WO2006109170A2

WO2006109170A2 - Combination therapy for treatment of cardiovascular diseases and related conditions

Info

Publication number: WO2006109170A2
Application number: PCT/IB2006/000968
Authority: WO
Inventors: Charles Larry Bisgaier; Roger Schofield Newton; Milton Lethan Pressler
Original assignee: Esperion Therapeutics Inc.
Priority date: 2005-04-14
Filing date: 2006-04-03
Publication date: 2006-10-19
Also published as: WO2006109170A3

Abstract

The invention provides combination regimens of an AIM-lipid complex, one or more statins and one or more anti-inflammatory agents for treating and preventing of cardiovascular disease and related conditions. The compositions and methods of the instant invention encompass a non-invasive combination therapy to treat cardiovascular disease and related conditions. Pharmaceutical compositions of the combination therapy are also provided.

Description

COMIBINATION THERAPY FOR TREATMENT OF CARDIOVASCULAR DISEASES AND RELATED CONDITIONS

FIELD OF INVENTION

The invention provides novel compositions and methods to treat or prevent cardiovascular disease or related disorders including but not limited to atherosclerosis, arteriosclerosis, acute coronary syndromes, myocardial or cerebral ischemia, restenosis, angina pectoris, stable angina, unstable angina, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, intermittent claudication, peripheral artery disease, bowel ischemia related to occlusion of mesenteric or celiac arteries, femora or iliac disease, Leriche's syndrome, sepsis, reduction or stabilization of atherosclerotic plaque, reduction of plaque in occluded vessels and promotion of cholesterol efflux. In a preferred embodiment, the method utilizes, or the composition comprises, a protein-lipid complex, a statin and an anti-inflammatory agent. Preferred triple combination methods and double or triple combination compositions are described further below. Also, quadruple, pentamerous, hexamerous and other combination methods and compositions are described further below.

BACKGROUND OF INVENTION 2.1. Apolipoprotein A-I Milano Human Apo-I Milano is a natural variant of Apo A-I (Weisgraber et al, J. Clin.

Invest. 66:901-907 (1980). In Apo A-I Milano the amino acid arginine (Arg173) is replaced by the amino acid cysteine (Cys173). Because the Apo A-I Milano contains one cysteine residue per polypeptide chain, it may exist in a monomeric, homodimeric, or heterodimeric form. (See, U.S. Patent No. 5,876,968). These forms are chemically interchangeable, and the term Apo A-I Milano does not discriminate between these forms. On the DNA level the variant form results from a C to T substitution in the gene sequence, i.e., the codon CGC changed to TGC, allowing the translation of a cysteine (Cys) instead of arginine (Arg) at amino acid position 173.

Methods for obtaining Apo A-I Milano are well-known in the art. For example, Apo A-I Milano can be separated from plasma, for example, by density gradient centrifugation followed by delipidation of the lipoprotein, reduction, denaturization and gel-filtration chromatography, ion-exchange chromatography, hydrophobic, e.g., phenyl sepharose, interaction chromatography or immunoaffinity chromatography, or produced synthetically, semi-synthetically, or using recombinant DNA techniques and subsequent purification techniques known to those skilled in the art. (See, e.g., U.S. Patent Nos. 6,107,467;

6,559,284; 6,423,830; 6,090,921 ; 5,834,596; 5,990,081 ; 6,506,879; 5,059528; 5,876,968; and 5,721 ,114; Mulugeta etal., J. Chromatogr. 798(1-2): 83-90 (1998); Chung etal., J. Lipid Res. 21(3): 284-91 (1980); Cheung et al., J. Lipid Res. 28(8): 913-29 (1987); Persson ef a/., J. Chromatogr. 711 : 97-109 (1998); and PCT Publication WO 86/04920 and WO 87/02062).

2.2. Statins

The statins are competitive inhibitors of 3-hydroxy-3-methyIglutaryl coenzyme A (HMG-CoA) reductase, which catalyzes an early, rate-limiting step in cholesterol biosynthesis. They lower cholesterol by slowing down the production of cholesterol and by increasing the liver's ability to remove the low density lipoprotein-cholesterol (LDL-C) already in the blood.

There are several statins currently available on the market in the United States: lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin and atorvastatin. Lovastatin (MEVACOR, Merck & Co., Inc.), a natural product derived from a strain of Aspergillus terreus, is the first statin that was approved for use in human beings. Lovastatin significantly reduces serum cholesterol and LDL-serum levels. It also slows progression of coronary atherosclerosis. However, serum high density lipoprotein (HDL) levels are only slightly increased following lovastatin administration. Pravastatin (PRAVACHOL, Bristol- Myers Squibb Co.) and simvastatin (ZOCOR, Merck & Co.) are chemically modified derivatives of lovastatin. They are fungal metabolites containing a hexahydronapthalene ring. Lovastatin and simvastatin are modified in the liver to active hydroxy acid forms. Being lactones, they are less soluble in water than are the other statins. Atorvastatin

(LIPITOR, Pfizer) and fluvastatin (LESCOL, Novartis) are synthetic compounds containing a heptanoic acid side chain. Atorvastatin and simvastatin in higher doses can also reduce triglyceride levels caused by elevated very low density lipoprotein (VLDL) levels. Pravastatin (an acid in the active form), fluvastatin (sodium salt), and atorvastatin (a calcium salt) are all administered in the active, open-ring form. Rosuvastatin (calcium salt) (CRESTOR, AstraZeneca) is one of the newer statins on the market.

Statins affect blood cholesterol levels by inhibiting cholesterogenesis in the liver, which results in increased expression of the LDL receptor gene. Increased synthesis of LDL receptors results in increased removal of LDL from the blood, thereby lowering LDL-C levels.

Some studies suggest that statins can also reduce LDL levels by enhancing the removal of LDL precursors (VLDL and IDL) and by decreasing hepatic VLDL production. The reduction in hepatic VLDL production induced by statins is thought to be mediated by reduced synthesis of cholesterol. This mechanism also likely accounts for the triglyceride- lowering effect of statins.

Triglyceride levels greater than 250 mg/dl are reduced substantially by statins. Hyperglyceridemic patients taking the highest doses (80 mg/day) of two of the most potent statins (atorvastatin and simvastatin) experience a 35% to 45% reduction in LDL-C and a similar reduction in fasting triglyceride levels. In studies of patients with elevated LDL-C levels and gender appropriate HDL-C levels, an increase in HDL-C of 5% to 10 % was observed, irrespective of the dose or statin employed. The effect depends on the dose and statin used with respect to lowering LDL-C levels. Statins also possess multiple cardioprotective effects other than LDL lowering.

These effects may relate to enhancing of synthesis of endothelial cell nitric oxide, reversing endothelial dysfunction, affecting atherosclerotic plaque stability, reducing susceptibility of lipoproteins to oxidation, reducing platelet aggregation and having an anti-inflammatory effect. Statins lower LDL-C by 20-55%, depending on the dose and statin used. In large trials comparing the effects of the various statins, equivalent doses appear to be 5 mg of simvastatin = 15 mg of lovastatin = 15 mg of pravastatin = 40 mg of fluvastatin (Pedersen and Tobert, Drug Saf., 14:11-24 (1996)) and 20 mg of simvastatin = 10 mg of atorvastatin (Jones et al., Am J Cardiol 81:582-587 (1998)). Analysis of dose-response relationships for all statins demonstrate that the efficacy of LDL-C lowering is log linear. LDL-C is reduced by 6% (from baseline) with each doubling of the dose (Pedersen and Tobert, 1996, supra; Jones et al., 1998, supra). Maximal effects on plasma cholesterol levels are achieved within 7 to 10 days.

As a result of their structural similarity to HMG-CoA, statins are reversible inhibitors of the enzyme's natural substrate, HMG-CoA. The inhibition constant of the statins is in the 1 nM range. The dissociation constant of HMG-CoA is three orders of magnitude higher than this value.

The mechanism of the LDL-lowering effect of statins may involve both reduction of VLDL concentration and induction of cellular expression of LDL-receptor, leading to reduced production and/or increased catabolism of LDL. Sometimes, the statins are used in combination therapy with bile-acid-binding resins. 2.2.1 Lipid Regulating Agents

Examples of other lipid regulating agents include a CETP inhibitor, a PPAR- activator, an MTP/Apo B secretion inhibitor, a cholesterol absorption inhibitor, LDL- lowering agent, HDL-cholesterol raising agent, triglyceride lowering agent, a cholesterol synthesis inhibitor, a cholesterol modulating agent, a fibrate, niacin, an ion-exchange resin, an antioxidant, an ACAT inhibitor, bile acid sequestrant. Specific examples of each of these agents include those known in the art, as well as those specificed below. Some of these agents may be useful to treat statin-intolerant patients.

Any cholesterol absorption inhibitor known in the art with the ability of a compound to prevent cholesterol contained within the lumen of the intestine from entering into the intestinal cells and/or passing from within the intestinal cells into the blood stream may be used. Such cholesterol absorption inhibition activity is readily determined according to standard assays (e.g., J. Lipid Res. (1993) 34: 377-395). Examples include, but are not limited to, ezetimibe (ZETIA™) as well as the cholesterol absorption inhibitors described in WO 94/00480.

Ezetimibe is a selective inhibitor of intestinal absorption of cholesterol and other phytosterols. Ezetimibe reduces total blood cholesterol, LDL-related cholesterol, triglycerides and Apolipoprotein B and elevates HDL-cholesterol via inhibition of intestinal cholesterol absorption. It works via a mechanism distinct from HMG-CoA reductase inhibitors, bile-acid sequestrants ('resins'), fibric acid derivatives and plant stanols. Its cholesterol and LDL-C lowering capacity adds to and acts complementary to co- administered statins.

Any cholesterol ester transfer protein ("CETP") inhibitor known in the art that inhibits the transfer of cholesteryl ester and triglyceride between lipoprotein particles, including high density lipoproteins (HDL), low density lipoproteins (LDL), very low density lipoproteins (VLDL), and chylomicrons may be used. The effect of a CETP inhibitor on lipoprotein profile is believed to be anti-atherogenic. Such inhibition may be determined by means known in the art (e.g., Crook et al. Arteriosclerosis 10, 625, 1990; U.S. Pat. No. 6,140,343). Examples of suitable CETP inhibitors include, but are not limited to, those described in U.S. Patent Nos. 6,197,786, 6,723,752 and 6,723,753. Additional examples of useful CETP inhibitors include the following compounds: [2R, 4S]4-[(3,5-bis- trifluoromethyl-benzyl)-methoxycarbonyl-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H- quinoline-1-carboxylic acid ethyl ester (Torcetrapib™), and 3-{[3-(4-Chloro-3-ethyl- phenoxy)-phenyl]-[3-(1 A ,2,2-tetrafluoro-ethoxy)-benzyl]-amino}-1 ,1 ,1 -trifluoro-propan-2- ol. To address the poor solubility of many of the CETP inhibitors, an appropriate dosage form such as one comprising (1) a solid amorphous dispersion comprising a cholesteryl ester transfer protein (CETP) inhibitor and an acidic concentration-enhancing polymer; and (2) an acid-sensitive HMG-CoA reductase inhibitor, may be necessary. This dosage form is more fully described in USSN 10/739,567.

Exemplary CETP inhibitors include [2R,4S]- 4-[(3,5-Bis-trifluoromethyl-benzyl)- methoxycarbonyl-amino^-ethyl-δ-trifluoromethyl-S^-dihydro^H-quinoline-i-carboxylic acid ethyl ester (torcetrapib); cis-(2R,4S)- 2-(4-{4-[(3,5-Bis-trifluoromethyl-benzyl)-(2- methyl-2H-tetrazol-5-yl)-amino]-2-ethyl-6-trifluoromethyl-3,4-dihydro-2H-quinoline-1- carbonyl}-cyclohexyl)-acetamide; (2R)-3-{[3-(4-Chloro-3-ethyl-phenoxy)-phenyl]-[[3- (1 ,1 ,2,2-tetrafluoro-ethoxy)-phenyl]-methyl]-amino}-1 ,1 ,1 -trifluoro-2-propanol; and (2R, 4R, 4aS)-4-[Amino-(3,5-bis-trifluoromethyl-phenyl)- methyl]-2-ethyl-6-trifluoromethyl-3,4- dihydro-2H-quinoline-1 -carboxylic acid isopropyl ester or a pharmaceutically acceptable salt of said compounds. Any peroxisome proliferator activated receptor ("PPAR") activator known in the art that activates or otherwise interacts with a human PPAR may be used. Three mammalian PPARs have been isolated and termed PPAR-alpha, PPAR- gamma, and PPAR-beta (also known as NUC1 or PPAR-delta). These PPARs regulate expression of target genes by binding to DNA sequence elements, termed PPAR response elements. These elements have been identified in the enhancers of a number of genes encoding proteins that regulate lipid metabolism suggesting that PPARs play a pivotal role in the adipogenic signaling cascade and lipid homeostasis. PPAR-gamma receptors are associated with regulation of insulin sensitivity and blood glucose levels. PPAR-α activators are associated with lowering plasma triglycerides and LDL cholesterol. PPAR-β activators have been reported to both increase HDL-C levels and to decrease LDL- C levels. Thus, activation of PPAR-β alone, or in combination with the simultaneous activation of PPAR-α and/or PPAR-gamma may be desirable in formulating a treatment for dyslipidemia in which HDL is increased and LDL lowered. PPAR-activation is readily determined by those skilled in the art by the standard assays (e.g. US 2003/0225158 and US 2004/0157885). Examples of suitable PPAR-activator compounds include, but are not limited to, those described in US 2003/0171377, US 2003/0225158, US 2004/0157885, and U.S. Pat. No. 6,710,063. Additional examples of useful PPAR-activator compounds include the following compounds: [5-Methoxy-2-methly-4-(4'-trifluoromethly-biphenyl- 4ylmethylsulfanyl)-phenoxy]-acetic acid; [5-Methoxy-2-methyl-4-(3'-trifloromethly-biphenyl- 4-ylmethylsulfanyl)-phenoxy]-acetic acid; [4-(4'Fluoro-biphenyl-4-ylmethylsulfanyl)-5- methoxy-2methy!-phenoxy]-acetic acid; {5-Methoxy-2methyl-4-[4-(4-trifluoromethyl- benzyloxy)-benzylsulfanyl]-phenoxy}-acetic acid; {{5-Methoxy-2-methyl-4-[4-(5- trifluoromethyl-pryidin-2-yl)-benzylsulfanyl]-phenoxy}-acetic acid; (4-{4-[2-(3-Fluoro-phenyl)- vinyl]-benzylsulfanyl}-5-methoxy-2-methyl-phenoxy)-acetic acid; [5-Methoxy-2-methyl-4-(3- methyl-4'-trifluoromethyl-biphenyl-4-ylmethylsulfanyl)-phenoxy]-acetic acid; [5-Methoxy-2- methyl-4-(4'-trif!uoromethyl-biphenyl-3-ylmethylsulfanyl)-phenoxy]- acetic acid; {5-Methoxy- 2-methyl-4-[2-(4- trifluoromethyl-benzyloxy)-benzylsu!fanyl]-phenoxy}acetic acid; 3-{5-[2-(-5- Methyl-2 phenyl-oxazol-4-yl-ethoxy] -indol- 1-yI} -propionic acid; 3-{4[2-(5-methy!-2- phenyl-1 ,3-oxazol-4-yl)ethoxy- 1H-indazol-1yl}propanoic acid; 2-Methyl-2-{3-[({2-(5-methyl- 2-phenyl-1 ,3-oxazol-4-yl)ethoxy]carbonyl}amino)methyl] phenoxyjpropionic acid; 1-{3'-[2-5- Methyl-2-phenyl-1 ,3-oxazol-4-y]-1 ,1'-biphenyl-3-yl}oxy)cyclobutanecarboxylic acid; 3-[3-(1- Carboxy-1 -methyl-ethoxy)-phenyl]-piperidine-1 -carboxylic acid 3-trifluoromethyl-benzyl ester; 3-[3-(1 -Carboxy-1 -methy!-ethoxy)-phenyl]-piperidine-1 -carboxylic acid 4- trifluoromethyl-benzyl ester; 5-[4-(4-Ethyl-benzyIsulfanyl)-phenylsulfamoyl]-2-methyl- benzoic acid; and 5-{2-[4-(3,4-Difluoro-phenoxy)-phenyl]-ethylsulfamoyl}-2-methyl-benzoic acid; 2-{2-methyl-4-[({4-methyl-2-[4-(trifluoromethyl)phenyl]-1 ,3-thiazol-5- yl}methyl)sulfany!]phenoxy}acetic acid; 2-{2-methyl-4-[({4-methyl-2-[4-(trifluoromethyl

