l||||||||||||||||||l|||||||||||||||||||||||||||lllll|||||||||||||||||||||||
`
`USll{l65445U4B1
`
`(12) United States Patent
`Grint et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 6,544,504 B1
`Apr. 8, 2003
`
`lsomaki p el al. Arthritis rhem 1996, Mar 39{3):f-386-95.
`ln1erleukin—l[l functions as an antiinflammtory cytokine in
`rheurnalois synovium.*
`Tremaine, W.J., “The Medical Treatment of Active Crohn‘s
`Disease”, Drugs of Tbdrry 35 (Suppl. A):89—96 (1999).
`Durex, P. et al., “Mclhotrexate inhibits I.PS—induced tumor
`necrosis factor production in vivo", Era: Cytokirtc Norm,
`9:669 (Dec. 1998).
`l.acki,J. ct al., “Circulating interleukin ID and iI‘tterleukin—6
`serum levels in rheumatoid arthritis patients treated with
`melltolrexale or gold salts: Preliminary report", lrtflrmmi.
`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’. Ciin. Irtve.s‘.', 1012783 (Feb. 1998}.
`Kremcr, .1. el al., “Clinical, Laboratory. Radiographic. and
`I1 istopathologic Features of Metho1rexate—Associatcd Lung
`Injury ln Patients with Rheurnatoirl Arthritis", Am'tr:'Iis &
`Rftettrrtarisrri, 402182‘) (Oct. 199?].
`Opal, SM. et al., “Interleukin—l0: Potential Benefits and
`Possible Risks in Clinical Infectious Diseases”, CIirticni
`Irtfectiorts i')iserrse.s 27:1-197, XP(l(lU91524-’l {I998}.
`Moritani M. et al., “Prevention of Adoplively Transferred
`Diabetes in Nonbese Diabetic Mice with II_—l0—'1‘ransduced
`lslel—specific Th 1 I..yrnphoeytes” J. Cfiri. :'niresr., 98:l85'l,
`Xl’—UG2l52U55 (1996).
`Weinhlatt, M. el al., “rHUIl..-I0 (TENOVIL) plus Metho1r-
`exate (MTX) in active rheumatoid arthritis (RA): A phase
`lill Stu:lv",ArrJtriri.s' & Ri:ewnrm‘.sm 42:Sl7'[l, XPO(l(I979 1 89
`(1999).
`International Search Report for International Appiieation
`No. l’(7T;’US 0(l.'2t)3(|4 dated Jan. 311, 2001, from European
`Patent Otfice.
`
`* cited by examiner
`
`Prirrmr_v Exnrm'ncr—(}ary Kunz
`.’\$.s'£St‘m'tt‘ E.rrmziner—Fozia llamud
`
`ABSTRACT
`(57)
`Acombination of interleukin 10 and methotrex-ate is used to
`suppress autoimmune diseases including arthritis and pso-
`riasis. It has been discovered that administration of a eornw
`hination of interleukin 10 and metholrcxale causes suppres-
`sion of '1' cell proliferation. Concurrent use of both agents
`avoids the toxicity associated with higher doses of melholr-
`exale.
`
`6 Claims, No Drawings
`
`(54) COMBINE!) USE 01*” lNTERl.l£Ul(lN ll] AND
`METHOTREXATE FOR IMMUNO-
`MODULATORY THERAPY
`
`(75)
`
`Inventors: Paul C. Grint, San Diego. CA (US);
`Satwant Nnrula, West Caldwell, NJ
`(US)
`
`(73) Assignee: Seltering 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 0 days.
`
`(21) Appl. No.: 09;6o2,949
`
`(22)
`
`Filed:
`
`Jun. 26, 2000
`
`(60)
`
`Related U.S. Application Data
`Provisional application No. eor14e.022.
`tiled on Jul. 28.
`1999.
`
`A61l( 38:20; AOIN 43354
`42411852; 514.1256; 514.3186
`424l"85.2; 514.-‘Z58,
`514E186
`
`Int. Ct.‘
`(51)
`(52) U.S. Cl.
