and patent documents 1 to 3). One of them is a humanized PM-1 antibody obtained by grafting Complementarity Determining Regions (CDRs) of mouse antibody PM-1 (non-patent document 9) to a human antibody (patent document 1).

Toslizumab, which is an anti-IL-6 receptor antibody, is currently used for the treatment of inflammatory diseases such as rheumatoid arthritis and Castleman's disease (non-patent document 10), and it has also been confirmed to be effective for diseases such as neuromyelitis optica (NMO) (non-patent document 11).

The therapeutic effect of IL-6 antibody on myasthenia gravis has also been reported (non-patent document 12).

Humanized antibodies such as tollizumab are the first generation antibody drugs. Second-generation antibody drugs are currently being developed by improving the drug efficacy, convenience, and cost of first-generation antibody drugs (patent document 2). As a second generation antibody drug, SA237, a novel anti-IL-6 receptor antibody, has been prepared by applying improved techniques such as those for enhancing effector function, antigen-binding ability, pharmacokinetics and stability, or those for reducing immunological risk, and has entered clinical trials.

Although various antibody treatments are currently being performed, the decline in therapeutic efficacy due to the development of anti-antibodies has been confirmed in alemtuzumab (alemtuzumab). In order to prevent this attenuation, it has been reported that it is effective to administer a non-cell-binding mutant that can be administered at a high dose, rather than to induce immune tolerance by administering high doses of alemtuzumab (non-patent document 13).

The prior art documents related to the invention of the present application are shown below.

Documents of the prior art

[ non-patent document ]

[ non-patent document 1] Hirano, T et al, Nature (1986)324,73-76

[ non-patent document 2] Akira, S, et al, adv.in Immunology (1993)54,1-78

[ non-patent document 3] Lotz, M et al, J.Exp.Med. (1988)167,1253-

[ non-patent document 4] Taga, T et al, J.Exp.Med. (1987)166,967-

[ non-patent document 5] Yamasaki, K et al, Science (1988)241,825-

[ non-patent document 6] Taga, T et al, Cell (1989)58,573-581

[ non-patent document 7] Novick, D, et al, Hybridoma (1991)10,137-

[ non-patent document 8] Huang, Y.W, etc., Hybridoma (1993)12,621-

[ non-patent document 9] Hirata, Y et al, J.Immunol. (1989)143,2900-

[ non-patent document 10] Nishimoto, N, et al, blood.2005Oct 15; 106(8):2627-32

[ Nonpatent document 11] Araki et al, Mod. Rheumatol. (2013)23(4), 827. su 831

[ non-patent document 12] Aricha, R, et al, J.Autoimmun. (2011)36(2),135-

[ non-patent document 13] Charlotte L et al, Nature Reviews Rheumatology (2010)6,558-

[ patent document ]

[ patent document 1] International patent application publication No. WO 92-19759

[ patent document 2] International patent application publication No. WO2010/035769

Summary of The Invention

[ problem to be solved by the invention ]

Even in the case of SA237 (antibody having the heavy chain sequence of SEQ ID NO:3 and the light chain sequence of SEQ ID NO:4) prepared by applying a technique for reducing immunogenicity, in a phase I study (SA-001JP) in which a single dose of 120mg of SA237 was subcutaneously administered to healthy adult male subjects, anti-SA 237 antibodies were produced in 54.2% of cases (39 out of 72 cases) and immunogenicity problems occurred. It is an object of the present invention to inhibit anti-antibody production and to provide more effective pharmaceutical compositions or dosing regimens for the treatment of IL-6 related diseases.

[ means for solving problems ]

In order to solve the above problems, the present inventors focused on immune tolerance, and found that anti-antibody production can be inhibited by administering a pharmaceutical composition in a predetermined administration method and dose.

More specifically, the present inventors have found that anti-antibody production can be inhibited to treat IL-6 related diseases by using a pharmaceutical composition administered at a predetermined dose and administration method, and thus have completed the present invention.

Specifically, the present invention includes the following:

[1] a pharmaceutical composition for treating an IL-6-related disease, comprising an IL-6 inhibitor as an active ingredient, wherein the pharmaceutical composition is conventionally administered after a short-spaced dosing period in which the same dose as a conventional dose is administered a plurality of times at intervals shorter than the conventional dosing interval.

[2] The pharmaceutical composition of [1], wherein the regular dosing interval is three to five weeks.

[3] The pharmaceutical composition of [1], wherein the regular dosing interval is four weeks.

[4] The pharmaceutical composition of any one of [1] to [3], wherein a dosage interval during the short-interval administration period in which the dose is administered multiple times at an interval shorter than a conventional dosage interval is one to two weeks.

[5] The pharmaceutical composition of any one of [1] to [3], wherein a dosing interval during the short interval dosing period in which the dose is administered multiple times at an interval shorter than a conventional dosing interval is two weeks.

[6] The pharmaceutical composition of any one of [1] to [5], wherein the short-interval administration period is four weeks from initial administration.

[7] The pharmaceutical composition of any one of [1] to [6], wherein the usual dose is 50mg to 800mg per administration.

[8] The pharmaceutical composition of any one of [1] to [7], wherein the usual dose is 120 mg/administration.

[9] The pharmaceutical composition of any one of [1] to [8], wherein the IL-6 inhibitor is an IL-6 receptor antibody.

[10] The pharmaceutical composition of [9], wherein the IL-6 receptor antibody is a chimeric antibody, a humanized antibody, or a human antibody.

[11] [9] the pharmaceutical composition, wherein the IL-6 receptor antibody comprises a heavy chain variable region having the sequence of SEQ ID NO. 1 and a light chain variable region having the sequence of SEQ ID NO. 2.

[12] [9] the pharmaceutical composition, wherein the IL-6 receptor antibody comprises a heavy chain having the sequence of SEQ ID NO.3 and a light chain having the sequence of SEQ ID NO. 4.

[13] The pharmaceutical composition of [9], wherein the IL-6 receptor antibody is SA 237.

[14] The pharmaceutical composition of any one of [1] to [13], wherein the IL-6-associated disease is rheumatoid arthritis, juvenile idiopathic arthritis, systemic onset juvenile idiopathic arthritis, Castleman's disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, Crohn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, Steyr disease, amyloidosis, multiple sclerosis, transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, Chronic Obstructive Pulmonary Disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetes, GVHD, endometriosis, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin's disease, kidney cancer, liver cancer, kidney cancer, and kidney cancer, Pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancer cachexia, cancer nerve invasion, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, panuveitis, anterior uveitis, intermediate uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry eye, post-operative inflammation, neuromyelitis optica, myasthenia gravis, or pulmonary hypertension.

[15] The pharmaceutical composition of any one of [1] to [14], wherein the pharmaceutical composition is a formulation for subcutaneous administration.

[16] A method for treating an IL-6-associated disease, said method comprising administering an IL-6 inhibitor, wherein said IL-6 inhibitor is routinely administered after a short-spaced dosing period, wherein the same dose as the conventional dose is administered multiple times at intervals shorter than the conventional dosing interval in said short-spaced dosing period.

[17] An IL-6 inhibitor for use in the treatment of an IL-6 related disease, wherein the IL-6 inhibitor is conventionally administered after a short-spaced dosing period in which the same dose as the conventional dose is administered multiple times at intervals shorter than the conventional dosing interval.

[18] Use of an IL-6 inhibitor for the manufacture of a medicament for the treatment of an IL-6 related disease, wherein the IL-6 inhibitor is conventionally administered after a short-spaced dosing period in which the same dose as the conventional dose is administered multiple times at shorter intervals than the conventional dosing interval.

Effects of the invention

The pharmaceutical composition or regimen of the present invention can solve the problem of immunogenicity due to anti-drug antibodies and provide the pharmaceutical composition in a manner that places less burden on the patient because it does not expose the patient to high doses.