)phenyl]-1 ,3-oxazol-5-y!}methyl)sulfanyl]phenoxy}acetic acid; methyl 2-{4-[({4-methyl-2-[4- (trifluoromethyl)phenyl]-1 ,3-thiazol-5-yl}methyl)sulfanyl]phenoxy}acetate; 2-{4-[({4-methyl-2- [4-(trifluoromethy!)phenyl]-1 ,3-thiazol-5-yl}methyl)suIfanyl]phenoxy}acetic acid; (E)-3-[2- methyl-4-({4-methyl-2-[4-(trifluoromethyl)phenyl]-1 ,3-thiazol-5-yl }methoxy)phenyl]-2- propenoic acid; 2-{3-chloro-4-[({4-methyl-2-[4-(trif luoromethyl)phenyl]-1 ,3-thiazol-5-yl}me thyOsulfanyOphenylJacetic acid^^-methyl-Φ^-methyl^-IS-fluoro^- (trifluoromethyl)phenyl]-1 ,3-thiazo l-5-yl}methyI)sulfanyl]phenoxy}acetic acid; and pharmaceutically acceptable salts thereof. Any MTP/Apo B secretion (microsomal triglyceride transfer protein and/or apolipoprotein B secretion) inhibitor known in the art which inhibits the secretion of triglycerides, cholesteryl ester and phospholipids may be used. Such inhibition may be readily determined according to standard assays (e.g., Wetterau, J. R. 1992; Science 258:999). Examples of suitable a MTP/Apo B secretion inhibitor include, but are not limited to, imputapride (Bayer) as well as those described in WO 96/40640 and WO 98/23593.

Any ACAT inhibitor known in the art that inhibits the intracellular esterification of dietary cholesterol by the enzyme acyl CoA: cholesterol acyltransferase may be used. Such inhibition may be determined readily according to standard assays, such as the method of Heider et al. described in Journal of Lipid

Research. 24:1127 (1983). Examples of suitable ACAT inhibitors include, but are not limited to, those described in U.S. Pat. No. 5,510,379 (carboxysulfonates),WO 96/26948 and WO 96/10559 (urea derivatives). Additional examples include Avasimibe (Pfizer), CS-505 (Sankyo) and Eflucimibe (EIi Lilly and Pierre Fabre). Any lipase inhibitor (e.g., pancreatic lipase inhibitor, a gastric lipase inhibitor) known in the art that inhibits the metabolic cleavage of dietary triglycerides into free fatty acids and monoglycerides may be used. Such lipase inhibition activity may be readily determined according to standard assays (e.g., Methods Enzymol. 286: 190-231). Examples of a suitable lipase inhibitor include, but are not limited to, lipstatin, (2S,3S,5S,7Z,10Z)-5-[(S)-2-formamido-4-methyl-valeryloxy]-2-hexyl-3-hydro- xy-7,10- hexadecanoic acid lactone, and tetrahydrolipstatin (orlistat), (2S,3S,5S)-5-[(S)-2- formamido-4-methyl-valeryloxy]-2-hexyl-3-hydroxy-hexa- decanoic 1 ,3 acid lactone, and the variously substituted N-formylleucine derivatives and stereoisomers thereof (U.S. Pat. No. 4,598,089); tetrahydrolipstatin U.S. Pat. Nos. 5,274,143; 5,420,305; 5,540,917; and 5,643,874; FL-386, 1-[4-(2-methy!propyl)cyclohexy!]-2-[- (phenylsulfonyl)oxy]-ethanone, and the variously substituted sulfonate derivatives related thereto (U.S. Pat. No. 4,452,813); WAY-121898, 4-phenoxyphenyl-4-methylpipe- ridin-1-yl-carboxylate, and the various carbamate esters and pharmaceutically acceptable salts related thereto (U.S. Pat. Nos. 5,512,565; 5,391 ,571 and 5,602,151); valilactone, and a process for the preparation thereof by the microbial cultivation of Actinomycetes strain MG147-CF2 (Kitahara, et al., J. Antibiotics, 40 (11), 1647-1650 (1987)); esterastin; ebelactone A and ebelactone B, and a process for the preparation thereof by the microbial cultivation of Actinomycetes strain MG7-G1 (Umezawa, et al., J. Antibiotics, 33, 1594-1596 (1980); Japanese Kokai 08-143457, published Jun. 4, 1996). The compound tetrahydrolipstatin is especially preferred. Additional examples include N-3-trifluoromethylphenyl-N'-3-chloro-4'- trifluoromethylphenylurea, and the various urea derivatives related thereto.U.S. Pat. No. 4,405,644; esteracin (U.S. Pat. Nos. 4,189,438 and 4,242,453); and cyclo-O,O'-[(1 ,6- hexanediyl)-bis-(iminoc- arbony!)]dioxime, and the various bis(iminocarbonyl)dioximes related thereto (Petersen et al., Liebig's Annalen, 562, 205-229 (1949).

Any bile acid sequestrant known in the art may be used. Examples of suitable bile acid sequestrants include, but are not limited to, Welchol^®, Colestid^®, LoCholest^® , Questran^® and fibric acid derivatives, such as Atromid^®, Lopid^® and Tricor^®' 2.2.2 Anti-hypertensive Agents Any antihypertensive agent known in the art may be used in a combination of the present invention. Antihypertensive activity may be determined according to standard tests (e.g. blood pressure measurements). Examples of suitable antihypertensive agents include, but are not limited to, (a) amlodipine and related dihydropyridine compounds (US Pat. Nos. 4,572,909 and 5,155,120) such as, but not limited to, amlodipine benzenesulfonate salt (also termed amlodipine besylate (Norvasc^®) ( U.S. Pat. No.

4,879,303) and other pharmaceutically acceptable acid addition salts of amlodipine (U.S. Pat. No. 5,155,120) (also, amolidpine besylate is combined with the statin, atorvastatin calcium, and marketed as Caduet®) (e.g., U.S. Pat. No. 6455574); (b) calcium channel blockers such as, but not limited to, bepridil (U.S. Pat. No. 3,962, 238 or U.S. Reissue No. 30,577), clentiazem (U.S. Pat. No. 4,567,175), diltiazem (U.S. Pat. No. 3,562), fendiline (U.S. Pat. No. 3,262,977), gallopamil (U.S. Pat. No. 3,261 ,859); mibefradil, prenylamine, semotiadil, terodiline, verapamil, aranipine, barnidipine, benidipine, cilnidipine, efonidipine, elgodipine, felodipine, isradipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine, cinnarizine, flunarizine, lidoflazine, lomerizine, bencyclane, etafenone, and perhexiline; (c) angiotensin converting enzyme inhibitors ("ACE-lnhibitors") such as, but not limited to, alacepril (U.S. Pat. No. 4,248,883), benazepril (U.S. Pat. No. 4,410,520), captopril, ceronapril, delapril, enalapril, fosinopril, imadapril, lisinopril, moveltopril, perindopril, quinapril, ramipril, spirapril, temocapril, and trandolapril; (d) angiotensin-ll receptor antagonists such as, but not limited to, candesartan (U.S. Pat. No. 5,196,444), eprosartan (U.S. Pat. No. 5,185,351), irbesartan, losartan, and valsartan; (e) beta-adrenergic receptor blockers (beta- or β-blockers) such as, but not limited to, acebutolol (U.S. Pat. No. 3,857,952), alprenolol, amosulalol (U.S. Pat. No. 4,217,305), arotinolol, atenolol, befunolol, betaxolol, metoprolol and carvedilol (COREG, GlaxoSmithKline); (f) alpha-adrenergic receptor blockers (alpha- or α-blockers) such as, but not limited to, amosulalol (U.S. Pat. No. 4,217,307), arotinolol (U.S. Pat. No. 3,932,400), dapiprazole, doxazosin, fenspiride, indoramin, labetolol, naftopidil, nicergoline, prazosin, tamsulosin, tolazoline, trimazosin, and yohimbine, which may be isolated from natural sources according to methods well known to those skilled in the art; and g) other marketed products such as eplerenone (INSPRA, Pfizer) and spironolactone (ALDACTONE, Pfizer). 2.2.3 Thrombosis Regulating Agents

Any thrombosis regulating agent known in the art may be used in a combination of the present invention. Thrombosis regulating activity may be determined according to standard tests known in the art. Examples of suitable thrombosis regulating agents include antithrombotic or antiplatelet agents, which prevent clots from forming, and fibrinolytic or thrombolytic agents, which dissolve existing clots. Examples of antithrombotic agents include Vitamin K antagonists (e.g., Warfarin), unfractionated heparin, low molecular heparin (e.g., enoxaparin (LOVENOX, Aventis)) and dalteparin (FRAGMIN, Pfizer). Examples of antiplatelet agents include aspirin, which is a cycloxygenase inhibitor and therefore blocks the formation of thromboxane A₂ (TXA₂). TXA₂ normally causes vasoconstriction and platelet aggregation. Other examples of antiplatelet agents include ticlopidine (TICLID, Roche), a thienopyridine derivative, which binds platelet ADP receptors and reduces platelet activation; clopidogrel (PLAVIX, Bristol-Myers Squibb), a thienopyridine derivative, which binds platelet ADP receptors and reduces platelet activation; cilostazol (PLETAL, Otsuka), a phosphodiesterase III inhibitor; dipyridamole

(PERSANTINE or AGGRENOX (in combination with aspirin), Boehringer Ingelheim), which is an adenosine reuptake inhibitor; and abciximab (REOPRO, Lilly), eptifibatide (INTEGRILIN, Millenium and Schering) and tirofiban (AGGRASTAT, Merck), which are antagonists of platelet receptor glycoprotein Gp llb/llla. Examples of fibrinolytic or thrombolytic agents include streptokinase (STREPTASE, Merck); urokinase

(ABBOKINASE, Abbot); and tissue plasminogen activator (TpA, ACTIVASE, Genentech). 2.3 Anti-Inflammatory Agents

The anti-inflammatory agents are a heterogeneous group of drugs used to diminish or suppress inflammation. They are divided into two major groups: non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs.

Examples of NSAIDs include, but not limited to aspirin, ibuprofen (ADVIL, Wyeth; MOTRIN, McNeil), celecoxib (CELEBREX, Pfizer), diclofenac (VOLTAREN, Novartis), etodolac (LODINE, Wyeth), fenoprofen (NALFON), indomethacin (INDOCIN, Merck), ketoralac (TORADOL), oxaprozin (DAYPRO), nabumetone (RELAFEN, GlaxoSmithKline), sulindac (CLINORIL, Merck), tolmentin (TOLECTIN), naproxen

(ALEVE, NAPROSYN, Roche), ketoprofen (ACTRON, ORUDIS, ORUVAIL, APO-KETO, RHODIS).

Steroidal anti-inflammatory drugs include, but not limited to glucocorticoids, dexamethasone (DECADRON, Merck), cortisone, hydrocortisone, prednisone (DELTASONE, Pharmacia), prednisolone, triamcinolone, azulfidine and eicosanoids such as prostaglandins, thromboxanes and leukotrienes.

NSAIDs employ their effect as inhibitors of prostaglandin biosynthesis by irreversible (e.g., aspirin) or reversible (the vast majority of NSAIDs) inhibition of cyclooxygenase activity, the first enzyme in the prostaglandin synthesis pathway. Cyclooxygenase converts arachidonic acid to the unstable intermediates prostaglandin G₂ (PGG₂) and prostaglandin G₃ (PGG₃). There are two forms of cyclooxygenase, termed cyclooxygenase-1 (COX-1 ) and cyclooxygenase-2 (COX-2) (Vane et al., Annu Rev Pharmacol Toxicol 38:97-120 (1998)). COX-1 is a constitutive isoform found in most normal cells and tissues, while COX-2 is induced in settings of inflammation by cytokines and inflammatory mediators (Seibert et al., Adv Exp Med Biol 400A:167-170 (1997)).

