(12) United States Patent
`Grint et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,544,504 Bl
`Apr. 8, 2003
`
`I IIIII IIIIIIII Ill lllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111
`US006544504Bl
`
`(54) COMBINED USE OF INTERLEUKIN 10 AND
`METHOTREXATE FOR IMMUNO(cid:173)
`MODULATORY THERAPY
`
`(75)
`
`Inventors: Paul C. Grint, San Diego, CA (US);
`Satwant Narula, West Caldwell, NJ
`(US)
`
`(73) Assignee: Schering Corporation, Kenilworth, NJ
`(US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 09/602,949
`
`(22) Filed:
`
`Jun.26,2000
`
`(60)
`
`(51)
`(52)
`(58)
`
`(56)
`
`Related U.S. Application Data
`Provisional application No. 60/146,022, filed on Jul. 28,
`1999.
`Int. Cl.7 ......................... A61K 38/20; AOlN 43/54
`U.S. Cl. ....................... 424/85.2; 514/256; 514/186
`Field of Search ......................... 424/85.2; 514/258,
`514/186
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`5,292,731 A *
`5,536,724 A *
`5,593,671 A
`5,753,218 A
`
`514/186
`3/1994 Love ..........................
`7/1996 DeGraw et al.
`............ 514/258
`1/1997 Kerwar et al.
`5/1998 Smith et al.
`
`Isomaki p et al. Arthritis rhem 1996, Mar 39(3):386-95.
`Interleukin-10 functions as an antiinflammtory cytokine in
`rheumatois synovium. *
`Tremaine, W.J., "The Medical Treatment of Active Crohn's
`Disease", Drugs of Today 35 (Suppl. A):89-96 (1999).
`Durez, P. et al., "Methotrexate inhibits LPS-induced tumor
`necrosis factor production in vivo", Eur. Cytokine Netw.,
`9:669 (Dec. 1998).
`Lacki, J. et al., "Circulating interleukin 10 and interleukin-6
`serum levels in rheumatoid arthritis patients treated with
`methotrexate or gold salts: Preliminary report", Inflamm.
`Res 44:24 (1995).
`Asadullah, K. et al., "IL-10 Is aKey Cytokine in Psoriasis.
`Proof of Principle by IL-10 Therapy: A New Therapeutic
`Approach", J. Clin. Invest, 101:783 (Feb. 1998).
`Kremer, J. et al., "Clinical, Laboratory, Radiographic, and
`Histopathologic Features of Methotrexate-Associated Lung
`Injury In Patients with Rheumatoid Arthritis", Arthritis &
`Rheumatism, 40:1829 (Oct. 1997).
`Opal, S.M. et al., "Interleukin-10: Potential Benefits and
`Possible Risks in Clinical Infectious Diseases", Clinical
`Infectious Diseases 27:1497, XP000915244 (1998).
`Moritani M. et al., "Prevention of Adoptively Transferred
`Diabetes in Nonbese Diabetic Mice with IL-10-Transduced
`Islet-specific Th 1 Lymphocytes" J. Clin. Invest., 98:1851,
`XP---002152055 (1996).
`Weinblatt, M. et al., "rHUIL-10 (TENOVIL) plus Methotr(cid:173)
`exate (MTX) in active rheumatoid arthritis (RA): A phase
`1/11 Study",Arthritis & Rheumatism 42:Sl 70, XP000979189
`(1999).
`International Search Report for International Application
`No. PCT/US 00/20304 dated Jan. 30, 2001, from European
`Patent Office.
`
`FOREIGN PATENT DOCUMENTS
`
`* cited by examiner
`
`WO
`WO
`WO
`
`93/18783
`98/05357
`98/24477
`
`9/1993
`2/1998
`6/1998
`
`. .. ... ... . A61K/37 /02
`......... A61K/39/395
`A61K/45/06
`
`OIBER PUBLICATIONS
`
`Opal et al. 1998. Clinical Infectious Diseases. , vol. 27: pp.
`1497-1507. Intereleukin-10: potential benefits and possible
`Risks in Clinical infectious diseases.*
`Kalden et al. 1997. Current Opinion in Rheumatlogy. vol. 9:
`pp. 206-212. Biologic agents in the treament of inflamma(cid:173)
`tory rheumatic diseases.*
`van Roon et al. Arthritis rheum 1996, May 36(5):829-35.
