Pharmaceutical compositions and methods for the treatment of chronic diseases

Abstract

Provided herein are pharmaceutical compositions that include a therapeutically effective amount of a compound or a pharmaceutically acceptable salt thereof. Also included herein is a method of regulating a disease governed by Fatty Acid Synthase (FAS) in a subject in need thereof, including atheroprogression and PAD.

Description

PHARMACEUTICAL COMPOSITIONS AND METHODS FOR THE TREATMENT OF CHRONIC DISEASES ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under HL153262 awarded by theNational Institutes of Health. The government has certain rights in the invention. CROSS REFERENCE TO RELATED APPLICATION

[0002] The present application claims priority to U.S. Patent Application No. 63 / 698,438,filed September 24, 2024, the disclosure of which is incorporated by reference in its entirety. BACKGROUND

[0003] Atherosclerosis is the predominant global underlying cause for cardiovascular disease.Its management predominantly revolves around mitigation of risk factors such as hyperlipidemia with pharmacological therapies that aim to reduce serum lipid levels. Both tissue Fatty Acid Synthase (FAS) and serum circulating FAS (cFAS) are elevated in individuals afflicted with severe cardiovascular disease. Even with effective reduction of total cholesterol using established treatment methods including statin monotherapy, individuals contending with cardiovascular co-morbidities continue to be disproportionately susceptible to atheroprogression. This heightened vulnerability translates into a persistently elevated risk of myocardial infarction, stroke, and major extremity amputations. Provided herein are solutions to regulate atheroprogression. BRIEF SUMMARY

[0004] Provided herein, inter alia, is a method of reducing atheroprogression in a subject inneed thereof. Further, provided herein, inter alia, is a method of regulating a disease governed by Fatty Acid Synthase (FAS) in a subject in need thereof. Provided herein, inter alia, are also compositions comprising a therapeutically effective amount of a Fatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof.

[0005] In one embodiment the method of reducing atheroprogression in a subject in needthereof includes administering to the subject a Fatty Acid Synthase (FAS) inhibitor or apharmaceutically acceptable salt thereof. In one embodiment, the FAS inhibitor is administered in combination with an additional therapeutic agent.

[0006] In one embodiment, the FAS inhibitor is a natural FAS inhibitor or a synthetic FASinhibitor. In another embodiment, the FAS inhibitor is selected from a small molecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof. In another embodiment, the FAS inhibitor is selected from GSK 2194069, imidazopyridine, epigallocatechin-3-gallate, BI 99179, TVB-2640, TVB-3664, TVB-3166, Cerulenin, C75, orlistat and platensimycin. In another embodiment, the FAS inhibitor is selected from GSK 2194069, GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat, G28UCM, Fasnall, imidazopyridine, epigallocatechin-3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, and 910249-49-9.

[0007] In one embodiment the FAS inhibitor inhibits tissue FAS and serum circulating FattyAcid Synthase (cFAS). In another embodiment, the FAS inhibitor reduces liver triglycerides, and Free Fatty Acids (FFAs).

[0008] In one embodiment, the FAS inhibitor is administered from about 100 mg / kg / day to asubject in need thereof. In another embodiment, the therapeutically effective amount of the FAS inhibitor is administered from about 50 to 200 mg to a subject in need thereof.

[0009] In one embodiment, the subject is a human patient.

[0010] In one embodiment, the FAS inhibitor inhibits tissue FAS and serum circulating FAS(cFAS). In another embodiment, the FAS inhibitor reduces liver triglycerides, and Free Fatty Acids (FFAs).

[0011] In one embodiment, the disease is selected from cancer, viral infections, nonalcoholicfatty liver disease or a metabolic disorder. In another embodiment, the metabolic disorder is diabetes. In another embodiment, the metabolic disorder is atherosclerosis. In another embodiment, the metabolic disorder is atherosclerotic cardiovascular disease.

[0012] Also provided herein is a method of treating peripheral artery disease (PAD) in asubject in need thereof including administering to the subject a glucagon-like peptide-1 (GLP- 1) receptor agonist or a pharmaceutically acceptable salt thereof. Further provided herein is a method of treating peripheral artery disease in a subject in need thereof comprisingadministering to the subject a peroxisome proliferator-activated receptor (PPAR) agonist or a pharmaceutically acceptable salt thereof.

[0013] In one embodiment, the GLP-1 receptor agonist is selected from a small moleculecompound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof. In another embodiment, the GLP-1 receptor agonist is a GLP-1 analogue. In another embodiment, the GLP-1 receptor agonist is selected from dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, and tirzepatide.

[0014] In one embodiment, the GLP-1 receptor agonist is administered in combination with anadditional therapeutic agent. In another embodiment, the therapeutic agent is metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, or sodium glucose co- transporters.

[0015] In one embodiment, the PPAR agonist is selected from a small molecule compound, anucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single- chain variable fragment (ScFv), and a fragment or variant thereof. In another embodiment, the PPAR agonist is a PPARα agonist or a PPARγ agonists. In another embodiment, the PPAR agonist is clofibrate, gemfibrozil, ciprofibrate, bezafibrate, fenofibrate, thiazolidinediones, pioglitazone or rosiglitazone.