Based on the existence of the two isoforms of cyclooxygenase, NSAIDs are classified as nonselective COX inhibitors and selective COX-2 inhibitors.

Nonselective COX inhibitors include, but are not limited to, salicylic acid derivatives (e.g., aspirin, sodium salicylate, choline magnesium trisalicylate, salsalate, diflunisal, sulfasalazine, olsalazine), para-aminophenol derivatives (e.g., acetaminophen), indole and indene acetic acids (e.g., indomethacin, sulindac), heteroaryl acetic acids (e.g., tolmetin, diclofenac, ketorolac), arylpropionoc acids (e.g., ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen, oxaprozin), anthranilic acids (e.g., mefenamic acid, meclofenamic acid), enolic acids (e.g., piroxicam, meloxicam) and alkanones (e.g., nabumetone).

Selective COX-2 inhibitors include, but are not limited to, diaryl-substituted furanones (e.g., rofecoxib), diaryl-substituted pyrazoles (e.g., celecoxib), indole acetic acids (e.g., etodolac) and sulfonanilides (e.g., numesulide). However, NSAIDs generally do not inhibit the formation of leukotrienes, which also contribute to inflammation.

NSAIDs find their chief clinical application in the treatment of musculoskeletal disorders, such as rheumatoid arthritis, osteoarthritis and ankylosing spondylitis. Chronic treatment of patients with celecoxib has been shown to be effective in suppressing inflammation without the gastric toxicity that is associated with treatment with nonselective NSAIDs (Simon et al., JAMA 282:1921-1928 (1999)). NSAIDs are also used for treating of dysmenorrhea, hypotension and vasodilation, humoral hypercalcemia, Banter's syndrome and colon cancer. Cardiovascular disorders usually involve a number of adverse events leading to a disease progression, such as vascular thrombosis, erosions, fissures or ruptures of atheromatous plaques, elevated levels of cholesterol, and inflammation. As such, early intervention is preferred. Otherwise, new therapies and therapeutic regimens are needed to treat these complex diseases and disease processes.

SUMMARY OF THE INVENTION

The present invention provides a method of treating or preventing a cardiovascular disease in a subject in need thereof, comprising administering, in any order, to the subject: an Apolipoprotein A-I Milano (Apo A-I Milano or AIM); a statin; and an anti-inflammatory drug.

More particularly, the present invention provides the above method wherein Apo A-I Milano is administered as a protein: lipid complex.

More particularly, the present invention provides the above method wherein the cardiovascular disease is selected from the group consisting of stroke, transient ischemic attacks, limb ischemia, bowel ischemia, femoral or iliac artery disease, Leriche's syndrome and sepsis. More particularly, the present invention provides the above method wherein the complex is administered at a dose of about 1 mg/kg to about 50 mg/kg. The present invention also provides the above method wherein the complex is administered at a dose of about 10 mg/kg to about 45 mg/kg. The present invention also provides the above method wherein the complex is administered at a dose of about 15 mg/kg to about 45 mg/kg. Most particularly, the present invention provides the above method wherein the complex is administered at the following doses: about 10 mg/kg, about 15 mg/kg, about 45 mg/kg or about 30 mg/kg

The present invention also provides the above method wherein the statin is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin and atorvastatin. More particularly, the present invention provides the above method wherein the statin is atorvastatin.

The present invention also provides the above method wherein the antiinflammatory drug is a nonsteroidal anti-inflammatory drug (NSAID). More particularly, the present invention provides the above method wherein the NSAID is a selective COX-2 inhibitor. More particularly, the present invention provides the above method wherein the NSAID is a selected from the group consisting of aspirin, celecoxib and etodolac. Most particularly, the present invention provides the above method wherein the NSAID is aspirin or celecoxib.

The present invention also provides the above method wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, arterosclerosis, acute coronary syndromes, restenosis, ischemic reperfusion injury, angina pectoris, stable angina, unstable angina, myocardial or cerebral ischemia and myocardial infarction.

The present invention provides the above method wherein the Apo A-I Milano is a recombinant Apo A-I Milano.

The present invention provides the above method wherein the lipid is a phospholipid, cholesterol, triglyceride, or cholesterol ester. More particularly, the present invention provides the above method wherein the lipid is a phospholipid. More particularly, the present invention provides the above method wherein the phospholipid is selected from the group consisting of phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dilaurylphosphatidylcholine,

1 -myristoyl^-palmitoylphosphatidylcholine, 1 -palnnitoyl-2-myristoylphosphatidylcholine, 1 -palmitoyl^-stearoylphosphatidylcholine, 1 -stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, 1 -palnnitoyl-2-oleoylphosphatidylcholine, 1 -oIeoyl-2- palmitylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, phosphatide acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, sphingomyelin, brain sphingomyelin, dipalmitoylsphingomyelin and distearoylsphingomyelin. Most particularly, the present invention provides the above method wherein the phospholipid is a 1 -palmitoyl-2-oleoylphosphatidylcholine (POPC). More particularly, the present invention provides the above method wherein the AIM:POPC complex has a ratio of about 1 :0.8 to about 1 :1.0 by weight. More particularly, the present invention provides the above method wherein the AlM:POPC complex has a ratio of about 1 :0.9 by weight or about 1 :1 by weight.

Also, the present invention provides the above method wherein the complex is administered as a pharmaceutical formulation. More particularly, the present invention provides the above method wherein the pharmaceutical formulation comprises a buffer comprising sucrose and mannitol, and wherein said Apo A-I Milano is recombinant Apo A-I Milano at a concentration of about 12 mg/ml to about 18 mg/ml of buffer. More particularly, the present invention provides the above method wherein the concentration is about 14.5 mg per ml of buffer. Also, the present invention provides the above method wherein the pharmaceutical formulation comprises an aqueous buffer comprising glucose and sodium phosphate, and wherein said buffer has a pH of about 7.0 to 7.8. More particularly, the present invention provides the above method wherein said buffer is about 2% glucose and about 4 mM of sodium phosphate, and wherein said pH is about 7.4. Also, the present invention provides the above method wherein the pharmaceutical formulation is a sterile liquid pharmaceutical formulation. The present invention provides the above method wherein the AIM:lipid complex, statin and anti-inflammatory drug are administered sequentially or concurrently. The present invention provides the above method wherein the AIM:lipid complex, statin and antiinflammatory drug are administered in the same pharmaceutical formulation or in different pharmaceutical formulations. In addition, the present invention provides a method of treating a cardiovascular disease, atherosclerosis, acute coronary syndromes, restenosis or ischemic reperfusion in a subject in need thereof comprising administering: an Apo A-I Milano and a lipid wherein the Apo A-I Milano and the lipid are in the form of an AIM:lipid complex; atorvastatin; and aspirin or celecoxib.

The present invention provides the above methods which further comprise an antihypertensive agent. More particularly, the present invention provides the above methods wherein the antihypertensive agent is amlodipine.

The present invention provides the above methods which further comprise a thrombosis regulating agent. More particularly, the present invention provides the above methods wherein the thrombosis regulating agent is dalteparin or clopidogrel.

The present invention provides the above methods which further comprise a lipid regulating agent other than a statin. More particularly, the present invention provides the above methods wherein the lipid regulating agent is a cholesterol absorption inhibitor. More particularly, the present invention provides the above methods where the cholesterol absorption inhibitor is ezetimibe.

In addition, the present invention provides a method of treating or preventing a cardiovascular disease in a subject in need thereof, comprising administering, in any order, to the subject: an Apolipoprotein A-I Milano (Apo A-I Milano or AIM); a lipid regulating agent other than a statin; and an anti-inflammatory drug. More particularly, the present invention provides the above method wherein the lipid regulating agent is an LDL lowering agent. More particularly, the present invention provides the above method wherein the LDL lowering agent is torcetrapib or ezetimibe. More particularly, the present invention provides the above method wherein the cardiovascular disease is selected from the group consisting of stroke, transient ischemic attacks, limb ischemia, bowel ischemia, femoral or iliac artery disease, Leriche's syndrome and sepsis.

More particularly, the present invention provides the above method which further comprises an antihypertensive agent. Most particularly, the present invention provides the above method wherein the antihypertensive agent is amlodipine. More particularly, the present invention provides the above method which further comprises a thrombosis regulating agent. Most particularly, the present invention provides the above method wherein the thrombosis regulating agent is dalteparin or clopidogrel.

More particularly, the present invention provides the above method which further comprises a second lipid regulating agent. In general, the invention encompasses triple combination therapies for prevention, treatment or amelioration of cardiovascular disease or related disorders including but not limited to atherosclerosis, arteriosclerosis, acute coronary syndromes, myocardial or cerebral ischemia, ischemic reperfusion injury, restenosis, angina pectoris, stable angina, unstable angina, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, intermittent claudication, peripheral artery disease, bowel ischemic related to occlusion of mesenteric or celiac arteries, occlusive femoral or iliace disease, Leriche's syndrome, sepsis, reduction or stabilization of atherosclerotic plaque, reduction of plaque in narrow or occluded vessels and promotion of cholesterol efflux in a subject. The methods comprise administering to a subject in need thereof an Apo A-I Milano ("AIM") or an AIM:lipid complex, one or more statins, and one or more anti-inflammatory agents, either simultaneously or sequentially (referred to herein as "triple combination therapy"). The invention encompasses a unique approach for the treatment and prevention of cardiovascular disease or related disorders using an exogenously produced HDL mimetic, such as Apo A-I Milano, preferably as an AIMrphospholipid complex in the combination therapy with one or more statins and one or more anti-inflammatory agents. Methods, compositions and dosage regimens are provided herein, and are believed to encompass safe, effective and non-surgical treatments, without being limited by theory, that rapidly promote cholesterol efflux and mobilization from atherosclerotic plaques, which reduces atheroma volume in one or more affected vessels. Reduced atheromas (the mass of plaque of degenerated thickened arterial intima occurring in atherosclerotic vessels) allow greater blood flow and a reduction in the risk of ischemic vascular events, including unstable angina, stroke, transient ischemic attacks, myocardial infarctions and acute coronary syndromes. The pharmaceutical compositions of the present invention may comprise a single active, two active or three active ingredients selected from one or more Apo A-I Milano or derivatives thereof, one or more peptiderlipid complexes such as AIM:lipid complex, one or more statins, and one or more an anti-inflammatory drugs.

In particular, the invention provides combination therapies for the prevention or treatment of cardiovascular disease or related disorders, most preferably utilizing a triple combination regimen of the ETC-216 (a specific AIM:lipid complex), the statin atorvastatin, and the anti-inflammatory agent aspirin or celecoxib. The invention also encompasses doses and dosing regimens for each component of the triple combination therapy to treat or prevent cardiovascular disease or related disorders in a subject. The compositions, methods and formulations described herein are believed to, without being limited by theory, cause the rapid reduction or stabilization of unstable atherosclerotic plaques which, if left untreated or treated by conventional methods, can rupture and lead to ischemic events including acute coronary syndromes. The present invention provides compositions and methods for the prevention or treatment of cardiovascular disease or related disorders comprising administering an Apo A-I Milano as part of a triple combination therapy to a subject in need thereof. The Apo A- I Milano can be any recombinant, synthetic or purified human or non-human Apo A-I Milano obtained from any source available by any method well-known in the art.

Preferably, the Apo A-I Milano is a recombinant protein. Similarly, it is preferred that the ApoA-l Milano is human ApoA-l Milano. In the methods and composition of the present invention an Apo A-I Milano can be complexed with a lipid. As used herein, Apo A-I Milano, or lipid complexes thereof, may or may not be in the form of a pharmaceutical composition. In the methods and compositions of the present invention, an Apo A-I Milano can be administered to a subject in need thereof in a dose of about 1 mg/kg to about 50 mg/kg.

The present invention provides compositions and methods for the prevention or treatment of cardiovascular disease or related disorders comprising administering an anti- inflammatory agent as part of a triple combination therapy to a subject in need thereof. The anti-inflammatory agent can be any non-steroidal or steroidal anti-inflammatory agent, but is preferably an oral dosage form. Preferably, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. More preferably, the anti-inflammatory agent is aspirin. Also, more preferably, the anti-inflammatory agent is a selective COX-2 inhibitor. Most preferably, the anti-inflammatory agent is celecoxib. As used herein, the antiinflammatory agent may or may not be administered in the form of a pharmaceutical composition. In the methods and compositions of the present invention, celecoxib can be administered to a subject in need thereof in a dose of about 1.5 mg/kg to about 3.0 mg/kg. In the methods and compositions of the present invention, aspirin can be administered to a subject in need thereof in a dose of about 75 to about 650 mg/day.

The present invention provides compositions and methods for the prevention or treatment of cardiovascular disease or related disorders comprising administering a statin as part of a triple combination therapy to a subject in need thereof. The statin can be any statin selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin and atorvastatin. Preferably, the statin is an oral dosage form, most preferably atorvastatin. As used herein, the statin may or may not be administered in the form of a pharmaceutical formulation. In the methods and compositions of the present invention, atorvastatin can be administered to a subject in need thereof in a dose of about 0.15 mg/kg to about 1.2 mg/kg.

In one embodiment, the patient in need of treatment is administered atorvastatin on a regular basis, i.e., daily, weekly or monthly for example, in accordance with its conventional use as a single agent; however, the patient is also administered an antiinflammatory agent on a regular basis, i.e., daily, weekly or monthly for example, in accordance with its conventional use as a single agent; and during the course of these continuous or intermittent courses of chronic treatments, the patient is infused with ETC- 216 weekly, monthly, semi-annually, annually or at times when the patient suffers an acute ischemic event (e.g., stroke or myocardial infarction). In certain embodiments, the compositions and methods of the invention comprise administration of a pharmaceutical composition of the ETC-216 at both high and low dose in conjunction with atorvastatin and aspirin or ceiecoxib at the dose in which they are used as single agents or a lower dose but in both cases are administered orally. In one embodiment encompassed by the present invention, a high dose of the ApoA-l Milano in a triple combination over a short dosing interval may be used to reduce atheroma volume in subjects with partially or fully occluded vessels. Alternatively, a low dose of the ApoA-l Milano in a triple combination over a long dosing interval, or intermittent courses of therapy with time breaks for adaptation, may be used as a preventive measure in a subject at risk to develop symptoms of cardiovascular disease or related disorders. In certain embodiments, the triple pharmaceutical combination can be administered before, during and/or after a surgical intervention for revascularization or reperfusion, such as Percutaneous Translumenal Coronary Angioplasty (PTCA), with and without stent placement, to unblock an occluded vessel. In certain embodiments, one or more intermittent doses can be administered to the subject to maintain the patency of a previously occluded vessel ("maintenance dose").