`Prevention and reversal of cartilage degradation in rheuma(cid:173)
`toid arthritis by interleukin-10 and interleukin-4. *
`
`Primary Examiner-Gary Kunz
`Assistant Examiner--Fozia Hamud
`
`(57)
`
`ABSTRACT
`
`A combination of interleukin 10 and methotrexate is used to
`suppress autoimmune diseases including arthritis and pso(cid:173)
`riasis. It has been discovered that administration of a com(cid:173)
`bination of interleukin 10 and methotrexate causes suppres(cid:173)
`sion of T cell proliferation. Concurrent use of both agents
`avoids the toxicity associated with higher doses of methotr(cid:173)
`exate.
`
`6 Claims, No Drawings
`
`Page 1 of 6
`
`KOIOS Exhibit 1003
`
`

`
`US 6,544,504 Bl
`
`1
`COMBINED USE OF INTERLEUKIN 10 AND
`METHOTREXATE FOR IMMUNO(cid:173)
`MODULATORY THERAPY
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a non-provisional application that
`claims the priority of provisional application U.S. Ser. No.
`60/146,022, filed Jul. 28, 1999. The Applicants' claim the
`benefits of this application under 35 U.S.C. §119(e).
`
`FIELD OF THE INVENTION
`
`The invention relates to a method for controlling autoim(cid:173)
`mune diseases, such as rheumatoid arthritis, inflammatory
`bowel disease, multiple sclerosis and psoriasis. In particular, 15
`the invention relates to the combined use of interleukin-10
`and methotrexate for immuno-modulatory therapy.
`
`2
`The following references describe the preparation of meth(cid:173)
`otrexate [see Seeger et al.,J Am.Chem.Soc., 1949, 71:1753];
`the metabolism of methotrexate [ see Freeman, J.Pharma(cid:173)
`col.Exp. Ther. 1958, 122:154; and Henderson et al., Cancer
`5 Res. 1965, 25:1008, 1018]; the toxicity of methotrexate
`[Condit et al., Cancer 1960, 13:222-249]; the pharmacoki(cid:173)
`netic models of methotrexate [Bischoff, et al., J.Pharm.Sci
`1970, 59:149]; the metabolism and pharmacokinetics of
`methotrexate [Evans,Appl.Pharmacokinet. 1980, 518-548];
`10 the clinical pharmacology of methotrexate [Bertino, Cancer
`Chemother, 1981, 3: 359-375; Jolivet et al., N.Engl.J.Med.,
`1983, 309: 1094-1104] and the clinical experience of meth(cid:173)
`otrexate in rheumatoid arthritis [J.Rheumatol., 1985, 12,
`Suppl, 12, 1-44].
`Methotrexate inhibits dihydrofolic acid reductase. Folic
`acid must be reduced to tetrahydrofolic acid by this enzyme
`in the process of DNA synthesis, repair and cellular repli(cid:173)
`cation. Therefore, methotrexate
`interferes with cellular
`reproduction.
`
`BACKGROUND OF THE INVENTION
`Interleukin 10 (IL-10), a cytokine produced by T 20
`lymphocytes, was first identified by its ability to inhibit
`interferon gamma (IFN-y) and IL-2 synthesis by mouse and
`human T lymphocytes [Fiorentino et al., 1989, J. Exp. Med.
`170:2081-2089;
`Moore
`et al.,
`1990,
`Science
`248:1230---1252; Vieira et al., 1991, Proc. Natl. Acad. Sci.
`USA 88:1172-1177]. IL-10 was subsequently shown to be
`produced by B cells [O'Garra et al., 1990, Internat. Immu(cid:173)
`nol. 2:821-828] and macrophages [Fiorentino et al., 1991, J.
`Immunol. 147:3815-3822].
`IL-10 exerts a wide range of effects on a variety of cell
`types. IL-10 inhibits the synthesis of a wide spectrum of
`cytokines produced by T cells and monocytes. In addition to
`inhibiting the synthesis of IFN-y and IL-2, IL-10 has also
`been shown to inhibit production of the monokines IL- la,
`IL-1~, IL-6 and TNFa [de Waal et al., 1991, J. Exp. Med.
