WO2016022543A1 - Lipoprotein lipase activator for mitigation of atherosclerosis and methods of using same - Google Patents

Abstract

Provided herein are methods of increasing LPL activity or ANGTPL4 inhibition of LPL; activating LPL; treating, preventing, or ameliorating atherosclerosis; treating hypertriglyceridemia; and improving lipid profile. The methods involve the use of a carboxamide having a general formula of R¹ONHR², wherein R¹ comprises a cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring. Also provided is a pharmaceutical composition having a carboxamide with a general formula of R¹ONHR², wherein R¹ comprises a cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring.

Description

TITLE

Lipoprotein Lipase Activator for Mitigation of Atherosclerosis and Methods of Using Same Inventors: Prabodh Sadana, Werner Geldenhuys

RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Ser. No. 62/034,242 filed August 7, 2014, and Ser/ No. 62/182,662 filed June 22,2015, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with no government support. The government has no rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The overall rate of death attributable to cardiovascular disease (CVD) in 2008 was 244.8 per 100,000. On the basis of mortality data from 2008, more than 2,200 Americans die of CVD each day, which is an average of 1 death every 39 seconds. About 150,000 of the Americans killed by CVD in 2008 were greater than 65 years of age. About 33% of the deaths due to CVD occurred before the age of 75, which is well before the average life expectancy of 77.9 years.

[0004] Coronary heart disease caused about 1 of every 6 deaths in the United States in 2008.

Coronary heart disease mortality in 2008 was 405,309. Each year, an estimated 785,000 Americans have a new coronary attack, and about 470,000 have a recurrent attack. It is estimated that an additional 195,000 silent first myocardial infarctions occur each year. Approximately every 25 seconds, an

American has a coronary event, and approximately every minute, someone dies of one.

[0005] Atherosclerosis, a systemic disease process in which fatty deposits, inflammation, cells, and scar tissue build up within the walls of arteries, is the underlying cause of the majority of clinical cardiovascular events. Individuals who develop atherosclerosis tend to develop it in a number of different types of arteries (e.g., large and small arteries and those feeding the heart, brain, kidneys, and

extremities), although they may have much more in some parts of the body than others. In recent decades, advances in imaging technology have allowed for an improved ability to detect and quantify atherosclerosis at all stages and in multiple different vascular beds. Still, there is a need for additional and improved therapies for the treatment or prevention of cardiovascular disease, coronary heart disease, and atherosclerosis. SUMMARY OF THE INVENTION

[0006] Provided herein is a method of increasing lipoprotein lipase (LPL) activity, the method comprising administering a compound to a subject and increasing LPL activity in the subject. The compound generally comprises a carboxamide having a general formula of R^ONHR², wherein R¹ comprises a cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring, or a salt, prodrug, stereoisomers, solvate, hydrate, polymorph, or racemate of such a compound. In certain embodiments, R¹ further includes an unsubstituted or substituted aromatic moiety attached to the cyclopropane ring. In particular embodiments, the aromatic ring is substituted with a halogen. In particular embodiments, the aromatic ring is a fluorinated benzyl group. In particular embodiments, the fluorinated benzyl group is bonded to the cyclopropane ring.

[0007] In certain embodiments, the imidazole ring is substituted with a halogen or methyl. In certain embodiments, the cyclopropane ring is substituted with at least one halogen, bromine, or methyl group. In particular embodiments, the cycloproprane ring is substituted with two halogens or bromines. In certain embodiments, the cyclopropane ring is substituted with two methyl groups. In certain embodiments, the imidazole ring or the morpholine ring is linked to the nitrogen atom by a carbon chain having from 1 to 10 carbons. In particular embodiments, the carbon chain has 3 carbons.

[0008] In certain embodiments, the compound is referred to as CIO and consists essentially of Formula I:

Formula I

[0009] In certain embodiments, the compound consists essentially of Formula II:

Formula II

wherein X is a halogen.

[0010] In particular embodiments, the compound is referred to as ClOd and consists essentially of Formula II A:

Formula IIA

[0011] In certain embodiments, the compound is referred to as ClOa and consists essentially of Formula III:

Formula III

[0012] In certain embodiments, the compound is referred to as ClOb and consists essentially of Formula IV:

Formula IV

[0013] In certain embodiments, the compound is referred to as ClOc and consists essentially of Formula V:

Formula V

[0014] Further provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound comprising a carboxamide having a general formula of R^CONHR², wherein R¹ comprises a cyclopropane ring; and R² comprises one of an imidazole ring or a morpholine ring.