The present invention provides pharmaceutical kits which comprise an ETC-216 dosage form in a combination therapy with a statin or an anti-inflammatory drug or both; preferably the statin and the anti-inflammatory drug are present as separate ■ dosage forms for oral administration. Alternatively, the oral dosage forms of statin and anti-inflammatory agent can be combined into a single unit dosage form.

The pharmaceutical formulations can be labeled and have accompanying labeling to identify the formulation contained therein and other information useful to health care providers and subjects in the prevention or treatment of acute coronary syndromes, including, but not limited to, instructions for use, dose, dosing interval, duration, indication, contradictions, warnings, precautions, handling and storage instructions and the like.

The invention encompasses double combination therapies for prevention, treatment or amelioration of cardiovascular disease or related disorders including but not limited to atherosclerosis, arteriosclerosis, acute coronary syndromes, myocardial or cerebral ischemia, ischemic reperfusion injury, restenosis, angina pectoris, stable angina, unstable angina, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, intermittent claudication, peripheral artery disease, bowel ischemic related to occlusion of mesenteric or celiac arteries, occlusive femoral or iliace disease, Leriche's syndrome, sepsis, reduction or stabilization of atherosclerotic plaque, reduction of plaque in narrow or occluded vessels and promotion of cholesterol efflux in a subject. In certain embodiments, the methods comprise administering to a subject in need thereof an Apo A- I Milano ("AIM") or an AIM:lipid complex and a CETP inhibitor, more specifically torcetrapib, either simultaneously or sequentially. In certain embodiments, the methods comprise administering to a subject in need thereof an Apo A-I Milano ("AIM") or an AIM:lipid complex and a PPAR-activated compound, either simultaneously or sequentially. In certain embodiments, the methods comprise administering to a subject in need thereof an Apo A-I Milano ("AIM") or an AIM:lipid complex and a cholesterol absorption inhibitor, either simultaneously or sequentially.

The present invention also encompasses the use of Apo A-I Milano ("AIM") or an

AIM:lipid complex for the treatment of sepsis. Sepsis, which includes severe sepsis and septic shock, is a systemic inflammatory response to infection or trauma, associated with and mediated by the activation of a number of host defense mechanisms including the cytokine network, leukocytes, and the complement and coagulation/fibrinolysis systems (Mesters, et al., Blood 88:881 -886, 1996). Disseminated intravascular coagulation (DIG), with widespread deposition of fibrin in the microvasculature of various organs, is an early manifestation of sepsis/septic shock. DIC is an important mediator in the development of the multiple organ failure syndrome and contributes to the poor prognosis of patients with septic shock (Fourrier, et al., Chest 101 :816-823, 1992). Current treatments for sepsis include Xigris® (Lilly) (drotrecogin alfa (activated)) (See, e.g., Physicians Desk Reference 2005. 59^th Ed., Thompson PDR, Montvale, NJ).

Sepsis may be caused by bacterial (either Gram negative or Gram positive), fungal, viral and other infections as well as by non-infective stimuli such as multiple trauma, severe burns, and organ transplantation. Additional information on sepsis is contained in the following references, which are hereby incorporated by reference: U.S. Patent No. 6,344,197; U.S. Patent No. 5,128,318; U.S. Patent No. 5,344,822; U.S. Patent No. 5,506,218; U.S. Patent No. 5,587,366; U.S. Patent No. 5,614,507; 5,674,855; International Publication No. WO 03/059270; D. M. Levine et al., "In vivo protection against endotoxin by plasma high density lipoprotein," Proc. Natl. Acad. Sci. USA, Vol. 90, pp. 12040-12044, December 1993; R.D. Goldfarb et al., " Protein-free phospholipid emulsion treatment improved cardiopulmonary function and survival in porcine sepsis," Am. J. Physiol. Regul. Integr. Comp. Physiol., 284: R550-R557, 2003; and B.R. Gordon et al., Neutralization of Endotoxin by a Phospholipid Emulsion in Healthy Volunteers," The Journal of Infectious Diseases, 2005:191 :1515-22 (1 May).

DETAILED DESCRIPTION OF THE INVENTION

4.1 Definitions

As used herein, the following terms shall have the following meaning: The terms "treat", "treating" or "treatment" refer to alleviating, reducing, abrogating, or otherwise modulating a disease, disorder and/or symptoms thereof, that is a therapeutic effect on an existing condition

The term "therapeutically effective amount" refers to that amount of an active ingredient sufficient to improve one or more of the symptoms of the condition or disorder being treated as compared to those symptoms that occur without treatment. The improvement may be temporary or permanent.

The term "prophylactically effective amount" refers to that amount of an active ingredient sufficient to result in the prevention, onset or recurrence of one or more symptoms of the condition or disorder.

The terms "prevent", "preventing" or "prevention" refer to barring, or reducing the risk of, a subject from acquiring a disease, disorder and/or symptoms thereof.

The term "acute coronary syndromes" refers to ischemic disorders, such as unstable angina, Q wave and non-Q wave myocardial infarction, NSTEMI or STEMI type myocardial infarction, or as provided by International Classification of Disease 9^th version (ICD-9) to be superceded by 10^th version (ICD-10). The myocardial infarction can present with or without an ST-segment elevation or depression on electrocardiograph.

The term "ischemia" refers to local inadequacy of blood flow to meet tissue demands due to mechanical or biologically induced, e.g., spasm, thrombosis, stenosis or obstruction (mainly arterial narrowing or disruption) of the blood supply. The term "myocardial ischemia" refers to inadequate circulation of blood to the myocardium to permit normal heart cell function, usually as a result of coronary artery disease.

The term "ischemic reperfusion" refers to reestablishment of an increased amount of oxygenated blood to the tissue, often with attendant ischemic byproducts in the blood. The term "cardiovascular diseases" refers to heart, blood vessel, and blood circulation diseases, such as myocardial infarction, acute coronary syndrome, atherosclerosis, angina, stroke, ischemic reperfusion injury and other related disorders described herein and known to those in the art.

The term "unstable angina" refers to an unstable change and/or worsening frequency, severity or duration of symptoms of chronic stable angina, often due to partial or complete coronary thrombosis. Chronic stable angina includes epigastric discomfort or pain, or pain in the chest, which can radiate to the jaws and arms of a subject that is induced by exercise, eating and/or stress and is relieved by rest or nitroglycerin without recent change in frequency or severity of activity necessary to produce symptoms. The term "surgical intervention" as used herein refers to manual, non- pharmacologic or operative methods (including arterial catheters) used for diagnosis, imaging (e.g., radiology), prevention or treatment of a disease or condition.

The term "combination therapy" refers to the use of more than one and preferably three drugs to treat or prevent disease in a patient. Each component of the combination therapy may or may not be in the form of a pharmaceutical composition. The drugs may be used simultaneously or sequentially. But they must be used in concert to treat the patient. Preferably, one or more of the combination agents are oral. More preferably, two of the components of the combinations are used simultaneously. The term "pharmaceutical formulation" refers to a composition comprising either an active ingredient and a suitable diluent, carrier, vehicle, or excipients suitable for administration to a subject. The pharmaceutical formulation or composition will comprise at least one of ApoA-l Milano, a statin or an anti-inflammatory drug. The terms "composition" and "formulation" are used interchangeably herein. The term is also meant to encompass situations wherein the components of the combination therapy are in the same or separate formulations. This term includes, but is not limited to oral, parenteral, mucosal and topical compositions as described below.

The term "statins" refers to a group of compounds, which inhibit cholesterol synthesis or preferably are HMG-CoA reductase inhibitors. Examples of statins include lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin, ezetimibe/simvastatin

(VYTORIN, Merck and Schering-Plough), atorvastatin or combinations thereof (Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ed. J. Hardman, L. Limbird and A. Goodman Gilman, McGraw-Hill Medical Publishing Division, 10^th Edition, 2001 , pp. 982-987). The term "anti-inflammatory drugs" refers to a group of compounds that counteract inflammation. As known by those in the art, inflammation can include, but is not limited to, a local response to cellular injury that is marked by capillary dilation, ■ leukocytic infiltration, redness, heat, and/or pain. The term "an anti-inflammatory drug" encompasses anti-inflammatory steroids and non-steroidal anti-inflammatory agents (NSAIDS). (Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ed. J.

Hardman, L Limbird and A. Goodman Gilman, McGraw-Hill Medical Publishing Division, 10^th Edition, 2001 , pp. 687-715).

The term "about" refers to a relative term denoting an approximation of plus or minus 10% of the nominal value it refers to, preferably plus or minus 5%, most preferably plus or minus 2%. For the field of this disclosure, this level of approximation is appropriate unless the value is specifically stated require a tighter range.

The term "label" refers to a display of written, printed or graphic matter upon the immediate container of an article, for example, the written material displayed on a vial containing a pharmaceutically active agent. The term "labeling" refers to all labels and other written, printed or graphic matter upon any article or any of its containers or wrappers or accompanying such article, for example, a package insert, instructional videotapes or instructional DVDs accompanying or associated with a container of a pharmaceutically active agent. 4.2 Pharmaceutical Compositions of the Invention The compositions and combination methods of the invention are meant to provide unique and effective approach to the treatment of cardiovascular disease or related disorders. In the compositions and methods of the invention, the triple combination therapy provides a non-surgical therapy that reverses the pathophysiologic basis of the disease and/or provides symptomatic relief. The compositions and methods of the invention provide administration of the triple combination therapy, which provides HDL- mimetic or HDL-like therapy, and which promotes cholesterol efflux, reverse cholesterol transport reduction in atherosclerotic plaque.

The compositions including doses provided herein, without being limited by theory are meant to act rapidly to reduce or stabilize atherosclerotic plaque in as little as a few weeks by promoting reverse cholesterol mobilization out of the arterial wall. In certain embodiments, the triple combination therapy described herein is meant to be used to treat a subject suffering from acute coronary syndromes or disorders associated with ischemia or vessel occlusion. In other embodiments, the triple combination therapy described herein may be used to treat or prevent disease progression once atherosclerotic plaques present a risk to the subject. The compositions and methods may be further used to treat or prevent an occlusion of a vessel or to treat or prevent the rupture of an unstable atherosclerotic plaque or plaques, which can lead to new or recurrent ischemic conditions including acute coronary syndromes, stroke and ischemic reperfusion injury.

The pharmaceutical composition of the present invention may comprise a single active, two active or three active ingredients selected from one or more ApoA-l Milano or a derivative thereof, one or more peptide:lipid complexes such as AIM:lipid complex, one or more statins, and one or more an anti-inflammatory drugs. Additional pharmaceutical compositions of the present invention may comprise the above active ingredients, plus four active, five active, six active or more active ingredients selected from one or more lipid regulating agents, one or more antihypertensive agents, and one or more thrombosis regulating agents.

The pharmaceutical composition can contain pharmaceutically acceptable excipients as required to approximate physiological conditions, such as pH adjusting agents, buffering agents, and tonicity adjusting agents (e.g., sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride). Antibacterial agents (e.g., phenol, benzalkonium chloride or benzethonium chloride) can be added to maintain sterility of a product, especially pharmaceutical formulations intended for multi-dose parenteral use. Suspending, stabilizing and/or dispersing agents can also be used in the compositions of the invention.

The pharmaceutical formulation can be in a variety of forms suitable for any route of administration which include, but are not limited to parenteral, intra-arterial or trans- arterial, intradermal, subcutaneous, oral, transdermal, transmucosal and rectal administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for parental administration. Parenteral administration refers to any route of administration that is not through the alimentary canal, including, but not limited to, injectable administration (i.e., intravenous, intraarterial intramuscular and the like as described herein) (see generally, Remington's Pharmaceutical Sciences. 18^th Edition, Gennaro et al., eds., Mack Printing Company, Easton, Pennsylvania, 1990). In another preferred embodiment, the pharmaceutical formulation is infused directly into the lumen of an occluded or diseased artery.

The injectable pharmaceutical formulations can be a pharmaceutically appropriate formulation for administration directly into the heart, pericardium or coronary arteries. In preferred embodiments, the pharmaceutical formulations are infused into a peripheral blood vessel of a subject, e.g., at the arm or antecubital fossa.

Injectable pharmaceutical formulations can be sterile suspensions, solutions or emulsions in aqueous or oily vehicles. The formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, and can comprise added preservatives. Preferred buffers for parenteral pharmaceutical formulations are phosphate, citrate and acetate, and may contain stabilizers (e.g., sucrose, mannitol, trehalose).

Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention.

In another preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for oral administration. Pharmaceutical compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, such compositions are presented in tablet or capsule form to facilitate, for example, accurate dosing. Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art (see generally, Remington's Pharmaceutical Sciences, 18^th Edition, Gennaro et al., eds., Mack Printing Company, Easton, Pennsylvania, 1990).

Typical oral dosage forms of the invention are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof. Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.

Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101 , AVICEL-PH-103 AVICEL RC-581 , AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, PA), and mixtures thereof. An specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103™ and Starch 1500 LM.

The pharmaceutical formulations can be formulated for a single, one-time use or can be formulated in multiple doses. The components of the combination therapy can be in the same or different pharmaceutical formulations and can be administered simultaneously or sequentially. In certain embodiments, the pharmaceutical formulation can be in sterile pre-filled syringes or sterile pre-filled bags which can be frozen or refrigerated. The formulation can also be lyophilized and reconstituted with a suitable vehicle.