[0016] In one embodiment, the PPAR agonist is administered in combination with anadditional a therapeutic agent including metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, or sodium glucose co-transporters. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 illustrates foam cell formation in vitro;

[0018] FIG. 2 illustrates effect of conditioned serum on intracellular and extracellular FAS inmacrophages;

[0019] FIG. 3 illustrates FAS inhibition affects murine lipid homeostasis;

[0020] FIG. 4 illustrates that conditional liver-specific knockdown of FAS and PTM treatmentimpacts serum cFAS content and activity;

[0021] FIG. 5 illustrates that targeting FAS reduces aortic atherosclerotic plaque burden;

[0022] FIG. 6 illustrates that targeting FAS reduced aortic root plaque burden;

[0023] FIG. 7 illustrates that FAS inhibition reduces tissue FAS and inflammation response;

[0024] FIG. 8 illustrates peripheral artery disease (PAD) condition;

[0025] FIG. 9 illustrates De novo Lipogenesis (DNL);

[0026] FIG. 10 illustrates experimental design to evaluate the impact of DNL signaling anddiabetes on peripheral atheroprogression;

[0027] FIG. 11 illustrates ilio-femoral peripheral arterial atherosclerosis model in diabeticmice maintained on a western diet;

[0028] FIG. 12 illustrates CEPT1 relative to PAD severity;

[0029] FIG. 13 illustrates the effect of conditional knockdown of Cept1 in the endothelium;

[0030] FIG. 14 illustrates the effect of conditional knockdown of Cept1 in the endothelium;

[0031] FIG. 15 illustrates the effect of conditional overexpression of Cept1 in theendothelium;

[0032] FIG. 16 illustrates the level of plaque FAS and serum cFAS in patients with diabetesand CLTI;

[0033] FIG. 17 illustrates the level of cFAS as per an embodiment described herein;

[0034] FIG. 18 illustrates the effect of conditional hepatic knockdown of Fasn and systemicpharmacological targeting of FAS;

[0035] FIG. 19 illustrates the effect of conditional hepatic knockdown of Fasn;

[0036] FIG. 20 illustrates the effect of conditional hepatic knockdown of Fasn;

[0037] FIG. 21 illustrates the effect of semaglutide;

[0038] FIG. 22 illustrates an experimental design as per an embodiment described herein; and

[0039] FIG. 23 illustrates foam cell and cFAS activity assays.DETAILED DESCRIPTION

[0040] Provided herein are methods of reducing atheroprogression in a subject in needthereof. Further, provided herein, is a method of regulating a disease governed by Fatty AcidSynthase (FAS) in a subject in need thereof. Also provided herein is a method of treating peripheral artery disease in a subject in need thereof. Furthermore, compositions including Fatty Acid Synthase (FAS) inhibitor compound or a GLP-1 receptor agonist compound or a PPAR agonist compound are also provided herein.

[0041] Composition

[0042] As described herein, a composition includes a therapeutically effective amount of aFatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof. The composition as described herein can also include a GLP-1 receptor agonist compound or a pharmaceutically effective salt thereof. Further, the composition can include a PPAR agonist compound or a pharmaceutically effective salt thereof.

[0043] Pharmaceutical compositions and dosage forms described herein typically include oneor more excipients. Suitable excipients are well known to those skilled in the art of pharmacy. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors such as, for example, the intended route of administration to the patient. Pharmaceutical compositions described herein can include other agents such as stabilizers, lubricants, buffers, and disintegrants that can reduce the rate by which an active ingredient can decompose in a particular formulation.

[0044] Pharmaceutical compositions described herein in certain instances include additionalactive agents other than those in the compositions described herein in a suitable amount.

[0045] The pharmaceutical composition described herein is suitable for administration via anyroute to a patient described herein including but not limited to oral, mucosal (e.g., nasal, inhalation, pulmonary, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intra-arterial), topical (e.g., eye drops or other ophthalmic preparations), transdermal or transcutaneous.

[0046] Exemplary dosage forms include tablets; caplets; capsules (e.g., gelatin capsules);cachets; lozenges; suppositories; powders; gels; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.

[0047] Furthermore, the compositions described herein are useful for treating a variety ofdiseases, disorder(s), and symptoms thereof, including for example, atherosclerosis and diabetes. In one embodiment, the composition is useful for treating peripheral arterial disease(PAD). In another embodiment, the composition is useful for treating atheroscleroticcardiovascular disease. In another embodiment, the composition is useful for regulating a disease governed by Fatty Acid Synthase (FAS) in a subject in need thereof.

