WO2017044607A1 - Activateurs de klf14 et leurs utilisations - Google Patents

Activateurs de klf14 et leurs utilisations Download PDF

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WO2017044607A1
WO2017044607A1 PCT/US2016/050748 US2016050748W WO2017044607A1 WO 2017044607 A1 WO2017044607 A1 WO 2017044607A1 US 2016050748 W US2016050748 W US 2016050748W WO 2017044607 A1 WO2017044607 A1 WO 2017044607A1
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klf14
composition
apoa
levels
mammal
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PCT/US2016/050748
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Yuqing Eugene CHEN
Jifeng Zhang
Yanbo FAN
Yanhong GUO
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The Regents Of The University Of Michigan
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4458—Non condensed piperidines, e.g. piperocaine only substituted in position 2, e.g. methylphenidate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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).
  • cardiovascular disease e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation.
  • 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.
  • Cardiovascular disease 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
  • 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,
  • HDL protective effect has 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 ATP-binding cassette transporter Al
  • 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.
  • compositions and methods utilizing the potent RCT pathway to mediate cholesterol efflux for stabilizing and regressing atherosclerotic plaques, i.e., for treating cardiovascular disease are provided.
  • 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.
  • plasma lipoproteins 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 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.
  • 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.
  • LCAT cholesterol acyltransferase
  • 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.
  • 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.
  • KLF14 Kruppel-like factor 14
  • HDL-C high-density lipoprotein cholesterol
  • 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
  • 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).
  • cardiovascular disease e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation.
  • 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.
  • 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.
  • compositions of the present invention 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.
  • 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.
  • 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.
  • 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
  • compositions comprising small molecule compounds structurally similar to perhexiline, which result in increased KFL14 activity, increased HDL-C levels, and increased apoA-1 levels.
  • the KLF 14 activator is subero lanilide hydroxamic acid
  • 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)
  • KLF activator
  • 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)ethylid
  • KLF activator e.g., perhexiline, suberoylanilide hydroxamic acid, LAQ824, Phorbol 12-myristate 13-acetate, Oxamflatin, F048- 0203,
  • 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
  • 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).
  • 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,
  • 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.
  • a mammalian subject e.g., a primate such as a human or chimpanzee or a rodent such as a rat or mouse
  • a composition comprising a compound capable of stimulating KFL14 activity.
  • 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.
  • 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-thienylmethyliden
  • 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
  • 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.
  • the mammal is a mammal diagnosed as having one or more symptoms of
  • the mammal is diagnosed as at risk for atherosclerosis.
  • the mammal is a human, but can also be a non-human animal.
  • the composition comprises perhexiline.
  • the compositions comprise a compound structurally similar to perhexiline.
  • the methods further comprise administering at least one additional therapeutic agent.
  • additional 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).
  • an additional agent may be administered concomitantly or one week, several days, 24 hours, 8 hours, or immediately before the administration of the composition.
  • an additional agent may be administered one week, several days, 24 hours, 8 hours, or immediately after the administration of the composition.
  • kits for treating or preventing a disease or condition associated with atherosclerosis, dyslipidemia, hypercholesterolemia or inflammation are provided.
  • 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.
  • 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,
  • 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.
  • compositions of the invention are also useful as a research tool and/or diagnostic tool.
  • a composition can be used to identify subjects having reverse cholesterol deficient plasma and those subjects that are responders to reverse cholesterol treatment.
  • compositions e.g., compositions capable of stimulating KLF14 activation
  • Such compositions can also be used to identify appropriate animal models for elucidation of lipid metabolic pathways.
  • such a composition can be used to identify animal models and gene and/or drug interactions that have an effect on reverse cholesterol transport.
  • 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.
  • 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.
  • FIG. 4A-K Overexpression of KLF14 increases both of HDL-C and apoA-I levels and cholesterol efflux capacity.
  • FIG. 5A-H Effects of KLF14 overexpression on the expression levels of genes involved in lipoprotein metabolism in vivo.
  • 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).
  • FIG. 6A-B Overexpression of KLFU does not regulate HDL-C and LDL-C levels in vivo.