Brief Description of Drawings

Figure 1 indicates the change in mean (and standard deviation) of serum SA237 concentrations. Fig. 1a shows the change in concentration of SA237 during the main evaluation period, fig. 1b shows the change in concentration of SA237 during the extension period, and fig. 1c shows the change in concentration of serum SA237 to week 8.

FIG. 2 indicates the variation of the mean (and standard deviation) of serum sIL-6R concentration as a pharmacodynamic marker of SA 237. FIG. 2a shows the change in concentration of sIL-6R during the primary evaluation period, and FIG. 2b shows the change in serum sIL-6R concentration during the extension period.

Fig. 3 indicates the change in the mean (and standard deviation) of serum CRP concentration as a pharmacodynamic marker of SA 237. Fig. 3a shows the change in CRP concentration during the main evaluation period, and fig. 3b shows the change in CRP concentration during the extension period.

Detailed Description

Hereinafter, the present invention will be described in detail.

The present invention relates to pharmaceutical compositions or dosing regimens for the treatment of IL-6 related diseases.

The "IL-6 inhibitor" of the present invention is a substance that blocks signal transduction by IL-6 and inhibits the biological activity of IL-6. The IL-6 inhibitor is preferably a substance having an inhibitory effect on the binding to any of IL-6, IL-6 receptor and gp 130.

Examples of the IL-6 inhibitor of the present invention include, but are not particularly limited to, an anti-IL-6 antibody, an anti-IL-6 receptor antibody, an anti-gp 130 antibody, an IL-6 variant, a soluble IL-6 receptor variant, or a partial peptide of IL-6 or IL-6 receptor, and a low molecular weight substance exhibiting similar activity. An example of the IL-6 inhibitor of the present invention may preferably be an antibody recognizing IL-6 receptor.

The source of the antibody of the present invention is not particularly limited, but it is preferably a mammal and more preferably a human.

The anti-IL-6 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using a known method. Monoclonal antibodies derived from mammals are particularly preferred for the anti-IL-6 antibodies used in the present invention. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by transforming hosts with expression vectors containing antibody genes using genetic engineering methods. By binding to IL-6, the antibody inhibits the binding of IL-6 to the IL-6 receptor and blocks the transduction of IL-6 biological activity into cells.

Examples of such antibodies include MH166 antibody (Matsuda, T. et al, Eur. J. Immunol. (1988)18,951-956) and SK2 antibody (Sato, K. et al, The antibodies of The 21st annular Meeting of The Japanese Society for Immunology (1991)21,166).

Basically, hybridomas that produce anti-IL-6 antibodies can be prepared as follows using known methods. Specifically, hybridomas can be prepared by: the immunization is carried out by a conventional immunization method using IL-6 as a sensitizing antigen, the resulting immune cells are fused with known parent cells by a conventional cell fusion method, and then cells producing monoclonal antibodies are screened using a conventional screening method.

Specifically, anti-IL-6 antibody can be prepared as follows. Human IL-6 used as a sensitizing antigen for obtaining an antibody can be prepared, for example, by using Eur.J.Biochem (1987)168, 543-550; J.Immunol. (1988)140, 1534-1541; and IL-6 gene and/or amino acid sequences as disclosed in Agr.biol.chem. (1990)54, 2685-2688.

After transformation of a suitable host cell with a known expression vector system into which the IL-6 gene sequence has been inserted, the target IL-6 protein is purified from inside the host cell or from the culture supernatant using known methods. The purified IL-6 protein can be used as a sensitizing antigen. Alternatively, a fusion protein of an IL-6 protein and another protein may be used as a sensitizing antigen.

The anti-IL-6 receptor antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using a known method. Monoclonal antibodies derived from mammals are particularly preferred for the anti-IL-6 receptor antibodies used in the present invention. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by transforming hosts with expression vectors containing antibody genes using genetic engineering methods. By binding to the IL-6 receptor, the antibody inhibits the binding of IL-6 to the IL-6 receptor and blocks the transduction of IL-6 biological activity into cells.

Examples of such antibodies include MR16-1 antibody (Tamura, T. et al Proc. Natl. Acad. Sci. USA (1993)90, 11924. Sci. 11928), PM-1 antibody (Hirata, Y. et al, J. Immunol. (1989)143, 2900. sup. 2906), AUK12-20 antibody, AUK64-7 antibody, and AUK146-15 antibody (International patent application publication No. WO 92-19759). Wherein the PM-1 antibody is listed as an example of a preferred monoclonal antibody directed against the human IL-6 receptor and the MR16-1 antibody is listed as an example of a preferred monoclonal antibody directed against the mouse IL-6 receptor.

Basically, hybridomas that produce anti-IL-6 receptor monoclonal antibodies can be prepared as follows using known methods. Specifically, hybridomas can be prepared by: the immunization is carried out by a conventional immunization method using an IL-6 receptor as a sensitizing antigen, the resulting immune cells are fused with known parent cells by a conventional cell fusion method, and then cells producing monoclonal antibodies are screened using a conventional screening method.

Specifically, anti-IL-6 receptor antibodies can be prepared as follows. The human IL-6 receptor or the mouse IL-6 receptor used as ｃA sensitizing antigen for obtaining an antibody can be obtained by, for example, using IL-6 receptor genes and/or amino acid sequences disclosed in European patent application publication No. EP 325474 and Japanese patent application publication No. Kokai (JP-A) H03-155795 (unexamined published Japanese patent application), respectively.

There are two types of IL-6 receptor proteins: one expressed on the cell membrane and the other isolated from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al, J.biochem. (1990)108, 673-676). The soluble IL-6 receptor consists essentially of the extracellular domain of the IL-6 receptor that is bound to the cell membrane, and differs from the membrane-bound IL-6 receptor in that it lacks a transmembrane region or both a transmembrane region and an intracellular region. Any IL-6 receptor can be used as the IL-6 receptor protein, so long as it can be used as a sensitizing antigen for producing an anti-IL-6 receptor antibody used in the present invention.

After transformation of a suitable host cell with a known expression vector system into which the IL-6 receptor gene sequence has been inserted, the target IL-6 receptor protein is purified from within the host cell or from the culture supernatant using known methods. The purified IL-6 receptor protein is useful as a sensitizing antigen. Alternatively, cells expressing the IL-6 receptor or a fusion protein of the IL-6 receptor protein with another protein may be used as the sensitizing antigen.

The anti-gp 130 antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody using known methods. Monoclonal antibodies derived from mammals are particularly preferred for the anti-gp 130 antibodies used in the present invention. Monoclonal antibodies derived from mammals include those produced by hybridomas and those produced by transforming hosts with expression vectors containing antibody genes using genetic engineering methods. By binding gp130, the antibody inhibits the binding of the IL-6/IL-6-receptor complex to gp130 and blocks the transduction of IL-6 biological activity into cells.

Examples of such antibodies include AM64 antibody (JP-A (Kokai) H03-219894), 4B11 and 2H4 antibody (US5571513), and B-S12 and B-P8 antibody (JP-A (Kokai) H08-291199).

Basically, hybridomas producing anti-gp 130 monoclonal antibodies can be prepared as follows using known techniques. Specifically, hybridomas can be prepared by: immunization was carried out by a conventional immunization method using gp130 as a sensitizing antigen, the resulting immune cells were fused with known parent cells by a conventional cell fusion method, and then cells producing monoclonal antibodies were screened using a conventional screening method.

Specifically, monoclonal antibodies can be prepared as follows. For example, gp130 used as a sensitizing antigen for obtaining an antibody can be obtained by using the gp130 gene and/or amino acid sequence disclosed in european patent application publication No. EP 411946.

After transformation of a suitable host cell with a known expression vector system into which gp130 gene sequences have been inserted, the target gp130 protein is purified from within the host cell or from the culture supernatant using known methods. The purified gp130 protein can be used as a sensitizing antigen. Alternatively, cells expressing gp130 or a fusion protein of gp130 protein with another protein can be used as a sensitizing antigen.