[0048] As described herein, the composition includes a therapeutically effective amount of aFatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof. The FAS inhibitor compound can be natural FAS inhibitor or a synthetic FAS inhibitor. In oneembodiment, the FAS inhibitor compound is selected from commercially available inhibitors comprising GSK 2194069, imidazopyridine, epigallocatechin-3-gallate, BI 99179, TVB-2640, TVB-3664, TVB-3166, Cerulenin, C75, orlistat or platensimycin. In another embodiment, the FAS inhibitor compound is selected from commercially available inhibitors comprising is GSK837149A, FT-113 or IPI-9119. In another embodiment, the FAS inhibitor compound is selected from 1309805-49-9, 1808260-84-5, 1375105-96-6 or 910249-49-9. Alternatively, GSK 2194069, GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat, G28UCM, Fasnall, imidazopyridine, epigallocatechin-3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, and 910249-49-9.

[0049] Alternatively, the FAS inhibitor compound is a small molecule compound, a nucleicacid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), or a fragment or variant thereof.

[0050] The composition as described herein can also include a GLP-1 receptor agonistcompound or a pharmaceutically effective salt thereof. The GLP-1 receptor agonist compound as described herein is a GLP-1 analogue. In an embodiment, the GLP-1 receptor agonist is selected from dulaglutide, exenatide, exenatide extended release, liraglutide, lixisenatide, semaglutide, or tirzepatide.

[0051] Alternatively, the GLP-1 receptor agonist compound is a small molecule compound, anucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single- chain variable fragment (ScFv), or a fragment or variant thereof.

[0052] Furthermore, the composition as described herein can also include a PPAR agonistcompound or a pharmaceutically effective salt thereof. In one embodiment, the PPAR agonist is selected from a small molecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), or a fragment or variant thereof. In another embodiment, the PPAR agonist is a PPARα agonist or a PPARγ agonist including clofibrate, gemfibrozil, ciprofibrate, bezafibrate, fenofibrate, thiazolidinediones, pioglitazone or rosiglitazone.

[0053] The FAS inhibitor compound, the GLP-1 receptor agonist compound and / or the PPARagonist compound described herein are individually present in the composition in an amount of at least about: 1 mg to about 10 mg, 1 mg to about 25 mg, 1 mg to about 50 mg, 5 mg to about10 mg, 5 mg to about 25 mg, 5 mg to about 50 mg, 10 mg to about 25 mg, 10 mg to about 50 mg, 50 mg to about 100 mg, or 100 mg to about 200 mg.

[0054] For example, the FAS inhibitor compound, the GLP-1 receptor agonist compoundand / or the PPAR agonist compound described herein are present in the composition in an amount of at least about 50 mg to about 500 mg, 100 mg to about 500 mg, 150 mg to about 500 mg, 200 mg to about 500 mg, 250 mg to about 500 mg, 300 mg to about 500 mg, 350 mg to about 500 mg, 400 mg to about 500 mg or 450 mg to about 500 mg.

[0055] The compounds described herein can be present in the composition relative to theweight of the patient (e.g., mg / kg). In some instances, FAS inhibitor compound, the GLP-1 receptor agonist compound and / or the PPAR agonist compound is present in an amount equivalent to about: 0.0001 mg / kg to about 200 mg / kg, 0.001 mg / kg to about 200 mg / kg, 0.01 mg / kg to about 200 mg / kg, 0.01 mg / kg to about 150 mg / kg, 0.01 mg / kg to about 100 mg / kg, 0.01 mg / kg to about 50 mg / kg, 0.01 mg / kg to about 25 mg / kg, 0.01 mg / kg to about 10 mg / kg, or 0.01 mg / kg to about 5 mg / kg, 0.05 mg / kg to about 200 mg / kg.

[0056] In another embodiment, the FAS inhibitor compound, the GLP-1 receptor agonistcompound and / or the PPAR agonist compound described herein is present in an amount equivalent to about: 0.05 mg / kg to about 150 mg / kg, 0.05 mg / kg to about 100 mg / kg, 0.05 mg / kg to about 50 mg / kg, 0.05 mg / kg to about 25 mg / kg, 0.05 mg / kg to about 10 mg / kg, or 0.05 mg / kg to about 5 mg / kg, 0.5 mg / kg to about 200 mg / kg, 0.5 mg / kg to about 150 mg / kg, 0.5 mg / kg to about 100 mg / kg, 0.5 mg / kg to about 50 mg / kg, 0.5 mg / kg to about 25 mg / kg, 0.5 mg / kg to about 10 mg / kg, or 0.5 mg / kg to about 5 mg / kg.

[0057] In another embodiment, the FAS inhibitor compound, the GLP-1 receptor agonistcompound and / or the PPAR agonist compound described herein is present in an amount equivalent to about: 1 mg / kg to about 200 mg / kg, 1 mg / kg to about 150 mg / kg, 1 mg / kg to about 100 mg / kg, 1 mg / kg to about 50 mg / kg, 1 mg / kg to about 25 mg / kg, 1 mg / kg to about 10 mg / kg, or 1 mg / kg to about 5 mg / kg.