  • FIG. 7A-E Adenoviral vectors containing shRNA-LacZ (AdshLacZ) or shRNA-KLF14 (AdshKLF14) (1 ⁇ 10 9 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.
  • 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.
  • 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.
  • FIG. 11A-G Liver specific deletion of Klfl4 showed decreased HDL-C level.
  • 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.
  • FIG. 12A-G Drug screening identifies perhexiline as an activator of KLF14.
  • A Diagram of the chemical structure of the perhexiline maleate salt.
  • FIG. 13A-C (A) HepG2 cells were incubated with 10 ⁇ perhexiline for 24 hours in
  • 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.
  • FIG. 15A-K Administration of perhexiline increased HDL cholesterol level in vivo.
  • the HDL-C (A), total cholesterol (B), LDL-C (C) and triglyceride (D) levels were measured. *, p ⁇ 0.05, Student's t test.
  • 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.
  • Plasma samples were collected and subjected individually to analytical chemistry to measure HDL-C (A), total cholesterol (B), LDL-C (C), triglycerides (D).
  • A total cholesterol
  • B LDL-C
  • D triglycerides
  • 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: ⁇ .
  • 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 50 ⁇ 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.
  • 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.
  • 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.
  • 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)).
  • cholesterol efflux and “cholesterol efflux activity” and “cholesterol efflux capacity” refer to efflux of cholesterol from any cell type.
  • macrophage foam-cells in the artery wall release (i.e., export) cholesterol to appropriate acceptors, such as
  • apolipoproteins and/or HDL apolipoproteins and/or HDL.
  • RCT Reverse Cholesterol Transport
  • ABCA1 and ACBG1 refer to membrane-associated proteins that mediate the efflux of cholesterol and phospholipids to lipid-poor apolipoproteins and HDL particles.
  • ameliorates means alleviate, lessen, or decrease the extent of a symptom or decrease the number of occurrences of episodes of a disease manifestation.
  • 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.
  • 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.
  • 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.
  • 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.
  • CHD coronary heart disease
  • ischemic stroke and peripheral arterial disease is the most common cause of death and disability worldwide.
  • CHDL-C high-density lipoprotein cholesterol
  • apoA-I apolipoprotein A-I
  • HDL-C low-density lipoprotein levels
  • apoA-1 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • cardiovascular disease e.g., atherosclerosis, dyslipidemia, hypercholesterolemia and inflammation
  • 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.
  • 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).
  • 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.
  • the present invention provides compositions comprising an agent capable of stimulating KLF14 activity.
  • the agent is a polypeptide or peptidomimetic.
  • the agent is a small molecule compound.
  • 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'
  • 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.
  • the compound is capable of stimulating KLF14 activity.
  • 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.
  • compositions of the present invention can be used to treat elevated cholesterol levels in a mammal, or to treat prophylactically a mammal at risk of developing elevated cholesterol levels.
  • compositions can also be used for improving the lipid parameters in a mammal.
  • 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.
  • 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.
  • compositions of the present invention 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.
  • 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).
  • 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.
  • 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.
  • the methods preferably involve administering to an organism, preferably a mammal and, more preferably, a human, a composition of the invention (
  • composition(s) is administered orally (e.g., as a syrup, capsule, tablet, etc.).
  • compositions of this invention 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.).
  • 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.
  • 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.
  • compositions of this invention 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
  • a composition of this invention 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);
  • 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
  • niacin/nicotinamide niacin/nicotinamide
  • PPAR activators Vitamin E
  • surgical intervention e.g., angioplasty, stents, stents, or endarterectomy
  • lifestyle changes e.g., low-fat diets, weight loss, and exercise.
  • compositions of this invention 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.
  • cytokines and growth factors such as interferon- ⁇
  • erythropoietins 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.
  • compositions of this invention can be used in combination with drugs commonly used to treat lipid disorders in, for example, diabetic patients.
  • 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.
  • HMG-CoA reductase inhibitors include lovastatin, pravastatin, simvastatin, rosuvastatin, fluvastatin and atorvastatin.