`17 4: 1209-1217]. IL-10 has growth promoting effects on
`murine thymocytes and T cells [MacNeil et al., 1990,
`Immunol. 145:4167] and mast cells [Thompson-Snipes et
`al., 1991, J. Exp. Med. 173:507-512], and it stimulates 40
`cytotoxic T-cell development [Chen and Zlotnik, 1991, J.
`Immunol. 147:528-533].
`Mouse and human IL-10 have high sequence similarity
`with a protein encoded by an open reading frame in the
`Epstein- Barr Virus. The expression product of this open
`reading frame, named viral IL-10, also has the capacity to
`inhibit cytokine synthesis [Moore et al., 1990, Science
`248:1230---1252; Vieira etal., 1991, Proc. Natl. Acad. Sci.
`USA 88:1172-1177].
`Several cytokines, including IL-2, IFN-y and TNF-a,
`have been shown to regulate the mixed lymphocyte reaction
`(MLR) [Shevach, 1985,Annu. Rev. Immunol. 3:397; Fidelus
`et al., 1982, Transplantation 34:308; Tadmori et al., 1985, J.
`Immunol. 134:4542-4550; Tadmori et al, 1986, J. Immunol.
`136:1155-1162; Novelli et al., 1991, 147:1445-1450;
`Landolfo et al., 1985, Science 229:176-180; Shalaby et al.,
`It has been reported that
`988, J. Immunol. 141:499-505].
`IFN-y may play an important role in MLR graft rejection
`[Novelli et al., 1991, J. Immunol. 147:1445-1450; Landolfo
`et al., 1985, Science 229:176-180]. Antibodies to IFN-y or 60
`to TNF [Shalaby et al., 1988, J. Immunol. 141:499-505]
`have been shown to block MLR-induced proliferation. In
`these studies it was found that antibodies to IFN-y sup(cid:173)
`pressed the MLR in human systems as well as allograft
`reactivity in vitro and in vivo in the mouse.
`Methotrexate
`is known as N-[ 4-[[(2,4-diamino-6-
`pteridinyl)methyl] methylamino ]benzoyl]-L-glutamic acid.
`
`SUMMARY OF THE INVENTION
`
`25
`
`The present invention provides a method for treating
`autoimmune disease comprising administering an effective
`amount of interleukin-10 (IL-10) and methotrexate (MTX)
`to a patient afflicted with an autoimmune disease.
`This invention also provides a method for treating rheu(cid:173)
`matoid arthritis comprising administering an effective
`amount of interleukin-10 and methotrexate to a patient
`30 experiencing arthritis. Other conditions treatable by the
`method of the present invention include but are not limited
`to psoriasis, inflammatory bowel disease and multiple scle-
`ros1s.
`Pharmaceutical compositions comprising a combination
`35 of IL-10 and MTX are also provided by this invention.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`
`In order that the invention described herein may be more
`fully understood, the following detailed description is set
`forth. All references cited herein are hereby incorporated in
`their entirety by reference.
`It has unexpectedly been discovered that the combined/
`45 concurrent administration of IL-10 and MTX, or IL-10 and
`a MTX analogue, causes an unexpectedly strong suppres(cid:173)
`sion of T cell proliferation. While the invention is discussed
`herein in terms of the combined use of IL-10 and MTX, it
`is to be understood that an analogue of MTX may also be
`50 combined with IL-10 to cause synergistic suppression of T
`cell proliferation, and that such combinations are contem(cid:173)
`plated for use in the practice of this invention.
`The combination of IL-10 and MTX can be advanta(cid:173)
`geously used in the suppression of pathology associated with
`T cell responses. For example, considering the diverse
`biological activities of IL-10, the concurrent use of IL-10
`and MTX provides long term treatment of inflammatory
`bowel disease and such autoimmune diseases as rheumatoid
`arthritis. The invention may also be used to treat autoim(cid:173)
`mune diseases such as diabetes mellitus, multiple sclerosis
`and myasthenia gravis; and to treat other diseases where
`MTX has been used, such as psoriasis.