[0015] Further provided is a method of reversing ANGTPL4 inhibition of LPL, the method comprising administering an effective amount of a compound to a subject, and reversing ANGTPL4 inhibition of LPL in the subject, the compound comprising a carboxamide having a general formula of R^ONHR², wherein R¹ comprises a cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring, or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate of such a compound.

[0016] Further provided is a method of activating LPL, the method comprising administering an effective amount of a compound to a subject, and activating LPL in the subject, the compound comprising a carboxamide having a general formula of R^CONHR², wherein R¹ comprises a cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring, or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate of such a compound.

[0017] Further provided is a method of treating, preventing, or ameliorating atherosclerosis in a subject with high triglycerides, the method comprising administering an effective amount of a compound to a subject, and treating, preventing, or ameliorating atherosclerosis in the subject, the compound comprising a carboxamide having a general formula of R^ONHR², wherein R¹ comprises a

cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring, or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate of such a compound.

[0018] Further provided is a method of improving lipid profile, the method comprising administering an effective amount of a compound to a subject, and improving lipid profile in the subject, the compound comprising a carboxamide having a general formula of R^ONHR², wherein R¹ comprises a

cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring, or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate of such a compound.

[0019] In certain embodiments of any method described herein, the compound is administered at a dose of about 10 mg/kg body weight.

BRIEF DESCRIPTION OF THE DRAWING

[0020] The patent or application file contains one or more drawings executed in color and/or one or more photographs. Copies of this patent or patent application publication with color drawing(s) and/or photograph(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fees.

[0021] FIG. 1: Illustration of compound ClOd reversing inhibition of LPL activity by ANGPTL4.

[0022] FIG. 2: Z-factor determination for LPL activity assay. Z-factor was determined for the LPL activity assay by measuring the minimum (min) and maximum (max) signal in the wells in the absence or presence of the substrate and performing the calculations as described.

[0023] FIGS. 3A-3B: LPL activity screen. Compound library was screened at 40 μΜ in the LPL activity assay. (FIG. 3A.) The values obtained for absorbance at 400 nM wavelength were determined and compared to DMSO control. The activity of compound CIO is indicated. FIG. 3B shows the structure of compound CIO.

[0024] FIG. 4: CIO dose response curve. An eight-point dose response relationship was determined for the compound CIO in the LPL activity assay. The data was plotted as a percentage of control (100%) at each dose point.

[0025] FIG. 5: Table 1. E-max values for a series of structure analogs of CIO (ClOa, ClOb, ClOc, ClOd) and NO-1886. The table shows compound structures, chembridge identification numbers where applicable, and the E-max values for the compounds.

[0026] FIG. 6: Effect of LPL agonists CIO and ClOd on ANGPTL4 inhibition of LPL, as compared to NO-1886.

[0027] FIGS. 7A-7F: Induced fit docking of LPL agonists. CIO (FIGS. 7A-7B), ClOd (FIGS. 7C-

7D) and NO-1886 (FIGS. 7E-7F) were docked on the homology model of human LPL generated using the crystal structure of human pancreatic lipase related protein 1 as a template.

[0028] FIG. 8: LPL activity in wild type animals following injection of ClOd.

[0029] FIGS. 9A-9C: Mice fed high fat diet for 4 months were administered ClOd in the last week of feeding. Liver was dissected and photographed (FIG. 9A). Hepatic histology was observed using H&E staining (FIG. 9B) and plasma TG and CHO were measured (FIG. 9C).

DETAILED DESCRIPTION OF THE INVENTION

[0030] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entirety to more fully describe the state of the art.

[0031] DEFINITIONS

[0032] For convenience, various terms used herein are defined prior to further description of the various embodiments of the present disclosure.

[0033] Unless stereochemistry is specifically indicated, all stereoisomers of the compounds herein are included, as pure compounds as well as mixtures thereof.

[0034] The term "composition" as used herein is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.

[0035] The term "alkyl" refers to monovalent alkyl groups having from 1 to 50 carbon atoms, preferably having from 1 to 10 carbon atoms, and more preferably having from 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, and the like.

[0036] The term "aromatic" refers to a compound or moiety having at least one unsaturated cyclic group with delocalized pi electrons. The term encompasses both hydrocarbon aromatic compounds and heteroaromatic compounds.