[0058] In one embodiment, the composition includes a FAS inhibitor compound present in anamount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg. In another embodiment, the composition includes a FAS inhibitor compound present in an amount of about: 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0059] In one embodiment, the composition includes a GLP-1 receptor agonist compoundpresent in an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg. In another embodiment, the GLP-1 receptor agonist compound is present in an amount of about: 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 800 mg, or 1000 mg. In an embodiment, the GLP-1 receptor agonist compound is present in an amount of about: 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, or 1 mg. In another embodiment, the GLP-1 receptor agonist compound present in the composition is present in an amount of about: 0.1 mg to 1 mg, 0.2 to 0.5 mg, 0.2 to 0.3 mg, or 0.2 to 0.2 to 0.4 mg. In another embodiment, the GLP-1 receptor agonist compound in the composition is present in an amount of about: 0.15 mg to 0.25 mg, 0.15 to 0.2 mg, or 0.2 to 0.25 mg.

[0060] In an embodiment, the composition includes a PPAR agonist compound present in anamount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

[0061] In an embodiment, the PPAR agonist compound is present in an amount of about: 0.1mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, or 1 mg. In another embodiment, the PPAR agonist compound in the composition is present in an amount of about: 0.1 mg to 1 mg, 0.2 to 0.5 mg, 0.2 to 0.3 mg, or 0.2 to 0.4 mg. In another embodiment, the PPAR agonist compound is present in an amount of about: 0.15 mg to 0.25 mg, 0.15 to 0.2 mg, or 0.2 to 0.25 mg.

[0062] Methods

[0063] The pharmaceutical composition comprising the compounds described herein areuseful for treating diseases, disorders, or alleviating or eliminating the symptoms of diseases and disorders such as, for example, atherosclerosis or diabetes. It is to be understood that the methods described herein pertain to administration of the pharmaceutical compositions and / or the compounds described herein.

[0064] Provided herein are methods of reducing atheroprogression in a subject in need thereofby administering to the subject a therapeutically effective amount of a Fatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof. Also, provided herein are methods treating diabetes in a subject in need thereof by administering to the subject a therapeutically effective amount of a Fatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof. Further provided herein are methods of reducingatherosclerotic cardiovascular disease in a subject in need thereof by administering to the subject a therapeutically effective amount of a Fatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof.

[0065] Alternatively, the method as described herein can be used for treating peripheralarterial disease (PAD) in a subject in need thereof by administering to the subject a therapeutically effective amount of a Fatty Acid Synthase (FAS) inhibitor compound or a pharmaceutically acceptable salt thereof.

[0066] In one embodiment, the method is used for treating peripheral arterial disease (PAD) ina subject in need thereof by administering to the subject a therapeutically effective amount of a GLP-1 receptor agonist compound or a pharmaceutically acceptable salt thereof.

[0067] In one embodiment, the GLP-1 receptor agonist compound or a pharmaceuticallyacceptable salt thereof impacts FAS expression in liver. In another embodiment, the GLP-1 receptor agonist compound or a pharmaceutically acceptable salt thereof reduces FAS expression in liver. In another embodiment, the GLP-1 receptor agonist compound or a pharmaceutically acceptable salt thereof reduces serum cFAS activity. In another embodiment, the GLP-1 receptor agonist compound or a pharmaceutically acceptable salt thereof reduces hepatic steatosis.

[0068] In one embodiment, the GLP-1 receptor agonist compound or a pharmaceuticallyacceptable salt thereof reduces fatty liver.

[0069] In one embodiment, the method is used for treating peripheral arterial disease (PAD) ina subject in need thereof by administering to the subject a therapeutically effective amount of a PPAR agonist compound or a pharmaceutically acceptable salt thereof.

[0070] In one embodiment, the subject is a mammal including humans.

[0071] Additionally, provided herein are methods of regulating a disease governed by FattyAcid Synthase (FAS) in a subject in need thereof by administering to the subject a therapeutically effective amount of a Fatty Acid Synthase (FAS) inhibitor compound.

[0072] The FAS inhibitor compound can be selected from commercially available inhibitorscomprising GSK 2194069, imidazopyridine, epigallocatechin-3-gallate, BI 99179, TVB-2640, TVB-3664, TVB-3166, Cerulenin, C75, orlistat or platensimycin. Alternatively, the FAS inhibitor compound can be selected from GSK 2194069, GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat,G28UCM, Fasnall, imidazopyridine, epigallocatechin-3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, or 910249-49-9. In another embodiment, the FAS inhibitor compound is a small molecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), or a fragment or variant thereof.

[0073] The FAS inhibitor compound used for the treatment of a disease is about 10 mg toabout 1000 mg, 50 mg to about 800 mg, 50 mg to about 700 mg, 50 mg to about 500 mg, 80 mg to about 100 mg, 80 mg to about 150 mg, 80 mg to about 200 mg, 100 mg to about 250 mg, 150 mg to about 200 mg, 100 mg to about 200 mg or 100 mg to about 300 mg.

