1015203035W0 98/091544CA 02264918 1999-03-03PCT/U S97/ 15308METHOD FOR LOWERING CHOLESTEROL LEVELSBACKGROUND OF THE INVENTIONThis invention relates to the lowering of serum cholesterol levels inmammals, especially humans. Elevated serum cholesterol levels are wellrecognized as a risk factor in heart disease. That is, the higher the serumcholesterol level, especially |owâdensity lipoprotein cholesterol (LDL-C), themore likely a patient is to develop heart disease. After a patient has alreadydeveloped heart disease and suffered a heart attack, the higher the serumcholesterol level, the more likely that patient will have another heart attack.Hence there are well recognized benefits to lowering serum cholesterollevels.There are many compounds available by prescription to lowerserum cholesterol. Probably the most commonly used are the HMGCoAreductase inhibitors, e.g., lovastatin, prevastatin, simvastatin and fluvastatin.While these compounds have some effect in lowering serum cholesterol, theydo not always achieve lowering to a desired level, and they have undesirableside effects in some people, such as muscle necrosis and hyperkalemia.The present invention lowers serum cholesterol with a well-toleratedactive ingredient. Furthermore, in its preferred aspects, the present inventionis believed to achieve significantly lower serum cholesterol levels than theHMGCoA reductase inhibitors.SUBSTITUTE SHEET (RULE 26)wo 98/09644 2101520l\)'J13035CA 02264918 1999-03-03PCT/US97/ 15308SUMMARY OF THE INVENTIONThe present invention may be summarized as a method for loweringserum cholesterol in a mammal, especially in a human, by administering aneffective amount of interleukinâ1O (IL-10). Preferably, the IL-10 isadministered to mammals diagnosed as having elevated blood serumcholesterol levels (hypercholesterolemia), or to those who have alreadydeveloped atherosclerotic heart or vascular disease. Preferably, themammals treated will be humans and the lL-10 will be recombinant human lL-1O (rhlL-10).The presently preferred method of administration is bysubcutaneous injection of from about 1 to 100 mcg/kg of patients body weight.More preferably the IL-10 is administered daily for a period of a least 3 days inan amount of 1 to 15 mcg/kg.BRIEF DESCRIPTION OF THE DRAWINGSFigure 1 illustrates average serum cholesterol levels for patientsadministered 8 mcg/kg rhlL-10 subcutaneously for 7 consecutive days and acontrol group receiving a placebo.Figure 2 illustrates the same data as figure 1 expressed as fractionalcholesterol levels with the initial screening level equal to 1.DETAILED DESCRIPTION OF THE INVENTIONThe invention provides a method for lowering blood cholesterollevels in mammals. e.g., mammals with hypercholesterolemia ornormocholesterolemia, by administering a serum cholesterol loweringeffective amount of IL-10. Elevated cholesterol levels can be associated withcardiovascular disease (e.g. atherosclerosis), cerebrovascular disease(stroke), and peripheral vascular disease.As used herein, interleukin 10 or IL-10 is defined as a proteinwhich (a) has an amino acid sequence substantially identical to a knownsequence of mature (i.e., lacking a secretory leader sequence) IL-10 asdisclosed in International Application Publication No. 91/003249, and (b)SUBSTITUTE SHEET (RULE 26)WO 98/09644 3101520253035CA 02264918 1999-03-03PCT/US97/ 15308has biological activity that is common to native IL-10. For the purposes ofthis invention, both glycosylated (e.g., produced in eukaryotic cells suchas yeast or CHO cells) and ungiycosylated (e.g., chemically synthesizedor produced in E. coli) IL-10 are equivalent and can be usedinterchangeably Also included are muteins and other analogs, includingviral IL-10, which retain the biological activity of lL-10.IL-10 suitable for use in the invention can be obtained from anumber of sources. For example, it can be isolated from culture media ofactivated T-cells capable of secreting the protein. Additionally, the IL-10or active fragments thereof can be chemically synthesized using standardtechniques known in the art. See, e.g., Merrifield, 1986, Science233:341-347 and Atherton at a/., Solid Phase Peptide Synthesis, APractical Approach, 1989, lFlL Press, Oxford.Preferably, the protein or polypeptide is obtained by recombinanttechniques using isolated nucleic acids encoding the IL-10 polypeptide.General methods of molecular biology are described, e.g., by Sambrooket a/., 1989, Molecular Cloning, A Laboratory Manual, , 2d Ed., ColdSpring Harbor, New York and Ausubel et al. (eds). Current Protocols inMolecular Biology, Green/Wiley, New York (1987 and periodicsupplements). The appropriate sequences can be obtained usingstandard techniques from either genomic or cDNA libraries. DNAconstructs