[0037] The term "solvate" refers to a pharmaceutically acceptable solid form of a specified compound containing solvent molecules as part of the crystal structure. A solvate typically retains at least some of the biological effectiveness of such compound. Solvates can have different solubilities, hygroscopicities, stabilities, and other properties. Examples of solvates include, but are not limited to, compounds in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine. Solvates are sometimes termed "pseudopolymorphs."

[0038] The term "hydrate" refers to a solvate with water.

[0039] The term "racemate" refers to a mixture that contains an equal amount of enantiomers.

[0040] It will be appreciated by one of ordinary skill in the art that asymmetric centers may exist in any of the compounds described herein. Thus, the compounds and pharmaceutical compositions thereof may be in the form of an individual enantiomer, diastereomer, or geometric isomer, or may be in the form of a mixture of stereoisomers. In certain embodiments, the compounds are enantiopure compounds. In certain other embodiments, mixtures of stereoisomers or diastereomer s are provided. Additionally, the compounds encompass both (Z) and (E) double bond isomers (or cis and trans isomers) unless otherwise specifically designated. Thus, compounds generally depicted in structures herein encompass those structures in which double bonds are (Z) or (E).

[0041] It will be appreciated that any of the compounds described herein may be substituted with any number of substituents or functional moieties. In general, the term "substituted" whether preceded by the term "optionally" or not, and substituents contained in formulas, refer to the replacement of hydrogen atoms (or other moieties, such as halogens) in a given structure with a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.

[0042] As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents or organic compounds. For purposes of explanation herein, heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. Furthermore, there is not any intention to be limited in any manner by the permissible substituents or organic compounds. Combinations of substituents and variables envisioned are preferably those that result in the formation of stable compounds useful in the treatment, for example, of diseases or disorders including, but not limited to, atherosclerosis, cardiovascular disease, or coronary heart disease.

[0043] The term "stable" as used herein refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.

[0044] The term "therapeutically effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal, or human that is being sought by a researcher, veterinarian, medical doctor, or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.

[0045] The term "pharmaceutically acceptable salt" means a salt of a compound. Suitable pharmaceutically acceptable salts of compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid.

[0046] The term "pharmaceutically acceptable carrier" means a medium that is used to prepare a desired dosage form of the compound. A pharmaceutically acceptable carrier includes solvents, diluents, or other liquid vehicles; dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents; preservatives; solid binders; lubricants; and the like.

[0047] GENERAL DESCRIPTION

[0048] Lipoprotein lipase (LPL) is a physiological regulator of triglycerides and atherosclerosis risk. LPL is the enzyme responsible for the hydrolysis of the core triglycerides in the triglyceride-rich lipoproteins. These lipoproteins are chylomicrons and very low density lipoproteins (VLDLs), which have 90% and 62% triglyceride content, respectively. The products of the hydrolysis reaction are free non-esterified fatty acids for tissue use and chylomicron remnants. LPL activity has been observed in heart tissue, adipose tissue, skeletal muscle, and kidneys, among other places, and LPL mRNA has been shown to be most abundant in heart and adipose tissue. The protein is synthesized and secreted by myocytes and adipocytes, and is found lining the lumen of capillaries in various tissues. At these sites, LPL performs the lipolytic processing of triglyceride-rich lipoproteins. Thus, LPL may hold promise as a drug target for hypolipidemic therapy.

[0049] The hydrolysis of triglycerides by LPL is a critical event in plasma lipid metabolism.

Hydrolysis of trigycerides by LPL also results in an increase in high density cholesterol (HDL-C) via the production of increased levels of free cholesterol, phospholipids, and apolipoproteins. A decrease in LPL activity is associated with an increase in plasma triglycerides and a decrease in plasma high density cholester (HDL-C). Hypertriglyceridemia and a decrease in HDL-C are both risk factors for

cardiovascular diseases such as atherosclerosis. Conversely, increased LPL activity is shown to have beneficial effects on lipid profile. The LPL agonist NO-1886, or ibrolipim, possesses potent LPL agonist activity.

[0050] Angiopoietin-like 4 (ANGPTL4) and other members of the ANGPTL family are

physiological regulators of LPL activity in vivo. ANGPTL4 potently inhibits LPL activity. Inhibition of LPL activity by ANGPTL3 and ANGPTL4 results in elevated triglycerides in the bloodstream and poor lipid profile. Injection of recombinant ANGPTL4, as well as peripheral over-expression of ANGPTL4, is associated with increased serum triglycerides, non-HDL cholesterol, and non-esterified fatty acids.