The mammal immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell for cell fusion. Typically, rodents such as mice, rats and hamsters are used.

Animals are immunized with the sensitizing antigen according to known methods. Typically, immunization is carried out by, for example, intraperitoneal or subcutaneous injection of the sensitizing antigen into the mammal. Specifically, it is preferable that the sensitizing antigen is diluted or suspended in Phosphate Buffered Saline (PBS), physiological saline, or the like to an appropriate volume and mixed and emulsified with an appropriate amount of a conventional adjuvant such as freund's complete adjuvant if necessary, and then administered to the mammal several times every 4 to 21 days. Suitable carriers may also be used for immunization with a sensitizing antigen.

After immunization of an animal in this manner and confirmation of an increase in serum levels of the desired antibody, the immunized cells are removed from the mammal and subjected to cell fusion. Splenocytes are particularly preferred as immune cells for cell fusion.

Myeloma cells from mammals are used as parent cells for fusion with immunized cells. At present, various Cell lines are known such as P3X63Ag8.653(Kearney, J.F. et al, J.Immunol (1979)123, 1548-.

Basically, the aforementioned cell fusion of immune cells with myeloma cells can be carried out according to known Methods such as the method of Milstein et al (Kohler, G., and Milstein, C., Methods Enzymol (1981)73, 3-46).

More specifically, cell fusion is carried out, for example, in a conventional nutrient medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG) or sendai virus (HVJ) is used as a fusion promoter, and if necessary, an adjuvant such as dimethyl sulfoxide may be further added for improving fusion efficiency.

The ratio of immune cells to myeloma cells used is preferably, for example, 1 to 10 immune cells to one myeloma cell. The medium used for cell fusion is, for example, RPMI1640 or MEM medium suitable for proliferation of myeloma cell lines. Other conventional media for this type of cell culture may also be used. In addition, serum supplements such as Fetal Calf Serum (FCS) may also be used in combination.

For cell fusion, the fused cells of interest (hybridomas) are formed by: a predetermined amount of the aforementioned immune cells and myeloma cells are sufficiently stirred in the aforementioned medium, a PEG solution (for example, a solution of PEG having an average molecular weight of about 1,000 to 6,000) at a concentration of usually 30% to 60% (w/v) preheated to about 37 ℃ is added, and then they are mixed. Then, cell fusion agents and the like unsuitable for hybridoma growth can be removed by repeating the following operations: appropriate media were sequentially added and the supernatant was removed by centrifugation.

Hybridomas are selected by culturing in a conventional selection medium, for example, HAT medium (a medium containing hypoxanthine, aminopterin, and adenosine). Culturing in HAT medium is carried out for a sufficient period of time, typically days to weeks, to kill cells other than the hybridoma of interest (unfused cells). Standard limiting dilution procedures were then performed to screen and clone hybridomas producing the antibody of interest.

In addition to obtaining hybridomas by immunizing non-human animals with antigens, desired human antibodies having binding activity to desired antigens or antigen-expressing cells can be obtained by: human lymphocytes are sensitized in vitro with a desired antigen protein or antigen-expressing cell, and the sensitized B lymphocytes are fused with human myeloma cells such as U266 (see, japanese patent application Kokoku publication No. (JP-B) H01-59878 (a examined and approved japanese patent application, published for objection)). In addition, an antigen or antigen-expressing cells may be administered to a transgenic animal having a human antibody gene bank (repotore), and then a desired human antibody may be obtained following the aforementioned method (see, international patent application publication nos. WO 93/12227, WO 92/03918, WO 94/02602, WO 94/25585, WO 96/34096 and WO 96/33735).

The monoclonal antibody-producing hybridomas thus prepared can be sub-cultured in conventional media and stored in liquid nitrogen for a long period of time.

For obtaining monoclonal antibodies from hybridomas, the following methods can be used: culturing the hybridoma according to a conventional method and obtaining the antibody as a culture supernatant or administering the hybridoma into a compatible mammal to proliferate and obtain the antibody from ascites; and so on. The former method is suitable for obtaining high purity antibodies, while the latter method is suitable for large scale antibody production.

For example, hybridomas producing anti-IL-6 receptor antibodies can be prepared by the methods disclosed in JP-A (Kokai) H03-139293. Such preparation can be carried out by: injecting the hybridoma producing the PM-1 antibody into the abdominal cavity of a BALB/c mouse to obtain ascites, and then purifying the PM-1 antibody from the ascites; or culturing the hybridoma in an appropriate medium (e.g., RPMI1640 medium containing 10% fetal bovine serum and 5% BM-conditioner H1(Boehringer Mannheim); hybridoma SFM medium (GIBCO-BRL); or PFHM-II medium (GIBCO-BRL)), and then purifying the PM-1 antibody from the culture supernatant.

A recombinant antibody can be used as the monoclonal antibody of the present invention, wherein the recombinant antibody is prepared by: using genetic recombination techniques, antibody genes are cloned from hybridomas, the genes are inserted into appropriate vectors, and the vectors are then introduced into a host (see, e.g., Borrebaeck, c.a.k. and Larrick, j.w., thermoeutic MONOCLONALANTIBODIES, published by MACMILLAN PUBLISHERS LTD in the uk, 1990).

More specifically, mRNA encoding the variable (V) region of an antibody is isolated from cells (e.g., hybridomas) that produce the antibody of interest. mRNA can be isolated by: total RNA was prepared according to known methods such as the guanidine ultracentrifugation method (Chirgwin, J.M. et al, Biochemistry (1979)18, 5294-. Alternatively, mRNA can be prepared directly using the QuickPrep mRNA purification kit (Pharmacia).

cDNA of the antibody V region was synthesized from the obtained mRNA using reverse transcriptase. The cDNA can be synthesized using AMV reverse transcriptase first strand cDNA synthesis kit or the like. Furthermore, for the synthesis and amplification of cDNA, the 5 '-RACE method using the 5' -Ampli FINDER RACE kit (Clontech) and PCR (Frohman, M.A. et al, Proc. Natl. Acad. Sci. USA (1988)85, 8998-. The DNA fragment of interest is purified from the obtained PCR product and then ligated with a vector DNA. Then, a recombinant vector is prepared using the above, and introduced into E.coli or the like, and then its clone is selected to prepare a desired recombinant vector. The nucleotide sequence of the DNA of interest is confirmed by a known method such as dideoxy method.

When a DNA encoding the V region of the antibody of interest is obtained, the DNA is ligated with a DNA encoding the constant region (C region) of the desired antibody, and inserted into an expression vector. Alternatively, the DNA encoding the antibody V region may be inserted into an expression vector comprising the DNA of the antibody C region.

To prepare the antibody for use in the present invention, an antibody gene is inserted into an expression vector to be expressed under the control of expression regulatory regions such as enhancers and promoters, as described below. The antibody can then be expressed by transforming a host cell with the expression vector.

In the present invention, a recombinant antibody, e.g., a chimeric antibody, a humanized antibody, or a human antibody, which is artificially modified, e.g., to reduce the antigenicity against humans, may be used. These modified antibodies can be prepared using known methods.

Chimeric antibodies can be obtained by: the DNA encoding the V region of the antibody obtained as above is ligated with the DNA encoding the C region of the human antibody, inserted into an expression vector, and the vector is introduced into a host to produce a chimeric antibody (see, European patent application publication No. EP 125023; International patent application publication No. WO 92-19759). This known method can be used to obtain chimeric antibodies useful in the present invention.

Humanized antibodies are also known as engineered human antibodies or antibodies made to human type. It is prepared by grafting Complementarity Determining Regions (CDRs) of an antibody from a non-human animal (e.g., mouse) into CDRs of a human antibody. General methods for this gene recombination are also known (see, European patent application publication No. EP 125023, International patent application publication No. WO 92-19759).