4.3 Use of Apolipoprotein A-I Milano

In one aspect, the present invention provides compositions and methods for the treatment or prevention of injury from acute coronary syndromes, stroke or related conditions by administering a composition comprising Apo A-I Milano to a subject in need thereof and also using one or more statins or antiinflammatories in concert. In certain embodiments the Apo A-I Milano can be complexed with a lipid. As used herein, Apo A-I Milano, or lipid complexes thereof, may or may not be in the form of a pharmaceutical formulation.

In certain embodiments, the Apo A-I Milano can be a variant or conservatively substituted Apo A-I Milano. By "conservative substitution" it is meant that certain amino acid residues of Apo A-I Milano can be replaced with other amino acid residues without significantly deleteriously affecting the activity of the protein. Thus, also contemplated by the present invention are altered or substituted forms of Apo A-I Milano, wherein at least one defined amino acid residue in the structure is substituted with another amino acid residue.

The Apo A-I Milano utilized in the invention can be obtained from any source available. For example, the Apo A-I Milano can be recombinant, synthetic, semi-synthetic or purified Apo A-I Milano. In a preferred embodiment, the Apo A-I Milano is a recombinant protein (rApo A-I Milano or rAIM) expressed in yeast or E. coli as described in U.S. Patent No. 5,721 ,114 and European Patents EP 0 469 017 and EP 0 267 703.

Methods for obtaining Apo A-I Milano, utilized by the invention are well-known in the art. For example, Apo A-I Milano can be separated from plasma, for example, by density gradient centrifugation followed by delipidation of the lipoprotein, reduction, denaturization and gel-filtration chromatography, ion-exchanging chromatography, hydrophobic, e.g., phenyl sepharose, interaction chromatography or immunoaffinity chromatography, or produced synthetically, semi-synthetically or using recombinant DNA techniques known to those skilled in the art and subsequent purification familiar to those skilled in the art. (See, e.g., U.S. Patent Nos. 6,107,467; 6,559,284; 6,423,830; 6,090,921; 5,834,596; 5,990,081 ; 6,506,879; 5,059,528; 5,876,968 and 5,721 ,114; Mulugeta et al., J. Chromatogr. 798(1-2): 83-90 (1998); Chung et al., J. Lipid Res. 21 (3):284-91 (1980); Cheung et al., J. Lipid Res. 28(8):913-29 (1987); Persson et al., J. Chromatogr. 711 :97-109 (1998); and PCT Publications WO 86/04920 and WO 87/02062). If the Apo A-I Milano is obtained from natural sources, it can be obtained from any animal source of any species. In certain embodiments, the Apo A-I Milano is obtained from a mammalian source. In certain embodiments, the Apo A-I Milano is obtained from a human source. In preferred embodiments of the invention, the Apo A-I Milano is derived from the same species as the subject to which the Apo A-I Milano is administered. 4.3.1 Lipid Complexes of Apolipoprotein A-I Milano

In certain embodiments, the compositions and combination therapies of the invention comprise lipid complexes of Apo A-I Milano. In some embodiments, the invention provides pharmaceutical formulations of AIM:phospholipid complexes. Efficacy can be enhanced by the complexing of lipids or phospholipids to Apo A-I Milano. Typically, the lipid or phospholipid is mixed with the Apo A-I Milano prior to administration. Apo A-I Milano and lipids can be mixed in an aqueous solution in appropriate ratios and can be complexed by methods known in the art including freeze-drying or freeze-thaw cycles, detergent solubilization followed by dialysis, microfluidization, sonication, and homogenization. Complex efficiency can be optimized, for example, by varying pressure, ultrasonic frequency, or detergent concentration. An example of a detergent commonly used to prepare AIM:phospholipid complexes is sodium cholate.

In some cases it is preferable to administer the Apo A-I Milano alone, essentially lipid-free, in the triple combination therapy to treat or prevent acute coronary syndromes. In preferred embodiments, a triple combination therapy comprising an AIM:lipid complex is administered to a subject in need thereof.

In one embodiment, the AIM:phospholipid complex is in solution with an appropriate pharmaceutical diluent. In another embodiment, freeze-dried or lyophilized preparations of AIM:phospholipid complexes can be hydrated or reconstituted with an appropriate pharmaceutical diluent prior to administration. In another embodiment, the AIM:lipid complexes can be frozen preparations that are thawed until a homogenous solution is achieved prior to administration to a subject in need thereof. Each of these preparations may or may not comprise the other components of the combination therapy.

The lipid can be any suitable lipid known to those of skill in the art. Non- phosphorus containing lipids can be used, including stearylamine, dodecylamine, acetyl palmitate, (1 ,3)-D-mannosyl-(1 ,3)diglyceride, aminophenylglycoside, 3-cholesteryl- 6'-(glycosylthio)hexyl ether glycolipids, N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N- trimethylammonium chloride and fatty acid amides.

In preferred embodiments, the lipid is a phospholipid. The phospholipid can be obtained from any source known to those of skill in the art. For example, the phospholipid can be obtained from commercial sources, natural sources or by synthetic or semisynthetic means known to those of skill in the art (see, e.g., Mel'nichuk et al., Ukr. Biokhim. Zh. 59(6):75-7 (1987); Mel'nichuk et al., Ukr. Biokhim. Zh. 59(5):66-70 (1987); Ramesh et al., J. Am. Oil Chem. Soc. 56(5):585-7 (1979); Patel and Sparrow, J. Chromatogr. 150(2):542-7 (1978); Kaduce et al., J. Lipid Res. 24(10):1398-403 (1983); Schlueter et al., Org. Lett. 5(3):255-7 (2003); Tsuji et al., Nippon Yakurigaku Zasshi 120(1 ):67P-69P (2002)).

The phospholipid can be any phospholipid known to those of skill in the art. For example, the phospholipid can be a small alkyl chain phospholipid, phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, soy phosphatidylglycerol, egg phosphatidylglycerol, distearoylphosphatidylglycerol, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dilaurylphosphatidylcholine, 1-myristoyl-2-palmitoylphosphatidylcholine, 1 -palmitoyl-2-myristoylphosphatidylcholine, 1 -palmitoyl-2-stearoylphosphatidylcholine, 1 -stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, 1 -paimitoyl-2- oleoylphosphatidylcholine, 1-oleoyl-2-palmitylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerol, phosphatidylserine, pTiosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, phosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, sphingomyelin, sphingolipids, brain sphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, galactocerebroside, gangliosides, cerebrosides, phosphatidylglycerol, phosphatidic acid, lysolecithin, lysophosphatidylethanolamine, cephalin, cardiolipin, dicetylphosphate, distearoyl-phosphatidylethanolamine and cholesterol and its derivatives.

The phospholipid can also be a derivative or analogue of any of the above phospholipids. In certain embodiments, the AIM:phospholipid complex comprises combinations of two or more phospholipids.

In preferred embodiments, the triple combination therapy comprises an AIMrphospholipid complex. In a more preferred embodiment, the lipid is a phospholipid, preferably, 1-palmitoyl-2-oleoyl phosphatidylcholine ("POPC") or ("1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine"). In yet a more preferred embodiment, the AIM:POPC complex comprises about a 1 :1 ratio by weight of AIM:POPC. In a most preferred embodiment, the AIM:POPC complex is a pharmaceutical formulation. The AIM:POPC complex is referred to as ETC-216 (see JAMA. 2003; 290: 2292-2300; also TV Broadcast, "Good Morning America", April 15, 2004).

The complex comprising Apo A-I Milano and a lipid can comprise any amount of lipid, preferably phospholipid, and any amount of Apo A-I Milano effective to treat or prevent acute coronary syndromes or stroke either alone or as a component of the combination therapy. In certain embodiments, the Apo A-I Milano comprises a complex of Apo A-I Milano and a phospholipid in a ratio of about 1 :1 by weight. However, the Apo A-I Milano can comprise complexes with other ratios of phospholipid to Apo A-I Milano, such as about 100:1 , about 10:1 , about 5:1 , about 3:1 , about 2:1 , about 1 :1 , about 1 :2, about 1 :3, about 1 :5, about 1 :10 or about 1 :100. In certain embodiments, a ratio by weight of between about 1 :0.5 to about 1 :3; in one embodiment, the ratio is about 1 :0.8 to about 1 :1.2, or even more specifically, about 1 :0.8 to about 1 :1.0, to produce the most homogenous population of lipoprotein particles and to produce stable and reproducible batches. In even more preferred embodiments, the ratio by weight of Apo A-I Milano to phospholipid is about 1 :0.9 or, more specifically, about 1 :0.95.

Additional lipids suitable for use in the compositions and methods of the invention are well known to persons of skill in the art and are cited in a variety of well known sources, e.g., McCutcheon's Detergents and Emulsifiers and McCutcheon's Functional Materials, Allured Publishing Co., Ridgewood, N.J., both of which are incorporated herein by reference. Generally, it is desirable that the lipids are liquid-crystalline at 37^s C, 35^s C, or 32^s C. Lipids in the liquid-crystalline state typically accept cholesterol more efficiently than lipids in the gel state. As subjects typically have a core temperature of about 37^s C, lipids that are liquid-crystalline at 37^s C are generally in a liquid-crystalline state during treatment.

In certain embodiments, the combination therapy comprises a concentration of ETC-216 sufficient to treat a subject in need thereof. In certain embodiments, the concentration of ETC-216 is of about 2.5 mg/ml to about 50 mg/ml, or, more specifically, about 5.0 mg/ml to about 50 mg/ml. In preferred embodiments, the ETC-216 concentration is of about 10 mg/ml to about 20 mg/ml. In a more preferred embodiment, the concentration of ETC-216 is about 13 mg/ml to about 16 mg/ml. The ETC-216 concentration can be determined by any suitable technique known to those of skill in the art. In certain embodiments, the ETC-216 concentration is determined by size exclusion high performance liquid chromatography (SE-HPLC).

In preferred embodiments, the pharmaceutical formulation comprises sucrose in an amount sufficient to make a pharmaceutically suitable formulation with ETC-216. In certain embodiments, the pharmaceutical formulation comprises about 0.5% to about 20% sucrose.

In certain embodiments, the pharmaceutical formulation comprises about 3% to about 12% sucrose. In other embodiments, the pharmaceutical formulation comprises about 5% to about 7% sucrose. In preferred embodiments, the pharmaceutical formulation comprises about 6.0% to about 6.4% sucrose. In a most preferred embodiment, the pharmaceutical formulation comprises 6.2% sucrose.

In preferred embodiments, the pharmaceutical formulation comprises mannitol in an amount sufficient to make a pharmaceutically suitable formulation of ETC-216. In some embodiments, the pharmaceutical formulation comprises about 0.01 % to about 5% mannitol. In other embodiments, the pharmaceutical formulation comprises about 0.1% to about 3% mannitol. In certain embodiments, the pharmaceutical formulation comprises about 0.5% to about 2% sucrose. In preferred embodiments, the pharmaceutical formulation comprises about 0.7% to about 1% mannitol. In a most preferred embodiment, the pharmaceutical formulation comprises 0.8% mannitol.

In certain embodiments, the pharmaceutical formulation comprises a buffer in an amount sufficient to make a pharmaceutically suitable formulation of Apo A-I Milano or AIM:lipid complex, statin, and/or anti-inflammatory drug, either alone or with the other combination therapy components. In some embodiments, the pharmaceutical formulation comprises a phosphate buffer. In other embodiments, the buffer concentration is about 3 mM to about 25 mM. In yet other embodiments, the buffer concentration is about 5 mM to about 20 mM. In preferred embodiments, the buffer concentration is about 8 mM to about 15 mM. In certain embodiments, an appropriate buffer is added to adjust the pH of the pharmaceutical formulation to a range suitable for administration to a subject. In certain embodiments, the pharmaceutical formulation has a pH of about 6.8 to about 7.8. In some embodiments, the pharmaceutical formulation has a pH of about 7.0 to about 7.8. In other embodiments, the pharmaceutical formulation can have a pH of about 7.2 to about 7.5. In a preferred embodiment, the pharmaceutical formulation has a pH of about 7.4. 4.3.2 Preparation of Lipid Complexes of Apolipoprotein A-I Milano

The AIMrlipid complexes can be made by any method known to one of skill in the art. In some cases it is desirable to mix the lipid and Apo A-I Milano prior to administration. Lipids can be in solution or in the form of liposomes or emulsions formed using standard techniques such as homogenization, sonication or extrusion. Sonication is generally performed with a tip sonifier, such as a Branson tip sonifier, in an ice bath. Typically, the suspension is subjected to several sonication cycles. Extrusion can be carried out by biomembrane extruders, such as the Lipex Biomembrane Extruder (Lipex Biomembrane Extruder, Inc., Vancouver, Canada). Defined pore size in the extrusion filters can generate unilamellar liposomal vesicles of specific sizes. The liposomes can also be formed by extrusion through an asymmetric ceramic filter, such as a CERAFLOW microfilter (Norton Company, Worcester, MA) or through a polycarbonate filter or other types of polymerized materials (i.e., plastics) known to those of skill in the art.

An AIMrlipid complex can be prepared in a variety of forms, including, but not limited to vesicles, liposomes or proteoliposomes, using methods well known in the art. A number of available techniques for preparing liposomes or proteoliposomes can be used. For example, Apo A-I Milano can be co-sonicated

(e.g., using a bath or probe sonicator) with the appropriate lipid to form lipid complexes. In certain embodiments, Apo A-I Milano can be combined with preformed lipid vesicles resulting in the spontaneous formation of an AIM:lipid complex. In another embodiment, the Apo A-I Milano can be made by a detergent dialysis method. For example, a mixture of Apo A-I Milano, lipid and a detergent, such as cholate, can be dialyzed to remove the detergent and reconstituted to make the lipid complexes. (See, e.g., Jonas et al., Methods Enzymol. 128, 553-82 (1986)). In another embodiment, the lipid complexes can be made by co-lyophilization.

