WO2017044607A1

WO2017044607A1 - Activators of klf14 and uses thereof

Info

Publication number: WO2017044607A1
Application number: PCT/US2016/050748
Authority: WO
Inventors: Yuqing Eugene CHEN; Jifeng Zhang; Yanbo FAN; Yanhong GUO
Original assignee: The Regents Of The University Of Michigan
Priority date: 2015-09-08
Filing date: 2016-09-08
Publication date: 2017-03-16

Abstract

The present invention provides compositions enhancing reverse cholesterol transport through increasing HDL-C and ApoA-1 levels and enhancing cholesterol efflux, and related methods of treating and/or preventing cardiovascular disease (e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation). In particular, the invention relates to compositions capable of increasing KLF14 activity for purposes of increasing levels and functions of HDL-C and ApoA-1 and cholesterol efflux in macrophage, and as a result, enhancing reverse cholesterol transport.

Description

ACTIVATORS OF KLF14 AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention claims benefit of U.S. Provisional Patent Application 62/215,410, filed September 8, 2015, which is incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL068878 and HL105114 awarded by the National Institutes of Health. The Government has certain rights in the invention.

FIELD OF THE INVENTION

INTRODUCTION

Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in the United States and throughout the world. The accumulation of cholesterol in macrophages in the artery wall promotes foam-cell formation and atherosclerosis constituting a main cause of CVD (see, e.g., Schmitz, G. and Kaminski, W. E., Curr Atheroscler Rep., 4(3):243-51 (2002)). Cholesterol accumulation in macrophages is largely dependent on the balance between the deposition by Apolipoprotein B-containing lipoprotein particles, such as VLDL, IDL and LDL, and the cholesterol removal by ApoA-I and ApoE particles-HDL. Lowering of plasma LDL

concentrations by statins and other cholesterol lowering medications prevents approximately one-third of the CVD events, while two-thirds of the events remain (see, e.g., Lancet,

344(8934): 1383-1389 (1994); Circulation, 197(15): 1440-5 (1998)). Elevated levels of plasma HDL cholesterol and high HDL cholesterol efflux capacity are associated with reduced risk of atherosclerosis (see, e.g., Gordon et al, Am. J. Med., 62:707-14 (1977) and Rohatgi et al, N Engl J Med., 371 :2383-93 (2014)). Epidemiological studies have been able to ascribe the HDL protective effect to its main apolipoprotein, Apo A-I (see, e.g., Walldius, G, et al, Lancet, 358(9298):2026-33 (2001); Yusuf et al, Lancet, 364(9438):937-52 (2004)). The beneficial effects of HDL are related, in part, to activity in mediating the antiatherogenic reverse cholesterol transport (RCT) pathway. RCT involves the transport of cholesterol from peripheral macrophages to the liver for excretion of sterol in feces (see, e.g. ,Lewis et al., Circ. Res., 96: 1221-32 (2005)). The rate-limiting step of RCT involves stimulation of cholesterol efflux from macrophages, mediated by native apolipoproteins such as Apo A-I and Apo E. This process of cholesterol efflux generates nascent HDL and requires the ATP-binding cassette transporter Al (ABCA1) or else atherosclerosis is developed (see, e.g., Calpe-Berdiel et al., Biochim. Biophys. Acta., 1738(l-3):6-9 (2005)). ABCA1 is the defective molecule in Tangiers disease, which is characterized by severe deficiency in plasma HDL and premature atherosclerosis (see, e.g., Artie et al., J Lipid Res., 42(11): 1717-26 (2001)). Apolipoproteins A and E also stabilize cellular ABCA1 protein by preventing its degradation, which ensures high- levels of cellular cholesterol export and HDL assembly.

Thus, there is a need in the art for compositions and methods utilizing the potent RCT pathway to mediate cholesterol efflux for stabilizing and regressing atherosclerotic plaques, i.e., for treating cardiovascular disease.

SUMMARY OF THE INVENTION

Cholesterol circulating in the human body is carried by plasma lipoproteins, which are particles of complex lipid and protein composition that transport lipids in the blood. Two types of plasma lipoproteins that carry cholesterol are low density lipoproteins ("LDL") and high density lipoproteins ("HDL"). LDL particles are believed to be responsible for the delivery of cholesterol from the liver (where it is synthesized or obtained from dietary sources) to extrahepatic tissues in the body. HDL particles, on the other hand, are believed to aid in the transport of cholesterol from the extrahepatic tissues to the liver, where the cholesterol is catabolized and eliminated. Such transport of cholesterol from the extrahepatic tissues to the liver is referred to as "reverse cholesterol transport."

The reverse cholesterol transport ("RCT") pathway has three main steps: (i) cholesterol efflux, i.e., the efflux of free cholesterol from the peripheral tissues such as macrophages in atherosclerotic plaques, and its binding by the HDL particle.; (ii) free cholesterol esterification by the action of lecithin: cholesterol acyltransferase ("LCAT") in HDL particles, thereby preventing a re-entry of effluxed cholesterol into cells; (iii) delivery of the HDL-cholesteryl ester complex to liver cells.

The RCT pathway is mediated by HDL particles. Each HDL particle has a lipid component and a protein component. The lipid component of HDL can be a phospholipid, cholesterol (or a cholesterol ester), or a triglyceride. The protein component of HDL is primarily made up of ApoA-I. ApoA-I is synthesized by the liver and small intestine as

preproapolipoprotein which is secreted as a proprotein that is rapidly cleaved to generate a mature polypeptide having 243 amino acid residues.

In cholesterol loaded macrophages, the cholesterol efflux activity is predominately controlled by ABCAl and ABCGl. ABCAl controls the rate-limiting step in cellular cholesterol and phospholipids efflux to lipid poor apoA-I/small HDL particles and ABCGl facilitates cholesterol efflux to mature HDL particles. Loss-of-function of ABCAl is characterized by impaired RCT and cholesterol accumulation in peripheral tissue macrophages both in humans (known as Tangier disease) and ABCAl -deficent mice.

Recent genome-wide association studies (GWAS) have revealed that variant near the gene locus encoding the transcription factor Kruppel-like factor 14 (KLF14) is strongly associated with high-density lipoprotein cholesterol (HDL-C) level, metabolic syndrome and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism, its impact on atherosclerosis, and its potential therapeutic targeting remain largely unexplored.

Experiments conducted during the course of developing embodiments for the present invention determined that the dysregulation of KLF14 was recapitulated in the liver of two dyslipidemia mouse models. Mechanistically, using both overexpression and genetic

inactivation, it was demonstrated that KLF14 regulates plasma HDL-C level and cholesterol efflux capacity by modulating hepatic apoA-I production. Hepatic specific klfl4 deficient mice show decreased HDL-C levels in the circulation. Experimental therapeutic efforts led to the identification of perhexiline, an approved therapeutic small-molecule presently in clinical use to treat angina and heart failure, as a novel KLF14 activator. Indeed, treatment with perhexiline was shown to increase HDL-C level and cholesterol efflux capacity via KLF14-mediated

upregulation of apoA-I expression in vivo. Additionally, it was demonstrated that perhexiline reduces atherosclerotic lesion development in apolipoprotein E-deficient (Apoe ^_/") mice. Furthermore, it was found that KLF14 regulates cholesterol efflux by upregulation of ABCA1 in macrophages, which contribute to the availability of cholesterol to HDL. These findings indicate that KLF14 enhances RCT pathway by two mechanisms: (1) control of the macrophage membrane transporters to mobilize intracellular cholesterol to HDL, and (2) regulation of the amount of cholesterol acceptor apoA-I and HDL particles in circulation, which contributes to enhanced RCT. Together, these results provide, for the first time, a comprehensive insight into the mechanisms and therapeutic intervention via the KLF14 pathway for treatment of atherosclerosis.

Accordingly, the present invention provides compositions having cholesterol efflux activity through increasing HDL-C and ApoA-1 levels and function, and related methods of treating and/or preventing cardiovascular disease (e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation). In particular, the invention relates to compositions capable of increasing KLF14 activity for purposes of increasing both of levels and functions of HDL-C and ApoA-1, and as a result, increasing cholesterol efflux capacity.

In one aspect, the invention therefore provides compositions capable of increasing KLF14 activity resulting in stimulation of cellular cholesterol efflux through increasing HDL-C levels and ApoA-1 levels.

In certain embodiments, the compositions of the present invention (e.g., compositions capable of increasing KLF14 activity) can be used therapeutically to promote both of levels and functions of HDL-C and ApoA-1. Such compositions can be used alone or, alternatively, in combination with other known pharmacological agents for the treatment of cardiovascular disease to reduce atherosclerosis. In addition, the compositions of the present invention can be used alone or, alternatively, in combination with other known pharmacological agents for the treatment of acute coronary syndrome to reduce plaque lipid content and to stabilize vulnerable plaques. Further, the compositions of the present invention can be used alone or, alternatively, in combination with other known pharmacological agents for the treatment of dyslipidemia, hypercholesterolemia and inflammation to raise plasma HDL concentrations and/or to promote reverse cholesterol transport.

The present invention contemplates that certain disorders in animals (e.g. humans) involving decreased KLF14 expression and/or decreased HDL-C levels and/or decreased apoA-1 levels can be treated, ameliorated, or prevented by exposure to therapeutically effective amounts of drug(s) capable of increasing KLF14 activity, which result in increased KFL14 activity, increased HDL-C levels, and increased apoA-1 levels. For example, in some embodiments, certain disorders in animals (e.g. humans) involving decreased KLF14 expression and/or decreased HDL-C levels and/or decreased apoA-1 levels can be treated, ameliorated, or prevented by exposure to therapeutically effective amounts compositions comprising perhexiline

increased KFL14 activity, increased HDL-C levels and function, and increased apoA-1 levels. In some embodiments, certain disorders in animals (e.g. humans) involving decreased KLF14 expression and/or decreased HDL-C levels and/or decreased apoA-1 levels can be treated, ameliorated, or prevented by exposure to therapeutically effective amounts compositions comprising small molecule compounds structurally similar to perhexiline, which result in increased KFL14 activity, increased HDL-C levels, and increased apoA-1 levels.

In some embodiments, the KLF 14 activator is subero lanilide hydroxamic acid (

Phorbol 12-

myristate 13 -acetate ( ),

F048-0203 (

), NSC 379543 ( ), N'4-(2- hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2-

ylhy drazino)carbothioyl] azepane-4-carbohy drazide ( ), C226-1860 (

fluoropheny l)-2-[ 1 -(2-fury l)ethy lidenejhy drazine- 1 -carbothioamide (

l -carbothiohy drazide

), compound 16 (

), and compound 18 (

In certain embodiments of the invention, combination treatment of animals with a therapeutically effective amount of a composition comprising a KLF activator (e.g., perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048- 0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, N 1 -(3 -fluoropheny l)-2-[ 1 -(2-fury l)ethy lidene]hy drazine- 1 -carbothioamide, N' 1 ,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) (e.g., a compound structurally similar to perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, N 1 -(3 -fluoropheny l)-2-[ 1 -(2-fury l)ethylidene]hy drazine- 1 -carbothioamide, N' 1 ,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18 ) and a course of an additional agent effective for treating and/or preventing disorders in animals (e.g. humans) involving decreased HDL-C levels and/or decreased apoA-1 levels and/or decreased cholesterol efflux is contemplated. Since the doses for all approved drugs and treatments for such additional agents are known, the present invention contemplates the various combinations of them with compositions comprising a KLF activator (e.g., perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048- 0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, N 1 -(3 -fluoropheny l)-2-[ 1 -(2-fury l)ethylidene]hy drazine- 1 -carbothioamide, N' 1 ,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) (e.g., a compound structurally similar to perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l 2-furyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18).

Examples of additional agents effective for treating and/or preventing disorders in animals (e.g. humans) involving decreased HDL-C levels and/or decreased apoA-1 levels and/or decreased cholesterol efflux include, but are not limited to, HMG-CoA reductase inhibitors (e.g., atorvastatin, pravastatin, simvastatin, rosuvastatin, pitavastatinm, lovastatin, fluvastatin), PCSK9 inhibitors (e.g., alirocumab, evolocumab), calcium channel blockers (e.g., amlodipine, nifedipine, verapamil, felodipine, diltiazem), ACE inhibitors (e.g., ramipril, quinapril, captopril, enalapril, lisinopril), platelet aggregation inhibitors (e.g., clopidogrel, abxiximab, aspirin), polyunsaturated fatty acids (e.g., omega-3 polyunsaturated fatty acid), fibric acid derivatives (e.g., fenofibrate, gemfibrozil), bile acid sequestrants (e.g., colestipol, cholestyramine), antioxidants (e.g., vitamin E), nicotinic acid derivatives (e.g., niacin), and antianginal agents (e.g., ranolazine).

In certain embodiments, the compounds of the invention are useful for the treatment, amelioration, or prevention of any disorder that is responsive to increased KFL14 activity, increased HDL-C levels and functions, and increased apoA-1 levels, and increased cholesterol efflux (e.g., disorders characterized by decreased KLF14 expression and/or decreased HDL-C levels and/or decreased apoA-1 levels and/or decreased ACBA1/ACBG1 expression). In certain embodiments, the compositions comprising a KLF activator (e.g., perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-fuiyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) (e.g., a compound structurally similar to perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl 3-fluorophenyl)-2-[l 2-furyl)ethylidene]hydrazine-l-carbothioamide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) can be used to treat, ameliorate, or prevent such disorders.