`Due to the activity of IL-10, MTX can be used in lower
`amounts, thereby avoiding or reducing the serious side
`65 effects normally associated with the use of this drug. The
`MTX/IL-10 combination therapy of the present invention is
`useful in treating patients who are non-responsive to MTX
`
`55
`
`Page 2 of 6
`
`KOIOS Exhibit 1003
`
`

`
`US 6,544,504 Bl
`
`3
`in
`is also useful
`therapy
`treatment alone. MTX/IL-10
`patients who have developed a resistance to MTX due to its
`long-term use.
`The methods of the invention can be used prophylacti(cid:173)
`cally or for treatment of established autoimmune disease.
`Individuals suitable for treatment by the methods of the
`invention include any individual at risk (predisposed) for
`developing rheumatoid arthritis, or an individual exhibiting
`clinical symptoms. Prophylactic use encompasses adminis(cid:173)
`tration prior to onset of clinical symptoms of arthritis, to
`prevent or postpone onset of disease.
`In the practice of the invention, IL-10 and-MTX are to be
`"concurrently"
`administered
`to a patient. Concurrently
`administering means the IL-10 and MTX are administered to
`the subject either (a) simultaneously in time (optionally by 15
`formulating the two together in a common carrier), or (b) at
`different times during the course of a common treatment
`schedule. In the latter case, the two compounds are admin(cid:173)
`istered sufficiently close in time to achieve the intended
`effect. The active agents may be administered together in a 20
`single pharmaceutical
`composition or separately. Both
`active agents (i.e., IL-10 and MTX) should be present in the
`patient at sufficient combined levels to be therapeutically
`effective. The routes of administration of the IL-10 and
`MTX may be the same or different. For any route of
`administration, single or divided doses may be used.
`Generally, IL-10 and MTX are administered as a phar(cid:173)
`maceutical composition comprising an effective amount of
`IL-10 and MTX in a pharmaceutical carrier. A pharmaceu(cid:173)
`tical carrier can be any compatible, non-toxic substance
`suitable for delivering the compositions of the invention to
`a patient.
`As used herein, "interleukin 10" or "IL-10" is defined as
`a protein which ( a) has an amino acid sequence substantially
`identical to a known sequence of mature (i.e., lacking a
`secretory leader sequence) IL-10 as disclosed in Interna(cid:173)
`tional Application Publication No. 91/003249, and (b) has
`biological activity that is common to native IL-10. For the
`purposes of this invention, both glycosylated ( e.g., produced
`in eukaryotic cells such as yeast or CHO cells) and ungly(cid:173)
`cosylated (e.g., chemically synthesized or produced in E.
`Coli) IL-10 are equivalent and can be used interchangeably.
`Also included are muteins and other analogs, including viral
`IL-10, which retain the biological activity of IL-10.
`IL-10 suitable for use in the invention can be obtained
`from a number of sources. For example, it can be isolated
`from culture media of activated T-cells capable of secreting
`the protein. Additionally,
`the IL-10 or active fragments
`thereof can be chemically synthesized using standard tech(cid:173)
`niques known in the art. See, e.g., Merrifield, 1986, Science
`and Atherton et al., Solid Phase Peptide
`233:341-347
`Synthesis, A Practical Approach, 1989, IRL Press, Oxford.
`Preferably,
`the protein or polypeptide
`is obtained by
`recombinant techniques using isolated nucleic acids encod(cid:173)
`ing the IL-10 polypeptide. General methods of molecular
`biology are described, e.g., by Sambrook et al., 1989,
`Molecular Cloning, A Laboratory Manual, 2d Ed., Cold
`Spring Harbor, N.Y. and Ausubel et al. (eds). Current
`Protocols in Molecular Biology, Green/Wiley, New York
`(1987 and periodic supplements). The appropriate sequences
`can be obtained using standard
`techniques
`from either
`genomic or cDNAlibraries. DNA constructs encoding IL-10
`may also be prepared synthetically by established standard
`methods, e.g., in an automatic DNA synthesizer, and then
`purified, annealed, ligated and cloned in suitable vectors.
`Atherton et al., 1989. Polymerase chain reaction (PCR)
`
`4
`techniques can be used. See e.g., PCR Protocols: A Guide to
`Methods andApplications, 1990, Innis et al, (ed.),Academic
`Press, New York.