[0074] In one embodiment, the FAS inhibitor compound is present at an amount present in thecomposition described herein relative to the weight of the patient (e.g., mg / kg). For instance, the FAS inhibitor compound is present in an amount equivalent to about: 1 mg / kg to about 200 mg / kg, 1 mg / kg to about 150 mg / kg, 1 mg / kg to about 100 mg / kg, 1 mg / kg to about 50 mg / kg, 1 mg / kg to about 25 mg / kg, 1 mg / kg to about 10 mg / kg, or 1 mg / kg to about 5 mg / kg.

[0075] In another embodiment, the composition includes a FAS inhibitor compound present inan amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

[0076] In one embodiment, the GLP-1 receptor agonist compound used for the treatment of adisease described herein is about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

[0077] In another embodiment, the GLP-1 receptor agonist compound used for the treatmentof a disease described herein is present in an amount of about: 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0078] In an embodiment, the GLP-1 receptor agonist compound used for the treatment of adisease described herein is present in an amount of about: 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, or 1 mg. In another embodiment, the GLP-1 receptor agonist compound used for the treatment of a disease described herein is present in an amount of about: 0.1 mg to 1 mg, 0.2 to 0.5 mg, 0.2 to 0.3 mg, or 0.2 to 0.4 mg. In another embodiment, the GLP-1 receptor agonist compound used for the treatment of a disease described herein is present in an amount of about: 0.15 mg to 0.25 mg, 0.15 to 0.2 mg, or 0.2 to 0.25 mg.

[0079] In an embodiment, the GLP-1 receptor agonist compound used for the treatment ofPAD is present in an amount of about: 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, or 1 mg. In another embodiment, the GLP-1 receptor agonist compound used for the treatment of PAD is present in an amount of about: 0.1 mg to 1 mg, 0.2 to 0.5 mg, 0.2 to 0.3 mg, or 0.2 to 0.4 mg. In another embodiment, the GLP-1 receptor agonist compound used for the treatment of PAD is present in an amount of about: 0.15 mg to 0.25 mg, 0.15 to 0.2 mg, or 0.2 to 0.25 mg.

[0080] In an embodiment, the GLP-1 receptor agonist compound used for the treatment ofPAD can be administered, for example, once a day (QD), twice daily (BID), once a week (QW), twice weekly (BIW), three times a week (TIW), or monthly (QM). For example, the GLP-1 receptor agonist compound can be administered QW. In certain instances, the GLP-1 receptor agonist compound used for the treatment of PAD is administered 1 to 2 times a week. The administration of GLP-1 receptor agonist compound can, in part, depend upon the tolerance of the patient and will be determined by the physician.

[0081] In certain instances, the GLP-1 receptor agonist compound used for the treatment isselected from the commercially available compounds including but not limited to exenatide, liraglutide, albiglutide, dulaglutide, lixisenatide, semaglutide, or tirzepatide.

[0082] In one embodiment, the PPAR agonist compound include, but are not limited torosiglitazone, pioglitazone, troglitazone, isaglitazone (known as MCC-555), 2-[2-[(2R)-4- hexyl-3,4-dihydro-3-oxo-2H-1,4-benzoxazin-2-yl]ethoxy]-benzene acetic acid, and the like.

[0083] In an embodiment, the PPAR agonist compound used for the treatment of a disease ispresent in an amount of about: 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 60 mg, 70 mg, 80 mg, 85 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, or 200 mg.

[0084] In an embodiment, the PPAR agonist compound used for the treatment of a diseasedescribed herein is present in an amount of about: 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, or 1 mg. In another embodiment, the PPAR agonist compound used for the treatment of a disease described herein is present in an amount of about: 0.1 mg to 1 mg, 0.2 to 0.5 mg, 0.2 to 0.3 mg, or 0.2 to 0.4 mg. In another embodiment, the PPAR agonist compound used for the treatment of a disease described herein is present in an amount of about: 0.15 mg to 0.25 mg, 0.15 to 0.2 mg, or 0.2 to 0.25 mg.

[0085] In an embodiment, the PPAR agonist compound used for the treatment of a diseasedescribed herein can be administered, for example, once a day (QD), twice daily (BID), once aweek (QW), twice weekly (BIW), three times a week (TIW), or monthly (QM). For example, the PPAR agonist compound can be administered BID. In certain instances, the PPAR agonist compound is administered 2 to 3 times daily. The administration of PPAR agonist compound can, in part, depend upon the tolerance of the patient and will be determined by the physician.

[0086] In an embodiment, the compounds of the instant specification can be administered incombination with other therapeutic agents to treat a disease, including PAD. In one embodiment, the GLP-1 receptor agonist compound is administered in combination with an additional a therapeutic agent including metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, or sodium glucose co-transporters.

[0087] In an embodiment, the additional therapeutic agents include the following:

[0088] a) Sulfonylureas. Suitable examples of sulfonylureas include, but are not limited tochlorpropamide, tolazamide, tolbutamide, glyburide, glipizide, glimepiride, and like.

[0089] (b) Meglitinides, another class of insulin secretagogues. Suitable examples ofmeglitinides include, but are not limited to repaglinide.