Without wishing to be bound by theory, it is believed this is due to stimulation of adipose tissue lipolysis. ANGPTL4 deficiency improves total cholesterol and triglycerides, and reduces foam cell formation, thereby protecting against atherosclerosis. Antagonism of ANGPTL4 inhibition of LPL is therefore one strategy in the management of dyslipidemias and associated cardio-metabolic conditions.

[0051] Provided herein are methods of utilizing compounds which, in certain embodiments, show greater LPL agonist activity than NO-1886, a known LPL agonist. The compounds share the scaffold of a carboxamide having a general formula of R^CONHR², wherein R¹ comprises a cyclopropane ring, and R² comprises one of an imidazole ring or a morpholine ring. In particular, compounds CIO and ClOd exhibit at least two-fold greater LPL activation than NO-1886. Unlike NO-1886, LPL agonists CIO and ClOd advantageously reverse the LPL inhibition by angiopoietin-like 4 (ANGPTL4), a physiological inhibitor of LPL.

[0052] The compound CIO has the structural formula of Formula I:

Formula I

CIO is also known as 2,2-dichloro-N-[3-(lH-irmdazol-l-yl)propyl]-l-methylcyclopropanecarboxamide. Structurally, CIO is a carboxamide with an imidazole ring warhead and a cyclopropane tail. Without wishing to be bound by theory, it is believed the cyclopropane of CIO extends towards a burrowed cavity in LPL. This is illustrated in FIG. 7B. CIO shows greater activation of LPL activity than the known LPL agonist NO-1886.

[0053] The addition of an aromatic ring, specifically a fluorinated benzyl group, removal of the chlorine substituents, and the addition of a second methyl substituent to the cyclopropane ring produces the compound ClOd, which has the structural formula of Formula IIA:

Formula IIA

ClOd is also known as l-(4-fluorophenyl)-N-[3-(lH-imidazol-l-yl)propyl]-2,2- dimethylcyclopropanecarboxamide. Without wishing to be bound by theory, it is believed the addition of the aromatic ring in ClOd increases the presence of the compound in a pocket of LPL by exploiting hydrophobic interactions. This is illustrated in FIG. 7C. The imidazole ring is involved in hydrogen bonding, and the cyclopropane tail of the compound is important for hydrophobic interactions. ClOd shows a dramatic increase in activation of LPL activity than CIO or NO-1886.

[0054] Compound ClOa is produced by replacing the imidazole ring of CIO with a morpholine ring, and replacing the chlorine substitutents on the cyclopropane ring with bromine atoms. Compound ClOa has the structural formula of Formula III:

Formula III

As described in the examples herein, compound ClOa shows slightly less LPL agonist activity than compound CIO.

[0055] Compound ClOb is produced by replacing the morpholine ring of ClOa with a chlorine- substituted imidazole ring. Compound ClOb has the structural formula of Formula IV:

Formula IV

[0056] Compound ClOc is produced by replacing the chlorine substituent in ClOb with a methyl group. Compound ClOc has the structural formula of Formula V:

Formula V

Compound 10c is only marginally weaker for LPL activity than compound 10b, indicating that substitution of the imidazole ring does not dramatically affect LPL activity.

[0057] PHARMACEUTICAL COMPOSITIONS

[0058] Pharmaceutical compositions of the present disclosure comprise an effective amount of a compound having the scaffold described herein, and/or additional agents, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical" or "pharmacologically acceptable" refer to molecular entities and compositions that produce no adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human. The preparation of a pharmaceutical composition that contains at least one compound or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it is understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.

[0059] A composition disclosed herein may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. Compositions disclosed herein can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically, intramuscularly, subcutaneously, mucosally, intraosseosly, periprosthetically, in utero, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference).

[0060] The actual dosage amount of a composition disclosed herein administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of

administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

[0061] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. Naturally, the amount of active compound(s) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. [0062] In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50

microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50

milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

[0063] In certain embodiments, a composition herein and/or additional agent is formulated to be administered via an alimentary route. Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. As such, these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsules, they may be compressed into tablets, or they may be incorporated directly with the food of the diet.