`the entire native
`The DNA constructs may contain
`5 sequence ofIL-10 or a homologue thereof. The term "homo(cid:173)
`logue" is intended to indicate a natural variant of the DNA
`sequence encoding IL-10 or a variant or fragment produced
`by modification of the DNA sequence. Examples of suitable
`modifications of the DNA sequence are nucleotide substi-
`10 tutions which do not give rise to another amino acid
`sequence or nucleotide substitutions which do give rise to a
`different amino acid sequence and therefore, possibly, a
`different protein structure. Other examples of possible modi(cid:173)
`fications are insertions of one or several nucleotides into the
`sequence, addition of one or several nucleotides at either end
`of the sequence, or deletion of one or several nucleotides at
`either end or within the sequence. Any homologous DNA
`sequence encoding a protein which exhibits IL-10 activity
`( e.g., with respect to suppression of T cell proliferation)
`similar to that of the naive protein is contemplated for use in
`the claimed invention.
`The nucleotide sequences used to transfect the host cells
`can be modified, as described above, to yield IL-10 muteins
`and fragments with a variety of desired properties. Such
`IL-10 can vary from
`the naturally-occurring
`25 modified
`sequence at the primary level, e.g., by amino acid insertions,
`substitutions, deletions and fusions. Preferably, amino acid
`substitutions will be conservative;
`i.e., basic amino acid
`residues will be replaced with other basic amino acid
`30 residues, etc. These modifications can be used in a number
`of combinations to produce the final modified protein chain.
`Amino acid sequence variants can be prepared with
`various objectives
`in mind,
`including
`increasing serum
`half-life, facilitating purification or preparation, improving
`35 therapeutic efficacy, and lessening the severity or occurrence
`of side effects during therapeutic use. The amino acid
`sequence variants are usually predetermined variants not
`found in nature, although others may be post-translational
`variants, e.g., glycosylation variants or proteins which are
`40 conjugated to polyethylene glycol (PEG), etc. Such variants
`can be used in this invention as long as they retain the
`biological activity of IL-10.
`Preferably, human IL-10 is used for the treatment of
`humans, although viral or mouse IL-10, or IL-10 from some
`45 other mammalian species, could be used instead. Most
`preferably, the IL-10 used is recombinant human IL-10.
`Recombinant production of human IL-10 is described in
`U.S. Pat. No. 5,231,012. Preparation of human and mouse
`IL-10 has been described in International Application Pub-
`50 lication No. WO 91/00349. The cloning and expression of
`viral IL-10 (BCRFI protein) from Epstein Barr virus has
`been disclosed by Moore et al. [Science 248:1230, 1990],
`and is described in EP O 506 836.
`Administration ofIL-10 is preferably parenteral by intra-
`55 peritoneal intravenous, subcutaneous or intramuscular injec(cid:173)
`tion or infusion or by any other acceptable systemic method.
`Administration by intramuscular or subcutaneous injection
`is most preferred. Alternatively, the IL-10 may be adminis(cid:173)
`tered by an implantable or injectable drug delivery system.
`60 See, e.g., Urquhart et al, 1984, Ann Rev. Pharmacol. Toxicol
`24:199; Lewis, ed., 1981, Controlled Release of Pesticides
`and Pharmaceuticals, Plenum Press, New York, N.Y.: U.S.
`Pat. Nos. 3,773,919, and 3,270,960. Oral administration
`may also be carried out, using well known formulations
`65 which protect the IL-10 from gastrointestinal proteases.
`Compositions useful for parenteral administration of such
`drugs are well known. See, e.g., Remington's Pharmaceu-
`
`Page 3 of 6
`
`KOIOS Exhibit 1003
`
`

`
`US 6,544,504 Bl
`
`5
`tical Science, 11th Ed., 1990, Mack Publishing Co., Easton,
`Pa. When administered parenterally, the IL-10 is typically
`formulated
`in a unit dosage
`injectable
`form (solution,
`suspension, emulsion) in association with a pharmaceutical
`carrier. Examples of such carriers are normal saline, Ring(cid:173)
`er's solution, dextrose solution, and Hank's solution. Non(cid:173)
`aqueous carriers such as fixed oils and ethyl oleate may also
`be used. A preferred carrier is 5% dextrose/saline. The
`carrier may contain minor amounts of additives such as
`substances that enhance isotonicity and chemical stability,
`e.g., buffers and preservatives. The IL-10 is preferably
`formulated in purified form substantially free of aggregates
`and other source proteins at a concentration in the range of
`about 100-2000 mg/ml. Any of the well known carrier
`proteins such as human serum albumin can also be added if
`desired.