`(58)
`Flteld of Search
`
`
`
`(56)
`
`References Cited
`U.S. l’A'I‘l:IN’I' DOCUMl:}N'l‘S
`
`5,292,731 A '°‘
`5,536,724 A *
`5,593,671 A
`5_.7"53.2|8 A
`
`3.31.994 Love
`'i'l’l99t’)
`l')e(}raw el al.
`U199‘.-' Kerwar et al.
`5;"l9‘J8 Sltiilli at al.
`
`514E186
`.......... .. 514E258
`
`l“ORI_ilCiN 1’/\Tl:'.N'l‘ DOCUMl:iN'l'S
`
`W0
`W0
`W0
`
`93.-’18'.-'83
`9Si"|'J535'.-'
`98.04477
`
`W1993
`2.31998
`M1998
`
`........ .. A<':tlK."3T;"CI2
`At’)IK4‘39.-“S95
`........ .. Af)lK,-"45x'fI6
`
`OTHER PUBLI CATIONS
`
`()pal et al. 1998. Clinical Infectious Diseases. , vol. 27: pp.
`1497-1507. inlcrclcukin—1(l: potential bcnellts and possible
`Risks in Clinical infectious diseases.*
`Kalden et al. 19.97. Current Opinion in Rheumatlogy. vol. 9:
`pp. 206-212. Biologic agents in the treantent of inflamma-
`tory rheumatic diseases.*
`van Roon et al. Arthritis rlteum I996, May 36(5):829—35.
`Prevention and reversal of cartilage degradation in rheuma~
`told arthritis by interleukin—l0 and interleukin—4.*
`
`MEDAC Exhibit 2008
`
`ANTARES v. MEDAC
`
`IPR2014-01901
`
`Page 00001
`
`MEDAC Exhibit 2008
`ANTARES v. MEDAC
`IPR2014-01901
`Page 00001
`
`

`
`US 6,544,504 B1
`
`1
`COMBINED USE OF IN'I‘ERI.EUKlN 10 AND
`MI‘l'I‘HOTREXATF. FOR IMMUNO-
`M()l)UI.A'I‘0RY 'l‘I-IlCR!\l’Y
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`This application is a non-provisional application that
`claims the priority of provisional application U.S. Ser. No.
`60,»'l46,022, lilcd Jul. 28, I999. 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-
`mune diseases, such as rheumatoid arthritis, inflammatory
`bowel disease, multiple sclerosis and psoriasis. In particular,
`the invention relates to the combined use of interleukin-10
`and methotrexate for immuno-modulatory therapy.
`BACKGROUND OF THE INVENTION
`
`Interleukin 10 (IL-10), a eytokine produced by T
`iymp!t0cytc.s, was first
`identified by its ability to inhibit
`interferon gamma (IFN-7) and IL-2 synthesis by mouse and
`human Tiyrttpiiocjvtes [Fiorentino et al., 1989, J. Exp. Med.
`17'O:2081—2{J89; Moore et al., 1990, Science
`248:l230—1252; Vicira et a1., 1991, Proc. Natl. Aerrd. Sci.
`USA 88:1 172-1177]. IL-10 was subsequently shown to be
`produced by B cells [0’Garra et al., 1990, Internet. [minte-
`not. 21821-828] and macrtophages [Fiorentino et al., l99l,J'.
`Imrttrinoi. [47:38 I 5-3822].
`1L-10 exerts a wide range of elIeets on a variety of cell
`types. IL-10 inhibits the synthesis of a wide spectrum of
`cytokines produced by '1‘ cells and monocytes. In addition to
`inhibiting the synthesis of ll~'N-y and IL-2, IL-10 has also
`been shown to inhibit production of the monokines ll..—I(J'.,
`IL-lfi, IL-6 and TNFa [de Waal et al.. 1991, J. Exp. Med.
`1?'4:I209—t217]. IL-l0 has growth promoting effects on
`murine thymocytcs and T cells [MacNeil et al., 1990,
`Immnnol. 145:4-167] and mast cells [Thompson-Snipes et
`al., 199], J’. Exp. Med.
`l73:507-512], and it stimulates
`cytotoxic T-cell development [Chen and Zlotnik, 1991, J’.
`I.-mttwtoi. 147:528—53 3].