[0064] In further embodiments, a composition described herein may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the alimentary tract. Specifically, the pharmaceutical compositions disclosed herein may be administered, for example but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally.

[0065] Solutions of the compositions disclosed herein as free bases or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.

Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some cases, the form is sterile and is fluid to the extent that easy injectability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and/or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, such as, but not limited to, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride.

Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption such as, for example, aluminum monostearate or gelatin.

[0066] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of

hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0067] Sterile injectable solutions are prepared by incorporating the compositions in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized compositions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, some methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. A powdered composition is combined with a liquid carrier such as, but not limited to, water or a saline solution, with or without a stabilizing agent.

[0068] In other embodiments, the compositions may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or via inhalation.

[0069] Pharmaceutical compositions for topical administration may include the compositions formulated for a medicated application such as an ointment, paste, cream, or powder. Ointments include all oleaginous, adsorption, emulsion, and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only. Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin. Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones, and luarocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorption, emulsion, or water-soluble ointment base. Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the composition and provide for a homogenous mixture.

Transdermal administration of the compositions may also comprise the use of a "patch." For example, the patch may supply one or more compositions at a predetermined rate and in a continuous manner over a fixed period of time.

[0070] It is further envisioned the compositions disclosed herein may be delivered via an aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. The typical aerosol for inhalation consists of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary according to the pressure requirements of the propellant. Administration of the aerosol will vary according to subject's age, weight and the severity and response of the symptoms.

[0071] In particular embodiments, the compounds and compositions described herein are useful for treating, preventing, or ameliorating atherosclerosis in a subject with high triglycerides; treating hypertriglyceridemia; treating poor lipid profile; treating high levels of non-HDL cholesterol; and treating high levels of non-esterified fatty acids. The compounds and compositions are particularly useful for decreasing the risk of atherosclerosis. In certain embodiments, the compounds and compositions herein are useful as part or parts of combination therapies with other drugs or therapies. Suitable other drugs or therapies include, but are not limited to: intestinal cholesterol absorption-inhibiting supplements such as ezetimibe; statins; niacin; smoking cessation; exercise; surgeries such as angioplasty procedures; changes in diet; anticoagulants; and combinations thereof.

[0072] KITS

[0073] It is envisioned that the compounds, compositions, and methods described herein could be embodied as parts of a kit or kits. A non-limiting example of such a kit is a kit comprising a compound herein and a pharmaceutically acceptable carrier, diluent, or adjuvant in separate containers, where the containers may or may not be present in a combined configuration. Many other kits are possible, such as kits further comprising a syringe. The kits may further include instructions for using the components of the kit to practice the subject methods. The instructions for practicing the subject methods are generally recorded on a suitable recording medium. For example, the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, such as a flash drive, CD-ROM, or diskette. In other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, such as via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0074] EXAMPLES

[0075] Identification ofLPL Agonists

[0076] An established in vitro assay was used for the identification of LPL agonists. Compounds were screened in an in vitro LPL activity assay using a 384 well plate. LPL activity was measured by an in vitro assay using p-nitrophenylbutyrate (PNPB, Sigma-N9876) as the substrate. The sample buffer consisted of 100 mM sodium phosphate buffer with 150 niM sodium chloride and 0.5% (v/v) Triton X- 100, pH 7.2. Bovine LPL (Sigma-Ca. no. L2254) was used at a concentration of 70 units/mL, and the PNPB substrate was used at a concentration of 0.5 mM. In assays incorporating ANGPTL4, 0.2 μg/well recombinant human ANGPTL4 (rhANGPTL4) was used. The compounds were tested at a single concentration of 40 μΜ in the primary screen. The assay was performed in a 384-well plate at a total volume of 25 \L. rhANGPTL4 was pre -incubated in sample buffer with LPL for 30 min at 25 °C before PNPB addition. Twenty minutes following the addition of PNPB, the plate was read at 400 nm. Z-factor determination was performed in the absence (min) or presence (max) of substrate. The robustness of the LPL assay for screening was determined by the Z-factor. As shown in FIG. 2, a Z-factor of 0.532 was observed.

[0077] Among the compounds screened, compound CIO showed the greatest increase in the LPL activity (FIGs. 3A-3B). Compounds C7 also showed agonist activity. A dose-response evaluation on the compound CIO was then conducted. A dose-dependent increase in LPL activity was observed in the presence of CIO (FIG. 4), with a maximum effect determined at 324% of control LPL activity.