`IL-10 can also be delivered by standard gene therapy
`techniques, including e.g., direct DNA injection into tissues,
`the use of recombinant viral vectors or phospholipid and
`implantation oftransfected cells. See, e.g., Rosenberg, 1992,
`J. Clin. Oneal. 10:180.
`MTX may be administered in a manner as is convention(cid:173)
`ally practiced. See, e.g., Goodman and Gilman's The Phar(cid:173)
`macological Basis of Therapeutics, 7th Ed, 1985, p. 1299.
`For example, methotrexate may be orally administered with
`an inert diluent or with an assimilable edible carrier, or it
`may be enclosed in hard or soft shell gelatin capsules, or it
`may be compressed into tablets, or it may be incorporated
`directly with the food of the diet. For oral therapeutic
`administration, methotrexate may be incorporated with
`excipients and used in the form of ingestible tablets, buccal
`tablets, troches, capsules, elixers, suspension, syrups, wafer,
`and the like. Such compositions and preparations should
`contain at least 0.5% of methotrexate. The percentage of the
`compositions and preparations may, of course, be varied and
`may conveniently be between about 2 to 60% of the weight
`of the unit. The amount of methotrexate in such therapeu(cid:173)
`tically useful compositions is such that a suitable dosage will
`be obtained. Preferred compositions or preparations accord(cid:173)
`ing to the present invention are prepared so that an oral
`dosage unit form contains between 0.025 and 35 mg of
`methotrexate.
`The tablets, troches, pills, capsules and the like may also
`contain the following: a binder, such as gum tragacanth,
`acacia, corn starch or gelatin; excipients such as dicalcium(cid:173)
`phosphate; a disintegrating agent such as corn starch, alginic
`acid and the like; a lubricant such as magnesium stearate;
`and a sweetening agent such as sucrose, lactose or saccharin
`may be added or a flavoring agent such as peppermint, oil of
`wintergreen or cherry flavoring. When the dosage unit form
`is a capsule, it may contain, in addition to material of the
`above type, a liquid carrier. Various other materials may be
`present as coating or to otherwise modify the physical form
`of the dosage unit. For instance, tablets, pills, or capsules
`may be coated with shellac, sugar or both. A syrup or elixer 55
`may contain methotrexate, sucrose as a sweetening agent,
`methyl and propylparabens as preservative, a dye and fla(cid:173)
`voring such as cherry or an orange flavor. Of course, any
`material used in preparing any dosage unit form should be
`pharmaceutically pure and substantially non-toxic in the 60
`amounts employed. In addition, methotrexate may be incor(cid:173)
`porated
`into sustained-release preparations and formula(cid:173)
`tions.
`Methotrexate may also be administered parenterally or
`intraperitoneally. Solutions of methotrexate can be prepared 65
`in glycerol,
`liquid polyethylene glycols, and mixtures
`thereof and in oils. Under ordinary conditions of storage and
`
`6
`use, these preparations contain a preservative to prevent the
`growth of microorganisms.
`The pharmaceutical
`forms suitable for injectable use
`include sterile aqueous solutions or dispersions and sterile
`5 powders
`for the extemporaneous preparation of sterile
`injectable solutions or dispersions. In all cases, the form
`must be sterile and must be fluid to the extent that easy
`syringability exists. The form must be stable under the
`conditions of manufacture and storage and must be pre-
`10 served against the contamination action of microorganisms
`such as bacteria and fungi. The carrier can be a solvent or
`dispersion medium containing, for example, water, ethyl
`alcohol, polyol (for example, glycerol, propylene glycol, and
`liquid polyethylene glycol and the like), suitable mixtures
`15 thereof, and vegetable oils. The proper fluidity can be
`maintained, for example, by the use of a coating such as
`lecithin, by the maintenance of the required particle size in
`the case of dispersion and the use of surfactants. The
`prevention of the action of microorganisms can be brought
`20 about by various antibacterial and antifungal agents, for
`example, parabens, chlorobutanol, phenol, sorbic acid,
`thimerosal and the like. In many cases, it will be preferable
`to include isotonic agents, for example, sugars or sodium
`chloride. Prolonged absorption of the injectable composi-
`25 tions can be brought about by the use in the compositions of
`agents delaying
`absorption,
`for example,
`aluminum
`monostearate and gelatin.