  • bile acid sequestrants include cholestyramine, colestipol and colesevelam.
  • fibric acid derivatives include gemfibrozil and fenofibrate.
  • compositions of this invention can also be used in combination with anti-hypertensive drugs, such as, for example, diuretics, ⁇ -blockers, cathepsin S inhibitors, methyldopa, a2-adrenergic agonists, guanadrel, reserpine, ⁇ -adrenergic receptor antagonists, a 1 -adrenergic receptor antagonists, hydralazine, minoxidil, calcium channel antagonists, ACE inhibitors and angiotensin II-receptor antagonists.
  • anti-hypertensive drugs such as, for example, diuretics, ⁇ -blockers, cathepsin S inhibitors, methyldopa, a2-adrenergic agonists, guanadrel, reserpine, ⁇ -adrenergic receptor antagonists, a 1 -adrenergic receptor antagonists, hydralazine, minoxidil, calcium channel antagonists, ACE inhibitor
  • Examples of ⁇ 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.
  • compositions of this invention 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.
  • 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.
  • 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.
  • 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.
  • compositions and the additional therapeutic agent can be administered simultaneously or sequentially.
  • the composition may be administered first, followed by the additional therapeutic agent.
  • the additional therapeutic agent may be administered first, followed by the composition of the invention.
  • the composition of the invention and the additional therapeutic agent are administered in the same formulation.
  • the composition and the additional therapeutic agent are administered in different formulations.
  • their administration may be simultaneous or sequential.
  • 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 (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.
  • 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
  • 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.
  • 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-carbothi
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • compositions of this invention 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • suitable skin contact adhesive materials include, but are not limited to, polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates, polyurethanes, and the like.
  • 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.
  • 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 is one that will provide for optimum drug delivery.
  • an ointment base should be inert, stable, nonirritating and nonsensitizing.
  • implanted devices e.g., arterial and intravenous stents, including eluting stents, and catheters
  • implanted devices are used to deliver the formulations comprising the polypeptides and peptidomimetics of the invention.
  • 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.
  • the stents and catheters may be coated with formulations comprising the compositions described herein.
  • the compositions will be in time-release formulations an eluted from the stents. Suitable stents are described in, e.g., U. S. Pat. Nos.
  • the pharmaceutical compositions are sustained release formulations.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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).
  • compositions of the present invention can be modified in a number of different ways.
  • 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.
  • acid addition salts are prepared from the free base using conventional methodology, which typically involves reaction with a suitable acid.
  • a suitable acid typically involves reaction with a suitable acid.
  • 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.
  • 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, cit
  • 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.
  • 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.
  • 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
  • Amides and prodrugs can also be prepared using techniques known to those skilled in the art or described in the pertinent literature.
  • 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.
  • kits for the treatment i.e., amelioration, or prevention of a disease or disorder, i.e., condition, associated with dyslipidemia,
  • 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 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).
  • agents include, but are not limited to, beta blockers, vasodilators, aspirin, statins, ace inhibitors or ace receptor inhibitors (ARBs) and the like.
  • 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.
  • 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.
  • electronic storage media e.g., magnetic discs, tapes, cartridges, chips, etc.
  • optical media e.g., CD ROM
  • Such media may include addresses to internet sites that provide such instructional materials.
  • compositions, and methods of the present invention are 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.
  • This example demonstrates that hepatic KLF14 expression is reduced in dyslipidemia mouse models.
  • 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.
  • 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 gtcccggtactc 24 acctcaa gatcata mMC4R melanocortin receptor 4 tcatctgtagcctg 25 ggtactggagcg 26 gctgtg cgtaaaa
  • 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
  • mARL15 ADP-ribosylation factorgttgctggctttttc 71 aagcgctcgaa 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
  • 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
  • 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
  • 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).
  • AdKLFU did not affect both of HDL-C and LDL-C levels in C57BL/6 mice fed HFD (Fig. 6).
  • efficient in vivo shRNA-based knock down of KLF 14 in the liver dramatically decreased plasma HDL-C level, but had no effect on
  • 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.
