`
`Frontier Therapeutics Exhibit 1003
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`US 6,544,504 B1
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`1
`COMBINED USE OF INTERLEUKIN 10 AND
`METHOTREXATE FOR IMMUNO-
`MODULATORY THERAPY
`
`CR()SS-RI£I<'ljRl_".N(_‘E TO RE|[AA.11E||)
`APPLICATIONS
`
`This application is a non-provisional application that
`claims the priority of provisional application U.S. Ser. No.
`6(I;’146,022, filed Jul. 28, 1999. The Applicants’ claim the
`benefiLs of this application under 35 U.S.C. §1l9(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.
`
`BACKGROUNI) 01" TIIE lNVENTI()N
`
`a cytokine produced by I’
`Interleukin 10 (IL-10),
`.fimipi10cyfe‘, was first identified by its ability to inhibit
`interferon gamma (IITN-1') and IL-2 synthesis by mouse and
`human T lymphocytes [Fiorentino et al., 1989, J. Exp. Med.
`170:2081—2089; Moore el al., 1990, Science
`2-’l8:l230—1252; Vieira et al., 199], Pmc. Nrr1l'.Acrrd. Sci.
`USA 88:ll72—ll'i'7]. IL-10 was subsequently shown to be
`produced by B cells [()’(iarra et al., 1990, Imemm‘. Immu-
`noi. 2821-828] and macrophages [Fiorentino et al., 1991, J.
`Irmmmoi. 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 '1' 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-lot,
`II..~] B, ll..-6 and 'I'NlioL [de Waal et al., 1991, J. Exp. Med.
`174:l2U9—1217]. II.-10 has growth promoting effects on
`murine thymocytes and ‘l' oells [MacNeil et al., 1990,
`Iimimnol. 14524167] and mast oetls [Thoinpson-Snipes et
`al., 1991, J. Exp. Med. 173:50'z'—512], and it stimulates
`cytotoxic '1'-cell development [Chen and Zlotnik, 1991, J.
`hrnmmoi’.
`l47:528—S33].
`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 II.-1U, also has the capacity to
`inhibit cytokine synthesis [Moore et al., 1990, Scierrce
`2-’l8:l230—1252; Vieira etal., 1991, Pmc. Natl‘. Acrrd. Sci.
`USA 88:1172—1177].
`including IL-2, IFN-7 and TNF—Ot,
`Several cytokines,
`have been shown to regulate the mixed lymphocyte reaction
`{MLR} [Shevach, 1985, Anmt. Rev. Inimmrrfl. 3:397; liidelus
`et al., 1982, Jl}‘rmspi’rrntri!iorr 3-4:308; Tadmori et al., 1985, J.
`Immimoi. 134:4542—4550; Tadmori et al, 1986, J. Inmmnei.
`136:1155—1162; Novelli et al., 1991, 14721445-1450;
`Landolfo et al., 1985, Science 229:1?'6—l80; Shalaby et al.,
`988, J. Iriyriurirfl. 141:-499-505]. It has been reported that
`IFN-y may play an important role in MLR graft rejection
`[Novelli et al., 1991, J. Immimoi. 147:1445—1450; Landolfo
`et al., 1985, Science 229176-180]. Antibodies to IFN-y or
`to TNI7 [Shalaby et al., 1988, J. .fm:mrrnr)l'. 141:499~5'[l5]
`have been shown to block MLR-induced proliferation. In
`these studies it was found that antibodies to IFN-7 sup-
`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-
`pteridinyI)rnethyl] methylamino]benmyl]-I.-glutamic acid.
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`The following references describe the preparation of meth-
`otrexate [see Seeger et al.,J/tm.Ci:em.S0c., 1949, 71:1753];
`the metabolism of methotrexate [see Freeman, J.Pharma-
`ce-i.Exp.1'I-‘ier. 1958, l22:154; and Henderson et al., Cancer
`Res. 1965, 2511008, 1018];
`the toxicity of methotrexate
`[Condit et al., Crmcer 1960, l3:222—249]; the pharmacoki-
`netic models of metholrexate [Bischof_F, et al.,J.Pimrm.ScrT
`1970, 59:149];
`the metabolism and pharmacokinetics of
`methotrexate [Evans,Appi’.P.-'rnrmricokinet. 1980, 518-548];
`the clinical phannacology of methotrexate [Bertino, Crmcer
`(fihemotirer, 1981, 3: 359-375; Jolivet et al., N.Ertgt'.I.Med.,
`1983, 309: 1094-1104] and the clinical experience of meth-
`otrexate in rheumatoid arthritis [J.Ri:emmn‘o!., 1985, 12,
`Suppl, 12, 1-4-4].
`Methotrexate inhibiLs dihydrofolic acid reductase. liolic
`acid must be reduced to tetrahydrofolic acid by this enzyme
`in the process of DNA synthesis, repair and cellular repli-
`cation. Therefore, methotrexate interferes with cellular
`reproduction.
`
`SUMMARY OF THE INVENTION
`
`invention provides a method for treating
`The present
`autoimmune disease comprising administering an elfective
`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-
`matoid arthritis comprising administering an effective
`amount of interleukin-1(} 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.