More specifically, a DNA sequence designed to link CDRs of a mouse antibody with Framework Regions (FRs) of a human antibody is synthesized by PCR from several oligonucleotides prepared to have overlapping portions at their ends. The obtained DNA is ligated with a DNA encoding a human antibody C region and inserted into an expression vector, and the expression vector is introduced into a host to prepare a humanized antibody (see, European patent application publication No. EP 239400, International patent application publication No. WO 92-19759).

Human antibody FRs linked via CDRs are selected so that the CDRs form a satisfactory antigen binding site. Amino acids within the framework regions of the antibody variable regions may be substituted, as desired, so that the CDRs of the engineered human antibody form the appropriate antigen-binding site (Sato, K., et al, Cancer Res. (1993)53,851 856).

Human antibody C regions are used for chimeric and humanized antibodies. Examples of the human antibody C region include C γ, and for example, C γ 1, C γ 2, C γ 3 or C γ 4 can be used. In addition, the human antibody C region may be modified in order to improve the stability of the antibody and its preparation.

The chimeric antibody is composed of a variable region of an antibody derived from a non-human animal and a C region derived from a human antibody; humanized antibodies consist of CDRs from an antibody derived from a non-human animal and framework regions and C regions derived from a human antibody. Its antigenicity in a human body is reduced, and therefore it can be used as an antibody for use in the present invention.

Preferred specific examples of the humanized antibody used in the present invention include humanized PM-1 antibodies (see, International patent application publication No. WO 92-19759).

In addition to the above-described methods for obtaining human antibodies, techniques for obtaining human antibodies by panning using human antibody libraries are also known. For example, the variable region of a human antibody can be expressed as a single chain antibody (scFv) on the phage surface by using a phage display method, and then a phage that binds to an antigen can be selected. By analyzing the genes of the selected phage, the DNA sequence encoding the variable region of a human antibody that binds to the antigen can be determined. After revealing the DNA sequence of the scFv that binds to the antigen, an appropriate expression vector containing the sequence can be prepared to obtain a human antibody. Such methods are known and publications WO 92/01047, WO 92/20791, WO93/06213, WO 93/11236, WO 93/19172, WO95/01438 and WO 95/15388 are available as references.

The antibody gene constructed as described above can be expressed according to a known method. When mammalian cells are used, the antibody gene can be expressed in the following manner: a DNA in which a commonly used effective promoter gene, an antibody gene to be expressed, and a polyadenylation signal at the 3' -side (downstream) of the antibody gene are operably linked together is used, or a vector containing the DNA is used. Examples of promoters/enhancers include the human cytomegalovirus immediate early promoter/enhancer.

In addition, other promoters/enhancers that may be used to express antibodies useful in the invention include viral promoters/enhancers from retroviruses, polyoma viruses, adenoviruses, simian virus 40(SV40), and the like; and promoters/enhancers derived from mammalian cells such as human elongation factor 1 alpha (HEF1 alpha).

Expression can be readily carried out, for example, following the methods in Mullingan et al (Mullingan, R.C. et al, Nature (1979)277, 108-one 114) when using the SV40 promoter/enhancer, or Mizushima et al (Mizushima, S. and Nagata S., Nucleic Acids Res. (1990)18,5322) when using the HEF1 α promoter/enhancer.

When Escherichia coli is used, the antibody gene can be expressed by operably linking a commonly used effective promoter gene, a signal sequence for antibody secretion, and the antibody gene to be expressed. Examples of the promoter include lacZ promoter and araB promoter. The lacZ promoter can be used according to the method of Ward et al (Ward, E.S. et al, Nature (1989)341, 544-546; Ward, E.S. et al, FASEB J. (1992)6, 2422-2427); and the araB promoter can be used according to the method of Better et al (Better, M. et al, Science (1988)240, 1041-1043).

The pel B signal sequence (Lei, S.P., et al, J.Bacteriol. (1987)169,4379-4383) can be used as a signal sequence for antibody secretion when antibodies are prepared in the periplasm of E.coli. Antibodies produced in the periplasm are isolated and then the antibody structure to be used is properly refolded (see, e.g., WO 96/30394).

As the replication origin, those derived from SV40, polyoma virus, adenovirus, Bovine Papilloma Virus (BPV), etc. can be used. In addition, in order to increase the gene copy number in the host cell system, the expression vector may contain an Aminoglycoside Phosphotransferase (APH) gene, an adenosine kinase (TK) gene, an escherichia coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene, a dihydrofolate reductase (dhfr) gene, etc. as a selection marker.

Any production system may be used to produce the antibodies for use in the present invention. Production systems for antibody production include in vitro and in vivo production systems. In vitro production systems include those using eukaryotic cells or those using prokaryotic cells.

When eukaryotic cells are used, the production system includes those using animal cells, plant cells or fungal cells. Such animal cells include (1) mammalian cells such as CHO, COS, myeloma, Baby Hamster Kidney (BHK), HeLa and Vero; (2) amphibian cells such as xenopus laevis oocytes; and (3) insect cells such as sf9, sf21 and Tn 5. Known plant cells include cells derived from tobacco (Nicotiana tabacum), which can be cultured in callus. Known fungal cells include yeasts such as Saccharomyces (Saccharomyces) (e.g.Saccharomyces cerevisiae) and mould fungi such as Aspergillus (Aspergillus) (e.g.Aspergillus niger).

When prokaryotic cells are used, production systems include those that use bacterial cells. Known bacterial cells include Escherichia coli and Bacillus subtilis.

The antibody can be obtained by introducing a gene of an antibody of interest into these cells by transformation and then culturing the transformed cells in vitro. The cells are cultured according to known methods. For example, DMEM, MEM, RPMI1640 or IMDM may be used as the culture medium, and a serum supplement such as Fetal Calf Serum (FCS) may be used in combination. Alternatively, cells into which antibody genes are introduced may be transferred into the abdominal cavity of an animal to prepare antibodies in vivo.

Meanwhile, in vivo preparation systems include those using animals or those using plants. When animals are used, preparation systems include those using mammals or insects.

Mammals that may be used include goats, pigs, sheep, mice and cattle (Vicki Glaser, spectrumb technology Applications, 1993). Further, insects that may be used include silkworms. When plants are used, tobacco and the like can be used.

Antibody genes are introduced into these animals or plants, and antibodies are produced in the animals or plants and then recovered. For example, an antibody gene can be prepared as a fusion gene by inserting it into the middle of a gene encoding a protein produced only in milk (e.g., goat beta casein). A DNA fragment containing a fusion gene including the inserted antibody gene was injected into a goat embryo, and the embryo was transferred into a ewe. The desired antibody is obtained from the milk of a transgenic goat born by a free-born goat or its offspring. Where appropriate, the transgenic goat may be given hormones to increase the amount of milk it produces containing the desired antibodies (Ebert, K.M., et al, Bio/Technology (1994)12, 699-702).

When silkworms are used, silkworms are infected with baculovirus into which a gene for the intended antibody is inserted, and the desired antibody is obtained from the body fluid of these silkworms (Maeda, S. et al, Nature (1985)315, 592-594). In addition, when tobacco is used, the gene of the antibody of interest is inserted into a plant expression vector such as pMON530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens (Agrobacterium tumefaciens). The bacterium is used to infect tobacco such as Nicotiana tabacum, and the desired antibody is then obtained from the leaves of that tobacco (Julian, K. -C.Ma et al, Eur.J.Immunol. (1994)24,131- "138).

When an antibody is produced using the in vitro or in vivo production system as described above, DNAs encoding the heavy chain (H chain) and light chain (L chain) of the antibody may be inserted into separate expression vectors, and then the host is co-transformed with the vectors. Alternatively, the DNA encoding the H chain and the DNA encoding the L chain may be inserted into a single expression vector for transformation of a host (see International patent application publication No. WO 94-11523).