`Mouse and human IL-10 have high sequence similarity
`with a protein encoded by an open reading frame in the
`ljpstein-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, Pmc. Nan’. Acad. Sci.
`USA 88:ll'r'2-1177].
`IFN-1' and TNF-oi,
`including II.-2.
`Several cytokines,
`have been shown to regulate the mixed lymphocyte reaction
`(MLR) [Shevach, 1985, Anna. Ret-: Irmnttrtof. 3:397; Fidelus
`et al., 1982, Irrrnaplnnrrtriori 34-:308; Tadmori et al., 1985, J.
`Irrirmtrtol. 134:4542—4-550; Tadmori et al, 1986, J , hmutmoi.
`136:11S5—l162; Novelli et al., 1991, 147:14-45-1450;
`Landolfo et al., 1985, Sciertce 229:176—180; Shalaby et al.,
`988, J. Imrmrrrol.
`l41:499-505]. It has been reported that
`IFN-y may play an important role in MLR graft rejection
`[Novellie1al., 199l,J’. Immurtoi. 147214-45-1450; Landolfo
`et al., 1985, Science 229:1'?6—l80]. Antibodies to lI~'N—y or
`to TNF [Shalahy et al., 1988, J. Imrmtrtoi. I4}:-I99-505]
`have been shown to block MLl{-induced proliferation. In
`these studies it was found that antibodies to Il'~‘N—y sup-
`pressed the MLR in human systems as well as allogralt
`reactivity in vitro and in vivo in the mouse.
`
`
`
`SUMMARY 017 THE INVENTION
`
`treating
`invention provides a method for
`The present
`autoimmune disease comprising administering an elfective
`amount of interleukin-10 (IL-10) and methotrexate {M‘t'X)
`to a patient alllicted with an autoimmune disease.
`This invention also provides a method for treating rheu-
`matoid arthritis comprising administering an elfective
`amount of interleukin-10 and methotrexate to a patient
`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-
`rosis.
`
`ll!
`
`15
`
`“
`
`30
`
`35
`
`Pharmaceutical compositions comprising a combination
`of IL-10 and MTX are also provided by this invention.
`
`DETAILED DESCRIPTION OF THE
`INVILNTION
`
`40
`
`45
`
`50
`
`_
`
`(i0
`
`05
`
`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!
`concurrent administration of IL-10 and MTX, or IL-10 and
`a MTX analogue, causes an unexpectedly strong suppres-
`sion ol’T cell proliferation. While the invention is discussed
`herein in tenns of the combined use of IL-10 and MTX, it
`is to be understood that an analogue of MTX may also be
`combined with IL-10 to cause synergistic suppression of T
`cell proliferation, and that such combinations are contem-
`plated for use in the practice of this invention.
`The combination of IL-10 and MTX can be advanta-
`geously used in the suppression of pathology associated with
`'l' cell
`responses. For example, considering the diverse
`biological activities of IL-10, the concurrent use of IL-10
`and MTX provides long tentt
`treatment of inflammatory
`bowel disease and such autoimmune diseases as rheumatoid
`arthritis. The invention may also be used to treat autoim-
`mune diseases such as diabetes mellitus, multiple sclerosis
`and myasthenia gravis; and to treat other diseases where
`M't'X 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
`effects normally associated with the use of this drug. The
`M'I'X;'II_-10 combination therapy of the present invention is
`useful in treating patients who are non-responsive to MTX
`
`Page 00002
`
`Page 00002
`
`

`
`US 6,544,504 B1
`
`ll!
`
`15
`
`30
`
`35
`
`40
`
`3
`in
`treatment alone. MTXHI.-I0 therapy is also useful
`patients who have developed a resistance to MTX due to its
`long-term use.
`The methods of the invention can be used prophylacti-
`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-
`tration prior to onset of clinical symptoms of arthritis, to
`prevent or postpone onset of disease.
`In the practice of the invention, II--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
`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-
`istered sutficiently close in time to achieve the intended
`effect. The active agents may be administered together in a
`single pharmaceutical composition or separately. Both
`active agents (i.e., IL-10 and MTX) should be present in the
`patient at suflicicnt 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-
`maceutical oomposition comprising an effective amount of
`ll.-l0 and MTX in a pharmaceutical carrier. A pharmaceu-
`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.c., lacking a
`secretory leader sequence) IL-l0 as disclosed in Interna-
`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 CIID cells) and ungly-
`cosylated (e.g., chemically synthesized or produced in E.