encoding IL-10 may also be prepared synthetically byestablished standard methods, e.g., in an automatic DNA synthesizer,and then purified, annealed, ligated and cloned in suitable vectors.Atherton et a/., 1989. Polymerase chain reaction (PCR) techniques canbe used. See e.g., PCFiâ Protocols: A Guide to Methods and Applications,1990, Innis eta/. (ed.), Academic Press, New York.The DNA constructs may contain the entire native sequence of IL-1O or a homologue thereof. The term "homologue" is intended to indicatea natural variant of the DNA sequence encoding IL-10 or a variant orfragment produced by modification of the DNA sequence. Examples ofsuitable modifications of the DNA sequence are nucleotide substitutionswhich do not give rise to another amino acid sequence or nucleotidesubstitutions which do give rise to a different amino acid sequence andtherefore, possibly, a different protein structure. Other examples ofpossible modifications are insertions of one or several nucleotides intoSUBSTITUTE SHEET (RULE 26)wo 98/09644 41015203035CA 02264918 1999-03-03PCT/US97/15308the sequence, addition of one or several nucleotides at either end of thesequence, or deletion of one or several nucleotides at either end orwithin the sequence. Any homologous DNA sequence encoding aprotein which exhibits IL-10 activity (e.g., with respect suppression of Tcell proliferation or cholesterol lowering) similar to that of the naiveprotein is contemplated for use in the claimed invention.The nucleotide sequences used to transfect the host cells can bemodified, as described above, to yield IL-10 muteins and fragments witha variety of desired properties. Such modified lL-10 can vary from thenaturally-occurring sequence at the primary level, e.g., by amino acidinsertions, substitutions, deletions and fusions. Preferably, amino acidsubstitutions will be conservative; i.e., basic amino acid residues will bereplaced with other basic amino acid residues, etc. These modificationscan be used in a number of combinations to produce the final modifiedprotein chain.Amino acid sequence variants can be prepared with variousobjectives in mind, including increasing serum half-life, facilitatingpurification or preparation, improving therapeutic efficacy, and lesseningthe severity or occurrence of side effects during therapeutic use. Theamino acid sequence variants are usually predetermined variants notfound in nature, although others may be post-translational variants, e.g.,glycosylation variants or proteins which are conjugated to polyethyleneglycol (PEG), etc. Such variants can be used in this invention as long asthey retain the biological activity of IL-10.Preferably, human lL-10 is used for the treatment of humans,although viral or mouse IL-10, or IL-10 from some other mammalianspecies, could be used instead. Most preferably, the IL-10 used isrecombinant human IL-10 (rhlL-10). Recombinant production of humanIL-10 is described in U.S. Patent No. 5,231,012. Preparation of humanand mouse lL-10 has been described in International ApplicationPublication No. WO 91/00349. The cloning and expression of viral IL-10(BCRFI protein) from Epstein Barr virus has been disclosed by Moore etal. (Science 248:1230, 1990), and is described in EP 0 506 836.Administration of IL-10 is preferably parenteral byintraperitoneal, intravenous, subcutaneous or intramuscular injection orSUBSTITUTE SHEET (RULE 26)wo 98/09644 5101520253035CA 02264918 1999-03-03PCT/US97/15308infusion or by an other acceptable systemic method. Administration bysubcutaneous injection is most preferred. Alternatively, the IL-10 may beadministered by an implantable or injectable drug delivery system. See,e.g., Urquhart eta/, 1984, Ann Rev. Pharacol. Toxico/242199; Lewis, ed.,1981, Controlled Release of Pesticides and Pharmaceuticals, PlenumPress, New York, New York: U.S. Patent Nos. 3,773,919, and 3,270,960.Oral administration may also be carried out, using well knownformulations which protect the IL-10 from gastrointestinal proteases.Compositions useful for parenteral administration of such drugsare well known. See, e.g., Remington's Pharmaceutical Science, 11thEd., 1990, Mack Publishing Co., Easton, PA. For parenteraladministration, the IL-10 is typically provided as a lyophilized powder, apreferred formula of which is given in the examples. The powder isreconstituted with sterile water for injection. The powder may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, e.g., buffers and preservatives. The IL-10 ispreferably formulated in purified form substantially free of aggregates andother source proteins at a concentration in the range of about 25 mcg/mlto 2 mg/ml. Any of the well known carrier proteins such as human serumalbumin can also be added if desired. Other injectable forms (solution,suspension, emulsion) in association with a pharmaceutical carrier maybe used. Examples of such carriers are normal saline, Ringer's solution,dextrose solution, and Hank's solution. Non-aqueous carriers such asfixed oils and ethyl oleate may also be used.IL-10 can also be delivered by standard gene therapy techniques,including e.g., direct DNA injection into tissues, the use of recombinantviral vectors or phospholipid and implantation of transfected cells. See,e.g., Rosenberg, 1992, J. Clin. Oncol. 