[0090] (c) Agents which modify insulin secretion such as Glucagon-like Peptide-1 (GLP-1)and mimetics thereof, Glucose-insulinotropic peptide (GIP) and mimetics thereof, Exendin and mimetics thereof, and Dipeptyl Protease Inhibitors (DPPIV).

[0091] (d) Biguanides. Suitable examples include, but are not limited to metformin.

[0092] (e) Thiazolidinediones. Suitable examples of PPAR-gamma agonists are thethiazolidinediones which include, but are not limited to rosiglitazone, pioglitazone, troglitazone, isaglitazone (known as MCC-555), 2-[2-[(2R)-4-hexyl-3,4-dihydro-3-oxo-2H-1,4- benzoxazin-2-yl]ethoxy]-benzene acetic acid, and the like. Additionally, the non- thiazolidinediones also act as insulin sensitizing drugs, and include, but are not limited to GW2570, and the like.

[0093] (f) Retinoid-X receptor (RXR) modulators, also insulin sensitizing drugs, whichinclude, but are not limited to targretin, 9-cis-retinoic acid, and the like.

[0094] (g) Other insulin sensitizing agents include, but are not limited to INS-1, PTP-1Binhibitors, GSK3 inhibitors, glycogen phosphorylase a inhibitors, fructose-1,6-bisphosphatase inhibitors, and the like.

[0095] (h) Alpha-glucosidase inhibitors which act to inhibit alpha-glucosidase. Suitableexamples include, but are not limited to, acarbose and miglitol.

[0096] (i) Insulins, including regular or short-acting, intermediate-acting, and long-actinginsulins, inhaled insulin, and insulin analogues such as insulin molecules with minor differences in the natural amino acid sequence. These modified insulins may have faster onset of action and / or shorter duration of action.

[0097] (j) Small molecule mimics of insulin, including, but not limited to L-783281, TE-17411, and the like.

[0098] (k) Na-glucose co-transporter inhibitors such as T-1095, T-1095A, phlorizin, and thelike.

[0099] (l) Amylin agonists which include, but are not limited to pramlintide, and the like.

[0100] (k) Glucagon antagonists such as AY-279955, and the like.

[0101] In an embodiment, the additional therapeutic agents include anti-obesity agents such asorlistat, a pancreatic lipase inhibitor, which prevents the breakdown and absorption of fat; or sibutramine, an appetite suppressant and inhibitor of the reuptake of serotonin, norepinephrine, and dopamine in the brain.

[0102] In another embodiment, the additional therapeutic agents include, but are not limitedto, appetite-suppressants acting through adrenergic mechanisms such as benzphetamine, phenmetrazine, phentermine, diethylpropion, mazindol, sibutramine, phenylpropanolamine or, ephedrine; appetite-suppressant agents acting through serotonergic mechanisms such as quipazine, fluoxetine, sertraline, fenfluramine, or dexfenfluramine; appetite-suppressant agents acting through dopamine mechanisms, e.g., apomorphine; appetite-suppressant agents acting through histaminergic mechanisms (e.g., histamine mimetics, H3 receptor modulators); enhancers of energy expenditure such as beta-3 adrenergic agonists and stimulators of uncoupling protein function; leptin and leptin mimetics; neuropeptide Y antagonists; melanocortin-1, 3 and 4 receptor modulators; cholecystokinin agonists; glucagon-like peptide-1 (GLP-1) mimetics and analogues (e.g., Exendin); androgens (e.g., dehydroepiandrosterone and derivatives such as etiocholandione), testosterone, anabolic steroids (e.g., oxandrolone), and steroidal hormones; galanin receptor antagonists; cytokine agents such as ciliary neurotrophic factor; amylase inhibitors; enterostatin agonists / mimetics; orexin / hypocretin antagonists; urocortin antagonists; bombesin agonists; modulators of protein kinase A; corticotropin- releasing factor mimetics; cocaine- and amphetamine-regulated transcript mimetics; calcitonin- gene related peptide mimetics; and fatty acid synthase inhibitors.

[0103] In an embodiment, the sulfonylurea or pharmaceutically acceptable salt thereof isadministered at a dosage level of from about 0.01 mg / kg to about 200 mg / kg of body weight per day, or any amount or range therein. Preferably, the range is from about 0.01 to about 50 mg / kg of body weight per day. In an embodiment, an effective amount of the sulfonylurea or pharmaceutically acceptable salt thereof is supplied at a dosage level of 1.0 mg, 2.5 mg, 5.0 mg, 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 250 mg, 500 mg, 100 mg, or any amount or range therein.