`Coli} ll.-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-
`niques known in the art. See, e.g., Merrifield, 1986, Scirmce
`2332341-347 and Athcrton et al., Solid Pltase Peptide
`Syrttltest‘s, A Practical Approach. i989, IRI. Press, Oxford.
`Preferably,
`the protein or polypeptide is obtained by
`recombinant techniques using isolated nucleic acids encod-
`ing the IL-10 polypeptide. General methods of molecular
`biology are described, e.g., by Sambrook et al., 1989,
`Molecular Clotting, A Laboratory Manual, 2d Ed., Cold
`Spring I-larbor, N.Y. and Ausubel ct al.
`(eds). Current
`Protocols in Molecular Biology, Grecnlwiley, New York
`(' 1987 and periodic supplements). The appropriate sequences
`can be obtained using standard techniques from either
`genomic or cDNA libraries. DNA eonst ructs 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)
`
`45
`
`50
`
`55
`
`60
`
`65
`
`4
`techniques can be used. See e.g., PCR Protocols.'A Guide to
`Methods’ nndApplt'catt'ort.s', 1990, Innis et al, {ed.), Academic
`Press, New York.
`The DNA constructs may contain the entire native
`sequence of IL-10 or a homologue thereof. The term "homo-
`loguc” 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-
`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-
`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 II.-10 activity
`(e.g., with respect
`to suppression of '1' 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 mortified, as described above, to yield IL-10 muteins
`and fragments with a variety of desired properties. Such
`moditled IL-10 can vary from the naturally-occurring
`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
`residues, etc. These modifications can be used in a number
`ofcombinations to produce the final modified protein chain.
`Amino acid sequence variants can be prepared with
`various objectives in mind,
`including increasing serum
`halt‘-lite, facilitating purification or preparation, improving
`therapeutic eficacy, 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
`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 ll.-10 from some
`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
`II.-10 has been described in lntemational Application Pub-
`lication No. W0 9ll00349. The cloning and expression of
`viral ll.-10 (BCRFI protein} from Epstein Barr virus has
`been disclosed by Moore et al. [Science 24811230. 1990],
`and is described in EP 0 S06 836.
`
`Administration of IL-10 is preferably parenteral by intra-
`peritoneal intravenous, subcutaneous or intramuscular injec-
`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-
`tered by an implantable or injectahle drug delivery system.
`See, e.g., Urquhart et al. 1984, Ann Rex: Pltartttrtcol. loxfcol
`24:l99; Lewis. ed., t98l, Controlled Release of Pesticides
`and Plmrmrtceuticals, 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
`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 00003
`
`Page 00003
`
`

`
`US 6,544,504 B1
`
`5
`tical Science, 11th E.d., 1990. Mack Publishing Co., Easton,
`Pa. When administered parenterally, the IL-l.U is typically
`formulated in a unit dosage injectable form (solution,
`suspension, emulsion) in association with a pharmaceutical
`carrier. Examples of such carriers are nonnal saline, Ring-
`er's solution, dextrose solution, and Hank’s solution. Non-
`aqueous carriers such as flxed oils and ethyl oleate may also
`be used. A preferred carrier is 5% dcxtrosetsaline. The
`carrier may contain minor amounts of additives such as
`substances that enhance isotonicity and chemical stability,
`e.g., bulliers 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 mgtml. 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 oflransfected cells. See, e.g.. Rosenberg, 1992,
`J. Clin. Oncol.
`l(l:18(t.
`
`ill
`
`6
`use, these preparations contain a preservative to prevent the
`growth of microorganisms.
`injectable use
`The pharmaceutical
`forms suitable for
`include sterile aqueous solutions or dispersions and sterile
`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-
`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 l.i.ke), suitable mixtures
`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
`about by various antibacterial and antifungal agents, for
`example, parabens. chlorobutanol, phenol, sorbic acid,
`thirnerosal 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-
`tions can be brought about by the use in the compositions of
`agents delaying absorption,
`for example, aluminum
`monostearate and gelatin.