[0078] Comparison of LPL Activity

[0079] FIG. 5 - Table 1 shows the maximum induction of LPL activity (E-max) compared between CIO, its structural analogs, and NO-1886. CIO showed greater activation of LPL activity as compared to NO-1886. (E-max: 196.39 ± 20.21 versus 169.57 ± 22.99.) Further, analogs ClOa, ClOb, and ClOc exhibited a decrease in LPL agonism activity when compared to CIO. However, compound ClOd (E- max: 342.43 ± 17.12) showed a dramatic increase in activity compared to CIO and NO-1886. From these compounds, a small trend in structure-activity relationship was observable. For instance, the compound 10a showed a reduction in LPL activity compared to the compound 10, with the replacement of halogens with bromine. On the other hand, the substitution on the imidazole ring did not dramatically affect activity, since compound 10c was only marginally weaker for LPL activity than 10b with the replacement of the chlorine in 10b with a larger methyl group in 10c. The most noticeable effect on E-max was observed upon addition of the fluorinated benzyl group in lOd as compared to compound 10.

[0080] The activity of the compounds in reversing the ANGPTL4 inhibition of LPL was determined in the in vitro LPL assay. ANGPTL4, when added at a concentration of 0.2 μg/well, inhibited the LPL activity by greater than 50%. Both compounds CIO and ClOd were able to rescue the LPL inhibition by ANGPTL4 in a dose-dependent manner, with ClOd exhibiting greater efficacy than CIO. (FIG. 6.) On the other hand, NO-1886 failed to rescue the LPL inhibition by ANGPTL4. Compounds CIO and ClOd are unexpectedly able to reverse ANGPTL4 inhibtion of LPL, which is a unique advantage over known LPL agonists.

[0081 ] ANGPTL4 Activity

[0082] Recombinant ANGPTL4 was added to the LPL activity assay at a concentration of 0.2 μg/well in the absence or presence of compounds CIO, ClOd, and NO-1886, added in a twelve -point dose response fashion. The LPL activity assay was conducted and the absorbance values obtained were plotted. These plots are shown in FIG. 6. As seen in FIG. 6, ClOd and CIO both increased LPL activity in the presence of ANGPTL4, as compared to NO-1886.

[0083] Molecular Modeling

[0084] A molecular modeling approach was used to elucidate the potential binding of the compounds described herein to LPL. The homology model of LPL was generated using YASARA, with default settings. The final model generated was used for docking studies in MOE 2012. The protein structure was protonated at a pH of 7.4 to correlate with the experimental conditions. Induced-fit docking was used to dock the compounds into LPL. The top five returned poses were evaluated visually.

[0085] Using the protein sequence of human LPL and the known structure of human pancreatic lipase-like protein, a homology model was constructed, and the compounds were docked on this structure. (FIGS. 7A-7F.) Findings in the docking evaluations corroborated the observations made in Table 1 and FIG. 6. The cyclopropane ring was observed extending towards a burrowed cavity in LPL (CIO, FIGS. 7A-7B). The addition of the aromatic ring in ClOd increased the presence of the compound in this pocket, exploiting hydrophobic interactions. (FIGS. 7C-7D.) These data indicate that the imidazole warhead is important for hydrogen bonding, and the tail cyclopropane is important for hydrophobic interactions. Furthermore, using protein-protein docking methods, it was determined that ANGPTL4 binds to LPL in such a way that NO-1886 may not have free access to the proposed binding pocket, whereas the CIO and ClOd compounds can still bind to LPL. (FIGS. 7E-7F.) These results create a structural scaffold for use in the design of LPL activators. Such activators can be used for the development of treatments for elevated triglycerides in a metabolic syndrome.

[0086] Animal Testing [0087] The ability of ClOd to activate LPL in wild-type mice was evaluated. ClOd was injected at 10 mg/kg body weight. As shown in FIG. 8, LPL activity was increased 4 hours after injection.

[0088] Liver Example

[0089] ClOd reverses fatty liver phenotype in high fat diet feeding model. When the mice are challenged with prolonged high fat diet (HFD,4 months), it was observed that mice receiving

supplementation of ClOd in the last week of feeding, showed significantly improved metabolic profile as compared to the mice who did not receive ClOd. Livers isolated from ClOd treated mice at the conclusion of study showed remarkable differences in liver morphology (FIG. 9A) and histology FIG. 9B).