(See, e.g., U.S. Patent Nos. 6,287,590 and 6,455,088). Other methods for the preparation of complexes are disclosed, for example, in U.S. Patent Nos. 6,004,925, 6,037,323 and 6,046,166. Other methods of preparing AIM:lipid complexes will be apparent to those of skill in the art. In a preferred embodiment, the lipid complexes are made by homogenization. In one embodiment, the AIM:lipid complexes are made by first diluting recombinant Apo A-I Milano to a concentration of 15 mg/ml in solution with water for injection. Sodium phosphate is then added to a final concentration of 9-15 mM to adjust the pH to between about 7.0 and about 7.8. Next, mannitol is added to a concentration of about 0.8% to about 1 % (w/v), and POPC is added to achieve a mixture of about 1 :0.95 (wt protein/wt lipid). The mixture is stirred at 5000 rpm for about 20 minutes using an overhead propeller and an ULTRA TURRAX dispensing instrument while maintaining the temperature between 37° C to 43° C. The feed vessel is stirred continuously at about 100 to about 300 rpm while the temperature is maintained between 32° C to 43° C with in-line heat exchangers (Avestin, Inc.). Homogenization for the first 30 minutes is carried out at 50MPa (7250 psi) and thereafter, the pressure is maintained at 80-120 MPa (11600-17400 psi) until in-process testing by gel permeation chromatography demonstrates the % AUC of >70% between protein standards. 4.3.3 Pharmaceutical Compositions of ETC-216 The pharmaceutical composition of ETC-216 comprises an Apo A-I Milano:lipid complex suitable for treatment or prevention a cardiovascular disease or related condition.

In one embodiment, the pharmaceutical composition of Apo A-I Milano:lipid complex comprises a pharmaceutically acceptable carrier or vehicle. Many pharmaceutically acceptable carriers or vehicles can be employed, such as sucrose- mannitol, normal saline, glucose, trehalose, sucrose, sterile water, buffered water, 0.45% saline (half normal saline), and 0.3% glycine, and may further comprise glycoproteins for enhanced stability, such as albumin. These formulations can be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized (freeze-dried). The lyophilized preparation can then be combined with a sterile aqueous solution prior to administration.

The pharmaceutical formulations comprising the ETC-216 can be in a salt form. For example, because proteins can comprise acidic and/or basic termini and/or side chains, the ETC-216 can be in the pharmaceutical formulation as either a free acid or base, or as a pharmaceutically acceptable salt. Suitable acids capable of forming pharmaceutically acceptable salts with the ETC-216 include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Suitable bases capable of forming salts with Apo A-I Milano can include, for example, inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di-and tri-alkyl amines (e.g., triethyl amine, diisopropyl amine, methyl > amine, dimethyl amine and the like) and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine and the like).

In another embodiment, the pharmaceutical formulations can be provided in powder form or lyophilized form for reconstitution before use with a suitable vehicle, including but not limited to sterile pyrogen free water, saline or dextrose. To this end, the ETC-216 can be lyophilized. In another embodiment, the pharmaceutical formulations can be supplied in unit dosage forms and reconstituted prior to use.

For prolonged delivery, the pharmaceutical formulation can be provided as a depot preparation, for administration by implantation; e.g., subcutaneous, intradermal, or intramuscular injection. Thus, for example, the composition of ETC-216 can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives; e.g., as a sparingly soluble salt form of the ETC-216. In certain embodiments, the pharmaceutical formulation of the ETC-216 is a unit dose package. As is known to those of skili in the art, a unit dose package provides delivery of a single dose of a drug to a subject. The compositions and methods of the invention provide for a unit dose package of a pharmaceutical formulation comprising, for example, 1050 mg of Apo A-I Milano protein per package. For example, 1050 mg of Apo A-I Milano protein is an amount that administers 15 mg/kg of Apo A-I Milano to a 70 kg subject. The unit can be, for example, a sterile single use vial, a sterile pre-filled syringe, a sterile pre-filled bag (i.e., piggybacks) and the like.

In other embodiments, the pharmaceutical formulation is a unit-of-use package. As is known to those of skill in the art, a unit-of-use package is a convenient, prescription size, patient ready unit labeled for direct distribution by health care providers. A unit-of- use package contains a pharmaceutical formulation in an amount necessary for a typical treatment interval and duration for a given indication. The compositions and methods of the invention provide for a unit-of-use package of a pharmaceutical formulation comprising the ETC-216 in an amount sufficient to treat an average sized adult male or female with, for example, 15 mg/kg of Apo A-I Milano protein intravenously once weekly for 5 weeks. Thus, a unit-of-use package may comprise five doses of the ETC-216 (available in a vial or pre-filled syringes). In one embodiment, the unit-of-use package comprises a pharmaceutical formulation comprising the ETC-216 in an amount sufficient to treat an average sized adult male or female with a dose of 15 mg/kg, 30 mg/kg or 45 mg/kg of Apo A-I Milano once weekly for 5 weeks. It will be apparent to those of skill in the art that the doses described herein are based on the subject's body weight.

The pharmaceutical formulations comprising the ETC-216 must be of a₌suitable pH, osmolality, tonicity, purity and sterility to allow safe administration to a subject. The pharmaceutical formulations of ETC-216 are described in U.S. published application US20050142180, published on June 30, 2005, and U.S. published application , US20030109442, published on June 12, 2003, and U.S. published application US20040038891 , published on February 26, 2004, which are hereby incorporated by reference in their entireties. 4.3.4 Use of ETC-216 in Therapy

The doses of ETC-216 encompassed by the invention are described herein. Doses useful for treatment or prevention include doses up to about 50 mg/kg of the ETC- 216 administered to a subject in need thereof. In certain embodiments, the compositions and methods comprise administration of the ETC-216 at a dose of about 1 mg/kg to about 50 mg/kg of a subject's body weight. In particular embodiments, the₌compositions and methods comprise administration of the ETC-216 at a dose of about 10 mg/kg to about 50 mg/kg. In a preferred embodiment, the compositions and methods comprise administration of a pharmaceutical formulation of the ETC-216 at a dose of about 15 mg/kg. In another preferred embodiment, the compositions and methods comprise administration of the ETC-216 at a dose of about 30 mg/kg. In another preferred embodiment, the compositions and methods comprise administration of the ETC-216 at a dose of about 45 mg/kg. The₌compositions and methods can also comprise the use of a 15 mg/kg dose or 30 mg/kg dose either alone or in combination with the 45 mg/kg dose for the treatment or prevention of cardiovascular disease or related disorders.

It is understood by those of skill in the art that the actual dose of the components of the combination therapies of the invention can vary with the height, weight, age, severity of illness of the subject, the presence of concomitant medical conditions and the like. For example, an elderly subject with compromised renal or liver function can be treated with a dose of the ETC-216 that is at the lower range of the about 1 mg/kg dose (e.g., 0.8 mg/kg or 0.9 mg/kg) as part of the triple combination therapy. A subject with severe cardiovascular disease or related disorders that is obese with good renal and liver function can be treated with a dose of the ETC-216 that is, for example, at the upper range of the about 45 mg/kg dose (e.g., 50 mg/kg, 48 mg/kg, 45 mg/kg and the like as part of the triple combination therapy. The dosage of the components of the combination therapies described herein have been shown to be effective to achieve the intended purpose. These doses achieve a range of circulating concentrations that include the effective dose with an acceptable risk to benefit profile.

The dose of each component of the combination therapy can vary over the duration of treatment. For example, a subject can be treated with 45 mg/kg of the pharmaceutical formulation of the ETC-216 in the combination therapy, once weekly for 3 weeks and then treated with 15 mg/kg of the ETC-216 in the combination therapy once every four months or once per year for the lifetime of the subject. Such intermittent doses can be administered to maintain the patency of a vessel. Intermittent doses during the lifetime of the subject to maintain a reduced atheroma volume and increased vessel lumen are within the scope of the invention.

In certain embodiments, a single high dose (e.g., 50 mg/kg or 45 mg/kg) of the ETC-216 is administered to the subject. In other embodiments, one or more high doses of the ETC-216 are administered to the subject followed by one or more of the same or lower doses of the ETC-216 (e.g., about 1 , 5, 10, 15, 45, or 50 mg/kg), wherein each dose of the ETC-216 is given either alone or in combination (sequentially or simultaneously) with one or more statins and one or more anti-inflammatory drugs. Additionally, the opposite regimen may be used comprising administration of one or more low doses of the ETC-216 (e.g., about 1, 5, 10 or 15 mg/kg) followed by one or more of the same or higher doses of the ETC-216 (e.g., about 1 , 5, 10, 15, 45 or 50 mg/kg) either alone or in combination (sequentially or simultaneously) with one or more statins and one or more anti-inflammatory drugs. For acute conditions, the high dose is preferably delivered first.

In certain embodiments, the ETC-216 is administered as an intravenous infusion. In certain embodiments, the compositions and methods comprise administration as a intravenous push infusion. By intravenous push infusion, it is preferred that the ETC-216 is administered intravenously over a short time period, such as up to 5 minutes, for example, 2-5 minutes. In certain embodiments, administration of the ETC-261 comprises a continuous intravenous infusion. By continuous intravenous infusion it is preferred that the ETC-216 is administered continuously over a period of time, for example, about 1 hour to 3 hours, preferably, about 30 minutes to 120 minutes. Continuous intravenous infusions can be administered with the aid of an infusion pump or device. In certain embodiments, administration of the ETC-216 can be a combination of continuous intravenous infusions and intravenous push infusions ("bolus doses") of the therapeutic combination. The bolus doses can be administered before, after or during the continuous infusion. In certain embodiments the administration of the ETC-216 can be in combination with other drugs that treat or prevent cardiovascular disease, concomitant, or comorbid diseases, or that provide symptomatic relief. The administration of other drugs can be concurrent or sequential.

The compositions and methods provide for intravenous infusion of the Apo A-I Milano component of the regimen. Any suitable blood vessel can be used for infusion, including peripheral vessels, such as the vessels in the antecubital fossa of the arm or a central line into the chest. In preferred embodiments, the pharmaceutical formulation is infused into the cephalic or median cubital vessel at the antecubital fossa in the arm of a subject.

The compositions and methods of the present invention can be used in conjunction with surgical intervention, i.e., before, during or after surgery. Surgical intervention can include angioplasty, IVUS, coronary artery bypass graft (CABG), coronary angiography, implantation of vascular stents, percutaneous coronary intervention (PCI) and/or stabilization of plaques. In one embodiment, the method comprises administering the combination therapy before or after surgical intervention to open an occluded vessel, or to reduce atherosclerotic plaque in a vessel. Surgical intervention refers to manual, non-pharmacologic or operative methods (including intraarterial catheters) used for diagnosis, imaging (radiology) prevent or treatment of disease or a condition. For example, IVUS and coronary angiography are procedures that can provide a quantitative assessment of plaque burden (diagnostic purpose), angiography can provide images of vessels (radiologic purpose) and angioplasty can open an occluded vessel (treatment purpose). All are included as surgical interventions as used herein.

4.3.5 Timing of Adminstration of ETC-216

In certain embodiments, the methods for the treatment or prevention of cardiovascular disease or related conditions comprise administering the ETC-216 as part of a triple combination therapy about every day, about every other day, about every 3, 4, 5, 6, 7, 8 to 10 or 11 to 14 days to a subject in need thereof. In a preferred embodiment, the ETC-216 can be administered about every 7 days. In certain embodiments, administration of the ETC-216 can be a one time administration every six months or every year. In certain embodiments, administration can continue for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7-12 weeks, about 13-24 weeks or about 25-52 weeks. In certain preferred embodiments, administration of the ETC-216 is about every 7 days for about 5 weeks. In certain embodiments, administration can be intermittent after, for example, about 5 weeks. For example, a subject can be treated once a week for about 5 weeks and then treated about 3 to about 4 times over the following year. In certain embodiments, the combination therapy pharmaceutical formulations described herein can be administered to the subject intermittently to maintain the patency of a vessel. For example, the ETC-216 in a dose of about 15 mg/kg can be administered in the combination therapy about every 10 days for about 7 weeks and then administered, for example, about 26 weeks later or about 52 weeks later or ad hoc after the occurrence of an acute ischemic event, acute coronary syndrome or stroke.

4.4 Use of Statins in a Combination Therapy The invention provides a triple combination therapy comprising ETC-216, a statin, and an anti-inflammatory drug for the treatment and prevention of cardiovascular disease or related disorders including but not limited to atherosclerosis, acute coronary syndromes, myocardial or cerebral ischemia, restenosis, angina pectoris, stable angina, unstable angina, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, intermittent claudication, peripheral artery disease, bowel ischemia related to occlusion of mesenteric or celiac arteries, occlusive femoral or iliac disease, Leriche's syndrome, 'reduction or stabilization of atherosclerotic plaque, reduction of plaque in occluded vessels and promotion of cholesterol efflux.

The statins encompassed by the instant invention include, but are not limited to lovastatin (MEVACOR, Merck & Co., Inc.), simvastatin (ZOCOR, Merck & Co.), pravastatin (PRAVACHOL, Bristol-Myers Squibb Co.), atorvastatin (LIPITOR, Pfizer), rovusvastatin (CRESTOR, Astra-Zeneca), fluvastatin (LESCOL, Novartis) and ezetimibe/simvastatin (VYTORIN, Merck & Co.). In the preferred embodiment, the statin is atorvastatin, simvastatin or ezetimibe/simvastatin. In the most preferred embodiment, the statin is atorvastatin.

In one embodiment, a statin can be administered in the form of oral pharmaceutical composition. The other components of the combination therapy may be administered simultaneously or sequentially as one or more individual pharmaceutical formulation preparations. In certain embodiments, the pharmaceutical composition can contain pharmaceutically acceptable excipients or auxiliary substances such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride and the like. In one embodiment, the combination therapy comprises lovastatin at a dose of about 10 mg, about 20 mg or about 40 mg. In a preferred embodiment, lovastatin is an oral dosage form such as a tablet having 10 mg, 20 mg or 40 mg of the active ingredient.

In another embodiment, the combination therapy comprises simvastatin at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg or about 80 mg. In a preferred embodiment, simvastatin is an oral dosage form such as a tablet having 5 mg, 10 mg, 20 mg, 40 mg or 80 mg of the active ingredient.