10:180.An effective amount of IL-10 is defined as any amount that willsignificantly lower the cholesterol level. A lowering of cholesterol by at least 5percent is considered significant. More preferably the cholesterol level will belowered by from 20 to 50 percent.The amount, frequency and period of administration will varydepending upon factors such as the cholesterol level, (e.g. severity of thecholesterol elevation), age of the patient, nutrition, etc. Usually, theSUBSTITUTE SHEET (RULE 26) g AWO 98/09644101520253035CA 02264918 1999-03-036 PCT/US97/15308administration will be daily initially and it may continue periodically during thepatients lifetime. Dosage amount and frequency may be determined duringinitial screenings of cholesterol levels and the magnitude of the effect of IL-10upon the lowering of the cholesterol levels. Dosage will be aimed to lower thecholesterol level to an acceptable level, which is currently believed to be lessthan about 200 milligrams per deciliter of blood serum. For patients with highrisk (e.g. previous evidence of established coronary artery disease, smokers,low HDL-C (high density lipoprotein cholesterol) levels, diabetics, etc.,) moreaggressive lowering of cholesterol, especially LDL-C is warranted.The currently preferred dosage of IL-10 for practice of this inventionis 1 to 100 mcg/kg, more preferably 1 to 15 mcg/kg administered daily for atleast 3 days, more preferably for at least 7 days. Other dosing schedules, suchas every other day, every third day, etc. are also believed effective. Thecurrently preferred administration method is by subcutaneous injection.However when orally effective lL-10 formulations of IL-10 are perfected, oraladministration would be more convenient.To complement the cholesterol lowering effect of IL-10, it may beuseful to administer it in conjunction with other pharmaceutically activecompounds. For example, it can be combined with other cholesterol loweringagents [e.g., lovastatin (1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2â(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)-ethyl]-1ânaphthalenyl 2-methylbutanoate,(U.S. Patent No. 4,231 ,938)) available from Merck, lnc., Rahway, NJ or otherHMG-CoA reductase inhibitors; gemfribrozil (5-(2,5-dimethylphenoxy)-2,2-dimethylpentanoic acid), available from Parke-Davis, |nc., Ann Arbor,Michigan; pravastatin, which is available from Squibb, lnc., Princeton NewJersey; niacin; nicotinic acid and its derivatives; cholestyramine; other anionexchange resins that bind to cholesterol; other cholesterol absorptioninhibitors; or bile acids]. For lowering cholesterol levels that may beassociated with acute manifestations of heart disease such as myocardialinfarction, lL-10 can be administered in conjunction with thrombolytic agents[e.g., tissue plasminogen activators (tPAs) (for example, those disclosed inUS. Patent Nos. 4,370,417, 4,752,603; U.K. Patent No. 2,119,804; PCTPatent Application Nos. 87/05934, 87/04722, 84/01786; Australian PatentApplication No. 55514/86; EPO Patent Application Nos. 227,462, 234,051,238,304, and 174,835, and the tPA is commercially available from Genentech,lnc., South San Francisco, California); eminase (available from Beecham |nc.,Bristol, Tennessee, and Upjohn Corporation, Kalamazoo, Michigan); andSUBSTITUTE SHEET (RULE 26)wo 98/09644 7101520253035CA 02264918 1999-03-03PCT/US97/ 15308streptokinase (for example, the materials disclosed in European PatentApplication Nos. 248,227, 28,489; and the streptokinase commerciallyavailable from Burroughs-Wellcome, |nc., Research Triangle, North Carolina)]or combinations of such thrombolytic agents (for example, see EuropeanPatent Application Nos. 91,240 and 28,489 for streptokinase/tPA complexes).These references are hereby incorporated by reference to illustrate examplesof other cholesterol lowering agents and thrombolytic agents that can be usedin combination with IL-10 in certain embodiments of the present invention.The specific cholesterol lowering agents and thrombolytic agents mentionedabove are merely examples of such agents known to those skilled in the artthat can be used in the practice of the present invention. It is believed thatadministering IL-10 in combination with