In certain embodiments, the invention provides methods of mediating cholesterol efflux in a mammalian subject (e.g., a primate such as a human or chimpanzee or a rodent such as a rat or mouse) by administering to the subject a composition comprising a compound capable of stimulating KFL14 activity. Based on their cholesterol efflux activity, such compositions of the present invention can be advantageously used to treat, ameliorate or prevent a disease or condition associated with atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation. Based on their cholesterol efflux activity, such compositions of the present invention can be advantageously used to treat, ameliorate or prevent a disease or condition associated reduced KLF14 activity. Based on their cholesterol efflux activity, such compositions of the present invention can be advantageously used to treat, ameliorate or prevent a disease or condition associated with reduced HDL-C levels and ApoA-1 levels. In some embodiments, the KLF14 activator is suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2- bicyclo[2.2.1]hept-5-en-2-ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766-0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l- carbothioamide, N'l,2-di(2-thienylmethylidene)hydrazine-l-carbothiohydrazide, and 7100-1079, compound 16, compound 17 and compound 18). In some embodiments, the compositions comprise a compound structurally similar to perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2- hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, and 7100-1079, compound 16, compound 17 and compound 18.

Still another aspect of the present invention provides methods for treating or preventing a symptom of atherosclerosis in a mammal by administering a composition comprising a compound capable of stimulating KFL14 activity to the subject. In one embodiment of this method, the mammal is a mammal diagnosed as having one or more symptoms of

atherosclerosis. In another embodiment, the mammal is diagnosed as at risk for atherosclerosis. Preferably, the mammal is a human, but can also be a non-human animal. In some embodiments, the composition comprises perhexiline. In some embodiments, the compositions comprise a compound structurally similar to perhexiline.

In another related embodiment, the methods further comprise administering at least one additional therapeutic agent. Examples of such therapeutic agents include, but are not limited to, an antibody, an enzyme inhibitor, an antibacterial agent, an antiviral agent, a steroid, a nonsteroidal anti-inflammatory agent, an anti-metabolite, a cytokine, or a soluble cytokine receptor. The enzyme inhibitor may be a protease inhibitor or a cyclooxygenase inhibitor. The additional agent may be added as a part of a pharmaceutical composition, or may be administered concomitantly or within a time period when the physiological effect of the additional agent overlaps with the physiological effect of the compositions of the present invention (e.g., compositions capable of stimulating KLF14 activation). More specifically, an additional agent may be administered concomitantly or one week, several days, 24 hours, 8 hours, or immediately before the administration of the composition. Alternatively, an additional agent may be administered one week, several days, 24 hours, 8 hours, or immediately after the administration of the composition.

The present invention also provides kits for treating or preventing a disease or condition associated with atherosclerosis, dyslipidemia, hypercholesterolemia or inflammation. In a preferred embodiment, the present invention provides kits for treating or preventing a symptom of atherosclerosis, the kit comprising a container containing a composition (e.g., a composition capable of stimulating KLF14 activation). The kit can further comprise a pharmaceutically acceptable carrier. The kit can further comprise additional therapeutic agents. In addition, the kit can further comprise instructional materials teaching the use of the composition for treating or preventing a disease or condition associated with atherosclerosis, dyslipidemia,

hypercholesterolemia or inflammation. In connection with such kits, instructional material can include a document or recorded media including a written or audible instruction for the use of a pharmaceutical composition. Instruction material includes, for example, a label on a bottle, a paper inserted in a box, printing on the box or carton, instructions provided by a website at an address given in any of these locations, etc.

Such compositions of the invention (e.g., compositions capable of stimulating KLF14 activation) are also useful as a research tool and/or diagnostic tool. For example, such a composition can be used to identify subjects having reverse cholesterol deficient plasma and those subjects that are responders to reverse cholesterol treatment. Also, such compositions (e.g., compositions capable of stimulating KLF14 activation) can be used to evaluate the anti- atherosclerotic potential of other compounds. Such compositions (e.g., compositions capable of stimulating KLF14 activation) can also be used to identify appropriate animal models for elucidation of lipid metabolic pathways. For example, such a composition can be used to identify animal models and gene and/or drug interactions that have an effect on reverse cholesterol transport.

Other features, objects and advantages of the invention and its preferred embodiments will become apparent from a reading of the detailed description, examples, claims and figures that follow.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A-E: Hepatic KLF14 expression is reduced in dyslipidemia mouse models. (A) Heat map of replicate experiments displays the HDL-C trait related gene expression in livers from C57BL/6 mice fed chow diet or HFD for 12 weeks. Expression of genes was determined by qRT-PCR and normalized with 18S RNA. (B and C) Klfl4 expression in liver from C57BL/6 mice fed chow diet or HFD for 12 weeks or wild-type or ob/ob mice, respectively, was determined by real-time qRT-PCR and normalized to 18S RNA (n = 4). (D and E) Hepatic KLF14 and GAPDH levels were determined in livers from the indicated animals by Western- blot (n = 3). *, p < 0.05, Student's t test. Chow, chow diet; HFD, high fat diet; WT, wild type C57BL/6 mice; ob/ob mice, leptin-deficient mice.

FIG. 2: Expression of KLF14 in mouse tissues. Expression of KLF14 was detected by Western-blot using whole-tissue lysates from wild-type C57BL/6 adult mice. Total cell lysates from HepG2 cells transfected with AdKLF14 were used as a positive control.

FIG. 3A-B: SREBPs inhibit the activation of KlfU. (A) The expression of SREBP1 was detected in the livers from C57BL/6 mice fed chow diet or HFD for 12 weeks by Western Blot. Chow, chow diet; HFD, high fat diet. (B) Luciferase activity of reporters was analyzed in HepG2 cells cotransfected wit KLF-luc and pcDNA3.1-SREBPla, pcDNA3.1-SREBPlc or pcDNA3.1- SREBP2 constructs after 24 hours. **, p < 0.01. Two-way ANOVA and Multiple comparisons. Values represent mean ± SEM; n = 3.

FIG. 4A-K: Overexpression of KLF14 increases both of HDL-C and apoA-I levels and cholesterol efflux capacity. Adenoviral vectors containing LacZ (AdLacZ) or human KLF14 (AdKLF14) (5 * 10⁸ pfu per mouse) were administered via tail vein injection to C57BL/6 mice fed HFD for 12 weeks (n = 10 per group). Serum samples were collected at day 6 and subjected individually to analytical chemistry to measure HDL-C (A), total cholesterol (B), LDL-C (C), triglycerides (D), fasting blood glucose (E) or to determine cholesterol and triglyceride levels from pooled samples by FPLC (fractions 1 to 40) (F and G). *, p < 0.05, Student's t test. (H) The ABC Al -mediated cholesterol efflux capacity of serum from AdKLF14- or AdLacZ-treated mice is expressed as the percentage cholesterol efflux of total cell cholesterol (n=10 per group). *, p < 0.05, Student's t test. Representative Western blot results show that AcL£ZJ74-treated mice exhibited increased expression of apoA-I levels in the liver (I) and serum (J). (K) Quantifications of apoA-I levels in the serum from AdLacZ and AdKLFl 4-treated mice by Western blot (n = 10 per group). Values represent mean ± SEM. **, p < 0.01, Student's t test.

FIG. 5A-H: Effects of KLF14 overexpression on the expression levels of genes involved in lipoprotein metabolism in vivo. Adenovirus containing LacZ (AdLacZ) or human KLF14 (AdKLF14) (5 x l0⁸ pfu per mouse) were administered via tail vein injection in C57BL/6 mice previously fed HFD for 12 weeks (n=10). Six days post-injection, liver samples of those animals were used in qRT-PCR to determine mRNA expression of lipoprotein metabolism genes including human KLF14 (A), ApoA-I (B), ApoC-III (C), ApoA-II (D), mouse Klfl4 (E), ApoB (F), HMGCR (G). Data are expressed relative to 18S RNA as mean ± SEM. *, p< 0.05; **, p< 0.01, Student's t test. Results were replicated in one or more independent experiments. apoA-I, Apolipoprotein A-I; apoA-II, Apolipoprotein A-II; apoC-III, Apolipoprotein C-III; HMGCR, 3- hydroxy-3-methylglutaryl-CoA reductase. (H) The serum apoC-III levels were determined by ELISA (n=10).

FIG. 6A-B: Overexpression of KLFU does not regulate HDL-C and LDL-C levels in vivo. Adenoviral vectors containing LacZ (AdLacZ) or human KLF 11 (AdKLFll) (5x 10^s pfu per mouse) were administered via tail vein injection to C57BL/6 mice fed HFD for 12 weeks. Plasma samples were collected at day 6 and subjected individually to analytical chemistry to measure HDL-C (A) and LDL-C (B) levels. Values represent mean ± SEM. n = 6 per group.

FIG. 7A-E: Adenoviral vectors containing shRNA-LacZ (AdshLacZ) or shRNA-KLF14 (AdshKLF14) (1 ^χ 10⁹ pfu per mouse) were administered via tail vein injection to C57BL/6 mice fed HFD for 12 weeks. Serum samples were collected at day 6. (A) KLF14 mRNA levels were determined by quantitative real-time PCR. Values represent mean ± SEM. **, p < 0.01, Student's t test. (B and C) Western blot analysis of apoA-I in 3 uL of serum samples from the mice injected with AdshLacZ or AdshKLF14. **, /? < 0.01, Student's t test. (D and E) Serum samples collected at day 6 after AdshLacZ or AdshKLF14 injection were pooled and the lipid profile was analyzed by FPLC followed by measurement of cholesterol levels in the fractions. FIG. 8A-F: KLF14 is a novel regulator of APOA-I expression. HepG2 cells were infected with AdLacZ or AdKLF14 for 24h and then incubated in medium containing ActD (5μg/ml) or DMSO for another 24h. KLF14 (A) and APOA-I (B) mRNA levels were determined by quantitative real-time PCR (n = 3). **, p < 0.01, Two-way ANOVA and Multiple comparisons. (C) The structure of human APOA-I promoter used in the luciferase assays indicating two putative CACCC-box KLF binding sites. Expression of KLF14 with human APOA-I promoter assay demonstrated that KLF14 significantly increased apoA-I luciferase activity (n = 3). **, p < 0.01 compared to control vector, Two-way ANOVA and Multiple comparisons. (D) Mutations of the two putative KLF binding sites demonstrated apoA-I expression is dependent on KLF 14 and CACCC-box binding sites (n = 3). **, p < 0.01 compared to pGL4-basic vector and ##, p<0.0\ compared to apoA-I promoter WT, Two-way ANOVA and Multiple comparisons. (E) ChIP assay revealed significant enrichment of KLF 14 protein on the human APOA-I promoter in HepG2 cells (n = 3). *, p < 0.05, Two-way ANOVA and Multiple comparisons. (F) Luciferase activity assay demonstrated that KLF14, not KLF2, KIF4, or KLF\ 1, led to an increase in apoA-I promoter activity in HepG2 cells (n = 3). **, p < 0.01, significant differences between groups were determined by Two-way ANOVA and Multiple comparisons. Representative of at least three experiments.

FIG. 9A-B: KLF14 regulates the transcription of mouse ApoA-I. (A) Primary hepatocytes from C57BL/6 mice were infected with AdLacZ or AdKLF14 for 24h and APOA-I mRNA levels were determined by quantitative real-time PCR. Values represent mean ± SEM. **, p < 0.01, Student's t test. (B) ChIP assay revealed significant enrichment of KLF 14 protein on the mouse ApoA -I promoter in primary hepatocytes isolated from C57BL/6 mice. Values represent mean ± SEM. n = 3. **, p < 0.01, Two-way ANOVA and Multiple comparisons.

FIG. lOA-C: Generation of liver specific knockout of Klfl4 in mice. (A) Strategy for conditional disruption of the Klfl4 gene. The wild-type Klfl4 gene is shown in the upper line. For conditional gene targeting, the only exon was flanked by loxP sites (triangles). Homologous recombination, subsequent Flp-mediated removal of the frt-flanked neo, and Cre-mediated deletion of the Klfl4 gene is outlined below. (B) Genotyping of mice harboring wild-type (WT), loxP flanked (floxed, KLF14 1) and^ft-Cre alleles. (C) qRT-PCR analysis revealed strong reduction of Klfl4 mRNA levels in the liver from KLF-LKO mice, but not the heart and intestine tissues. Values represent mean ± SEM. n = 3.

FIG. 11A-G: Liver specific deletion of Klfl4 showed decreased HDL-C level. (A) Western blot of KLF14 and apoA-I expression in the livers of KLF14-LKO and littermate control (WT) mice. The total cholesterol (B), HDL cholesterol (C) and triglyceride (D) levels were determined in KLF14-LKO and littermate control mice fasted overnight (n = 5-7 for each genotype). *, p < 0.05, Student's t test. (E and F) Pooled serum samples from KLF14-LKO and WT mice were assayed by HPLC and cholesterol and triglyceride levels (fractions 1 to 32) were determined. G, Representative Western blot and quantifications of apoA-I levels by Western blot analysis in 1 of serum samples from WT and KLF14-LKO mice and values represent mean ± SEM (5-7 for each genotype). *, p < 0.05, Student's t test.