Collectively, the data indicates an attenuation of hepatic steatosis. Further, mice receiving ClOd showed significantly reduced plasma triglyceride and cholesterol (CHO) levels (FIG. 9C).

[0090] Certain embodiments of the compounds, compositions, and methods disclosed herein are defined in the above examples. It should be understood that these examples, while indicating particular embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope thereof, can make various changes and modifications to adapt the compositions and methods described herein to various usages and conditions. Various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the disclosure. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the disclosure without departing from the essential scope thereof.

Claims

CLAIMS What is claimed is:

1. A method of increasing LPL activity, the method comprising administering a compound to a subject and increasing LPL activity in the subject, the compound comprising a carboxamide having a general formula of R^CONHR², wherein:

R¹ comprises a cyclopropane ring; and

R² comprises one of an imidazole ring or a morpholine ring;

or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate thereof.

2. The method of claim 1 , wherein R¹ further comprises an aromatic ring.

3. The method of claim 2, wherein the aromatic ring is substituted with a halogen.

4. The method of claim 3, wherein the aromatic ring is a fluorinated benzyl group.

5. The method of claim 4, wherein the fluorinated benzyl group is bonded to the cyclopropane ring.

6. The method of claim 1, wherein the imidazole ring is substituted with a halogen or methyl group.

7. The method of claim 1, wherein the cyclopropane ring is substituted with at least one halogen, bromine, or methyl group.

8. The method of claim 7, wherein the cyclopropane ring is substituted with two halogens or bromines.

9. The method of claim 7, wherein the cyclopropane ring is substituted with two methyl groups.

10. The method of claim 1, wherein the imidazole ring or the morpholine ring is linked to the nitrogen atom by a carbon chain having from 1 to 10 carbons.

11. The method of claim 10, wherein the carbon chain has 3 carbons.

wherein the compound consists essentially of Formula II:

Formula II; wherein X is a halogen.

The method of claim 12, wherein the compound consists essentially of Formula IIA:

Formula IIA.

The method of claim 1, wherein the compound consists essentially of Formula I:

Formula I.

The method of claim 1, wherein the compound consists essentially of Formula III:

Formula III.

The method of claim 1, wherein the compound consists essentially of Formula IV:

Formula IV.

17. The method of claim 1, wherein the compound consists essentially of Formula V:

Formula V.

18. A pharmaceutical composition comprising:

a pharmaceutically acceptable carrier; and

a compound comprising a carboxamide having a general formula of R^CONHR², wherein:

R¹ comprises a cyclopropane ring; and

R² comprises one of an imidazole ring or a morpholine ring.

19. A method of reversing ANGTPL4 inhibition of LPL, the method comprising administering an effective amount of a compound to a subject, and reversing ANGTPL4 inhibition of LPL in the subject, the compound comprising a carboxamide having a general formula of R^ONHR², wherein:

R¹ comprises a cyclopropane ring; and

R² comprises one of an imidazole ring or a morpholine ring;

or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate thereof.

20. A method of activating LPL, the method comprising administering an effective amount of a compound to a subject, and activating LPL in the subject, the compound comprising a carboxamide having a general formula of R^ONHR², wherein:

R¹ comprises a cyclopropane ring; and

R² comprises one of an imidazole ring or a morpholine ring;

or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate thereof.

21. A method of treating, preventing, or ameliorating atherosclerosis in a subject with high triglycerides, the method comprising administering a therapeutically effective amount of an LPL agonist to a subject and treating, preventing, or ameliorating atherosclerosis in the subject, the LPL agonist comprising a carboxamide having a general formula of R^ONHR², wherein:

R¹ comprises a cyclopropane ring; and R² comprises one of an imidazole ring or a morpholine ring;

or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate thereof.

22. A method of improving lipid profile in a subject, the method comprising administering an effective amount of an LPL agonist to a subject and improving lipid profile in the subject, the LPL agonist comprising a carboxamide having a general formula of R^ONHR², wherein:

R¹ comprises a cyclopropane ring; and

R² comprises one of an imidazole ring or a morpholine ring;

or a salt, prodrug, stereoisomer, solvate, hydrate, polymorph, or racemate thereof.

23. The method of any preceding claim, wherein the compound is administered at a dose of about 10 mg/kg body weight.

24. A method of reversing fatty liver phenotype in a subject in need thereof, comprising administering an effective amount of compound ClOd.