The antibody used in the present invention may be an antibody fragment or a modified product thereof as long as it is applicable to the present invention. For example, antibody fragments include Fab, F (ab') 2, Fv and single chain Fv (scFv), wherein the Fv of the H and L chains are linked via an appropriate linker.

Specifically, antibody fragments were prepared by: the antibodies are treated with enzymes such as papain or pepsin, or alternatively, genes encoding these antibody fragments are constructed and introduced into expression vectors, which are then expressed in appropriate host cells (see, e.g., Co, M.S. et al, J.Immunol. (1994)152, 2968-.

scFv can be obtained by linking the H-chain V region and L-chain V region of an antibody. In this scFv, the H-chain V region and the L-chain V region are linked via a linker, preferably a peptide linker (Huston, J.S. et al, Proc. Natl.Acad.Sci.USA (1988)85, 5879-. The V regions of the H and L chains in the scFv may be derived from any of the above-described antibodies. Peptide linkers for linking the V regions include, for example, any single chain peptide consisting of 12 to 19 amino acid residues.

DNA encoding scFv can be obtained by: amplifying the portion in the template sequence using PCR using a primer pair defining the end of a DNA portion encoding a desired amino acid sequence, wherein a DNA encoding the H chain or H-chain V region of the aforementioned antibody and a DNA encoding the L chain or L-chain V region of the aforementioned antibody are used as templates, and then further amplifying the amplified DNA portion using a DNA encoding a peptide linker moiety and a primer pair defining both ends of the linker so that it can be ligated to each of the H and L chains.

After preparing the DNA encoding the scFv, an expression vector comprising the DNA and a host transformed with the expression vector can be obtained according to a conventional method. Further, the scFv can be obtained by using a host according to a conventional method.

Similarly to the above, the antibody fragment can be prepared by obtaining the gene thereof, expressing it, and then using a host. As used herein, "antibody" encompasses such antibody fragments.

Antibodies conjugated to a variety of molecules, such as polyethylene glycol (PEG), may also be used as modified antibodies. "antibody" as used herein encompasses such modified antibodies. These modified antibodies can be obtained by chemically modifying the obtained antibodies. Such methods have been established in the art.

The antibody prepared and expressed as above may be isolated from the inside or outside of the cell or from the host, and then purified to homogeneity. The antibodies used in the present invention can be isolated and purified by affinity chromatography. Columns for affinity chromatography include protein a columns and protein G columns. Supports for protein a columns include HyperD, POROS and Sepharose F.F. Other methods for the isolation and/or purification of common proteins may be used without limitation.

For example, the antibody used in the present invention can be isolated and purified by appropriately selecting and combining chromatography other than the above-mentioned affinity chromatography, filtration, ultrafiltration, salting out, dialysis, and the like. Examples of chromatography include ion exchange chromatography, hydrophobic chromatography, and gel filtration. These chromatographs can be applied to high-energy liquid chromatography (HPLC). Alternatively, reverse phase HPLC may be used.

The concentration of the antibody obtained as above can be determined by absorbance measurement, ELISA, or the like. Specifically, when using absorbance measurements, the concentration can be determined by: the antibody solution was appropriately diluted with PBS (-), the absorbance thereof at 280nm was measured, and the concentration was calculated using the conversion factor 1.35OD/1 mg/ml. Alternatively, when ELISA is used, the concentration can be determined as follows. Specifically, 100. mu.l of goat anti-human IgG (TAG) diluted to 1. mu.g/ml with 0.1M bicarbonate buffer (pH 9.6) was added to a 96-well plate (Nunc) and incubated overnight at 4 ℃ to immobilize the antibody. After blocking, 100 μ l of an appropriately diluted antibody to be used in the present invention or an appropriately diluted sample containing the antibody or human igg (cappel) as a standard was added and the plate was incubated at room temperature for one hour.

After washing, 100 μ l of alkaline phosphatase-labeled anti-human igg (bio source) diluted 5,000x was added, and the plate was incubated at room temperature for one hour. After another wash, substrate solution was added, the plate was incubated, and absorbance at 405nm was measured using a Microplate Reader Model 3550(Bio-Rad) to calculate the concentration of the antibody of interest.

IL-6 variants used in the present invention are substances that have binding activity to the IL-6 receptor and do not transmit IL-6 biological activity. That is, the IL-6 variant competes with IL-6 for binding to the IL-6 receptor, but does not conduct IL-6 biological activity, and thus blocks IL-6 mediated signal transduction.

IL-6 variants by replacing in the amino acid sequence of IL-6 in amino acid residues introduced mutation to prepare. Any IL-6 from which the IL-6 variant is derived can be used, but human IL-6 is preferred in view of antigenicity and the like.

More specifically, amino acid substitutions are made by predicting the secondary structure of IL-6 from the IL-6 amino acid sequence using known molecular modeling programs such as WHATIF (Vriend et al, J.mol.graphics (1990)8,52-56) and further assessing the effect of the substituted amino acid residue on the overall molecule. After the appropriate amino acid residues for substitution are determined, mutations are introduced by a conventionally performed PCR method using a vector comprising a nucleotide sequence encoding the human IL-6 gene as a template to cause amino acid substitutions, and thereby a gene encoding an IL-6 variant is obtained. If desired, the gene is inserted into a suitable expression vector, and the IL-6 variant can be obtained according to the methods described above for expression, preparation and purification of recombinant antibodies.

Specific examples of IL-6 variants are disclosed in Brakenhoff et al, J.biol.chem. (1994)269, 86-93; savino et al, EMBO J. (1994)13, 1357-; WO 96-18648; and WO 96-17869.

The IL-6 or IL-6 receptor partial peptide used in the present invention is a substance which has a binding activity to the IL-6 receptor or IL-6, respectively, and does not conduct IL-6 biological activity. That is, IL-6 or a partial peptide of the IL-6 receptor binds to and captures the IL-6 receptor or IL-6, and thereby specifically inhibits the binding of IL-6 to the IL-6 receptor. Thus, IL-6 biological activity is not conducted, and thus, IL-6 mediated signal transduction is blocked.

IL-6 or IL-6 receptor partial peptides are peptides which consist of the entire amino acid sequence of a region of the IL-6 or IL-6 receptor amino acid sequence or a part thereof which is involved in the binding between IL-6 and the IL-6 receptor. Such peptides typically consist of 10 to 80, preferably 20 to 50, more preferably 20 to 40 amino acids.

IL-6 or partial peptides of the IL-6 receptor can be prepared by: specifying a region of the amino acid sequence of IL-6 or IL-6 receptor involved in the binding between IL-6 and the IL-6 receptor, and applying generally known methods such as genetic engineering techniques and peptide synthesis methods to the entire amino acid sequence of the specified region or a portion thereof.

In order to prepare IL-6 or a partial peptide of IL-6 receptor by genetic engineering methods, a DNA sequence encoding a desired peptide is inserted into an expression vector, and then the peptide can be obtained by applying the aforementioned method for expression, preparation and purification of a recombinant antibody.

For the preparation of IL-6 or a partial peptide of IL-6 receptor by a peptide synthesis method, a commonly used peptide synthesis method such as a solid phase synthesis method and a liquid phase synthesis method can be used.

Specifically, The Peptide can be synthesized according to The method described in "The sequence of Development of Pharmaceuticals (Zoku Iyakuhin no Kaihatsu), Vol.14, Peptide Synthesis (edited Haruaki Yajima,1991, Hirokawa Shoten)". As the solid phase synthesis method, the following methods and the like can be employed: binding an amino acid corresponding to the C-terminus of the peptide to be synthesized to a support soluble in an organic solvent, and then extending the peptide chain by alternately repeating (1) a reaction of condensing an α -amino group and an amino acid whose branched functional group is protected by an appropriate protecting group, one at a time in the C-terminal to N-terminal direction; and (2) a reaction of removing a protecting group from an α -amino group of the resin-bound amino acid or peptide. Solid phase peptide synthesis is largely classified into Boc method and Fmoc method according to the type of protecting group used.