FIG. 12A-G: Drug screening identifies perhexiline as an activator of KLF14. (A) Diagram of the chemical structure of the perhexiline maleate salt. (B and C) Luciferase activity of reporters was analyzed in HepG2 cells transfected with pGL4-i LF-luc or pGL4-apoA-I-Luc constructs after 12 hours treatment with 10 μΜ perhexiline or DMSO. **, p < 0.01. Student's t test. Values represent mean ± SEM; n = 3. (D) HepG2 cells were infected with AdshLacZ or AdshKLF14 for 48 hours and then incubated with 10 μΜ perhexiline for 24 hours in DMEM containing 0.2% BSA. The apoA-I concentrations in the medium were detected by ELISA. *, p < 0.05. Two-way ANOVA and Multiple comparisons. Values represent mean ± SEM; n = 6. (E) HepG2 cells were treated with DMSO or perhexiline at 10 μΜ for indicated time points in DMEM containing 0.2% BSA and apoA-I production were detected by Western blot. (F) HepG2 cells were treated with DMSO or perhexiline at indicated dosage for 24 hours in DMEM containing 0.2% BSA and apoA-I production were detected by Western blot. (G) HepG2 cells were treated with DMSO, perhexiline, RVX-208 or etomoxir at 10 μΜ for 24 hours in DMEM containing 0.2% BSA and apoA-I production were detected by Western blot. Quantifications from three independent experiments were shown in E-G and values represent mean ±

SEM.*, /? < 0.05; **, p < 0.01, Significant differences between groups were determined by Two- way ANOVA and Multiple comparisons.

FIG. 13A-C: (A) HepG2 cells were incubated with 10 μΜ perhexiline for 24 hours in

DMEM containing 0.2% BSA. The apoA-I concentrations in the medium were detected by ELISA. Values represent mean ± SEM; n = 6. *, p < 0.05. (B) HepG2 cells were infected with AdshLacZ or AdshKLF14 for 48 hours and the knockdown efficiency of Klfl4 was detected by qRC-PCR. Values represent mean ± SEM; n = 3. **, p < 0.01, Student's t test. (C) HepG2 cells were infected with AdshLacZ or AdshKLF14 for 72 hours and then changed to DMEM containing 0.2% BSA. The apoA-I concentrations in the medium were detected by ELISA. Values represent mean ± SEM; n = 6. *, p < 0.05, Student's t test. FIG. 14A-B: Perhexiline upregulates KLF14 and apoA-I expression in Caco2 cells. qRT- PCR analysis showing the expression levels of Klfl4 (A) and ApoA-I (B) in Caco2 cells in the presence of perhexiline (10 μΜ) or DMSO for 30 hours. Data are expressed relative to 18S RNA. Values represent mean ± SEM; n = 3. *, p < 0.05, Student's t test.

FIG. 15A-K: Administration of perhexiline increased HDL cholesterol level in vivo.

C57BL/6J mice placed on HFD for 12 weeks were treated with DMSO or perhexiline maleate salt (lOmg/Kg/day) for five consecutive days by gavage administration and plasma samples collected at day 7 (n = 10 per group). The HDL-C (A), total cholesterol (B), LDL-C (C) and triglyceride (D) levels were measured. *, p < 0.05, Student's t test. (E) The ABC Al -mediated cholesterol efflux capacity of serum from DMSO- or perhexiline-treated mice is expressed as the percentage cholesterol efflux of total cell cholesterol (n = 10 per group). *, p < 0.05, Student's t test. Pooled serum samples from DMSO- or perhexiline-treated mice were assayed by HPLC and cholesterol (F) and triglyceride (G) levels (fractions 1 to 32) were determined. (H to K) KLF14- LKO and littermate control mice were treated with DMSO or perhexiline maleate salt

(lOmg/Kg/day) for five consecutive days by gavage administration and plasma samples collected at day 7 (n = 5-8 for each genotype). HDL cholesterol levels were determined (H) and (I) quantifications of apoA-I levels by Western blot analysis (n = 5-8 for each genotype). J and K, qRT-PCR analysis showing the expression levels of Klfl4 and ApoA-I in indicated groups. Data are expressed relative to 18S RNA (n = 5-8 for each genotype). Values represent mean ± SEM. *, p < 0.05; **, p < 0.01, Two-way ANOVA and Multiple comparisons.

FIG. 16A-B: C57BL/6J mice placed on HFD for 12 weeks were treated with DMSO or perhexiline maleate salt (lOmg/Kg/day) for five consecutive days by gavage administration and samples collected at day 7 (n = 10 per group). Total RNA was isolated from liver and the expression of Klfl4 (A) and ApoA -1 (B) were determined qRT-PCR. Values represent mean ± SEM. *, p < 0.05; **, p < 0.01, Student's t test.

FIG. 17A-H: Administration of perhexiline increased HDL-C and apoA-I levels and enhanced serum cholesterol efflux capacity in Apoe ^A mice. Apoe^~ mice were placed on HFD for 12 weeks and then were treated with DMSO or perhexiline at 10 mg/kg for 6 weeks (three times a week) via gavage administration with continuous HFD (n = 15 per group). Plasma samples were collected and subjected individually to analytical chemistry to measure HDL-C (A), total cholesterol (B), LDL-C (C), triglycerides (D). *, p < 0.05, Student's i test. (E) The ABCA1- mediated cholesterol efflux capacity of serum from DMSO- or perhexiline-treated mice is expressed as the percentage cholesterol efflux of total cell cholesterol (n = 15 per group). *, p < 0.05, Student's t test. The pooled serum from DMSO- or perhexiline-treated mice were analyzed by HPLC (fractions 1 to 32) and the cholesterol (F) and triglyceride (G) levels in each fraction were measured. (H) Representative Western blot and quantifications of apoA-I levels by Western blot analysis in 3 of plasma samples from the mice treated with DMSO or perhexiline (n = 14 per group). *, p < 0.05, Student's t test. Values represent mean ± SEM.

FIG. 18A-D: Administration of perhexiline reduces atherosclerosis development Apoe ^" mice. Apoe^~ mice were placed on HFD for 12 weeks and then were treated with DMSO or perhexiline at 10 mg/kg for 6 weeks (three times a week) via gavage administration with continuous HFD. Perhexiline-treat mice exhibited decreased oil red O-stained lesions in the whole aorta (A) as well as reduced cross-sectional plaque area in the aortic sinus (C). Scale bars: ΙΟΟμιτι. Quantified en face (B) and histology (D) data are shown. Data represent mean±SEM (n = 11-12). Student's t test.

FIG. 19A-D: KLF14 upregulates ABCA1 in macrophages. A, Western blot analysis shows that adenoviral mediated verexpression of KLF14 upregulated ABCA1 expression in J774A.1 cells. B, Knockdown of KLF14 downregulated ABCA1 expression detected by qRT- PCR. acLDL at 50ug/ml and cAMP at 50μΜ were used to induce ABCA1 expression. C, Overexpression of KLF14 in J774A.1 macrophages increased ABC Al -meditated cholesterol efflux to HDL. D, KLF14 regulate ABCA1 transcription analyzed by human ABCA1 promoter luciferase activities. *, p < 0.05, **, p < 0.01.

FIG. 20A-C: Perhexiline upregulates ABCA1 expression by activation of KLF14 in macrophages. A and B, qRT-PCR and Western blot analysis of KLF14 expression in J774.1 cells treated with DMSO or perhexiline. C, Perhexiline increased ABCA1 -mediated cholesterol efflux in J774.1 macrophages *, p < 0.05.

FIG. 21A-B: KLF14 activators up regulate KLF14 and ApoA-I expression in HepG2 cells. qRT-PCT assay of KLF14 and apoA-I expression in HepG2 cells treated with DMSO or KLF14 activators.

DEFINITIONS

The term "apolipoprotein" or "Apo" or "exchangeable apolipoprotein" refers to any one of several water soluble proteins that combine with a lipid (i.e., solubilize the lipid) to form a lipoprotein and are constituents of chylomicrons, HDL, LDL and VLDL. Apolipoproteins exert their physiological effect on lipid metabolism by binding to and activating specific enzymes or lipid-transfer proteins or cell-surface receptors or ATP binding cassette transporters (e.g., ABC transporters). For example, the interaction between apolipoproteins and ABCA1 produces cholesterol efflux and HDL particle assembly. Apolipoproteins include, e.g., Apo A-I, Apo A-II, Apo A-IV, Apo C-I, Apo C-II, Apo C-III, Apo E, and serum amyloid proteins such as, serum amyloid A.

The term " Apolipoprotein Al" or Apo A-I refers to a polypeptide comprising 243 amino acids forming N- and C-terminal domains (see, e.g., Saito et al., J. Biol. Chem, 278:23227- 23232 (2003); Saito et al, Prog. Lipid Res., 43:350-380 (2004)). The tertiary structure of apoA-I comprises an N-terminal four-helix bundle domain and a C-terminal domain that binds lipid strongly (see, e.g., Saito et al, Prog. Lipid Res., 43:350-380 (2004); Mishra et al., Biochemistry, 37: 10313-10324 (1998)). Residues 44-243 of apoA-I contain the necessary structural determinants for mediating cholesterol efflux (see, e.g., Chroni et al, J. Biol. Chem., 278:6719- 6730 (2003); Natarajan et al., J. Biol. Chem., 279:24044-24052 (2004)).

The terms "cholesterol efflux" and "cholesterol efflux activity" and "cholesterol efflux capacity" refer to efflux of cholesterol from any cell type. For example, macrophage foam-cells in the artery wall release (i.e., export) cholesterol to appropriate acceptors, such as

apolipoproteins and/or HDL.

"Reverse Cholesterol Transport (RCT)," as used herein, refers to the process of removing cholesterol from macrophage foam cells and the lipid rich plaque from the arterial wall, with subsequent transfer through plasma to the liver for uptake, processing and excretion as neutral sterols (cholesterol) or acidic sterols (hydroxylated cholesterol/bile) in feces. The efflux of cholesterol from macrophage foam cells is a requirement for RCT benefit in itself even though the cholesterol may be shifted to other less vulnerable adjacent cells. However, the further disposal of such cholesterol by transport in HDL-like particles to the liver for excretion is a favorable aspect of treatment. Such complete RCT provide a general rejuvenation of the arterial tree by actual net removal of the cholesterol content in the arteries.

The terms "ABCA1" and "ACBG1" refer to membrane-associated proteins that mediate the efflux of cholesterol and phospholipids to lipid-poor apolipoproteins and HDL particles.

As used herein, "ameliorates" means alleviate, lessen, or decrease the extent of a symptom or decrease the number of occurrences of episodes of a disease manifestation.

The term "preventing" is art-recognized, and when used in relation to a condition, such as recurrence or onset of a disease such as hypercholesterolemia or atherosclerosis, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

As used herein, "treating" means either slowing, stopping or reversing the progression of the disorder or disease. In a preferred embodiment, "treating" means reversing the progression to the point of eliminating the disorder or disease.

As used herein, "inhibits" means that the amount is reduced as compared with the amount that would occur in a control sample. In a preferred embodiment, inhibits means that the amount is reduced by more than 50%, even more preferably by more than 75% or even 100%.

A "subject," "patient" or "mammal" to be treated by the methods disclosed herein can mean either a human or non-human animal.

DETAILED DESCRIPTION OF THE INVENTION

Atherosclerosis-related cardiovascular disease, including coronary heart disease (CHD), ischemic stroke and peripheral arterial disease, is the most common cause of death and disability worldwide. Epidemiologic studies and experimental observations have consistently shown that decreased high-density lipoprotein cholesterol (HDL-C) levels and apolipoprotein A-I (apoA-I), the major protein component of HDL-C, are powerful, independent predictors of CHD (see, e.g., Gordon T, et al, The American journal of medicine. 1977;62(5):707-14; Gordon DJ, et al., Circulation. 1989;79(1):8-15; McQueen MJ, et al, Lancet. 2008;372(9634):224-33; Yusuf S, et al., Lancet. 2004;364(9438):937-52). High levels of HDL-C and apoA-1 are strongly associated with a reduced cardiovascular risk, even among attain-treated patients achieving low-density lipoprotein levels (LDL-C) of less than 50 mg/dL (see, e.g., Boekholdt SM, et al, Circulation. 2013;128(14): 1504-12). Indeed, both HDL and apoA-I have cardiovascular protective effects, including reverse cholesterol transport (RCT), anti-inflammatory and anti-oxidative effects. HDL cholesterol efflux capacity, a new biomarker that characterizes a key step in RCT, is a strong inverse predictor of coronary disease events (see, e.g., Khera AV, et al, The New England journal of medicine. 2011;364(2):127-35; Hafiane A, et al., The American journal of cardiology.