another cholesterol-lowering agentmay allow use of significantly less of the other agent, thereby eliminating orreducing the other agent's undesirable side effects.Administration of the dose can be intravenous, nasal,parenteral, oral, subcutaneous, intramuscular, topical, transdermal or anyother acceptable method. The IL-10 could be administered in any number ofconventional dosage forms. Parenteral preparations include sterilesolutions or suspensions. Inhalation administration can be in the form of anasal or oral spray, or by insuffulation. Topical dosage forms can becreams, ointments, lotions, transdermal devices (e.g., of the conventionalreservoir or matrix patch type or iontophoresis) and the like.The formulations and pharmaceutical compositionscontemplated by the above dosage forms can be prepared withconventional pharmaceutically acceptable excipients and additives, usingconventional techniques.Preferably the IL-10 is administered via the subcutaneous route.The solutions to be administered may be reconstituted from lyophilizedpowders and they may additionally contain preservatives, buffers, dispersants,etc. Preferably, IL-10 is reconstituted with a medium normally utilized forintravenous injection, e.g., preservative-free sterile water. The maximumconcentration of IL-10 preferably should not exceed 2000 micrograms permilliliter. Administration may be accomplished by continuous intravenous orsubcutaneous infusion or by intravenous injection. For continuous infusion,the daily dose can be added to normal saline or other solution and thesolution infused by mechanical pump or by gravity.SUBSTITUTE SHEET (RULE 26)W0 98l0964410152030CA 02264918 1999-03-038 PCT/US97/ 15308The following examples illustrate the effect of administration ofhuman recombinant IL-10 (rhlL-10) on the serum cholesterol levels in healthyhuman volunteers.ExamplesExample 1: Daily Dosing for Seven Davs.A group of patients having an initial serum cholesterol levelbetween 150 and 160 mg/dl were dosed daily with 8 mcg of IL-10 per kg ofbody weight for 7 days. The IL-10 was administered by subcutaneousinjection. The IL-10 was initially furnished as a lyophilized powder preparedfrom the following formulationingredient mg/vialârhlL-10 0.8Sodium Citrate Dihydrate, 3USP/Ph. EurSucrose, NF/Ph. Eur 50Glycine, USP/Ph. Eur 10Water for Injection, USP 1.0 ml***upon reconstitution the amount in 1.0 ml**Sublimed during lyophilizationEach vial of lyphilized powder was reconstituted with 1.0 ml ofsterile water for injection.The results are shown in figures 1 and 2.A second group of patients having an average initial serumcholesterol level of between 130 and 140 mg/dl were injected with a placebowith the same regimen as the first group, except that the injected compositioncontained no IL-10. The results of this control experiment are also illustratedin figures 1 and 2. It is immediately evident that IL-10 causes a rapid and verysignificant drop in serum cholesterol levels. Future experiments are plannedto determine which type of cholesterol-- LDL cholesterol (the so called badSUBSTITUTE SHEET (RULE 26)W0 98/0964141015CA 02264918 1999-03-039 PCT/US97/15308cholesterol) or HDL cholesterol (the so called good cholesterol) -- is mostaffected by IL-10. Future experiments should also determine the duration ofthe drop in cholesterol levels, and whether follow-up cholesterolmeasurements and further dosing with IL-10 are necessaryExample 2: Effect of a Single Dose of IL-10Randomized, third-party blind, rising single-dose, placebo-controlled experiments were performed with the following dosages:placebo (18 participants)1, 2.5, 5, 10, 25, and 50 mcg/kg of IL-10 (6 participants at eachdosage level).The results are shown in the following tables:SUBSTITUTE SHEET (RULE 26)CA 02264918 1999-03-03WO 98/09644 1 O 9 PCT/U S97] 15308Percent of Baseline (pretreatment) Cholesterol concentrationsGroup Baseline 2 hours 24 hours 48 hoursplacebo 100 96 95 1001 mcg/ml 100 94 91 982.5 mcg/ml 100 98 94 1005 mcg/ml 100 91 85 8810 mcg/ml 100 91 77 8225 mcg/ml 100 97 83 8550 mcg/ml 100 97 86 85Cholesterol Concentrations by Treatment (mg/dl)Group Baseline 2 hours 24 hours 48 hoursplacebo 160 155 153 1601 mcg/ml 177 167 162 1752.5 mcg/ml 175 171 164 1755 mcg/ml 200 181 170 17510 mcg/ml 170 155 131 13925 mcg/ml 185 178 152 15650 mcg/ml 196 191 168 1675Change from Baseline (pretreatment) Cholesterol Concentrations(mg/dl)Group Baseline 2 hours 24 hours 48 hoursplacebo O -5 -7 O1 mcg/ml O -10 -15 -22.5 mcg/ml O -4 -11 15 mcg/ml O -18 -30 -2510 mcg/ml 0 -15 -39 -3125 mcg/ml O -6 -33 -2950 mcg/ml O -5 -28 -29Based on the above, it is clear that IL-10 is has the surprising10 effect of significantly lowering serum cholesterol levels in human patients.SUBSTITUTE SHEET (RULE 26) V