After the synthesis of the desired peptide as described above, a deprotection reaction and a reaction for cleaving the peptide chain from the support are carried out. For the reaction of cleaving peptide chain, hydrogen fluoride or trifluoromethanesulfonic acid is generally used for Boc method, and TFA is generally used for Fmoc method. In the Boc method, for example, the resin bound to the protected peptide is treated with hydrogen fluoride in the presence of anisole. The peptide is then recovered by removing the protecting group and cleaving the peptide from its support. By lyophilizing the recovered peptide, a crude peptide can be obtained. In the Fmoc method, the deprotection reaction and the cleavage of a peptide chain from a support can be carried out in TFA or the like by a similar operation as described above.

The crude peptide obtained can be isolated and purified by application of HPLC. Elution can be performed under optimal conditions using a water-acetonitrile solvent system commonly used for protein purification. The fractions corresponding to the peaks of the obtained chromatographic characteristic curve were collected and lyophilized. The peptide fraction purified in this way was identified by molecular weight analysis via mass spectrometry, amino acid composition analysis, amino acid sequence analysis, and the like.

Specific examples of IL-6 and partial peptides of IL-6 receptor are disclosed in JP-A (Kokai) H02-188600, JP-A (Kokai) H07-324097, JP-A (Kokai) H08-311098, and U.S. Pat. publication No. US 5210075.

The antibodies used in the present invention may be conjugated antibodies that bind to a variety of molecules such as polyethylene glycol (PEG), radioactive substances and toxins. Such conjugated antibodies can be obtained by chemical modification of the obtained antibodies. Methods for antibody modification are well established in the art. Thus, the term "antibody" as used herein encompasses such conjugated antibodies.

In the present invention, "IL-6-associated disease" refers to a disease associated with IL-6, and examples include rheumatoid arthritis, juvenile idiopathic arthritis, systemic onset juvenile idiopathic arthritis, Castleman's disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, Crohn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, still's disease, amyloidosis, multiple sclerosis, transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, Chronic Obstructive Pulmonary Disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin's lymphoma, and the like, Pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancer cachexia, cancer nerve invasion, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, panuveitis, anterior uveitis, intermediate uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry eye, post-operative inflammation, neuromyelitis optica, myasthenia gravis, and pulmonary hypertension.

In the present invention, "conventional dosing interval" refers to a dosing interval generally used for the above-mentioned drug (pharmaceutical composition of the present invention), for example, a dosing interval of conventional administration which can be described as "a subsequent dose should be administered at four-week intervals" or the like in the inner page of the package. The conventional administration interval in the present invention is not particularly limited, but examples include one day to 24 weeks, preferably two weeks to eight weeks, more preferably three to five weeks, and even more preferably four weeks. Conventional dosing intervals may have a range.

In the present invention, the "conventional dose" is a dose generally used for the above-mentioned drug (pharmaceutical composition of the present invention), for example, a dose which can be described as a usual administration of "generally, a single dose of 8mg/kg body weight" in a package inner page. The conventional dose in the present invention is not particularly limited, but the dose per administration may be, for example, 2 to 20mg of IL-6 inhibitor per kg of body weight (2-20mg/kg) or 50mg to 800mg of IL-6 inhibitor, preferably 2 to 8mg of IL-6 inhibitor per kg of body weight (2-8mg/kg) or 80 to 160mg of IL-6 inhibitor, or more preferably 8mg of IL-6 inhibitor per kg of body weight (8mg/kg) or 120mg of IL-6 inhibitor.

In the present invention, the "short interval administration period" refers to an administration period in which immunological tolerance against a drug (the pharmaceutical composition of the present invention) is induced to suppress the production of anti-drug antibodies due to immunogenicity. The short interval administration period in the present invention refers to an administration period in which the same dose as the conventional dose is administered multiple times at intervals shorter than the conventional administration interval. Although the short interval period is not particularly limited as long as it is a period that causes immune tolerance, the period is preferably 1 to 8 weeks from initial administration, and more preferably 4 weeks from initial administration. "the same dose as the conventional dose" includes a dose that provides the same blood concentration of the IL-6 inhibitor as the conventional dose. The "shorter interval than the regular administration interval" is not particularly limited as long as it is shorter than the regular administration interval, and is preferably half of the regular administration interval, for example, two weeks (when the regular administration interval is four weeks). For example, the short interval administration period may have a range such as one to two weeks. "(multiple) administration" means two or more administrations including the initial administration, and preferably 2 to 5 administrations including the initial administration, more preferably 3 administrations including the initial administration. Whether or not immune tolerance is induced can be determined by observing whether or not anti-drug antibody production is inhibited.

The "conventional administration" in the present invention means administration commonly used for the above-mentioned drugs (pharmaceutical composition of the present invention), for example, administration in the above-mentioned "conventional dose" and "conventional dosing interval".

Preferred examples of the "IL-6 receptor antibody" of the present invention include truzumab which is a humanized anti-IL-6 receptor IgG1 antibody, and a humanized anti-IL-6 receptor antibody prepared by modifying the variable region and the constant region of truzumab, specifically, an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO:1 and a light chain variable region comprising the sequence of SEQ ID NO: 2. A more preferred example is an antibody comprising a heavy chain comprising the sequence of SEQ ID NO.3 (heavy chain of SA 237) and a light chain comprising the sequence of SEQ ID NO. 4 (light chain of SA 237). SA237 is particularly preferred.

Such antibodies may be obtained according to the methods described in WO2010/035769, WO2010/107108, WO2010/106812, etc. Specifically, antibodies can be prepared based on the sequences of the above IL-6 receptor antibodies using genetic recombination techniques known to those skilled in the art (see, e.g., Borrebiack CAK and Larrick JW, THERAPEUTIC MONOCLONALANTIBIDIES, published by MACMILLAN PUBLISHERS LTD in the UK, 1990). Recombinant antibodies can be obtained by: the antibody-encoding DNA is cloned from a hybridoma-or antibody-producing cell, such as an antibody-producing sensitized lymphocyte, inserted into an appropriate vector, and the vector is introduced into a host (host cell) to produce an antibody.

Such an antibody can be isolated and purified using an isolation and purification method conventionally used for antibody purification without limitation. For example, the antibody can be isolated and purified by appropriately selecting and combining column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and the like.

In the present invention, the conventional administration period starts with the last administration of the short interval administration period. More specifically, the last administration in the short-interval administration period is followed by a regular administration interval, followed by the first administration in the regular administration period.

The pharmaceutical composition of the present invention is preferably a pharmaceutical composition in which the same dose of the IL-6 inhibitor as the conventional dose is administered 2 to 5 times at an interval of 1 to 3 weeks from the first administration in a short-interval administration period, and then the IL-6 inhibitor is administered at an interval of 2 to 8 weeks from the last administration in the short-interval administration period, using a conventional dose of 50mg to 800mg per administration; or more preferably a pharmaceutical composition in which SA237 is administered 3 times at the same dose as the conventional dose at 2-week intervals from the first administration in a short-interval administration period (i.e., at week 0, week 2 and week 4), and then SA237 is conventionally administered at 8-week intervals from the last administration in the short-interval administration period (i.e., at week 12, week 20, week 28, and so on, at 8-week intervals, counted from the first administration in the short-interval administration period), using 120 mg/administration of the conventional dose.

The preferred timing of administration for the IL-6 inhibitor can be adjusted, for example, by monitoring the disease state and the change in blood test values to appropriately extend the dosing interval.