2014;113(2):249-55). Recently, a population-based cohort study has demonstrated that a high HDL cholesterol efflux capacity is associated with a decreased risk of coronary disease, even after adjustment for traditional cardiovascular risk factors including HDL particle numbers and size (see, e.g., Rohatgi A, et al, The New England journal of medicine. 2014;371(25):2383-93).

As HDL particles and apoA-I are the key acceptors of cholesterol efflux, it may be necessary to develop therapeutic strategies to raise functional HDL and/or apoA-I levels and enhance their antiatherogenic functions. Most of the orally administration of HDL-raising agents, such as niacin, cholesteryl ester transfer protein (CETP) inhibitors and fibrates, has yielded convincing results to increase HDL-C levels, but the effects on reducing cardiovascular risk and enhancing RCT need to be further investigated (see, e.g., Digby JE, et al, Arteriosclerosis, thrombosis, and vascular biology. 2012;32(3):582-8; Kappelle PJ, et al, Cardiovascular therapeutics. 2011 ;29(6):e89-99; Goldenberg I, et al, The American journal of cardiology.

2006;97(4):466-71). These observations indicate that increasing the cholesterol content of HDL particles does not necessarily reduce CVD events. Turning on endogenous production of apoA-I to facilitate new HDL particles formation is becoming one of the most attractive approaches, which is strongly supported by results from human apoA-I transgenic mice and virus-mediated overexpression of apoA-I in mice model of experimental atherosclerosis (see, e.g., Goldenberg I, et al, The American journal of cardiology. 2006;97(4):466-71; Jahagirdar R, et al,

Atherosclerosis. 2014;236(1):91-100). RVX-208, a bromodomain and extraterminal domain inhibitor, is an orally active small molecule that upregulates apoA-I production (see, e.g., Jahagirdar R, et al, Atherosclerosis. 2014;236(1):91-100; McLure KG, et al., PloS one.

2013;8(12):e83190; Picaud S, et al, Proceedings of the National Academy of Sciences of the United States of America. 2013;110(49): 19754-9). However, in patients with CHD,

administration of RVX-208 did not statistically reduce cardiovascular events and the percentage of coronary atheroma volume due to its small effect on HDL-C level and significant side effects in the ASSURE study (see, e.g., Rvx 208. Drugs in R&D. 2011;11(2):207-13; Nicholls SJ, et al, Journal of the American College of Cardiology. 2011;57(9): 1111-9). Therefore, the identification of novel molecules that regulate apoA-I production is essential to increase apoA-I and HDL production and to confer protection against atherosclerosis.

Large scale GWA studies have identified that a genetic variant on chromosome 7 is strongly associated with both HDL trait and CHD (see, e.g., Teslovich TM, et al., Nature.

2010;466(7307):707-13; Chen X, et al, Journal of thrombosis and haemostasis : JTH.

2012;10(8): 1508-14; Chasman DI, et al, PLoS genetics. 2009;5(l l):el000730; de Assuncao TM, et al, The Journal of biological chemistry. 2014;289(22): 15798-809; Huang P, et al., BioMed research international. 2013;2013(231515)). This variant lies in a noncoding region in the vicinity of KLF14 and TSGA13 which encode Kruppel-like factor 14 and Testis specific gene A13, respectively. KLF14 is a member of a large family of zinc-finger transcription factors which have been widely studied in embryogenesis, cell proliferation, differentiation and development. The eighteen KLFs described in mammals possess highly conserved cysteine and histamine zinc fingers, critical for recognition and binding to CACCC or CGCCC DNA motifs (see, e.g., McConnell BB, and Yang VW. Physiological reviews. 2010;90(4): 1337-81). KLF14, a maternally expressed imprinted gene without introns, is robustly associated with HDL-C levels, CHD, type 2 diabetes, obesity and cancer (see, e.g., Teslovich TM, et al, Nature.

2010;466(7307):707-13; Chen X, et al., Journal of thrombosis and haemostasis : JTH.

2012;10(8): 1508-14; de Assuncao TM, et al, The Journal of biological chemistry.

2014;289(22): 15798-809; Huang P, et al, BioMed research international. 2013;2013(231515); Chen G, et al., Human molecular genetics. 2012;21(20):4530-6; Ohshige T, et al, PloS one. 2011 ;6(10):e26911; Rees SD, et al., Diabetologia. 2011 ;54(6): 1368-74; Small KS, et al, Nature genetics. 2011 ;43(6):561-4; Stacey SN, et al, Nature genetics. 2009;41(8):909-14; Steegenga WT, et al, Age. 2014;36(3):9648). In fact, KLF14 has been recently proposed as a master trans- regulator of multiple genes which are associated with metabolic phenotypes in adipose tissue (see, e.g., Small KS, et al, Nature genetics. 2011 ;43(6):561-4), T regulatory cell differentiation (see, e.g., Sarmento OF, et al, Cellular and molecular gastroenterology and hepatology.

2015;l(2): 188-202 e4) and lipid-mediated signaling through a distinct epigenetic mechanism (see, e.g., de Assuncao TM, et al., The Journal of biological chemistry. 2014;289(22): 15798- 809), though its role in lipid metabolism and cardiovascular disease remains to be determined. Fortunately, through a combination of animal models, genetic tools, and pharmacological screening, here, experiments conducted during development of embodiments for the present invention demonstrated a mechanistic role for KLF14 in regulating HDL metabolism and cholesterol efflux capacity by modulation of apoA-I production. Moreover, a small drug, perhexiline, was identified as a novel KLF14 activator, for which administration reduces atherosclerosis development in Apoe^_/" mice. Collectively, such experiments demonstrate a novel molecular mechanism by which KLF14 pathway serves as a therapeutic target for the treatment of cardiovascular disease (e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation).

Accordingly, the present invention provides compositions having cholesterol efflux activity through increasing HDL-C and ApoA-1 levels and functions, and related methods of treating and/or preventing cardiovascular disease (e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation). In particular, the invention relates to compositions capable of increasing KLF14 activity for purposes of increasing HDL-C levels and ApoA-1 levels, and as a result, increasing cholesterol efflux capacity. Indeed, the present invention provides compositions having cholesterol efflux activity through increasing cholesterol efflux through regulation of ABCA1 and ABCG1 expression in macrohage, and related methods of treating and/or preventing cardiovascular disease (e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation). In particular, the invention relates to compositions capable of increasing KLF14 activity for purposes of increasing ABCA1 and ABCG1 levels, and as a result, increasing cholesterol efflux.

Thus, in certain embodiments, the present invention provides compositions comprising an agent capable of stimulating KLF14 activity. In some embodiments, the agent is a polypeptide or peptidomimetic. In some embodiments, the agent is a small molecule compound. In some embodiments, the compound is perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3- methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2-ylhydrazino)carbothioyl]azepane-4- carbohydrazide, C226-1860, C301-6842, C301-3879, C766-0584, Nl-(3-fluorophenyl)-2-[l-(2- furyl)ethylidene]hydrazine-l-carbothioamide, N'l,2-di(2-thienylmethylidene)hydrazine-l- carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18.

In some embodiments, the compound is structurally similar to perhexiline,

suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048- 0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l-carbothioamide, N'l,2-di(2- thienylmethylidene)hydrazine-l -carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18. In some embodiments, the compound is capable of stimulating KLF14 activity.

In addition to being potent and selective stimulator of KLF14 activity, such compositions of the present invention also serve to increase HDL-C levels, increase ApoA-1 levels, enhance cholesterol efflux activity, increase ABCA1 and ABCG1 levels, enhance cholesterol efflux, any combination of these activities and, preferably, all of these activities.

In view of their biological activities and, in particular, their ability to mediate cholesterol efflux, the compositions of the present invention (e.g., compositions capable of stimulating KLF14 activity) can be used to treat elevated cholesterol levels in a mammal, or to treat prophylactically a mammal at risk of developing elevated cholesterol levels. In addition, the compositions can also be used for improving the lipid parameters in a mammal. An improvement in "lipid parameters" includes, for example, one or more of a decrease in the propensity of lipoproteins to adhere to a blood vessel, a decrease in the amount of atherosclerotic plaque (even though plasma LDL and/or HDL concentrations may not significantly changed), a reduction in the oxidative potential of an HDL or LDL particle, a regression in atherosclerosis (e.g., as measured by carotid angiography or ultrasound) and a reduction in cardiac events. Thus, the compositions of the present invention (e.g., compositions capable of stimulating KLF14 activity) can be used to treat or prevent (i.e., prophylactically treat) diseases and conditions associated with atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation, or diseases and conditions that are treatable by altering lipid parameters, such as those diseases and conditions disclosed herein.

In one embodiment, the present invention provides methods for treating, ameliorating and/or preventing one or more symptoms of atherosclerosis. The methods preferably involve administering to an organism, preferably a mammal and, more preferably, a human, a composition of the invention (e.g., a composition capable of stimulating KLF14 activity). The compositions can be administered, as described herein, according to any of a number of standard methods including, but not limited to, injection, suppository, nasal spray, time-release implant, transdermal patch, orally and the like. In one particularly preferred embodiment, the

composition(s) is administered orally (e.g., as a syrup, capsule, tablet, etc.).

The methods of the present invention are not limited to treating humans or non-human animals having one or more symptom(s) of atherosclerosis (e.g., hypertension, narrowing of vessels, plaque formation and rupture, heart attack, angina, or stroke, high levels of plasma cholesterol, high levels of low density lipoprotein, high levels of very low density lipoprotein, or inflammatory proteins, etc.), but are also very useful in a prophylactic context. Thus, the compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) can be administered to an organism, such as a human or non-human animal, to prevent the onset, i.e., development, of one or more symptoms of atherosclerosis. Suitable candidate subjects for prophylactic treatment include, for example, those subjects having one or more risk factors for atherosclerosis (e.g., family history, genetic markers that correlate with atherosclerosis, hypertension, obesity, high alcohol consumption, smoking, high blood cholesterol, high blood triglycerides, elevated blood LDL, VLDL, IDL, or low HDL, diabetes, or a family history of diabetes, high blood lipids, heart attack, angina or stroke, etc.).

Treatment can complement or obviate the need for vascular surgery making anti- atherosclerosis treatment systemic and sustainable. Thus, the composition can be given before intervention to optimize circulation before surgery, during surgery for regional administration in the vasculature or its vicinity, or post-surgery to lessen inflammation and atherosclerosis caused by mechanical trauma by surgical intervention.

In some embodiments, the compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) are administered in combination with one or more additional therapeutic agents for treating or preventing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation, such as cardiovascular disease, including

atherosclerosis. For instance, in one embodiment, a composition of this invention (e.g., a composition capable of stimulating KLF 14 activity) is administered in conjunction with any of the standard treatments for atherosclerosis including, for example, statins (e.g., atorvastatin, lovastatin, pravastatin, simvastatin, fluvastatin, or rosuvastatin); a Nieman-Pick CI -Like 1 sterol transporter channel inhibitor (e.g., Ezetimibe); bile acid binders (e.g., cholestyramine or colestipol); platelet clumping inhibitors (e.g., aspirin, ticlopidine, or clopidogrel);

niacin/nicotinamide; PPAR activators; Vitamin E; surgical intervention (e.g., angioplasty, stents, stents, or endarterectomy); and lifestyle changes (e.g., low-fat diets, weight loss, and exercise).

More particularly, the compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) can be used in combination, either as separate units or fixed combinations, with one or more of the following: an antibody which binds to an unwanted inflammatory molecule or cytokine such as interleukin-6, interleukin-8, granulocyte macrophage colony stimulating factor, and tumor necrosis factor-a; an enzyme inhibitor such as a protease inhibitor aprotinin or a cyclooxygenase inhibitor; an antibiotic such as amoxicillin, rifampicin, erythromycin; an antiviral agent such as acyclovir; a steroidal anti-inflammatory such as a glucocorticoid; a non-steroidal anti-inflammatory such as aspirin, ibuprofen or acetaminophen; or a non-inflammatory cytokine such as interleukin-4 or interleukin-10. Other cytokines and growth factors such as interferon-β, tumor necrosis factors, antiangiogenic factors,

erythropoietins, thrombopoietins, interleukins, maturation factors, chemotactic protein, and their variants and derivatives that retain similar physiological activities may also be used as an additional therapeutic agents.

The compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) can be used in combination with drugs commonly used to treat lipid disorders in, for example, diabetic patients. Such drugs include, but are not limited to, HMG-CoA reductase inhibitors, nicotinic acid, ezetimide, bile acid sequestrants, fibric acid derivatives, MTP inhibitor, AC AT inhibitor and CETP inhibitors. Examples of HMG-CoA reductase inhibitors include lovastatin, pravastatin, simvastatin, rosuvastatin, fluvastatin and atorvastatin. Examples of bile acid sequestrants include cholestyramine, colestipol and colesevelam. Examples of fibric acid derivatives include gemfibrozil and fenofibrate.

The compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) can also be used in combination with anti-hypertensive drugs, such as, for example, diuretics, β-blockers, cathepsin S inhibitors, methyldopa, a2-adrenergic agonists, guanadrel, reserpine, β-adrenergic receptor antagonists, a 1 -adrenergic receptor antagonists, hydralazine, minoxidil, calcium channel antagonists, ACE inhibitors and angiotensin II-receptor antagonists. Examples of β blockers include acebutolol, bisoprolol, esmolol, propanolol, atenolol, labetalol, carvedilol and metoprolol. Examples of ACE inhibitors include captopril, enalapril, lisinopril, benazepril, fosinopril, ramipril, quinapril, perindopril, trandolapril and moexipril.

The compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) can also be used in combination with cardiovascular drugs such as calcium channel antagonists, .beta. -adrenergic receptor antagonists and agonists, aldosterone antagonists, ACE inhibitors, angiotensin II receptor antagonists, nitrovasodilators, and cardiac glycosides. The compositions of the invention can also be used in combination with anti-inflammatory drugs such as HI -receptor antagonists, H2-receptor mediated agonists and antagonists, COX-2 inhibitors, NSAID, salicylates, acetaminophen, propionic acid derivatives, enolic cids, diaryl substituted fuanones, cyclooxygenase inhibitors, and bradykinin agonists and antagonists.

The composition and the additional therapeutic agent can be administered simultaneously or sequentially. For example, the composition may be administered first, followed by the additional therapeutic agent. Alternatively, the additional therapeutic agent may be administered first, followed by the composition of the invention. In some cases, the composition of the invention and the additional therapeutic agent are administered in the same formulation. In other cases, the composition and the additional therapeutic agent are administered in different formulations. When the composition and the additional therapeutic agent are administered in different formulations, their administration may be simultaneous or sequential.

In order to carry out the methods of the invention, one or more compositions of this invention (e.g., compositions capable of stimulating KLF 14 activity) are administered to an individual diagnosed as having or at risk of having a disease or disorder associated with dyslipidemia, hypercholesterolemia and inflammation (e.g., to an individual diagnosed as having one or more symptoms of atherosclerosis, or as being at risk for atherosclerosis). The compositions can be administered in their "native" form or, if desired, in the form of, for example, salts, esters, amides, prodrugs, derivatives, and the like, provided that the salt, ester, amide, prodrug or derivative is suitable pharmacologically, i.e., effective in the methods of the present invention.

In one embodiment of the methods described herein, the route of administration can be oral, intraperitoneal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, infusion; liposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art as one skilled in the art may easily perceive. Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method/mode of

administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, etc.

As such, in another aspect, the present invention provides pharmaceutical compositions comprising a pharmaceutically effective amount of a KLF14 activator (e.g., perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048- 0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l-carbothioamide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) (e.g., a compound structurally similar to perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) and an acceptable carrier and/or excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the KLF14 activator. Preferably, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include, but are not limited to, wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. One skilled in the art will appreciate that the choice of pharmaceutically acceptable carrier(s), including a physiologically acceptable compound depends, for example, on the route of administration of the KLF 14 activator and on the particular physiochemical characteristics of the KLF 14 activator.

In some embodiments, the pharmaceutically acceptable carrier is physiological saline. Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington's Pharmaceutical Science (18.sup.th Ed., ed. Gennaro, Mack Publishing Co., Easton, Pa., 1990). Various pharmaceutically acceptable excipients are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipients (4.sup.th ed., Ed. Rowe et al, Pharmaceutical Press, Washington, D.C.). Again, the

pharmaceutical composition can be formulated as a solution, microemulsion, liposome, capsule, tablet, or other suitable form. The active component may be coated in a material to protect it from inactivation by the environment prior to reaching the target site of action.

In certain preferred embodiments, the compositions of this invention (e.g., compositions capable of stimulating KLF14 activity) can be administered orally (e.g., via a tablet) or as an injectable in accordance with standard methods well known to those of skill in the art. In other preferred embodiments, the compositions can also be delivered through the skin using conventional transdermal drug delivery systems, i.e., transdermal "patches," wherein the composition is typically contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is typically contained in a layer, or "reservoir," underlying an upper backing layer. It will be appreciated that the term "reservoir" in this context refers to a quantity of "active ingredient(s)" that is ultimately available for delivery to the surface of the skin. Thus, for example, the "reservoir" may include the active ingredient(s) in an adhesive on a backing layer of the patch, or in any of a variety of different matrix formulations known to those of skill in the art. The patch may contain a single reservoir, or it may contain multiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.

Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. The backing layer in these laminates, which serves as the upper surface of the device, preferably functions as a primary structural element of the "patch" and provides the device with much of its flexibility. The material selected for the backing layer is preferably substantially impermeable to the active agent(s) and any other materials that are present.

Other preferred formulations for topical drug delivery include, but are not limited to, ointments and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent are typically viscous liquid or semisolid emulsions, often either oil-in-water or water-in-oil. Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the "internal" phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. The specific ointment or cream base to be used, as will be appreciated by those skilled in the art, is one that will provide for optimum drug delivery. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and nonsensitizing.

In some embodiments, implanted devices (e.g., arterial and intravenous stents, including eluting stents, and catheters) are used to deliver the formulations comprising the polypeptides and peptidomimetics of the invention. For example, aqueous solutions comprising the compositions of this invention (e.g., compositions capable of stimulating KLF 14 activity) are administered directly through the stents and catheters. In some embodiments, the stents and catheters may be coated with formulations comprising the compositions described herein. In some embodiments, the compositions will be in time-release formulations an eluted from the stents. Suitable stents are described in, e.g., U. S. Pat. Nos. 6,827,735; 6,827,735; 6,827,732; 6,824,561 ; 6,821,549; 6,821,296; 6,821,291; 6,818,247; 6,818,016; 6,818,014; 6,818,013;

6,814,749; 6,811,566; 6,805,709; 6,805,707; 6,805,705; 6,805,704; 6,802,859; 6,802,857;

6,802,856; and 6,802,849. Suitable catheters are described in, e.g., U.S. Pat. Nos. 6,829,497; 6,827,798; 6,827,730; 6,827,703; 6,824,554; 6,824,553; 6,824,551; 6,824,532; and 6,819,951.

In certain embodiments of the present invention, the pharmaceutical compositions are sustained release formulations. For example, in some embodiments, the KLF14 activators may be admixed with biologically compatible polymers or matrices which control the release rate of the copolymers into the immediate environment. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also contemplated by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. Acceptable carriers include carboxymethyl cellulose (CMC) and modified CMC.

The pharmaceutical composition of the present invention is preferably sterile and non- pyrogenic at the time of delivery, and is preferably stable under the conditions of manufacture and storage. These pharmaceutical compositions can be sterilized by conventional, well known sterilization techniques.

In therapeutic applications, the compositions of this invention are administered to an individual diagnosed as having or at risk of having a disease or disorder associated with dyslipidemia, hypercholesterolemia and inflammation (and, in preferred embodiments, to an individual diagnosed as having one or more symptoms of atherosclerosis or as being at risk for atherosclerosis) in an amount sufficient to cure or at least partially prevent or arrest the disease, condition and/or its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions can be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the active agents (e.g., KLF14 activators) of the formulations of this invention to effectively treat (ameliorate one or more symptoms) the individual or patient.

The concentration of the KLF14 activator within the composition can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, circulating plasma levels of the polypeptide, polypeptide toxicities, progression of the disease (e.g., atherosclerosis), the production of antibodies that specifically bind to the polypeptide, and the like in accordance with the particular mode of administration selected and the patient's needs. Typically, the dose equivalent of a KLF14 activating compound (e.g., perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2- hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl 3-fluorophenyl)-2-[l 2-fuiyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) (e.g., a compound structurally similar to perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-fuiyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hydrazine-l-carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18) is from about 0.1 to about 50 mg per kg, preferably from about 1 to about 25 mg per kg, most preferably from about 1 to about 20 mg per kg body weight. It will be appreciated that such dosages may be varied to optimize a therapeutic regimen in a particular subject or group of subjects. Administration can be accomplished via single or divided doses, e.g., doses administered on a regular basis (e.g., daily) for a period of time (e.g., 2, 3, 4, 5, 6, days or 1-3 weeks or more).

As explained herein, the compositions of the present invention can be modified in a number of different ways. For example, to enhance delivery and/or biological activities in vivo, salts, esters, amides, prodrugs and other derivatives of the KLF14 activator can be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base using conventional methodology, which typically involves reaction with a suitable acid. Generally, the base form of the drug is dissolved in a polar organic solvent such as methanol or ethanol and the acid is added thereto. The resulting salt either precipitates or may be brought out of solution by addition of a less polar solvent. Suitable acids for preparing acid addition salts include both organic acids, e.g., acetic acid, propionic acid, gly colic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like, as well as inorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. An acid addition salt may be reconverted to the free base by treatment with a suitable base. Particularly preferred acid addition salts of the polypeptides described herein are halide salts, such as may be prepared using hydrochloric or hydrobromic acids. Conversely, preparation of basic salts of KLF 14 activators are prepared in a similar manner using a pharmaceutically acceptable base such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like. Particularly preferred basic salts include alkali metal salts, e.g., sodium salts and copper salts.

Preparation of esters typically involves functionalization of hydroxyl and/or carboxyl groups that may be present within the polypeptides or peptidomimetics of the present invention. The esters are typically acyl-substituted derivatives of free alcohol groups, i.e., moieties that are derived from carboxylic acids of the formula RCOOH, wherein R is alkyl and, preferably, lower alkyl. Esters can be reconverted to the free acids, if desired, by using conventional

hydrogenolysis or hydrolysis procedures.

Amides and prodrugs can also be prepared using techniques known to those skilled in the art or described in the pertinent literature. For example, amides may be prepared from esters, using suitable amine reactants, or they may be prepared from an anhydride or an acid chloride by reaction with ammonia or a lower alkyl amine. Prodrugs are typically prepared by covalent attachment of a moiety that results in a compound that is therapeutically inactive until modified by an individual's metabolic system.

The foregoing formulations and administration methods are clearly intended to be illustrative and not limiting in any way. It will be appreciated that, using the teaching provided herein, other suitable formulations and modes of administration can be readily devised.

In another aspect, the present invention provides kits for the treatment, i.e., amelioration, or prevention of a disease or disorder, i.e., condition, associated with dyslipidemia,

hypercholesterolemia and inflammation. In a preferred embodiment, the present invention provides kits for the treatment, i.e., amelioration, of one or more symptoms of atherosclerosis or for the prophylactic treatment of a subject (e.g., human or animal) at risk for atherosclerosis. The kits preferably comprise a container containing one or more of the compositions of this invention (e.g., compositions capable of stimulating KLF14 activity). The compositions can be provided in a unit dosage formulation (e.g., tablet, caplet, patch, suppository, etc.) and/or can be optionally combined with one or more pharmaceutically acceptable excipients. The kit can, optionally, further comprise one or more other agents used in the treatment of a disease or condition associated with dyslipidemia, hypercholesterolemia and inflammation (such as heart disease and/or atherosclerosis). Such agents include, but are not limited to, beta blockers, vasodilators, aspirin, statins, ace inhibitors or ace receptor inhibitors (ARBs) and the like.

In addition, the kits can optionally include labeling and/or instructional materials providing directions (i.e., protocols) for the practice of the methods or use of the "therapeutics" or "prophylactics" of this invention. Preferred instructional materials describe the use of one or more compositions of this invention, for example, to mitigate one or more symptoms of atherosclerosis and/or to prevent the onset or increase of one or more of such symptoms in an individual at risk for atherosclerosis. The instructional materials can also, optionally, teach preferred dosages/therapeutic regiment, counter indications and the like.

While the instructional materials typically comprise written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips, etc.), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

One of ordinary skill in the art will readily recognize that the foregoing represents merely a detailed description of certain preferred embodiments of the present invention. Various modifications and alterations of the compositions and methods described above can readily be achieved using expertise available in the art and are within the scope of the invention.

EXAMPLES

The following examples are illustrative, but not limiting, of the compounds,

compositions, and methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.

Example I.

This example demonstrates that hepatic KLF14 expression is reduced in dyslipidemia mouse models. In an attempt to identify genes that may play an critical role in regulation of lipid metabolism, using real-time PCR analysis, the expression profile of 43 candidate genes which are associated with HDL-C-trait and CHD were first examined, as indicated by previous GWAS studies (see, e.g., Teslovich TM, et al, Nature. 2010;466(7307):707-13; Chen X, et al, Journal of thrombosis and haemostasis : JTH. 2012;10(8): 1508-14; Chasman DI, et al, PLoS genetics. 2009;5(l l):el000730), in the liver from two dyslipidemia mouse models. It was found that Klfl 4 mRNA expression was significantly decreased by approximately 70% in the liver of C57BL/6 mice in response to a high-fat diet (HFD) (Fig. 1 A and IB). Klfl 4 mRNA expression was next assessed in the liver of leptin-deficient (ob/ob) mouse, a well-accepted model characterized by obesity, insulin resistance, and dyslipidemia. It was found that the levels of this gene are reduced by 52% (Fig. 1C). Among the tissues of healthy adult C57BL/6 mice examined, KLF14 protein expression was detected in liver and kidney, while heart showed the highest level of KLF14 (Fig. 2). Consistent with mRNA expression, KLF14 protein levels were decreased in the livers from mice fed HFD and ob/ob mice compared to control animals (Fig. ID and IE).