`
`Pharmaceutical compositions comprising a combination
`of IL-10 and MTX are also provided by this invention.
`
`I)I_".'l"/\II..l_iI) IJESCRIPTION OI" TIIE
`INVl3NI‘ION
`
`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 entirely 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 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
`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
`T cell
`responses. For example, considering the diverse
`biological activities of IL-10, the Concurrent use of IL-10
`and MTX provides long tenn treatment of inllamrnatflry
`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’I'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
`MTXIII.-10 combination therapy of the present invention is
`useful in treating patients who are non-responsive to MTX
`
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`US 6,544,504 B1
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`in
`treatment alone. MTX/ll.-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
`forrrlulating the two together in a common caJTier), or (b) at
`different times during the course of a common treatment
`schedule. In the latter case, the two compou nds are admin-
`istered sufiiciently close ir1 time to achieve the intended
`effect. The active agenLs may be administered together in a
`single pharmaceutical composition or separately. Both
`active agents (i.e., IL-l.[|' 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-
`maceutical composition comprising an effective amount of
`IL-10 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.e., lacking a
`secretory leader sequence) II.-I0 as disclosed in Interna-
`tional Application Publication No. 91 ,=’{l03249, and (b) has
`biological activity that is common to native [L-10. For the
`purposes of this invention, both glycosylated (e.g., produced
`in etlkaryotic cells such as yeast or CIIO cells) and ungly-
`cosylated (e.g., chemically synthesized or produced in E.
`Coir") 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 '1'-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., Merrifleld, 1986, Scr'e-rice’
`233:34]—34? and Atherton et al., Solid P.-'trr.s'c I"pfr'de
`Synthesis, A Practical /lppmaclr, 1989, IRL 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,
`Mrflecnirrr Cfrmirtg, A Laborrrtr)I}‘ Mrrnrraf, 2d L3d., Cold
`Spring Harbor, N.Y. and Ausubel et al.
`(eds). Current
`Protocols in Molecular Biology, Greenx'Wi1ey, New York
`(1987 and periodic supplements). The appropriate sequences
`can be obtained using standard techniques from either
`genomic or cDNA libraries. 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)
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`techniques can be used. See e.g., PCR I’rotoco.’s.‘ A (hide 10
`Methods cmdApph'crm'ons, 1990, Innis et al, (ed), Academic
`Press, New York.
`The DNA constructs may contain the entire native
`sequence ot‘lI..-10 or a homologue thereot‘. The term “homo-
`logue" is intended to indicate a natural variant of the DNA
`sequence encoding IL-10 or a variant or fragment produced
`by modification ofthe DNAsequence. 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 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 II.-10 muteins
`and fragments with a variety of desired properties. Such
`modified 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
`of combinatiorts 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
`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
`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
`other mammalian species, could be used instead. Most
`preferably,
`the IL-10 used is recombinant human IL-10.
`Recombinant production of human lI..-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-
`lication No. WO 91200349. The cloning and expression of
`viral II.-IU (BCRFI protein) from Epstein Barr virus has
`been disclosed by Moore et al. [Science 24811230, 1990],
`and is described in EP 0 506 836.
`
`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 injectable drug delivery system.
`See, e.g., Urquhart et al, 1984, Arm Rev. Plmmmcol. Toxicol
`M1199; levvis, ed., 1981, Cr)m‘roHed Release of !’e.vtr'cr'a'c.s'
`and l":'rarmrrcerrticrrr's', 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 lI.—l(l from gastrointestinal proteases.
`Compositions useful for parenteral administration of such
`drugs are well known. See, e.g., Remington’s Pharmaceu-
`
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`tical Science, 11th lE,d., 1990, Mack Publishing Co., Easton,
`Pa. When administered parcnterally, the IL-10 is typically
`formulated in a unit dosage injectable form (solution,
`suspension, emulsion) in association with a pharmaceutical
`carrier. Lixamples of such carriers are normal saline, Ring-
`er’s solution, dextrose solution, and Hank’s solution. Non-
`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
`l0't}—2DOU mglml. 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 of transfected cells. See, e.g., Rosenberg, 1992,
`J. Ctirz. Oncol. 10:180.
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`MTX may be administered in a manner as is convention-
`ally practiced. See, e.g., Goodman and Gilman’s The Phar-
`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
`excipicnts and used in the form of ingestible tablets, buecal
`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 met hotrexate 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.
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`The tablets, troches, pills, capsules and the like may also
`contain the following: a binder, such as gum tragaeanth,
`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 font‘!
`of the dosage unit. For instance, tablets, pills, or capsules
`may be coated with shellac, sugar or both. A syrup 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, methotrexate 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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`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 for111
`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 like), suitable mixtures
`thereof, and vegetable oils. The proper lluidity 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,
`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-
`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
`solvent with various of the other ingredients enumerated
`above, as required,
`followed by littered 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 prefe1Ted
`methods of preparation are vacuum drying and the freeze-
`drying technique which yield a powder of methotrexate, plus
`any additional desired ingredient from a previoLLsly 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 agertLs and the like. The use of such media and
`agents for phamiaceutically active substances is well known
`in the art. llxcept 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 fon11ulate 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-
`duce the desired therapeutic efl'ect in association with the
`required pharmaceutical carrier.