`Sterile injectable solutions are prepared by incorporating
`methotrexate
`in the required amount in the appropriate
`30 solvent with various of the other ingredients enumerated
`above, as required,
`followed by filtered sterilization.
`Generally, dispersions are prepared by incorporating meth(cid:173)
`otrexate
`into a sterile vehicle which contains
`the basic
`dispersion medium and the required other ingredients from
`35 those enumerated above. In the case of sterile powder, for
`the preparation of sterile injectable solutions, the preferred
`methods of preparation are vacuum drying and the freeze(cid:173)
`drying technique which yield a powder of methotrexate, plus
`any additional desired ingredient from a previously sterile
`40 filtered solution thereof.
`As used herein, "pharmaceutically acceptable carriers"
`includes any and all solvents, dispersion media, coating,
`antibacterial and antifungal agents, isotonic and absorption
`45 delaying agents and the like. The use of such media and
`agents for pharmaceutically active substances is well known
`in the art. Except insofar as any conventional media or agent
`is incompatible with the active ingredient, its use in the
`therapeutic compositions
`is contemplated. Supplementary
`50 active ingredients can also be incorporated into the compo(cid:173)
`sitions.
`It is especially advantageous to formulate parenteral com(cid:173)
`positions in dosage unit form for case of administration and
`uniformity of dosage. Dosage unit form as used herein refers
`to physically discrete units suited as unitary dosages for the
`mammalian subjects to be treated; each unit containing a
`predetermined quantity of active material calculated to pro(cid:173)
`duce the desired therapeutic effect in association with the
`required pharmaceutical carrier.
`Methotrexate is compounded for convenient and effective
`administration in effective amounts with a suitable pharma(cid:173)
`ceutically acceptable carrier in dosage unit form as herein(cid:173)
`tofore disclosed. A unit dosage form can, for example,
`contain methotrexate in amounts ranging from about 0.1 to
`400 mg, with from 1 to 35 mg being preferred, and 10 to 25
`being most preferred. Expressed in proportions, methotrex(cid:173)
`ate is generally present in from about 0.1 to about 40 mg/ml
`
`Page 4 of 6
`
`KOIOS Exhibit 1003
`
`

`
`US 6,544,504 Bl
`
`7
`of carrier. In the case of compositions containing supple(cid:173)
`mentary active ingredients, the dosages are determined by
`reference to the usual dose and manner of administration of
`said ingredients.
`A single intravenous dosage, slow constant infusion, or
`repeated daily dosages can be administered. Daily dosages
`up to about 1 to 10 days are often sufficient. It is also
`possible to dispense one daily dosage or multiple daily doses
`or one dose on alternate or less frequent days. As can be seen
`from the dosage regimens,
`the amount of methotrexate
`administered is to be sufficient to relieve the autoimmune
`disease symptoms prevalent in diseases such as arthritis and
`psoriasis.
`IL-10 and MTX are concurrently administered to a human
`patient in an amount effective to provide an immunosup(cid:173)
`pressive effect. As used herein "effective amount" means an
`amount sufficient to reduce or prevent rheumatoid arthritis,
`an autoimmune disease or psoriasis, and refers to the com(cid:173)
`bined effects of the two agents working in concert. One or
`both agents may, for example, be used at a dose which, if
`used alone, would be considered suboptimal for the intended
`purpose.
`Based on the judgment of the clinician, the amount of
`IL-10 and/or MTX will, of course vary. The effective
`amount for a particular patient will depend on such factors
`as the overall health and age of the patient, the route of
`administration, the severity of observed side-effects, and the
`like. The effective dose of IL-10 typically will range from
`about 0.1-100 µg/kg/day, preferably about 1-20 µg/kg/day
`in a single or divided doses. More preferably, the effective
`dose of IL-10 will be 8 µg/kg three times a week [TIW], 8
`µg/kg daily or 20 µg/kg TIW. The effective dose of MTX
`typically range from about 1-100 mg/week, more preferably
`from about 5-35 mg/week, and most preferably from about
`10-25 mg/week. The length of administration may vary and, 35
`in some cases, may continue over the remaining lifetime of
`a patient, to control autoimmune symptoms or graft rejection
`processes.