Table 1. Primer Pairs used for real-time qRT-PCR Experiments

mSR-BI scavenger receptor class attcccacgtatc 17 gctcctttgggtta 18 B member 1 gcttcac gggttc hapoA-I apolipoprotein A-I tggatgtgctcaa 19 aggccctctgtct 20 agacagc ccttttc hapoA-II apolipoprotein A-II gagctttggttcg 21 tgtgttccaagttc 22 gagacag cacgaa hKLF14 kruppel-like factor 14 tacaagtcgtcgc 23 gtccccggtactc 24 acctcaa gatcata mMC4R melanocortin receptor 4 tcatctgtagcctg 25 ggtactggagcg 26 gctgtg cgtaaaag mMVK mevalonate kinase gaagcaggctga 27 cagatggtgctg 28 ccaagttc gttcatgt mANGPTL4 angiopoietin-like 4 tccaatttcccatc 29 ggctcttggcaca 30 catttg gttaagg mNAT2 arylamine N- ctgggctttgaaa 31 ctgaggctgatcc 32 acetyltransferase ccacaat tttccag mSORTl sortilin 1 caggagacaaat 33 ccttccgccacag 34 gccaaggt acatatt mTRIBl tribbles homolog 1 gaggtgctccttg 35 tcggtggagaag 36 gtgagag acgaactt mHNFla hepatocyte nuclear tcacagacacca 37 gaggacactgtg 38 factor 1 -alpha acctcagc ggactggt mPCSK9 proprotein convertase tccattgggaagt 39 acctgctctgaag 40 subtilisin/kexin type 9 ggaagac gacctga mGCKR glucokinase (hexokinase cagcgtgagttaa 41 tcagtgatggagc 42

4) regulator gcaccaa acctgag mLIPC lipase, hepatic tctcggagcaaa 43 tatgaatggcgtc 44 gttcacct cacaaaa mLIPG lipase, endothelial cttccagtgcaca 45 gggtgtccccact 46 gactcca gttattg mTOPl topoisomerase (DNA) I gccaaggtgttcc 47 cccttcgagcatc 48 gtaccta tgctaac mMYLIP myosin regulatory light tagagtggcatgc 49 ctccttggtgacg 50 chain interacting protein tgtgagg gtcaagt mST3GAL4 ST3 beta-galactoside cgatggacttcca 51 gcagaggtgtag 52 alpha-2,3- ctggatt agccaagg sialyltransferase 4

mCOBLLl cordon-bleu WH2 repeat ctgtgccacaag 53 ctggcgatgctgt 54 protein-like 1 cacagatt tagatga mKLHL8 kelch-like family tgggtgtgatctct 55 tctccacgtcact 56 member 8 gtggaa gaagcac mTSPAN8 tetraspanin 8 ctggccatatggg 57 tttcacagctcca 58 tgagagt cagcatc mPABPC4 poly (A) binding protein, ccagggggtgaa 59 ccagggggtgaa 60 cytoplasmic 4 tctctaca tctctaca mLCAT lecithin-cholesterol aaagaggagca 61 gcccacaccgta 62 acyltransferase gcgcataac gagacaat mSLC39A Zinc transporter ZIP 11 agcctaacggac 63 agtacaagatgc 64 acatccac cccaatcg mPPPlR3B protein phosphatase 1, tgctgaaggataa 65 gccgttacactcg 66 regulatory subunit 3B ggccatc tagcaca mTTC39B tetratricopeptide repeat acaggtggatgg 67 cctcagccttctc 68 domain 39B tctgaagc cacagtc mSTARD3 StAR-related lipid ggcagggaaag 69 cctgatacaccag 70 transfer (START) gaagctact ctcagca domain containing 3

mARL15 ADP-ribosylation factorgttgctggctttttc 71 aagcgctcgaaa 72 like 15 aggag acacagat mPLTP phospholipid transfer aaatcagtctgcg 73 gcaggacggttct 74 protein ctggagt tgtcaat mGALNT2 UDP-N-acetyl-alpha-D- ctggacaccttgg 75 gagttgccttcga 76 galactosamine :polypepti gacactt tctgctc de N- acetylgalactosaminyltran

sferase 2 (GalNAc-T2)

mPGSl CDP-diacylglycerol-- acgctgattggct 77 ttcttgattagcgg 78 glycerol-3 -phosphate 3- ctcctaa ggtcac phosphatidyltransferase

mHNF4a hepatic nuclear factor 4 gattgccaacatc 79 aggagcagcac 80 alpha acagacg gtccttaaa mUBE2L3 ubiquitin-conjugating agcttgaagagat 81 tgtgatcttgggtg 82 enzyme E2L 3 ccgcaaa gtttga mCITED2 Cbp/p300-interacting tgggcgagcaca 83 ggtaggggtgat 84 transactivator, with tacactac ggttgaaa

Glu/ Asp-rich carboxy- terminal domain, 2

mTRPSl trichorhinophalangeal gcccagggttcat 85 gggtgttttgcag 86 syndrome I tgactaa gtctcat mAMPD3 adenosine ctgcccctgttca 87 agcaccatgatgt 88 monophosphate aagctac tggcata deaminase 3

mLRP4 low density lipoprotein ccaagccagccg 89 tgctctgtctccgt 90 receptor-related protein tgtataat gtcatc 4

mPDE3A phosphodiesterase 3A, gaggacgaagc 91 ctcttggcttcccc 92 cGMP-inhibited ctgtgaaag ttctct mSBNOl strawberry notch accaaacactgg 93 cacttttgtccaga 94 homolog 1 gaagcaac cgctca mZNF664 zinc finger protein 664 catattcattggcg 95 agctccagttgaa 96 agacca ggctttg mSCARBl scavenger receptor class tcgaattctgggg 97 aatgccttcaaac 98

B, member 1 tcttcac acccttg mLCATB lactamase, beta tgctgacaactgt 99 tcacccactgtgg 100 ccaggag acagaaa mCMIP c-Maf inducing protein ctgctgtccgact 101 cagggctgtaga 102 acgatga gctggaac mABCA8 ATP -binding cassette, caggaccagctg 103 ccctgattgcttgc 104 sub-family A (ABC 1), aagtctcc catatt member 8

mAMPD3 adenosine ctgcccctgttca 105 agcaccatgatgt 106 monophosphate aagctac tggcata deaminase 3

* m, in front of the gene name indicates mouse; h, indicates human.

FP: Forward primer; RP: Reverse primer. Among the tissues of healthy adult C57BL/6 mice examined, KLF14 protein expression was detected in the liver (Fig. 2). Sterol-response element-binding proteins (SREBPs), transcription factors that regulate the expression of genes involved in the synthesis of cholesterol, fatty acids and triglycerides in mammalian cells, are upregulated in the liver of both the dyslipidemia mouse models (see, e.g., Shimomura I, et al, The Journal of biological chemistry. 1999;274(42):30028-32; Mei M, et al, Lipids in health and disease. 2011 ;10(110)). A significant upregulation of SREBP1 in the liver from C57BL/6 mice fed HFD was also observed (Fig. 3A). Next, to test the effects of SREBPs on KLF14 expression, luciferase reporter gene assays using human KLF14 promoter (KLF14-luc, spanning -1567 to +65, relative to the transcription start site) construct were performed. Fitting with the effects observed for endogenous Klfl4 expression, SREBPlc and SREBP2 significantly repressed the transcription at human KLF14 promoter (Fig. 3B). Since KLF14 expression is reduced in the liver of dyslipidemia models, it was postulated that hepatic KLF14 plays an important role in lipid metabolism and performed additional experimentations to test the validity of this idea.

Example II.

This example demonstrates that overexpression of KLF14 increases HDL-C level and cholesterol efflux capacity.

To investigate whether KLF14 contributes to lipid metabolism, as inferred from

Genotype-to-Phenotype correlations previously made in humans, C57BL/6 mice were fed HFD for 12 weeks and recombinant adenoviruses encoding human KLF14 (AdKLFl 4) or β- galactosidase (AdLacZ) were injected via tail vein. After 6 days, AcL£ZJ74-treated animals showed a 29% increase in HDL-C compared to AdLacZ -treated group (Fig. 4A), whereas total cholesterol, LDL-C levels, triglycerides and fasting blood glucose (Fig. 4B-E) were not affected. Fast protein liquid chromatography (FPLC) analysis of pooled sera from each experimental group confirmed that AdKLFl 4-treated mice had increased circulating HDL cholesterol levels (Fig. 4F), but not triglycerides (Fig. 4G). An increase in apoA-I protein levels in both the liver and serum of AdKLFl 4-treated mice (Fig. 4I-K) was also observed. It was detected that KLF14- induced marked mRNA increases in Klfl4 and ApoA-I in livers from mice treated with AdKLFl 4 (Fig. 5A and B). Although a slight upregulation of ApoC-LLL mRNA was observed (Fig. 5C), which is linked in a genetic cluster with ApoA-L (see, e.g., Shimomura I, et al, The Journal of biological chemistry. 1999;274(42):30028-32), the circulation ApoC-III levels did not increase dramatically as measured by enzyme-linked immuno assay (ELISA) (Fig. 5H). As expected, overexpression of human KLF14 did not affect the mouse endogenous Klfl4 expression (Fig. 5E). The expression of genes related to cholesterol metabolism, including apolipoprotein A-II, apolipoprotein B, and 3-hydroxy-3-methylglutaryl-CoA reductase, did not change in livers from AcL£ZJ74-treated mice (Fig. 5D, F and G). Tail vein injection with adenovirus containing human KLF11 was also performed, which is a member of the same family of metabolic regulator KLF proteins and found that treatment with AdKLFU did not affect both of HDL-C and LDL-C levels in C57BL/6 mice fed HFD (Fig. 6). Conversely, efficient in vivo shRNA-based knock down of KLF 14 in the liver dramatically decreased plasma HDL-C level, but had no effect on

triglycerides (Fig. 7A, D and E). A reduced circulating level of apoA-I by Western blot was also observed (Fig. 7B and C).

ApoA-I and HDL particles play critical roles in the process of RCT, in which cholesterol from non-hepatic peripheral tissues is transferred to HDL particles and returns to the liver for biliary excretion (see, e.g., Khera AV, et al., The New England journal of medicine.

2011 ;364(2): 127-35; Hellerstein M, and Turner S. Current opinion in lipidology. 2014;25(1):40- 7). HDL functionality is critical for the assessment of HDL-mediated atheroprotective effects. Thus, subsequently, the ATP -binding cassette transporter ABCA1 -mediated cholesterol efflux capacity of serum from AdKLF14- or AdLacZ -treated mice was quantified. It was found that, concomitant with increased HDL-C and apoA-I levels, cholesterol efflux capacity increased significantly in the KLF14-treated group (Fig. 4H). Therefore, collectively, these data demonstrate that KLF14 regulates lipid metabolism and establish that KLF14 expression directly modulates the levels of apoA-I and HDL-C in vivo.

Example III.

This example demonstrates that KLF 14 is a novel regulator of ApoA-I expression.

In accordance with the in vivo observations, overexpression of KLF 14 in HepG2 cells resulted in increased apoA-I transcription and this KLF14-induced apoA-I upregulation was blocked by actinomycin D (Fig. 8 A and B), a transcriptional inhibitor, suggesting that KLF 14 regulated apoA-I at the transcriptional level. These results led to investigation of whether KLF 14 functions as a transcriptional regulator of this protein. Indeed, initial evidence derived from analysis of the 5' flanking regions of human APOA -I identified a sequence (CACCC box), similar to the recently described functional KLF14 binding site (see, e.g., de Assuncao TM, et al, The Journal of biological chemistry. 2014;289(22): 15798-809). As shown in Fig. 8C, the region from the apoA-I promoter containing these sites (located at nt -491/-486 and -1943/-1938) displayed increased reporter activity relative to control vector. Interestingly, mutation of the nt - 491/-486 site greatly reduced promoter activity, while a similar change in the nt -1943/-1938 site had no significant change (Fig. 8D). Chromatin immunoprecipitation (ChIP) assay revealed that KLF14 binds the promoter region that harbors the proximal CACCC box (-491/-486) (Fig. 8E), demonstrating that this is a functional KLF14 binding site in human APOA-I promoter. Similarly, adeno virus-mediated overexpression of KLF14 significantly upregulated apoA-I mRNA expression and ChIP assay revealed that KLF14 was bound to the promoter region that harbors the CACCC boxes (-499A494 and -451/-446) in primary hepatocytes (Fig. 9A and B). Given the similar DNA-binding preferences of KLF family members (see, e.g., McConnell BB, and Yang VW. Physiological reviews. 2010;90(4): 1337-81), whether other KLF transcription factors could regulate apoA-I expression was considered. While AdKLF14 cotransfection upregulated apoA-I promoter activity in HepG2 cells, adenoviral vectors containing KLF2, KLF4 or KLF\ 1 failed to increase ApoA-I promoter reporter activity (Fig. 8F), indicating that these effects are specific for KLF 14.

Example IV.