[0104] In an embodiment, the compounds or pharmaceutically acceptable salts thereof areadministered at a dosage level of from about 0.01 mg / kg to about 200 mg / kg of body weight per day, or from about 0.5 mg / kg to about 50 mg / kg of body weight per day, or any amount or range therein. In another embodiment, the compounds or pharmaceutically acceptable salts thereof are administered at a dosage level from about 1.0 to about 50.0 mg / kg of body weight per day, or any amount or range therein, more preferably, from about 5 mg / kg to about 30 mg / kg, or any amount or range therein, more preferably, from about 5 to about 20 mg / kg of body weight per day, or any amount or range therein. In an embodiment, an effective amount of the compound or pharmaceutically acceptable salts thereof are supplied at a dosage level of 250 mg, 500 mg, 750 mg, 1000 mg or 2000 mg, or any amount or range therein.

[0105] In an embodiment, the compounds or pharmaceutically acceptable salts thereof areadministered at a dosage level of from about 0.01 mg / kg to about 0.5 mg / kg of body weight per day, or from about 0.01 mg / kg to about 0.3 mg / kg of body weight per day, or any amount or range therein. In an embodiment, an effective amount of the compound or pharmaceutically acceptable salts thereof are administered at a dosage level of 1.0 mg, 2.5 mg, 5.0 mg, 7.5 mg, 10.0 mg, 12.5 mg, 15 mg, or 20 mg, or any amount or range therein.

[0106] In an embodiment, the compounds or pharmaceutically acceptable salts thereof areadministered at a dosage level of about 0.01 mg / kg to about 500 mg / kg of body weight per day, or 0.01 mg / kg to about 200 mg / kg of body weight per day, or any amount or range therein. Preferably, the range is from about 0.01 to about 50 mg / kg of body weight per day, or any amount or range therein, more preferably, from about 0.05 mg / kg to about 10 mg / kg, or any amount or range therein, more preferably, from about 1 to about 5 mg / kg of body weight per day, or any amount or range therein.

[0107] In an embodiment, the compounds or pharmaceutically acceptable salts thereof areadministered at a dosage level of 10 mg, 25 mg, 50 mg, 100 mg, 150 mg or 300 mg, or any amount or range therein.

[0108] In an embodiment, the additional therapeutic agent also known as the co-therapy of thepresent invention may be administered on a regimen of 1 to 4 times per day.

[0109] The compositions and / or compounds described herein can be administered in aregimen. The regimen can be structured to provide therapeutically effective amounts of the compounds of the present invention over a predetermined period of time (e.g., an administration time). The regimen can be structured to limit or prevent side-effects or undesired complications of each of the components of the composition described herein. Administration periods can be broken by a rest period that includes no administration of at least one therapy. For example, a regimen can include administration periods that include 2, 3, 5, 7, 10, 15, 21, 28, or more days. These periods can be repeated. For example, a regimen can include a set number of days as previously described where the regimen is repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more times.

[0110] Regimens can include a rest period of at least 1, 2, 3, 5, 7, 10, or more days, where atleast one therapy is no longer administered to a patient. The rest period can be determined by, for example, monitoring the reaction of the patient to the drug or by measuring the efficacy of the treatment.

[0111] Optimal dosages to be administered cany be determined by those skilled in the art, andwill vary with for example, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages.

[0112] In some embodiments, the compound described, or composition thereof, isadministered in combination with another therapeutic agent. In some embodiments, the compound described, or composition thereof, is administered in combination with one or more other therapeutic agents described herein.

[0113] In some embodiments, compounds described, or composition thereof as describedherein can be administered together with a biguanide selected from metformin, phenformin, or buformin, to a patient in need thereof. In some embodiments, the patient is administered acombination of a compound of the invention and a biguanide. In some embodiments, the patient is suffering from cancer, obesity, liver disease, diabetes or two or more of the above.

[0114] In the compositions which comprise an additional therapeutic agent, that additionaltherapeutic agent and the compound as described in may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent.

[0115] It is understood that modifications which do not substantially affect the activity of thevarious embodiments of this invention are also included within the definition of the invention provided herein. Accordingly, the following examples are intended to illustrate but not limit the present invention. EXAMPLES EXAMPLE 1

[0116] Methods

[0117] Human serum

[0118] Native human serum was obtained from an institutional review board (IRB)-approvedvascular biobank. Fresh human serum aliquots were collected from at least 12 hour fasting study participants and concentrated using a 100 kDa ultrafiltration centrifuge tube at 15,000 g, for 15 minutes. A resultant minimum volume of 250 μL of concentrated serum was collected for each patient. Concentrated serum samples were stored in aliquots at -80 ºC for subsequent use for in vitro macrophage-foam cell experiments.

[0119] Serum used for conditioned media studies contained either low (<90 mg / dL), medium(90-180 mg / dL), or high (>180 mg / dL) LDL content, and undetectable levels of cFAS (Table 1A). Alternatively, media was also conditioned with serum containing either low (<0 μg / μl), medium (5-6 μg / μl), or high (>17 μg / μl) levels of cFAS, and low levels of LDL (<90 mg / dL; Table 1A). Serum LDL content was determined by the Washington University in St. Louis Core Laboratory for Clinical Studies (CLCS), utilizing a N-Geneous® LDL cholesterol kit using a Roche Cobas c501 analyzer. Serum cFAS content was determined using a commercial ELISA according to manufacturer’s instructions.