The pharmaceutical composition of the present invention for therapeutic or prophylactic purposes may be formulated into a lyophilized preparation or a solution preparation by mixing with suitable pharmaceutical carriers, excipients and the like, if necessary. Suitable pharmaceutical carriers and excipients include, for example, sterile water, physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphates, citrates, histidine and other organic acids), antimicrobials, surfactants (such as PEG and Tween), chelating agents (such as EDTA) and binders. Other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, amino acids such as glycine, glutamine, asparagine, glutamic acid, aspartic acid, methionine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugar alcohols such as mannitol and sorbitol may also be present. When preparing an aqueous solution for injection, physiological saline and isotonic solution containing glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride; and suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol and PEG), and nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer 188 and HCO-50) may be used in combination. Larger fluid volumes can be administered subcutaneously by mixing hyaluronidase into the formulation (Expert opin. drug deliv.2007 jul; 4(4): 427-40). In addition, the syringe may be pre-filled with the pharmaceutical composition of the present invention. Solution formulations may be prepared according to the methods described in WO 2011/090088.

If desired, the pharmaceutical compositions of the present invention may be encapsulated in microcapsules (e.g., those made of hydroxymethylcellulose, gelatin, and poly (methylmethacylate)), or incorporated into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, e.g., "Remington's pharmaceutical science 16th edition," Oslo Ed. (1980)). Methods for preparing agents into controlled release agents are also known and such methods can be applied to the pharmaceutical compositions of the present invention (Langer et al, J.Biomed.Mater.Res.15:267-277 (1981); Langer, Chemtech.12:98-105 (1982); U.S. Pat. No.3,773,919; European patent application publication No. EP 58,481; Sidman et al, Biopolymers 22:547-556 (1983); and EP 133,988).

The pharmaceutical compositions of the present invention may be administered to a patient via any suitable route. For example, it may be administered to a patient intravenously (by bolus injection or continuous infusion), intramuscularly, intraperitoneally, intracerebroventricularly, transdermally, subcutaneously, intraarticularly, sublingually, intrasynovially, orally, by inhalation, by topical or external continuous infusion for a specified length of time. Intravenous administration or subcutaneous administration is preferred.

All prior art documents cited herein are incorporated by reference in this specification.

Examples

Hereinafter, the present invention will be specifically described with respect to embodiments, but the present invention is not to be construed as being limited to the embodiments.

EXAMPLE 1 preparation of IL-6 inhibitor

The IL-6 receptor antibody SA237 described in patent document WO2010/035769 (the antibody in WO2010/035769 containing a heavy chain having the sequence of SEQ ID NO:26 of WO2010/035769 (SEQ ID NO:3 herein) and a light chain having the sequence of SEQ ID NO:29 of WO2010/035769 (SEQ ID NO:4 herein)) was prepared according to the description in the above patent document. The prepared antibody was used to prepare a formulation for subcutaneous administration by the method in patent document WO 2011/090088.

Example 2 detection by single subcutaneous administration to healthy adult male Japanese and Caucasian subjects (SA001JP)

Safety, tolerability, pharmacokinetics and bioavailability of SA237 when administered subcutaneously to healthy adult male japanese and caucasian subjects were evaluated. In this study, SA237 was administered subcutaneously or intravenously to 48 japanese subjects by instillation, and SA237 was administered subcutaneously to 24 caucasian subjects. The safety and tolerability of a single administration of SA237 was almost satisfactory in 24 cases. The absolute bioavailability of SA237 administered subcutaneously at 60mg and 120mg was 64.6% and 69.4%, respectively. Development of anti-SA 237 antibody was observed in 39 of 72 subjects administered with SA 237.

Example 3 parallel group comparison study by open label of multiple subcutaneous administrations to Japanese rheumatoid arthritis patients (SA-105JP)

Patients who meet the following criteria were selected as subjects:

(1) diagnosed as having Rheumatoid Arthritis (RA) according to the 1987 American College of Rheumatology (ACR) criteria;

(2) duration of RA disease above 6 months;

(3) (ii) exhibits a level of C-reactive protein (CRP) above the upper limit of the laboratory reference range in a test performed within two weeks prior to the start of administration of the study pharmaceutical product (IMP);

(4) age 20 years old or older with informed consent;

(5) signing on an informed consent form by the person;

(6) no treatment with Methotrexate (MTX) 16 weeks or after IMP administration was initiated;

(7) leflunomide (leflunomide) therapy was not received 12 weeks or later before IMP administration was initiated (or 4 weeks or later before study drug administration was initiated if standard cholestyramine (cholestyramine) therapy or drug removal with activated charcoal had been performed);

(8) (ii) receives no treatment with a DMARD or an immunosuppressive agent other than the above 4 weeks or after the study agent administration is initiated; and is

(9) No treatment was received with more than 10 mg/day of prednisolone (prednisolone) equivalent 2 weeks or after the study medication was initiated.

Subjects were randomized into three groups (A, B, C groups) according to the centralized enrollment method, and an open-label, parallel group comparison study was performed (see table 1). Randomization was graded by body weight. The clinical study included a primary evaluation period, an extension period, and a follow-up period.

In the main evaluation period, 120mg of SA237 was administered at week 0, week 2 and week 4; and 120mg, 60mg and 30mg of SA237 were administered to A, B, C groups at four week intervals from week 8 to week 16, respectively. Thereafter, essentially, A, B, C groups through week 32, week 28, and week 24, respectively, were observed, with the serum SA237 concentration in each group expected to be at undetectable levels (the observation included anti-SA 237 antibody measurements) at the time indicated.

Over an extended period, 120mg of SA237 was administered at week 0, week 2, and week 4; and 120mg of SA237 was administered at four week intervals from week 8 to week 20 and observed for additional weeks 32.

The test drug was in the form of a vial containing 1.0mL of a solution containing 120mg of SA 237. The solution contains L-histidine, L-arginine, L-aspartic acid and polyoxyethylene (160) polyoxypropylene (30) diol as additives and is adjusted to pH 5.5 to 6.5. Basically, the drug is administered subcutaneously to the abdominal region.

[ Table 1]

Number of cases

In pharmacokinetic and pharmacodynamic assessments, and in the examination of efficacy (in the complete analysis setting (FAS)) and safety of repeat administrations of SA237 to RA patients, the background of subjects in each 11 case group (33 total cases) subjected to each analysis was: the age was 59.0 to 65.0 years (median range for each group; the same applies hereinafter) and the body weight was 50.30 to 57.90 kg. The percentage of women in each group was high and was 81.8% in group a (9/11 cases), 90.9% in group B (10/11 cases), and 63.6% in group C (7/11 cases). Subjects who received study agent until the end of the primary evaluation period were 10/11 cases (90.9%) in group a, 10/11 cases (90.9%) in group B, and 9/11 cases (81.8%) in group C; and the subjects that could be observed throughout the period (main evaluation period and extension period) were 10/11 cases (90.9%) in group a, 7/11 cases (63.6%) in group B, and 7/11 cases (63.6%) in group C.

(1) Pharmacokinetics

The evaluation method comprises the following steps: observations and tests were performed according to tables 2 and 3. When not specifically indicated, the evaluation was performed prior to administration of the study agent. Even if the defined primary evaluation period has not reached completion, subsequent observations and testing of the primary evaluation period are determined to be unnecessary when the evaluation is performed on or after the day of initial administration of the extended period. The test period is defined as follows.

The main evaluation period: in principle, for the A, B, C group, the observation and testing period started on the first day of study agent administration, and by weeks 32, 28, and 24, respectively, serum SA237 concentrations were expected to be eliminated at that time. However, when the serum SA237 concentration was confirmed to be an undetectable level and administration in the extended period started before the end of the above period, the main evaluation period will be set as the period from before the first administration in the extended period until observation and testing.

And (3) prolonging the period: after completion of the main evaluation period, starting with the first administration in the extension period and through observation and testing at week 24 of the extension period.

And (3) post-observation period: starting from observation and completion of the test at week 24 of the extended period and up to week 32.