This example demonstrates that loss of hepatic KLF 14 induces decreased HDL-C levels. To study the role of hepatic KLF 14 in lipid metabolism, liver-specific Klfl ^-knockout (LKO) mice were generated using the Cre-loxP strategy (Fig. 10A). Mice harboring floxed Klfl 4 alleles in which the only one exon of Klfl 4 was flanked by loxP sites (Κ 14?^/β) were generated. To ablate Klfl 4 in the liver, the Klfl4^m mice were crossed with mice harboring a Cre transgene under the control of the promoter for the albumin (Alb) gene (Alb-Cre mice). Deletion of Klfl 4 in the liver was confirmed at the genomic DNA (Fig. 10B), mRNA (Fig. IOC) and protein levels (Fig. 11 A). It was found that deletion of Klfl 4 was specific to the liver, as its mRNA levels in other tissues were comparable to those in the control mice (Fig. IOC). As shown in Fig. 11, total cholesterol and triglycerides levels were comparable between wild-type (WT) and KLF14-LKO mice at 8 weeks of age (Fig. 11B, D and F). However, deletion of Klfl 4 in the liver resulted in an approximately 14.9% decrease in HDL-C level compared with those οΐΚΙ/14^¹ littermates (Fig. 11C). In addition, a decrease in the levels of apoA-I was detected in both the liver and the circulation of the KLF14-LKO mice (Fig. 11A and G). These results reveal that hepatic KLF14 contributes to HDL metabolism.

Example V. This example demonstrates that the KLF14 upregulates ABCA1 expression in macrophages. Adenovirus-mediated overexpression of KLF14 upregulates ABCA1 expression and increase cholesterol efflux in J774.1 macrophages (Fig. 19A and B). Knockdown of KLF14 downregualted ABCA1 expression in J774.1 macrophages (Fig. 19C). Initial evidence derived from analysis of the 5' flanking regions of human ABCA1 identified three sequences (CACCC box) similar to the recently described functional KLF14 binding site and overexpression of KLF14 significantly increased human ABCA1 promoter luciferase activity (Fig. 19D).

Example VI.

This example demonstrates that the perhexiline upregulates ABCA1 expression and increase cholesterol efflux in macrophages. Perhexiline have been identified as KLF14 activator. Perhexiline upregulates KLF14 and ABCA1 expression in macrophages, results in increased cholesterol efflux (Fig. 20 A-C). Example VII.

This example demonstrates that drug screening identified novel inducers of KLF14 expression: perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13- acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2- bicyclo[2.2.1]hept-5-en-2-ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766-0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l- carbothioamide, N' 1 ,2-di(2-thienylmethylidene)hydrazine- 1 -carbothiohy drazide, 7100- 1079, compound 16, compound 17 and compound 18.

Example VIII.

This example demonstrates that novel inducers of KLF14 upregulate KLF14 and its target gene expression in hepatocytes and macrophages.

The preceding data demonstrate that upregulation of KLF14 expression results in increased apoA-I and HDL-C levels, underscoring a fundamental role for KLF14 in maintaining the homeostasis of lipid metabolism. Therefore, efforts were initiated toward identifying pharmacological interventions that can activate endogenous Klfl4 and, thereby, increase apoA-I and HDL-C levels, ABCA1 and ABCG1 levels and cholesterol efflux. For this purpose, a high- throughput screening was designed using a uman KLF14 promoter-driven luciferase reporter, KLF14- xc. From the high throughput screening of chemical libraries including 150,000 compounds and 35,960 Natural Extracts, 18 compounds were identified to activate KLF14-Xuc activity two-fold or more (Table 2). Noteworthy, it was confirmed that perhexiline significantly increased KLF14 promoter activity, but not APOA-I promoter activity after incubation for 12 hours (Fig. 12A-C). To investigate the effect of perhexiline on apoA-I production, the apoA-I levels in the medium was detected by ELISA and Western blot. Following treatment of HepG2 cells with perhexiline (10 μΜ) for 24 hours, the production of apoA-I in the medium was increased by 28% compared with DMSO-treated cells (Fig. 13 A). Efficient knockdown of KLF14 significantly decreased the production of apoA-I induced by perhexiline, suggesting that this effect was largely dependent on the KLF14 in hepatocytes (Fig. 12D, Fig. 13B and C). Next, the experiments demonstrated that perhexiline induced apoA-I production in a time-dependent and dose-dependent manners (Fig. 12E and F). Previous pharmacological studies indicate that perhexiline is a potent inhibitor of mitochondrial carnitine palmitoyltransferase I (CPT-1). To identify whether the effect of perhexiline on apoA-I production is dependent on the inhibition of CPT-1 pathway, another well-established CPT-1 inhibitor, etomoxir (10μΜ), was used which did not induce apoA-I production (Fig. 12G). RVX-208 was used as a positive control because this drug upregulates apoA-I production in hepatocytes (see, e.g., Jahagirdar R, et al.,

Atherosclerosis. 2014;236(1):91-100; McLure KG, et al, PloS one. 2013;8(12):e83190; Picaud S, et al, Proceedings of the National Academy of Sciences of the United States of America. 2013; 110(49): 19754-9) (Fig. 12G). As apoA-I is synthesized mainly in the liver and

small intestine of mammals (see, e.g., Glickman RM, et al, The New England journal of medicine. 1978;299(26): 1424-7), it was also found that perhexiline activated KLF14 and apoA-I expression in Caco2 cells, a human enterocytic cell line (Fig. 14A and B), indicating that perhexiline stimulates apoA-I production both in liver and intestine, which shows an additional potential mechanistic and pharmacological importance.

Table 2.

LAQ824 13.8

Phorbol 12-myristate 13 -acetate 12.9

Oxamflatin 3.7

F048-0203 3.2

NSC 379543 4.1

N'4-(2-hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- 2.9 ylhydrazino)carbothioyl]azepane-4-carbohydrazide

C226-1860 9.6

C301-6842 3.1

C301-3879 4.0

C766-0584 2.4

Nl -(3-fluorophenyl)-2-[ 1 -(2-furyl)ethylidene]hy drazine- 1 -carbothioamide 4.5

N' 1 ,2-di(2-thienylmethylidene)hy drazine- 1 -carbothiohydrazide 3.5

7100-1079 3.1

Compound 16 2.4

Compound 17 3.7

Compound 18 2.9

KLF14-luc-transfected 293 cells were used for high-throughput screening of chemical libraries including 150,000 compounds and 35,960 Natural Extracts. The luciferase activities were measured 24h after compound treatment. From the high throughput screening, 18 compounds were identified that activate KLF14-luc activity 2-fold or more in dose-dependent manner. The effects of some compounds on KLF14 and AapoA-I expression were determined in HepG2 cells (Fig. 20 A and B).

Example VI. This example demonstrates that administration of perhexiline increases KLF14, HDL and apoA-I levels in vivo.

To examine the potential role of perhexiline on upregulation of apoA-I in vivo, C57BL/6 mice were treated with perhexiline at lOmg/Kg/day via gavage administration for five consecutive days. At day seven, perhexiline-treated animals showed 18.3% increase in HDL-C levels and slightly decreased triglyceride levels compared to the DMSO-treated group, whereas the total cholesterol, LDL-C and triglyceride levels did not significantly change (Fig. 15A-D). An increase in HDL-C level was also confirmed by HPLC using pooled serum from DMSO- or perhexiline-treated mice, but not triglycerides (Fig. 15F and G). These effects were associated with approximately 4.1 -fold upregulation of KLF14 and 2-fold upregulation of apoA-I in the liver by real-time qPCR compared with mice treated with vehicle only (Fig. 16A and B). As expected, the ABC Al -mediated cholesterol efflux capacity of serum from DMSO- or perhexiline-treated mice was quantified and found that cholesterol efflux capacity markedly increased in the perhexiline-treated group (Fig. 15E). Thus, perhexiline behaves as the first KLF14 activator having a beneficial impact on the regulation of apoA-I and HDL-C levels and function. To determine whether hepatic KLF14 deficiency interferes with perhexiline-induced increase in HDL-C levels, KLF14-LKO and littermate control mice were administrated with perhexiline or DMSO as control via gavage for five days. A significant increase in HDL-C and apoA-I levels in perhexiline-treated control mice was found, though not in the perhexiline- treated KLF14-LKO counterparts (Fig. l5H and I). Subsequently, using real time qPCR, KLF14 and APOA-I mRNA levels were measured in livers obtained from mice belonging to each of these groups. Perhexiline-treated control mice showed a significant upregulation ofKIF 4 and APOA-I expression in the liver as compared with KLF14-LKO mice (Fig. 15J and K). Thus the systemic administration of perhexiline increased HDL-C levels in a manner that is largely dependent on hepatic KLF14.

Example VII.

This example demonstrates that administration of perhexiline reduces atherosclerosis development in Apoe^~ mice.

The effect of perhexiline-mediated activation of the KLF14 pathway on atherosclerosis in

Apoe^~A mice was tested. After 10 weeks challenge of high cholesterol diet (HCD), Apoe^~ mice were treated three times a week (Monday, Wednesday, and Friday) with either perhexiline (10 mg/kg) or DMSO for 6 weeks via gavage administration. The circulating HDL-C levels were significantly increased in perhexiline-treated mice, but no significant differences were found in total cholesterol, triglyceride, and LDL-C levels relative to control animals (Fig. 17A-D). Next, the ABC Al -mediated cholesterol efflux capacity was quantified and it was found that this process is markedly increased in the perhexiline-treated group (Fig. 17E). HPLC analysis of pooled sera from each experimental group confirmed that perhexiline-treated mice had increased circulating HDL cholesterol levels, but no changes in triglycerides (Fig. 17F and G). An increase in apoA-I protein level in plasma from perhexiline-treated mice was also detected (Fig. 17H). Most importantly, perhexiline treatment significantly inhibited atherosclerotic lesion formation by 27.3% as measured by the fraction of the surface area in en face aorta trees stained by Oil Red O (Fig. 18A and B). The cross-sectional plaque area in the aortic sinus was also attenuated by 30.2% in perhexiline-treated Apoe^~ mice compared to DMSO-treated animals (Fig. 18C and D). These findings indicate that perhexiline, the KLF14 activator, inhibited atherosclerosis development. Example VIII.

This example describes the materials and methods for Examples I-VII.

Conditional disruption ofKlfl4 in mice. The only one exon οΐΚΙ/14 gene was flanked by loxP sites. Germline transmission of the loxP-flanked allele and Flp recombinase-mediated removal of the frt-flanked selection marker in vivo yielded mice (C57BL/C) harboring &KI/14 allele with one fit and two loxP sites {Klfl4^fl/fl). Klfl4^m mice were crossed with ,4/6-Cre transgenic mice (Stock Number: 003574) purchase from The Jackson Laboratories. Two-month- old male mice were used for experiments. Genomic DNA was extracted from mice tails and was used for genotyping. Genotyping of liver specific knockout mice was performed using 2 sets of primers. The first primer set was designed to amplify the _^4/Z>-Cre construct (forward, 5'- gaagcagaagcttaggaagatgg-3'; reverse, 5'-ttggccccttaccataactg-3'). Genotyping oiKlf ^ mice was performed by PCR amplification (forward, 5'-tagtgaggaaaggaagagcaggtagga-3'; reverse, 5'- tcacatgaggaaacagacaagcaaaag-3 ').

Animals and diets. C57BL/6 mice, ob/ob mice (leptin-deficient mice), Apoe^~ mdAlb- Cre transgenic mice were purchased from the Jackson Laboratories and were housed at 22 ± 1°C in a 12: 12-h light-dark cycle. C57BL/6 mice had free access to water and rodent chow before switch to adjusted Kcal high-fat diet (HFD, 44% from fat, Harlan, T.D. 06415). For hepatic overexpression of LacZ or KLF14, mice were administered AdKLF14 or AdLacZ at a dose of 5 x lO⁸ plaque-forming units via tail vein injection after 12 weeks of HFD feeding. For knockdown of Klfl4 in liver, mice were administered AdshKlfl4 or AdshLacZ at a dose of 1 x 10⁹ plaque- forming units via tail vein injection after 12 weeks of HFD feeding. Six days after the adenoviral infection, the animals were fasted for 12 hours and then sacrificed. Collected serum and liver tissues were stored at -80°C until processed. Mouse atherosclerosis model was generated by feeding 8-week-old male Apoe ^{" "} mice an atherogenic diet (HCD, 21% fat, 34% sucrose, and

0.2% cholesterol, Harlan, T.D. 88137) for 10 weeks and then the mice were treated three times a week (Monday, Wednesday, and Friday) with perhexiline (10 mg/kg) or DMSO for 6 weeks via gavage administration with continuous HCD.

Blood Biochemical Tests. Direct LDL-cholesterol (LDL-C), direct HDL-cholesterol (HDL-C), and enzymatic-colorimetric assays used to determine serum total cholesterol (TC) and triglycerides (TG) were carried out at the Chemistry Laboratory of the Michigan Diabetes Research and Training Center. Blood glucose was measured using an ACCU-CHEK glucometer and glucose strips.