`Methotrexate is compounded for convenient and elfective
`administration in effective amounLs with a suitable phan'na-
`ceulically 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 10 to 25
`being most preferred. Expressed in proportions, methotrex-
`ate is generally present in from about 0.] to about 40 mgfml
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`US 6,544,504 B1
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`of carrier. In the case of compositions containing supple-
`mentary active ingredients, the dosages are determined by
`reference to the usual dose and manner of administration of
`
`therapy given daily or 'l'IW plus MTX (oral.iintramtLscular/
`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
`Activity, and to detennine changes in the circulating levels
`of soluble p55 and p75 TNIV receptors and IL-1 receptor
`antagonist. Protocol—defined responders were defined as
`those patients with at least 20% 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’s assessment of pain, disease activity
`or physical function and physician’s global assessment of
`disease activity.
`Fifty patients were enrolled and sequentially randomized
`to receive one of the five dosing regimens of IL-10 (SC) (1
`Jttgfkg daily, 4 Jugflcg daily, 8 ,.t.tgfl~:g 'l'IW, 8 Jrtgfkg daily and
`20 ,tig;"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
`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
`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 IL-10 treatment groups
`than for the placebo group, with the higher dose groups (8
`Jttgfkg TIVV, 8 ,ug_,f'kg daily and 20 Jug/kg TIVV) showing the
`highest percent of both 20 ACR and 50 ACR responders. A
`trend towards decreased production of ex-vivo induced
`proinflammatory cytokines (TNliu. and IL-13) and a trend
`towards increased circulating serum levels of soluble TNF
`p55 and TNI’ p75 receptors and IL-1 receptor antagonists
`occu rred 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-
`tis. Trends indicate that IL-10 in combination with MTX
`
`may have beneficial effects on rheumatoid arthritis Disease
`Activity. This elfect was greatest for the 8 ,ngx'kg TIW, 8
`,.t.tga’kg daily and 2t) ,ttg;’l<g 'l'IW II.-ltl dosing regimens. The
`dosing regimen which maximizes safety and ellicacy results
`is 8 _ugt'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-
`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 specific embodiments that have been pre-
`sented herein by way of example.
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`said ingredients.
`A single intravenous dosage, slow oonstant infusion, or
`repeated daily dosages can be administered. Daily dosages
`up to about
`1
`to 10 (lays are often sutficient.
`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 elfective to provide an immunosup-
`pressive etfect. As used herein “effective amount” means an
`amount sufiicient 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, if
`used alone, would be considered suboptimal for the intended
`purpose.
`Based on the judgment of the clinician, the amount of
`IL-10 andfor MTX will, of course vary. The efiective
`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-elfects, and the
`like. The elfective dose of IL-10 typically will range from
`about 0.l—100 gigfkgtday, preferably about 1-20 ;:g"kg,r‘day
`in a single or divided doses. More preferably, the effective
`dose of IL-10 will be 8 yg,r'kg three times a week ['l'IW], 8
`tigfkg daily or 20 ,ug,»’l<g TIW. The effective dose of MTX
`typically range from about 1-100 mgfweek, more preferably
`from about 5-35 mgfweek, and most preferably from about
`10-25 mgfweek. The length ofadministration may vary and,
`in some cases, may continue over the remaining lifetime of
`a patient, to control autoimmune symptoms or graft rejection
`processes.
`
`40
`
`50
`
`60
`
`EXAMPLE 1
`
`Safety and Tolerance Study of IL-10 in
`Combination with a Stable Dosing Reqimen of
`MTX in Patients with Active Rheumatoid Arthritis
`
`A multinational, mu lticenter, sequentially randomized,
`double-blirtd, 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 five dosing regimens
`of IL-10 (SC) (1 ,t 0 g daily, 4 gtgfkg daily, 8 pgfkg three
`times a week [TIW], 8 ygfkg daily and 20 grgflig 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 12.5—25
`mglweek (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 close
`cohorts starting with the lowest dose of II..-10. Safety was
`assessed for each dose level prior to progressing to the next
`higher dose. Ten patients were assessed at each of the 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)
`
`Page 5 of 6
`Page 5 of 6
`
`
`
`9
`
`10
`
`US 6,544,504 B1
`
`We claim:
`lv AlTlC1h0d Of treating l'h¢Uma10i(| flflhfilis. Said m0lh0('
`‘30ml31'i5ifl8 aC'mW5‘°1'i'18 3“ °ECC1i"° 31710" '1‘ 0f im‘3l'l°Uki‘1
`10 and methotrexate to an individual afllieted 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 ll).
`
`5
`
`4. A pharmaceutical eornposition comprising interleukin
`10, rnelholrexale and a pharrnaceutical 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
`is viral interleukin 10.
`
`*
`
`*
`
`*
`
`*
`
`*
`
`Page 6 of 6
`Page 6 of 6