  • HDL functionality is critical for the assessment of HDL-mediated atheroprotective effects.
  • 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.
  • KLF 14 is a novel regulator of ApoA-I expression.
  • KLF14 significantly upregulated apoA-I mRNA expression
  • 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).
  • 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.
  • 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 ( ⁇ 14? / ⁇ ) 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).
  • Alb albumin
  • 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).
  • 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).
  • This example demonstrates that novel inducers of KLF14 upregulate KLF14 and its target gene expression in hepatocytes and macrophages.
  • 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.
  • 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).
  • 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).
  • the systemic administration of perhexiline increased HDL-C levels in a manner that is largely dependent on hepatic KLF14.
  • 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).
  • mice Conditional disruption ofKlfl4 in mice. The only one exon ⁇ /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 ').
  • mice 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).
  • HFD Kcal high-fat diet
  • mice were administered AdKLF14 or AdLacZ at a dose of 5 x lO 8 plaque-forming units via tail vein injection after 12 weeks of HFD feeding.
  • mice were administered AdshKlfl4 or AdshLacZ at a dose of 1 x 10 9 plaque- forming units via tail vein injection after 12 weeks of HFD feeding.
  • AdshKlfl4 or AdshLacZ a dose of 1 x 10 9 plaque- forming units via tail vein injection after 12 weeks of HFD feeding.
  • 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
  • mice 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.
  • LDL-C Direct LDL-cholesterol
  • HDL-C direct HDL-cholesterol
  • 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.
  • Plasma lipoprotein separation by FPLC or HPLC Lipoprotein separation by FPLC or HPLC. Plasma lipoprotein profiles were determined by fast-performance liquid chromatography (FPLC) or by high-performance liquid
  • HPLC chromatography
  • 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).
  • 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).
  • siRNA oligo which targets a region 100% conserved between human and mouse, was purchased from Invitrogen.
  • 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:
  • the 293 AD cells were transfected with Pad linearized recombinant adenoviruses. After propagation, the recombinant adenoviruses were purified by CsCl 2 density gradient
  • Adenovirus titration was performed using the Adeno-XTM 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.
  • 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 ⁇ / ⁇ 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.
  • 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
  • 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.
  • 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.
  • Luciferase activity was measured as described before (see, e.g., Fan Y, et al., The Journal of biological chemistry. 2011;286(47):40584-94).
  • 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 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.

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Abstract

La présente invention concerne des compositions améliorant le transport inverse du cholestérol grâce à une augmentation des niveaux de cholestérol HDL et d'ApoA-1 et à une amélioration de l'efflux du cholestérol, ainsi que des méthodes apparentées de traitement et/ou de prévention des maladies cardiovasculaires (par exemple l'athérosclérose, la dyslipidémie, l'hypercholestérolémie et l'inflammation). En particulier, l'invention concerne des compositions capables de renforcer l'activité de KLF14 à des fins d'augmentation des niveaux et de l'activité du cholestérol HDL et de l'ApoA-1, ainsi que de l'efflux du cholestérol dans les macrophages, et par conséquent, à des fins d'amélioration du transport inverse du cholestérol.
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* Cited by examiner, † Cited by third party
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US20040110733A1 (en) * 2002-09-04 2004-06-10 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Methods and agents for treating cardiovascular diseases
US20110212081A1 (en) * 2009-02-23 2011-09-01 New York Blood Center Krüppel-like factors and fat regulation
WO2013149258A2 (fr) * 2012-03-30 2013-10-03 Charles Drew University of Medicine and Science Compositions et procédés de traitement ou de prévention de troubles du syndrome métabolique

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040110733A1 (en) * 2002-09-04 2004-06-10 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Methods and agents for treating cardiovascular diseases
US20110212081A1 (en) * 2009-02-23 2011-09-01 New York Blood Center Krüppel-like factors and fat regulation
WO2013149258A2 (fr) * 2012-03-30 2013-10-03 Charles Drew University of Medicine and Science Compositions et procédés de traitement ou de prévention de troubles du syndrome métabolique

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