In yet another embodiment, the combination therapy comprises ezetimibe/simvastatin at a dose of about 10 mg of ezetimibe combined with about 10 mg, about 20 mg, about 40 mg or about 80 mg of simvastatin. In a preferred embodiment, ezetimibe/simvastatin is an oral dosage form such as a tablet having 10 mg of ezetimibe and 10 mg, 20 mg, 40 mg or 80 mg of simvastatin.

In another embodiment, the combination therapy comprises pravastatin at a dose of about 10 mg, about 20 mg, about 40 mg or about 80 mg. In a preferred embodiment, pravastatin is an oral dosage form such as a tablet having 10 mg, 20 mg, 40 mg or 80 mg of the active ingredient.

In another embodiment, the combination therapy comprises fluvastatin at a dose of about 20 mg, about 40 mg or about 80 mg. In a preferred embodiment, fluvastatin is an oral dosage form such as a tablet having 20 mg, 40 mg or 80 mg of the active ingredient.

In another embodiment, the combination therapy comprises rosuvastatin at a dose of about 5 mg, about 10 mg, about 20 mg or about 40 mg. In a preferred embodiment, rosuvastatin is an oral dosage form such as a tablet having 5 mg, 10 mg, or 20 mg of the active ingredient. In a preferred embodiment, the combination therapy comprises atorvastatin at a dose of about 10 mg, about 20 mg, about 40 mg or about 80 mg. More preferably, atorvastatin is an oral dosage form such as a tablet having 10 mg, 20 mg, 40 mg or 80 mg of the active ingredient. As mentioned above, the statin delivery may be chronic on a daily or weekly basis while the patients also undergo a periodic administration of an Apo A-I Milano and a periodic or chronic administration of an anti-inflammatory agent.

4.4.1 Methods of Use of Statins

In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of lovastatin in a dose of about 0.15 mg/kg to about 0.6 mg/kg, or about 0.15 mg/kg to about 0.3 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of lovastatin in a dose of about 0.15 mg/kg, about 0.3 mg/kg or about 0.6 mg/kg.

In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of pravastatin in a dose of about 0.15 mg/kg to about 1.2 mg/kg, about 0.15 mg/kg to about 0.6 mg/kg, or about 0.15 mg/kg to about 0.3 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of pravastatin in a dose of about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg or about 1.2 mg/kg. In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of fluvastatin in a dose of about 0.3 mg/kg to about 1.2 mg/kg, or about 0.3 mg/kg to about 0.6 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of fluvastatin in a dose of about 0.3 mg/kg, about 0.6 mg/kg or about 1.2 mg/kg. In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of simvastatin in a dose of about 0.08 mg/kg to about 1.2 mg/kg, about 0.08 mg/kg to about 0.6 mg/kg, about 0.08 mg/kg to about 0.3 mg/kg, or about 0.08 mg/kg to about 0.15 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of simvastatin in a dose of about 0.08 mg/kg, about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg or about 1.2 mg/kg.

In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of ezetimibe/simvastatin in a dose of about 0.15 mg/kg to about 1.2 mg/kg, about 0.15 mg/kg to about 0.6 mg/kg, or about 0.15 mg/kg to about 0.3 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of ezetimibe/simvastatin in a dose of about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg or about 1.2 mg/kg. In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of rosuvastatin in a dose of about 0.07 mg/kg, about 0.14 mg/kg, about 0.29 mg/kg and about 0.57 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of rosuvastatin in a dose of about 0.07 mg/kg, about 0.14 mg/kg and about 0.29 mg/kg. In a preferred embodiment, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of atorvastatin in a dose of about 0.15 mg/kg to about 1.2 mg/kg, about 0.15 mg/kg to about 0.6 mg/kg, or about 0.15 mg/kg to about 0.3 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of atorvastatin in a dose of about 0.15 mg/kg, about 0.3 mg/kg, about 0.6 mg/kg or about 1.2 mg/kg.

In certain embodiments, statins are administered as a unit dosage form. As is known to those of skill in the art, a unit dosage form provides delivery of a single dose of a drug to a subject. In one embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 10 mg to about 40 mg or about 10 mg to about 20 mg of lovastatin.

In another embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 10 mg to about 80 mg, about 10 mg to about 40 mg or about 10 mg to about 20 mg of pravastatin.

In yet another embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 20 mg to about 80 mg or about 20 mg to about 40 mg of fluvastatin. In another embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 5 mg to about 80 mg, about 5 mg to about 40 mg, about 5 mg to about 20 mg or about 5 to about 10 mg of simvastatin.

In another embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 10 mg of ezetimibe and about 10 mg to about 80 mg of simvastatin, about 10 mg of ezetimibe and about 10 mg to about 40 mg of simvastatin or about 10 mg of ezetimibe and about 10 mg to about 20 mg of simvastatin.

In another embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 5 mg to about 20 mg or about 10 to about 40 mg of rosuvastatin.

In a preferred embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 10 mg to about 80 mg, about 10 mg about 40 mg, or about 10 mg to about 20 mg of atorvastatin. In particular embodiment, the present invention provides a unit dosage form that comprises about 10 mg, about 20 mg, about 40 mg or about 80 mg of atorvastatin per tablet. These unit dosage forms can be based on 5 mg or 10 mg dose regimens. For example, 10 mg of atorvastatin is an amount that administers 0.15 mg/kg of atorvastatin to a 70 kg subject. In one embodiment, tablets for oral administration contain 10 mg, 20 mg or 40 mg of lovastatin and the following inactive ingredients: cellulose, lactose, magnesium stearate, starch and iron oxides. (See, e.g., Physicians Desk Reference 2005. 59^th Ed., Thompson PDR, Montvale, NJ).

In another embodiment, tablets for oral administration contain 10 mg, 20 mg, 40 mg or 80 mg of pravastatin and the following inactive ingredients: croscarmellose sodium, lactose, magnesium oxide, magnesium stearate, microcrystalline cellulose, and povidone and iron oxides. (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ). In yet another embodiment, tablets for oral administration contain 20 mg, 40 mg or 80 mg of fluvastatin and the following inactive ingredients: gelatin, magnesium stearate, microcrystalline cellulose, pregelatinized starch (corn), red iron oxide, sodium lauryl sulfate, talc, titanium dioxide and yellow iron oxide. (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ).

In another embodiment, tablets for oral administration contain 5 mg, 10 mg, 20 mg, 40 mg or 80 mg of simvastatin and the following inactive ingredients: cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, iron oxides, lactose, magnesium stearate, starch, talc and titanium dioxide. (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ).

In yet another embodiment, tablets for oral administration contain 10 mg of ezetimibe and 10 mg of simvastatin, 10 mg of ezetimibe and 20 of simvastatin, 10 mg of ezetimibe and 40 mg of simvastatin or 10 mg of ezetimibe and 80 mg of simvastatin. The inactive ingredient include: butylated hydroxyanisole NF, citric acid monohydrate USP, croscarmellose sodium NF, hydroxypropyl methylcellulose USP, lactose monohydrate NF, magnesium stearate NF, microcrystalline cellulose NF and propyl gallate NF. (See, e.g., Physicians Desk Reference 2005. 59^th Ed., Thompson PDR, Montvale, NJ).

In another embodiment, tablets for oral administration contain 5 mg, 10 mg, 20 mg, 40 mg or 80 mg of rosuvastatin and the following inactive ingredients: microcrystalline cellulose NF, lactose monohydrate NF, tribasic calcium phosphate NF, crospovidone NF, magnesium stearate NF, hypromellose NF, triacetin NF, titanium dioxide USP, yellow ferric oxide, and red ferric oxide NF (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ).

In a preferred embodiment, tablets for oral administration contain 10 mg, 20 mg, 40 mg or 80 mg of atorvastatin and the following inactive ingredients: calcium carbonate, candelilla wax, croscarmellose sodium, hydroxypropyl cellulose, lactose monohydrate, magnesium stearate, microcrystalline cellulose, hypromellose, polyethylene glycol, talc, titanium dioxide, polysorbate 80, and semithicone emulsion. (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ). 4.4.2 Timing of Administration of Statins

In certain embodiments, the compositions and methods for the treatment or prevention of cardiovascular disease or related conditions comprise administering atorvastatin as part of a triple combination therapy about every day or about every other day to a subject in need thereof. In a preferred embodiment, atorvastatin can be taken daily for about 1 month, about 2 months, about 3 months, about every 4 months, about 5 months, or about 6 months. This scheme can be repeated after the interval of 1 or 2 months. In certain embodiments, administration of atorvastatin can continue for about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months or about 8-12 months. In certain embodiments, the combination therapy pharmaceutical formulations described herein can be administered to the subject intermittently to maintain the desirable cholesterol blood level. For example, atorvastatin in a dose of about 0.6 mg/kg can be taken daily for about 6 months and then 4 weeks later can be taken, for example, for another 6 months. In one embodiment, the patient in need of treatment is administered atorvastatin on a regular basis, i.e., daily, weekly or monthly, in accordance with its use as a single agent or combination therapy drug; however, the patient is also administered an antiinflammatory agent on a regular basis, i.e., daily or weekly; and during the course of these continuous or chronic treatments, the patient is infused with ETC-216 weekly, monthly, semi-annually or annually or ad hoc after the occurrence of an acute ischemic event like an acute coronary syndrome or stroke, as described in Sections 4.3.4 and 4.3.5.

Atorvastatin can be administered before, after, or at the same time as the other components of the combination therapy. In one embodiment, atorvastatin is administered sequentially with the other components of the combination therapy. In another embodiment, atorvastatin is administered concurrently with the other components of the combination therapy. Atorvastatin can be administered in any order with the other components of the combination therapy. 4.5 Use of Anti-Inflammatory Drugs in a Combination Therapy

The invention provides a triple combination therapy comprising the ETC-216, a statin, and an anti-inflammatory drug for the treatment and prevention of cardiovascular disease or related disorders including but not limited to atherosclerosis, acute coronary syndromes, myocardial or cerebral ischemia, restenosis, angina pectoris, stable angina, unstable angina, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, intermittent claudication, peripheral artery disease, bowel ischemia related to occlusion of mesenteric or celiac arteries, occlusive femoral or iliac disease, Leriche's syndrome, reduction or stabilization of atherosclerotic plaque, reduction of plaque in occluded vessels and promotion of cholesterol efflux. The anti-inflammatory agents encompassed by the invention include, but are not limited to aspirin (ECOTRIN, GlaxoSmithKline), naproxen (ALEVE, NAPROSYN, Roche Laboratories), and celecoxib (CELEBREX, Pfizer). In a preferred embodiment, the antiinflammatory drug is aspirin. In another preferred embodiment, the anti-inflammatory drug is celecoxib. In certain embodiments, anti-inflammatory drugs as part of the triple combination therapy can be administered in the form of an oral pharmaceutical composition. The other components of the combination therapy may be administered simultaneously or sequentially as one or more individual pharmaceutical formulation preparations. In one embodiment, the combination therapy comprises aspirin at a dose of about 75 mg, about 325 mg or about 650 mg. In a preferred embodiment, aspirin is an oral dosage form such as a tablet having about 81 mg, 325 mg or 650 mg of the active ingredient. In yet another embodiment, the combination therapy comprises naproxen at a dose of about 250 mg, about 375 mg or about 500 mg. In a preferred embodiment, naproxen is an oral dosage form such as a tablet having 250 mg, 375 mg or 500 mg of the active ingredient.

In a preferred embodiment, the combination therapy comprises celecoxib at a dose of about 100 mg, about 200 mg or about 400 mg. In a preferred embodiment, celecoxib is an oral dosage form such as a tablet having 100 mg, 200 mg or 400 mg of the active ingredient.

4.5.1 Methods of Use of Anti-Inflammatory Drugs In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of aspirin in a dose of about 1.2 mg/kg to about 7.0 mg/kg or about 1.2 mg/kg to about 5.0 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of aspirin in a dose of about 1.2 mg/kg, about 5.0 mg/kg or about 7.0 mg/kg. In certain embodiments, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of naproxen in a dose of about 4.0 mg/kg to about 7 mg/kg or about 4.0 to about 5.5 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of naproxen in a dose of about 4 mg/kg, about 5.5 mg/kg or about 7 mg/kg.

In a preferred embodiment, the methods for the treatment and prevention of cardiovascular disease or related disorders comprise administering of celecoxib in a dose of about 1.5 mg/kg to about 6.0 mg/kg or about 1.5 mg/kg to about 3.0 mg/kg. In particular embodiment, the methods of the instant invention comprise administering of celecoxib in a dose of about 1.5 mg/kg, about 3.0 mg/kg or about 6.0 mg/kg.

In certain embodiments, anti-inflammatory agents are administered as unit dosage forms. The compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 75 mg to about 650 mg of aspirin, about 12.5 to about 50 mg of celecoxib or about 250 mg to 500 mg of naproxen.

In a preferred embodiment, the compositions and methods of the invention provide a unit dosage form of a pharmaceutical formulation comprising, for example, about 100 mg to about 400 mg of celecoxib. In particular embodiment, the present invention provides a unit dosage form that comprises about 100 mg, about 200 mg or about 400 mg of celecoxib. For example, 100 mg of celecoxib is an amount that administers 1.5 mg/kg of celecoxib to a 70 kg subject

In one embodiment, tablets for oral administration contain about 81 mg, 325 mg or 650 mg of aspirin. The inactive ingredients include: Carnuba Wax, colloidal silicon dioxide, hypromellose, methacrylic acid copolymer, microcrystalline cellulose, pregelatinized starch, propylene glycol, simethicone, sodium starch glycolate, stearic acid, talc, titanium dioxide, triethyl citrate, D&C Yellow No.10 and FD&C Yellow No.6. (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ).

In yet another embodiment, tablets for oral administration contain 250 mg, 375 mg or 500 mg of naproxen. The inactive ingredients include: croscarmellose sodium, iron oxides, povidone and magnesium stearate. (See, e.g., Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale, NJ).

In a preferred embodiment, tablets for oral administration contain 100 mg, 200 mg or 400 mg of celecoxib. The inactive ingredients include: croscarmellose sodium, edible inks, gelatin, lactose monohydrate, magnesium stearate, povidone, sodium lauryl sulfate and titanium dioxide. (See, e.g., Physicians Desk Reference 2005. 59^th Ed., Thompson PDR, Montvale, NJ).