`Sterile injectahie solutions are prepared by incorporating
`methotrexate in the required amount
`in the appropriate
`solvent with various of the other ingredients enumerated
`above, as required,
`followed by filtered sterilization.
`Generally, dispersions are prepared by incorporating meth-
`otrexate into a sterile vehicle which contains the basic
`dispersion medium and the required other ingredients from
`those enumerated above. In the case of sterile powder, for
`the preparation of sterile injectable solutions, the prefened
`methods of preparation are vacuum drying and the freeze-
`drying technique which yield a powder of rnethotrexate, plus
`any additional desired ingredient from a previously sterile
`filtered solution thereof.
`
`As used herein, “pharmaceutically acceptable carriers”
`includes any and all solvents. dispersion media, coating,
`antibacterial and antifungal agents, isotonic and absorption
`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
`active ingredients can also be incorporated into the compo-
`sitions.
`
`It is especially advantageous to formulate parenteral com»
`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-
`duee the desired therapeutic elfeet in association with the
`required pharmaceutical carrier.
`Methotrexate is compounded for convenient and effective
`administration in elfective amounts with a suitable pharma-
`ceutically acceptable carrier in dosage unit form as herein-
`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 lit to 2'5
`being most preferred. Expressed in proportions, methotrex—
`ate is generally present in from about 0.1 to about 40 mgfml
`
`Page 00004
`
`MTX may be administered in a manner as is convention-
`ally practiced. Sec, e.g., Goodman and Gilman‘s The Phar-
`macological Basis of Therapeutics, 'r'th Ed, 1985, p. 1399.
`For example, methotrexate may be orally administered with _
`an inert diluent or with an assintilablc 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, methotrexatc 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 (1.5% 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-
`tically useful compositions is such that a suitable dosage will
`be obtained. Preferred compositions or preparations accord-
`ing to the present
`invention are prepared so that an oral
`dosage unit form contains between 0.025 and 35 mg of
`methotrexate.
`
`30
`
`35
`
`40
`
`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-
`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. Asyrup or elixer
`may contain methotrexate, sucrose as a sweetening agent,
`methyl and propylparabens as preservative, a dye and fla-
`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
`amounts employed. In addition, metholrexate may be incor-
`porated into sustained-release preparations and formula-
`tions.
`
`Methotrexate may also be administered parenterally or
`intraperitoneally. Solutions of methotrexate can be prepared
`in glycerol,
`liquid polyethylene glycols. and mixtures
`thereof and in oils. Under ordinary conditions of storage and
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`US 6,544,504 B1
`
`Ill
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`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—
`pressivc elfect. As used herein “elfective amount" means an
`amount sufficient to reduce or prevent rheumatoid arthritis,
`an autoimmune disease or psoriasis, and refers to the com-
`bined effects of the two agents working in concert. One or
`both agents may, for example, be used at a dose which, it‘
`used alone, would be considered suboptimal for the intended
`purpose.
`
`ill
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`15
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`'
`
`Based on the judgment of the clinician, the amount of
`IL-10 andfor 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-etfects, and the
`like. The effective dose of IL-10 typically will range from
`about 0,l—10(I grgtkgtday, preferably about 1—20 gtgfltgtday
`in a single or divided doses. More preferably, the effective
`dose of IL-10 will be 8 gtgfleg three times a week [TIW], 8
`lrtgfkg daily or 20 pgfkg TIW. The cfiective dose of MTX
`typically range from about 1-10!) mgtweek, more preferably
`from about 5-35 mglweek, and most preferably from about
`10-25 mgxweek. The length of administration may vary and,
`in some cases, may continue over the remaining lifetime of
`a patient, to control autoimmune symptoms or graft rejection
`processes.
`
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`35
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`EXAMPLE 1
`
`Safety and Tolerance Study of IL-10 in
`Combination with a Stable Dosing Reqimcn of
`MTX in Patients with Active Rheumatoid Arthritis
`
`A multinational, multicenter, sequentially randomized,
`double-blind, placebo-controlled, rising multiple—dose study
`of IL-10 plus methotrexate (MTX) treatment was completed
`in patients with active rheumatoid arthritis.