[ Table 2]

Observation and inspection scheduling (Main evaluation period)

[ Table 3]

Observation and examination scheduling (extension and follow-up period)

As a result: a chart indicating the pharmacokinetics in this study is shown in figure 1. Trough levels of serum SA237 concentrations were approximately constant from week 4 in the main evaluation period of group a and in the extension period. On the other hand, serum SA237 concentrations in groups B and C decreased from week 8 during the main evaluation period. Since the main evaluation and extension periods did not show serum SA237 concentrations and AUC_0-2W(to week 8), so that when administration of SA237 was discontinued and continued thereafter, the pharmacokinetics did not change.

(2) Pharmacodynamic evaluation

As a result: graphs from pharmacodynamic assessments in this study are shown in figures 2 and 3. In group a, the serum SA237 concentration was maintained at a constant level and the serum sIL-6R concentration was also maintained at an approximately constant level during the main assessment period from week 8 to week 20, and during the extension period from week 8 to week 24. On the other hand, in groups B and C, serum concentration of sIL-6R as a PD marker of IL-6 inhibition decreased as the concentration of SA237 decreased from week 8 during the main evaluation period.

During the main evaluation period, CRP as a PD marker of IL-6 inhibition was below the lower limit of quantification (0.005mg/dL) in nearly half of the subjects in group a from week 4 to week 20, and the mean value also remained low, about 0.01 mg/dL. The values increased above 0.1mg/dL from week 16 (in group B) and from week 8 (in group C). The percentage of CRP normalization (below 0.3 mg/dL) also showed a similar trend to the change in mean value. The percentage of each group at week 4 was 81.8% to 90.9%; and thereafter, when comparing the percentage of week 20 to the percentage of week 8, group a showed no change from 100%, group B showed a change from 81.8% to 80.0% and was approximately the same, and group C showed a decrease from 90.9% to 33.3%. In most subjects and time points, CRP was considered to have decreased from baseline as long as serum SA237 concentration was quantifiable (0.2 μ g/mL).

(3) Efficacy

The evaluation method comprises the following steps: DAS28 (improved disease activity score based on 28 joint counts) is an indicator for assessing rheumatoid arthritis activity, which was calculated from the following equation using Tender Joint Count (TJC) and Swollen Joint Count (SJC) in 28 joints, ESR, and "patient global assessment". Changes in DAS28 were examined from the beginning of administration until the last day of observation. Summary statistics (mean, standard deviation, median, minimum and maximum) were calculated for each group and each period. In addition, clinical remission rates were calculated.

28 joints examined for DAS28

ACR 20%, 50%, 70% improvement criteria (ACR20, ACR50, ACR70) were evaluated as follows.

ACR improvement criteria

As a result: the time course of DAS28 scoring during the primary assessment period (which indicates efficacy in this examination) is shown in table 4 below.

[ Table 4]

DAS28 showed improvement at week 8. After the initial administration of different doses (at week 8) in the main evaluation period, a showed further improvement in DAS28, group B showed no significant change, and group C showed a trend back to baseline scores.

At week 8, in each group, the frequency of 20% improvement according to ACR criteria was 70.0% to 81.8%, the frequency of 50% improvement was 40.0% to 50.0%, and the frequency of 70% improvement was 18.2% to 30.0%. At week 20, the 20% improvement frequency was maintained in groups a and B, but decreased in group C, compared to week 8. At week 20, the frequency of 50% and 70% improvement increased to 72.7% (8/11 cases) and 54.5% (6/11 cases), respectively, in group a compared to week 8 values; however, no significant change was observed in groups B and C.

(4) Immunogenicity and pharmacokinetics, pharmacodynamic evaluation, efficacy and safety in antibody positivity cases

anti-SA 237 antibodies were detected in one single case each of groups B and C (i.e., 2 out of 33 total). In both cases, the serum SA237 concentration was below the lower limit of quantification during the extended period after detection of the anti-SA 237 antibody, and from the time of detection of the antibody, an increase in the soluble IL-6 receptor (sIL-6R) concentration and a decrease in CRP concentration due to SA237 administration was not observed, and DAS28, CDAI and SDAI were increased. After detection of the antibody, an adverse event, i.e. mild diabetes, was observed in one of the two cases. The adverse event is not an allergy but rather a worsening of complications. No safety issues were observed in repeated administration of SA237 to both patients after detection of the antibody.

(5) Conclusion

When 120mg of SA237 was administered to RA patients three times at 2-week intervals and then three 120mg administrations at 4-week intervals from week 8 onward, stable serum drug concentrations were maintained from week 4 to four weeks after the last administration. This resulted in high serum sIL-6R concentrations and low CRP, as well as stable improvement of all efficacy assessment programs including DAS 28. The incidence of anti-SA 237 antibodies throughout the clinical study was 6.1% (2/33 cases), and upon detection of anti-SA 237 antibodies, serum SA237 concentrations were found to decrease after detection of anti-SA 237 antibodies, but no safety issues were observed and immunogenicity was considered acceptable. Thus, there are no safety concerns in this administration regimen.

Industrial applicability

The pharmaceutical composition or regimen of the present invention can solve the problem of immunogenicity generated by anti-drug antibodies, reduce side effects, and provide a pharmaceutical composition exhibiting higher therapeutic effects with less burden on patients because it does not expose patients to high doses.

Sequence listing

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Claims (15)

1. A pharmaceutical composition for treating an IL-6-related disease, comprising an IL-6 inhibitor as an active ingredient, wherein the pharmaceutical composition is conventionally administered after a short-spaced dosing period, wherein the same dose as a conventional dose is administered a plurality of times at intervals shorter than the conventional dosing interval in the short-spaced dosing period.

2. The pharmaceutical composition of claim 1, wherein the regular dosing interval is three to five weeks.

3. The pharmaceutical composition of claim 1, wherein the regular dosing interval is four weeks.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the dosage interval during the short interval dosing period during which the dose is administered multiple times at shorter intervals than conventional dosing intervals is one to two weeks.

5. The pharmaceutical composition of any one of claims 1 to 3, wherein the dosing interval during the short interval dosing period for which the dose is administered multiple times at shorter intervals than the conventional dosing interval is two weeks.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the short interval dosing period is four weeks from initial administration.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein the conventional dose is from 50mg to 800mg per administration.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the conventional dose is 120 mg/administration.

9. The pharmaceutical composition of any one of claims 1 to 8, wherein the IL-6 inhibitor is an IL-6 receptor antibody.

10. The pharmaceutical composition of claim 9, wherein the IL-6 receptor antibody is a chimeric antibody, a humanized antibody, or a human antibody.

11. The pharmaceutical composition of claim 9, wherein the IL-6 receptor antibody comprises a heavy chain variable region having the sequence of SEQ ID No. 1 and a light chain variable region having the sequence of SEQ ID No. 2.

12. The pharmaceutical composition of claim 9, wherein the IL-6 receptor antibody comprises a heavy chain having the sequence of SEQ ID No.3 and a light chain having the sequence of SEQ ID No. 4.

13. The pharmaceutical composition of claim 9, wherein the IL-6 receptor antibody is SA 237.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the IL-6 related disease is rheumatoid arthritis, juvenile idiopathic arthritis, systemic onset juvenile idiopathic arthritis, castleman's disease, Systemic Lupus Erythematosus (SLE), lupus nephritis, crohn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, kawasaki disease, still's disease, amyloidosis, multiple sclerosis, transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, Chronic Obstructive Pulmonary Disease (COPD), IgA nephropathy, osteoarthritis, asthma, diabetic nephropathy, GVHD, endometriosis, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-hodgkin's disease, Pancreatic cancer, lung cancer, esophageal cancer, colon cancer, cancer cachexia, cancer nerve invasion, myocardial infarction, myopic choroidal neovascularization, idiopathic choroidal neovascularization, uveitis, chronic thyroiditis, delayed-type hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, panuveitis, anterior uveitis, intermediate uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry eye, post-operative inflammation, neuromyelitis optica, myasthenia gravis, or pulmonary hypertension.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the pharmaceutical composition is a formulation for subcutaneous administration.