4.5.2 Timing of Administration of Anti-Inflammatory Drugs

The invention provides methods of treating or preventing cardiovascular disease or related disorders with a dosing administration schedule sufficient to treat or prevent cardiovascular disease or related disorders in a subject in need thereof.

In certain embodiments, the compositions and methods for the treatment or prevention of cardiovascular disease or related conditions comprise administering celecoxib as part of a triple combination therapy about twice a day for about 3 days, about 5 days, about 7 days or about 10 days to a subject in need thereof. In a preferred embodiment, celecoxib can be taken daily for one week, two weeks, three weeks or four weeks. This scheme can be repeated after the interval of 4 or 8 weeks. In certain embodiments, administration of celecoxib can continue for about 1 week, about 2 weeks, about 3 weeks or about 4 weeks. In one embodiment, the patient in need of treatment is administered celecoxib on a regular basis, i.e., daily or weekly; however, the patient is also administered atorvastatin on a regular basis, i.e., daily or monthly, in accordance with its use as a single agent or combination therapy drug; and during the course of these continuous or chronic treatments, the patient is infused with ETC-216 weekly, monthly, semi-annually or annually.

Celecoxib can be taken before, after, or at the same time as the other components of the combination therapy. In one embodiment, celecoxib is administered sequentially with the other components of the combination therapy. In another embodiment, celecoxib is administered concurrently with the other components of the combination therapy. Celecoxib can be administered in any order with the other components of the combination therapy.

In another embodiment, the patient in need of treatment is administered aspirin on a regular basis, i.e., daily or weekly; however, the patient is also administered atorvastatin on a regular basis, i.e., daily or monthly, in accordance with its use as a single agent or combination therapy drug; and during the course of these continuous or chronic treatments, the patient is infused with ETC-216 weekly, monthly, semi-annually or annually.

Aspirin can be taken before, after, or at the same time as the other components of the combination therapy. In one embodiment, aspirin is administered sequentially with the other components of the combination therapy. In another embodiment, aspirin is administered concurrently with the other components of the combination therapy. Aspirin can be administered in any order with the other components of the combination therapy. 4.5 Patients and Diseases to be Treated by the Therapy The invention provides novel compositions and methods to treat or prevent cardiovascular disease or related disorders including but not limited to atherosclerosis, acute coronary syndromes, myocardial or cerebral ischemia, restenosis, angina pectoris, stable angina, unstable angina, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, intermittent claudication, peripheral artery disease, bowel ischemia related to occlusion of mesenteric or celiac arteries, occlusive femoral or iliac disease, Leriche's syndrome, reduction or stabilization of atherosclerotic plaque, reduction of plaque in occluded vessels and promotion of cholesterol efflux.

In some embodiments, the compositions and methods treat cardiovascular disease or related disorders in subjects with signs or symptoms thereof. In one embodiment, subjects have signs and/or symptoms of myocardial ischemia, for instance, pain or discomfort in the chest, jaw, arms, or epigastric region, palpitations, shortness of breath, diaphoresis, nausea and/or vomiting. In another embodiment, the compositions and methods treat cardiovascular disease or related disorders in subjects exhibiting signs or symptoms of cardiovascular disease or related disorders in conjunction with changes in electrocardiogram ("ECG" or "EKG"), such as ST segment elevations or depression; T wave changes, such as inversions; increases in creatine kinase MB fraction, troponin I or troponin T.

In one embodiment, the compositions and methods provide for prevention of cardiovascular disease or related disorders in subjects at risk for developing cardiovascular disease or related disorders. Subjects at risk can include, subjects of varying ages (for example, about 18-24, 25, 30, 40, 50, 60, 70, 80 or 90 years of age); subjects with a family history of cardiovascular disease, or genetic predisposition to cardiovascular disease; subjects that have diabetes, hypertension, multiple-vessel or left- mainstem coronary disease; or subjects that have had a previous myocardial infarction. In one embodiment, the compositions and methods of the instant invention treat cardiovascular disease or related disorders in subjects previously treated for these conditions. In certain embodiments, the previous treatment includes atorvastatin or other statins. In certain embodiments, the previous treatment includes aspirin, celecoxib or other anti-inflammatory agents. In another embodiment, the compositions and methods treat cardiovascular disease or related disorders in subjects previously not treated for these conditions.

In one embodiment, the compositions and methods of the instant invention treat cardiovascular disease or related disorders in subjects scheduled for surgical intervention, which can include angioplasty, IVUS, coronary artery bypass graft (CABG), coronary angiography, implantation of vascular stents, percutaneous coronary intervention (PCI) and Percutaneous Translumenal Coronary Angioplasty (PTCA). In another embodiment, the compositions and methods of the instant invention treat cardiovascular disease or related disorders in post-surgery subjects. EXAMPLES

The present invention will be further understood by reference to the following non-limiting examples.

5.1. Example 1 : Formulation of ETC-216

ETC-216 is a recombinant apolipoprotein A-I Milano/1 palmitoyl-2-oleoyl phosphatidylcholine complex in a 1 to 0.9 ratio by weight. Stock solutions of ETC-216 contain 14.5 mg protein/ml in a sucrose mannitol buffer.

5.2. Example 2: Formulation of Atorvastatin

Atorvastatin (LIPITOR, Pfizer) tablets contain 10 mg, 20 mg, 40 mg or 80 mg of atorvastatin and the following inactive ingredients: calcium carbonate, candelilla wax, croscamnellose sodium, hydroxypropyl cellulose, lactose monohydrate, magnesium stearate, microcrystailine cellulose, hypromellose, polyethylene glycol, talc, titanium dioxide, polysorbate 80, and simethicone emulsion. (See, e.g., Physicians Desk Reference 2005. 59^th Ed., Thompson PDR, Montvale, NJ).

5.3. Example 3: Formulations of Celecoxib Celecoxib (CELEBREX, Pfizer) tablets contain 100 mg, 200 mg or 400 mg of celecoxib. The inactive ingredients include: croscarmellose sodium, ediblr inks, gelatin, lactose monohydrate, magnesium stearate, povidone, sodium lauryl sulfate and titanium dioxide. (See, Physicians Desk Reference 2005, 59^th Ed., Thompson PDR, Montvale NJ).

5.4. Example 4. Triple Combination Therapy This example provides an exemplary dosing regimen for a triple combination therapy including ETC-216, a statin and an anti-inflammatory agent. Formula Dose

ETC-216 15-45 mg/kg/per administration Statin 0.15-1.2 mg/kg/day Anti-inflammatory agent 0.15-6.0 mg/kg/day

5.5. Example 5. Specific Triple Combination Therapy

This example provides a specific triple combination regimen including ETC-216, atorvastatin and celecoxib.

Formula Dose

ETC-216 15 mg/kg

Atorvastatin 0.15 mg/kg Celecoxib 1.5 mg/kg

5.6. Example 6. Specific Triple Combination Therapy

This example provides a specific triple combination regimen including ETC-216, atorvastatin and celecoxib. Formula Dose

ETC-216 45 mg/kg

Atorvastatin 0.15 m g/kg

Celecoxib 3.0 mg/kg

5.7. Example 7. Specific Triple Combination Therapy

Formula Dose

ETC-216 15 mg/kg

Atorvastatin 0.3 mg/kg

Celecoxib 1.5 mg/kg

5.8. Example 8. Specific Triple Combination Therapy

Formula Dose

ETC-216 45 mg/kg

Atorvastatin 0.3 mg/kg

Celecoxib 6.0 mg/kg

5.9. Example 9. Specific Triple Combination Therapy

Formula Dose

ETC-216 15 mg/kg

Atorvastatin 0.6 mg/kg

Celecoxib 1.5 mg/kg 5.10. Example 10. Specific Triple Combination Therapy

Formula Dose

ETC-216 45 mg/kg

Atorvastatin 0.6 mg/kg

Celecoxib 3.0 mg/kg

5.11. Example 11. Specific Triple Combination Therapy

Formula Dose

ETC-216 15 mg/kg

Atorvastatin 1.2 mg/kg

Celecoxib 1.5 mg/kg

5.12. Example 12. Specific Triple Combination Therapy

ETC-216 45 mg/kg

Atorvastatin 1.2 mg/kg

Celecoxib 6.0 mg/kg

5.13. Example 13. Specific Triple Combination Therapy

This example provides a specific triple combination regimen including ETC-216, atorvastatin and aspirin. Formula Dose

ETC-216 15 mg/kg

Atorvastatin 0.15 mg/kg

Aspirin 2 mg/kg

5.14. Example 14. Specific Triple Combination Therapy

ETC-216 45 mg/kg

Atorvastatin 0.15 mg/kg

Aspirin 2 mg/kg 5.15. Example 15. Specific Triple Therapy Combination

This example provides a specific triple combination regimen including ETC-216, atorvastatin and aspirin.

Formula Dose

ETC-216 15 mg/kg

Atorvastatin 0.3 mg/kg

Aspirin 2 mg/kg

5.16. Example 16. Specific Triple Combination Therapy

Formula Dose

ETC-216 45 mg/kg

Atorvastatin 0.3 mg/kg

Aspirin 2 mg/kg

5.17. Example 17. Specific Triple Combination Therapy

ETC-216 15 mg/kg

Atorvastatin 0.6 mg/kg

Aspirin 2 mg/kg

5.18. Example 18. Specific Triple Combination Therapy

This example provides a specific triple combination regirnen including ETC-216, atorvastatin and aspirin. Formula Dose

ETC-216 45 mg/kg

Atorvastatin 0.6 mg/kg

Aspirin 2 mg/kg

5.19. Example 19. Specific Triple Combination Therapy

Formula Dose

ETC-216 15 mg/kg

Atorvastatin 1.2 mg/kg

Aspirin 2 mg/kg 5.20. Example 20. Specific Triple Combination Therapy

This example provides a specific triple combination regimen including ETC-216, atorvastatin and valdecoxib.

Formula Dose

ETC-216 45 mg/kg

Atorvastatin 1.2 mg/kg

Aspirin 2 mg/kg

5.21. Example 21. Specific Triple Combination Therapy

Formula Dose

ETC-216 30 mg/kg

Atorvastatin 0.15 mg/kg

Aspirin 2 mg/kg

5.22. Example 22. Specific Triple Combination Therapy

Formula Dose

ETC-216 30 mg/kg

Atorvastatin 0.3 mg/kg

Aspirin 2 mg/kg

5.23. Example 23. Specific Triple Combination Therapy

Formula Dose

ETC-216 30 mg/kg

Atorvastatin 0.6 mg/kg

Aspirin 2 mg/kg

5.24. Example 24. Specific triple combination therapy

Formula Dose

ETC-216 30 mg/kg

Atorvastatin 1.2 mg/kg

Aspirin 2 mg/kg 5.25. Example 25. In vivo Model of Sepsis

An in vivo model of sepsis is used to study the effect of ETC-216 on animal survival. Female ob/ob mice (8-9 weeks) are challenged with an i.p. injection of lipopolysaccharide (LPS) (27.5 ug LPS/g mouse in 100ul PBS). ETC-216 or human serum albumin (50ug per injection) are injected BID by s.c. injection in 20OuI of PBS beginning at 1 hour post LPS and continuing for 48 hours. The mice are monitored 3 times daily for survival over a 54 hour time period.

Various embodiments of the invention have been described. The descriptions and examples are intended to be illustrative of the invention and not limiting. Indeed, it will be apparent to those of skill in the art that modifications may be made to the various embodiments of the invention described without departing from the spirit of the invention or scope of the appended claims set forth below. All references cited herein are hereby incorporated by reference in their entireties.

Claims

What is claimed:

1. Use of Apolipoprotein A-I Milano (Apo A-I Milano), a statin and an anti- inflammatory agent for the manufacture of a medicament for the treatment or prevention of a cardiovascular disease in a subject in need thereof.

2. The use of Claim 1 , 8 or 12, wherein the Apo A-I Milano is administered as a protein:lipid complex.

3. The use of Claim 2, wherein said complex is administered at a dose of about 1 mg/kg to about 50 mg/kg.

4. The use of Claim 1 or 13, wherein the statin is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, rosuvastatin and atorvastatin.

5. The use of Claim 1 , 11 or 14, wherein the anti-inflammatory agent is selected from the group consisting of aspirin, celecoxib and etodolac.

6. The use of Claim 1 or 8, wherein the cardiovascular disease is selected from the group consisting of atherosclerosis, arterosclerosis, acute coronary syndromes, restenosis, ischemic reperfusion injury, angina pectoris, stable angina, unstable angina, myocardial or cerebral ischemia, myocardial infarction, stroke, transient ischemic attacks, limb ischemia, bowel ischemia, femoral or iliac artery disease, Leriche's syndrome and sepsis.

7. The use of Claim 2, wherein the lipid is a phospholipid selected from the group consisting of phosphatidylcholine, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dilaurylphosphatidylcholine,

1 -myristoyl-2-palmitoylphosphatidylchoIine, 1 -palmitoyl-2-myristoylphosphatidylchoIine, 1 -palmitoyl-2-stearoylphosphatidylcholine, 1 -stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine, 1 -palmitoyl-2-oleoylphosphatidylcholine, 1 -oleoyl-2- palmitylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglyceroi, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, phosphatide acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylseriπe, brain phosphatidylserine, sphingomyelin, brain sphingomyelin, dipalmitoylsphingomyelin and distearoylsphingomyelin.

8. Use of Apolipoprotein A-I Milano (Apo A-I Milano or AIM) and a lipid regulating agent other than a statin for the manufacture of a medicament for the treatment or prevention of a cardiovascular disease in a subject in need thereof.

9. The use of Claim 8, wherein the lipid regulating agent is a CETP inhibitor, a PPAR-activator compound or a cholesterol absorption inhibitor.

10. The use of Claim 9, wherein the lipid regulating agent is selected from the group consisting of torcetrapib and ezetimibe.

11. The use of Claim 8, which further comprises an anti-inflammatory agent.

12. Use of Apolipoprotein A-I Milano (AIM) for the manufacture of a medicament for the treatment of sepsis in a subject in need thereof.

13. The use of Claim 12, which further comprises a statin.

14. The use of Claim 12 or 13, which further comprises an anti-inflammatory agent.