`Fifty patients were to receive one of live dosing regimens
`of IL-10 (SC) ('1 ,ugx'i<g daily, 4 ,ug,t'kg daily, 8 ,t¢g.t'kg three
`times a week [TIW], 8 ;rg,r’lcg daily and 20 gigfkg TIW) or
`placebo for 28 days, in addition to stable dosing with MTX
`(Treatment Phase). The patients were followed for 8 weeks
`after the end of IL-10 dosing (Follow-up Phase). Patients
`received MTX at therapeutic doses for at least 4 months
`prior
`to study entry. The dose of MTX was 125-25
`Irtgtweek (oral, subcutaneous or
`intramuscular) and
`remained constant throughout the study (Screening, Treat-
`ment and Follow-up Phases).
`Patients were sequentially enrolled into the study in dose
`cohorts starting with the lowest dose of IL-10. Safety was
`assessed for each dose level prior to progressing to the next
`higher dose. Ten patients were assessed at each ofthe IL-10
`dose cohorts: 8 received IL-10 and 2 received placebo (4: 1).
`There was no replacement of patients.
`in a dose-
`The primary objective was to evaluate,
`escalating manner, the safety and tolerance of IL-10 (SC)
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`8
`therapy given daily or TIW plus MTX (oralfiritramuscularf
`SC) over a % day period to patients with active rheumatoid
`arthritis. The secondary objectives were to evaluate the
`elfect of IL-10 on measures of rheumatoid arthritis Disease
`Activity, and to determine changes in the circulating levels
`of soluble p55 and p75 'I'Nt~' receptors and IL-1 receptor
`antagonist. Protocol—defined responders were defined as
`those patients with at least 2(l'% ACR criteria, ie. at least
`20% improvement in number of tender joints, number of
`swollen joints and in at least 3 of 5 RA Disease Activity
`measures {i.e. subject’.-3 assessment of pain, disease activity
`or physical function and physiciaI1’s global assessment of
`disease activity.
`I-‘ifty patients were enrolled and sequentially randomized
`to receive one of the five dosing regimens of IL-10 (SC) {I
`gtgfkg daily, 4 grgfkg daily, 8 yugfkg TIW, 8 gig/kg daily and
`2(J,ng;’1;g 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
`were similar in demographic characteristics except for slight
`diflerenccs 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
`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
`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-
`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
`(ACR 50) was higher for each of the II.-I0 treatment groups
`than for the placebo group, with the higher dose groups (8
`gigfkg TIW, 8 ,ttg;’kg daily and 20 gtgfkg TIW) showing the
`highest percent of both 20 ACR and 50 ACR responders. A
`trend towards decreased production of cx—vivo induced
`proinflammatory cytokines (TNF(1 and IL-16) and a trend
`towards increased circulating serum levels of soluble ’I'Nl-'
`p55 and Tlalli p75 receptors and IL-1 receptor antagonists
`occurred in nearly all IL-1U 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-
`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 grgfkg TIW, 8
`gigflcg daily and 20 ygfltg TIW IL-10 dosing regimens. The
`dosing regimen which maximizes safety and eflicacy results
`is 3 pgfkg II.-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-
`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
`which such claims are entitled; and the invention is not to be
`limited by the S]2It‘:C'lll(.‘ embodiments that have been pre-
`sented herein by way of example.
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`US 6,544,504 B1
`
`9
`
`We claim:
`1. A rnelhod of treating rheumatoid arthritis, said method
`comprising administering an ctfectivc amount of interleukin
`10 and methotrexale to an incliviclual afflicted with rheuma-
`toid arthritis.
`2. The method of claim 1 wherein the interleukin 10 is
`human interleukin 10.
`3. The method of claim 1 wherein the interleukin 10 is
`viral interleukin 10.
`
`10
`4. A pharmaceutical composition comprising interleukin
`I0, metholrexatc and a pharmaceutical acceptable carrier.
`5. The composition of claim 4 wherein the interleukin 10
`is human interleukin 10.
`
`6. The composition of claim 4 wherein the interleukin 10
`L: viral interleukin 10.
`
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