`
`8
`therapy given daily or TIW plus MTX (oral/intramuscular/
`SC) over a 28 day period to patients with active rheumatoid
`arthritis. The secondary objectives were to evaluate the
`effect of IL-10 on measures of rheumatoid arthritis Disease
`5 Activity, and to determine changes in the circulating levels
`of soluble p55 and p75 TNF receptors and IL-1 receptor
`antagonist. Protocol-defined
`responders were defined as
`those patients with at least 20% ACR criteria, i.e. at least
`20% improvement
`in number of tender joints, number of
`10 swollen joints and in at least 3 of 5 RA Disease Activity
`measures (i.e. subject's assessment of pain, disease activity
`or physical function and physician's global assessment of
`disease activity.
`Fifty patients were enrolled and sequentially randomized
`15 to receive one of the five dosing regimens of IL-10 (SC) (1
`µg!kg daily, 4 µg!kg daily, 8 µg!kg TIW, 8 µg!kg daily and
`20 µg/kg TIW) or placebo which formed the intent-to-treat
`population (ITT). Mean duration of treatment was at least 26
`days for each of the treatment groups. The treatment groups
`20 were similar in demographic characteristics except for slight
`differences in age. Baseline characteristics of RA Disease
`Activity were similar for treatment groups.
`IL-10 was generally well tolerated. No anti-dsDNA or
`anti IL-10 antibodies were present at any time during the
`25 study. The most frequently reported adverse events were
`headache, injection site reaction, nausea, musculoskeletal
`pain, with no dose-response relationship seen.
`Protocol-defined response was evaluated after 28 days of
`30 dosing versus baseline for the ITT population. Results
`showed a trend toward a greater percentage of responders in
`patients
`treated with IL-10 compared with the placebo
`group. Similar trends were seen for mean change from
`baseline for individual clinical measures of rheumatoid
`arthritis disease activity, with IL-10 treatment groups gen(cid:173)
`erally showing a greater percentage of responders than in
`placebo group. The percent of patients having a 20%
`improvement
`in disease activity (ACR 20) and that of
`patients having a 50% improvement
`in disease activity
`40 (ACR 50) was higher for each of the IL-10 treatment groups
`than for the placebo group, with the higher dose groups (8
`µg!kg TIW, 8 µg/kg daily and 20 µg!kg TIW) showing the
`highest percent of both 20 ACR and 50 ACR responders. A
`trend towards decreased production of ex-vivo
`induced
`45 proinflammatory cytokines (TNFa and IL-1~) and a trend
`towards increased circulating serum levels of soluble TNF
`p55 and TNF p75 receptors and IL-1 receptor antagonists
`occurred in nearly all IL-10 treatment groups compared with
`placebo.
`The following conclusions can be drawn from this study.
`IL-10, in combination with stable dosing of MTX, was safe
`and well tolerated in patients with active rheumatoid arthri(cid:173)
`tis. Trends indicate that IL-10 in combination with MTX
`may have beneficial effects on rheumatoid arthritis Disease
`Activity. This effect was greatest for the 8 µg/kg TIW, 8
`µg!kg daily and 20 µg/kg TIW IL-10 dosing regimens. The
`dosing regimen which maximizes safety and efficacy results
`is 8 µg/kg IL-10 TIW.
`Many modifications and variations of this invention can
`be made without departing from its spirit and scope, as will
`be apparent to those skilled in the art. The specific embodi(cid:173)
`ments described herein are offered by way of example only,
`and the invention
`is to be limited by the terms of the
`appended claims, along with the full scope of equivalents to
`65 which such claims are entitled; and the invention is not to be
`limited by the specific embodiments
`that have been pre(cid:173)
`sented herein by way of example.
`
`EXAMPLE 1
`
`Safety and Tolerance Study of IL-10 in
`Combination with a Stable Dosing Regimen of
`MTX in Patients with Active Rheumatoid Arthritis
`A multinational, multicenter, sequentially randomized,
`double-blind, placebo-controlled, rising multiple-dose study
`of IL-