Lipoprotein separation by FPLC or HPLC. Plasma lipoprotein profiles were determined by fast-performance liquid chromatography (FPLC) or by high-performance liquid

chromatography (HPLC). For FPLC assay, 180μί of serum pooled from mice were loaded and eluted at a constant flow rate of 0.50 mL/minute. The 40 fractions per sample were collected after running 36 minutes. For HPLC assay, 50μί of serum pooled from mice were loaded and eluted at a constant flow rate of 1.0 mL/minute. The 32 fractions per sample were collected after running 5 minutes. Sample elution was monitored spectrophotometrically at an optical density of 280 nm. The cholesterol and triglyceride contents in each fraction were measured with a fiuorometric enzymatic assay (Cayman, MI) and triglyceride colorimetric assay in a GloMax Multi Plus plate reader (Promega, WI).

Cells. The cell lines 293 AD, HepG2, J774.1 and Caco2 were obtained from ATCC and cultured according to ATCC protocols. Adenovirus-mediated gene transfer was performed by exposing 70% confluent HepG2 cells to the adenoviruses at a multiplicity of infection of 20 for 2 hours. Primary hepatocytes were isolated from 6-10-week old mice as described previously (58). In brief, mice were anesthetized and the liver was exposed. The liver was perfused with liver perfusion medium and liver digestion medium (Invitrogen) and hepatocytes were washed and separated from other types of cells with Percoll (Sigma). Hepatocytes were seeded on rat tail type I collagen-coated plates or dishes in Williams' E medium supplemented with 10% FBS for 3 hours, followed by change to fresh DMEM containing 10% FBS. Preparation of adenoviral vectors. The full-length human KLF14 cDNA encoding KLF14 was subcloned into pCR8/GW/TOPO entry vector (Invitrogen). After sequencing, the LR recombination reaction was carried out between the entry clone pCR GW TOVO/KLFl 4 and destination vector (pAd/CMV/V5-DEST) according to the manufacturer's protocol (Invitrogen). For knockdown experiments, a siRNA oligo, which targets a region 100% conserved between human and mouse, was purchased from Invitrogen. To prepare adenovirus containing shRNA for KLF14, synthesized oligos were annealed and inserted into BLOCK-iT U6 entry vector. The U6 promoter and shRNA were cloned into the adenoviral plasmid pAd/BLOCK-iT-DEST according to the manufacturer's instructions. The sequences for shRNA are as follow:

s Klfl4, 5'-caccggcatccaagcgacatcagtcgaaactgatgtcgcttggatgc-3',

5 ' -aaaagcatccaagcgacatcagtttcgactgatgtcgcttggatgcc-3 ' ;

shLacZ, 5 ' -caccgctacacaaatcagcgatttcgaaaaatcgctgatttgtgtag-3 ' ,

5 ' -aaaactacacaaatcagcgatttttcgaaatcgctgatttgtgtagc-3 ' . The 293 AD cells were transfected with Pad linearized recombinant adenoviruses. After propagation, the recombinant adenoviruses were purified by CsCl₂ density gradient

ultracentrifugation. Adenovirus titration was performed using the Adeno-X™ qPCR Titration Kit (Clontech).

RNA isolation and RT-PCR. Total RNA from tissues and cells was purified using Qiagen's RNeasy kits (Qiagen). cDNA was synthesized using superscript III (Invitrogen), and qPCR was performed using SYBR Green reagents (Bio-Rad). Primer pairs for RT-PCR are shown in Table S2. Gene expression was presented as fold increase compared with RNA isolated from the control group by the comparative CT (2^~AACT) method with 18S RNA as the reference gene.

Cholesterol efflux capacity assays. The sera from AdKLF14- or AdLacZ -treated mice and

DMSO or perhexiline-treated mice were used for cholesterol efflux studies (see, e.g., Khera AV, et al, The New England journal of medicine. 2011 ;364(2): 127-35). J774.1 murine macrophages were labeled with 2 μΟί/ιηΕ H cholesterol for 24 hours in the presence of ACAT inhibitor (Sando 58-035) and equilibrated overnight with 0.3 mM 8-(4-chlorophenylthio)-cyclic AMP in the present of ACAT inhibitor. ApoB-depleted serum was obtained by PEG precipitation. 2.8% v/v ApoB-depleted serum from mice was used as efflux acceptor for 4 hours. Efflux was quantified by liquid scintillation and expressed as a percentage of total cell H-cholesterol content. All assays were performed in duplicate. Cholesterol efflux assays. J774.1 murine macrophages were labeled with 2 μθί/ηιί H cholesterol for 24 hours in the presence of ACAT inhibitor (Sando 58-035) and equilibrated overnight with KLF14 activators in the present of ACAT inhibitor (see, e.g., Khera AV, et al, The New England journal of medicine. 2011;364(2): 127-35). ApoA-I at 50μg/ml or HDL at 80 μg/ml was used as efflux acceptor for 4 hours. Efflux was quantified by liquid scintillation and expressed as a percentage of total cell H-cholesterol content. All assays were performed in duplicate.

Immunoblotting. Protein was extracted from the cells or liver tissues with lysis buffer (Thermo Scientific) supplemented with protease inhibitor cocktail (Roche Applied Science). The lysates were resolved by 4-12% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. Antibodies used in this study were obtained from the following sources: apoA-I (Sigma, SAB3500270, 1 :2000 working dilution; Santa Cruz Biotechnology, sc-30089, 1 : 1000 working dilution), KLF14 antibody (Santa Cruz

Biotechnology, sc- 104345, 1 : 1000 working dilution was used to detect the overexpression samples; Thermo Scientific, PA5-23784, 1 : 1000 working dilution was used to detect mouse samples), SREBPl antibody (Santa Cruz Biotechnology, sc-366, 1 : 1000 working dilution), actin and GAPDH antibody (Santa Cruz Biotechnology, sc-1616 and sc-25778, 1 :2000 working dilution). IRDye 680RD and 800CW secondary antibodies (LI-COR Biotechnology, 926-32212, 926-32213, 926-32214, 926-68074, 1 : 10000 working dilution) were used as second antibodies. Western blots were visualized and quantified using an Odyssey Infrared Imaging System (LI- COR Biosciences, Version 2.1). The apoA-I concentrations and apoC-III in the serum were quantitated by ELISA according to the manufacturer's protocol (apoA-I ELISA kits are from Abeam (abl08804) and MyBioSource (MBS702111); apoC-III ELISA kit is from Abnova (KA1030).

Plasmids and transient transfection assays. 7he genomic fragments harboring the putative KLF binding sites in human APOA-I promoter were cloned by PCR from the human genomic DNA. The amplified products of 2.1 and 0.7 kb upstream of the translation start site of human APOA-I gene were ligated into the pGL4-luciferase reporter vector (Promega) to generate pGL4-1979/+163-Luc, pGL4-710/+163-Luc, and pGL4-94/+163-Luc plasmids. Promoter activity was further validated by mutation of the two putative KLF14-binding sites on the promoter at -1943/-1938 or -491/-486 by replacing CACCC to CAtaC using the Quickchange site-directed mutagenesis kit (Stratagen, La Jolla, CA). The numbers indicate the distance in nucleotides from the transcription start site (+1) of the human apoA-I gene. To prepare human KLF14 promoter-driven luciferase reporter, the amplified product of 1.6Kb upstream of the translation start site of human KLF14 gene (-1567 to +65) was ligated into the pGL4-luciferase reporter vector to generate KLF14-luc plasmid. All PCR-generated constructs were verified by sequencing the DNA. Luciferase activity was measured as described before (see, e.g., Fan Y, et al., The Journal of biological chemistry. 2011;286(47):40584-94). In brief, HepG2 cells were transfected with pGL4-luciferase reporter plasmids and pRenilla-null as internal control (Promega) using Lipofectamine 2000 (Life Technologies). Cells were cultured for 24 hours after transfection, and cell lysates were measured using the Dual Luciferase Reporter Assay System Kit (Promega). For the screen to identify compounds activating KLF14, HepG2 cells were cultured for 24 hours after transfection with KLF14-luc and stimulated with compounds for another 24 hours. Luciferase activity was measured.

Chromatin immunoprecipitation. ChIP assays were performed according to the manufacturer's protocol with minor modifications using the EZ ChIP kit (Millipore) (see, e.g., Fan Y, et al, The Journal of biological chemistry. 2011;286(47):40584-94). In brief, HepG2 cells or mouse primary hepatocytes were infected with AdKLF14 or AdLacZ for 24 hours and then crosslinked with 1% formaldehyde and quenched prior to harvest and sonication. The sheared chromatin was immunoprecipitated with anti-KLF14 antibody (or control

immunoglobulin G) conjugated to protein A/G Sepharose beads. The eluted immunoprecipitates were digested with proteinase K, and DNA was extracted and underwent PCR with primers (Table 1). flanking the putative KLF14 binding site within apoA-I. The supernatant of the control group was used as an input control.

Analysis of atherosclerotic lesions (see, e.g., Chang L, et al., Circulation.

2012;126(9): 1067-78. (59). Two quantitative methods were used in this study. (1) En face analysis of atheromatous plaques: after stained with Oil red O (Sigma) and removal of the adventitia of the whole aorta, aortas were opened longitudinally and pinned flat onto a black-wax plate. The percentage of the plaque area stained by oil red O with respect to the total luminal surface area was quantified with Image J analysis software. (2) The extent of the atherosclerotic lesions in the aortic root: the atherosclerotic lesions in the aortic sinus region were examined at 5 locations, each separated by 80 μιτι, with the most proximal site starting after the appearance of at least two aortic valve leaflets. The largest plaque of the three valve leaflets were adopted for morphological analysis. All morphometric analyses were performed in a double-blinded manner.

Statistical Analysis. Statistical analyses were performed using GraphPad Prism 6 software (GraphPad Software, Inc). Statistical comparisons and analyses between two groups were performed by two-tailed unpaired Student's t test, and among three groups or more, with one-way analysis of variance followed by a Newman-Keuls test or Two-way ANOVA and Multiple comparisons. Ap value < 0.05 was considered statistically significant. Data are presented as mean ± S.E.M..

Having now fully described the invention, it will be understood by those of skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications and publications cited herein are fully incorporated by reference herein in their entirety.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

What Is Claimed Is: 1. A composition comprising an agent capable of stimulating KLF14 activity.

2. The composition of Claim 1, wherein the agent is a polypeptide.

3. The composition of Claim 1, wherein the agent is a peptidomimetic.

4. The composition of Claim 1, wherein the agent is small molecule compound.

5. The composition of Claim 4, wherein the small molecule compound is selected from the group consisting of perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12- myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2-hydroxy-3- methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2-ylhydrazino)carbothioyl]azepane-4- carbohydrazide, C226-1860, C301-6842, C301-3879, C766-0584, Nl-(3-fluorophenyl)-2-[l-(2- furyl)ethylidene]hy drazine-1 -carbothioamide, N' 1 ,2-di(2-thienylmethylidene)hy drazine- 1 - carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18.

6. The composition of Claim 4, wherein the small molecule compound has chemical structure that is similar to the chemical structure of perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048-0203, NSC 379543, N'4-(2- hydroxy-3-methoxybenzylidene)-l-[(2-bicyclo[2.2.1]hept-5-en-2- ylhydrazino)carbothioyl]azepane-4-carbohydrazide, C226-1860, C301-6842, C301-3879, C766- 0584, Nl-(3-fluorophenyl)-2-[l-(2-furyl)ethylidene]hydrazine-l-carbothioarnide, N'l,2-di(2- thienylmethylidene)hy drazine-1 -carbothiohydrazide, 7100-1079, compound 16, compound 17 and compound 18.

7. The composition of Claim 1, further comprising a pharmaceutically acceptable carrier.

8. The composition of Claim 7, further comprising a therapeutic agent for treating cardiovascular disease.

9. A method for mediating reverse cholesterol transport in a mammal, the method comprising administering to the mammal a composition of Claim 1, whereby reverse cholesterol transport is mediated.

10. A method for treating a symptom of atherosclerosis wherein the symptom is high levels of low density lipoprotein in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a composition of Claim 1.

11. A method for increasing HDL-C levels in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a composition of Claim 1.

12. A method for increasing ApoA-1 levels in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a composition of Claim 1.

13. A method for increasing ABCA1 and ABCG1 levels in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a composition of Claim 1.

14. A method for increasing cholesterol efflux in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a composition of Claim 1.

15. A method for stimulating KLF14 activity in a mammal, the method comprising administering to the mammal a therapeutically effective amount of a composition of Claim 1.

16. A kit for treating atherosclerosis, the kit comprising a container containing a composition of Claim 1.

17. The kit of Claim 16, further comprising a pharmaceutically acceptable carrier.

18. The kit of Claim 16, wherein the composition is combined with a pharmaceutically acceptable carrier in a unit dosage formulation.

19. A method of treating, ameliorating, or preventing atherosclerosis in a subject comprising administering to said patient a therapeutically effective amount of the composition of Claim 1.

20. A method of treating, ameliorating, or preventing dyslipidemia in a subject comprising administering to said patient a therapeutically effective amount of the composition of Claim 1.

21. A method of treating, ameliorating, or preventing hypercholesterolemia in a subject comprising administering to said patient a therapeutically effective amount of the composition Claim 1.