[0120] Table 1A. Human Serum Lipid Profile. Human serum demographics and lipidconcentration from Zayed’s Lab BioBank utilized for foam cell lipid formation experiments.

[0121] Tissue culture and foam cell formation assessment

[0122] Human U-937 (ATCC # CRL-1593.2) were cultured for 48 hours in 10% FBS RPMI,and then differentiated to macrophages in 5% FBS RPMI treated with 1 µl / ml Phorbol 12- myristate 13-acetate, 95%. Monocytes were allowed 24 hours to differentiate into macrophages. Differentiated macrophages were then washed 3x with PBS and incubated for 48 hours in cell culture media RPMI conditioned with 10% of human serum containing either low, medium, and high levels of cFAS or LDL on fibronectin treated coverslips (Figure 1A & Table 1A). Similarly, macrophages conditioned with serum containing either higher levels cFAS or LDL, were also simultaneously treated with Platensimycin (PTM, 20 mM; Cayman Chemical, #15507) for 48 hours. Macrophages were then stained in oil-Red O working solution (3:2 dilution with distilled water of a stock solution of 0.5 mg / µl in 100% isopropanol) and hematoxylin and eosin (H&E). Stained coverslips were imaged using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope. Area positively stained with oil-Red O was quantified using ImageJ color threshold toolkit. The intracellular lipid droplet area stained with oil-Red O was expressed as a percentage of the total cell area.

[0123] Following conditioned media treatments, macrophages were also lysed with standardfreeze thaw method in PBS and lysates were standardized to protein concentration using Bradford Protein Assay. Acetyl-CoA concentration was determined by using a commercial ELISA according to the manufacturer’s instructions. Protein was loaded onto Bis-Tris gel and transferred to polyvinylidene fluoride membranes for content analysis. Anti-FAS 1:500 and anti-β actin 1:10,000 were used for Western blotting. Band densitometry analysis wasperformed using ImageJ software, and FAS band densities were expressed as ratios relative to the β actin loading control.

[0124] Animal regulations

[0125] All animal protocols were approved by the local institutional animal care and usecommittee (IACUC). Mouse housing, breeding, and experimental procedures were conducted in accordance with national and institutional guidelines.

[0126] Mouse models

[0127] Conditional liver-specific knockdown of Fasn was achieved using previously reportedFasnfl / flmice that also express an albumin-Cre promoter (Cre+). Liver Fasnfl / flCre+mice were crossed with Apoe- / -knockout mice (Jackson lab, strain #002052) to yield Liver Fasnfl / flCre+Apoe- / -mice. At 7 weeks of age, Fasnfl / flCre+Apoe- / -mice and Fasn+ / +Cre-Apoe- / -littermates were maintained on a continuous 42% high-fat diet for 16 weeks. On a weekly basis, body weights were recorded, and blood serum samples were collected from the tail vein. Similarly, Fasn+ / +Cre-Apoe- / -littermates were maintained on a 42% high-fat diet with and without PTM (100 mg / kg / day infused into the diet) for a 16-week treatment period. After 16 weeks, mice were sacrificed and serum, hearts, aorta, liver, and white adipose tissue were collected for immediate analysis, embedded in OCT, or stored at -80 °C for later use.

[0128] FAS enzyme activity and content assay

[0129] FAS enzyme activity was measured as previously described with some modifications.Liver and white adipose tissue were digested in freshly prepared lysis buffer (250 mM sucrose, 20 mM HEPES buffer (pH 7.2), 2 mM MgCl2, 1 mM DTT, 1 mM EDTA, and 1× protease inhibitor cocktail (PIC)) and homogenates were centrifuged at 5,000 g for 5 min at 4 °C. The supernatant and serum were standardized to 100 µg of total protein and added to 200 µL of assay buffer (200 mM potassium phosphate buffer (PPB) at pH 6.6, 1 mM DTT, 1 mM EDTA, 0.24 mM NADPH (Millipore Sigma, N1630), 0.031 mM Acetyl-CoA.

[0130] The rate of NADPH oxidation was monitored by measuring absorbance at 340 nm at37 °C for 15 minutes in the absence, and then in the presence of 10 µL of the rate-limiting substrate malonyl-CoA (5.8 mM stock) for another 15 minutes. Data was analyzed by calculating the rate of optical density (OD) decrease with malonyl CoA, after correcting for the nonspecific background rate without malonyl CoA substrate. FAS enzyme was defined as µmoles NADPH consumed·min-1·mg-1. An extinction coefficient of 6220 M-1cm-1was used in the specific activity calculation utilizing Beer’s law, as previously described. Tissue and serumFAS protein content were evaluated using a commercial ELISA kit according to manufacturer’s instructions.

[0131] Aortic atherosclerotic burden assessment

[0132] Murine hearts were harvested en bloc at time of sacrifice after 16-weeks of diettreatment. The tissue was fixed in OCT compound (Fisher Scientific), and the aortic valve region was sectioned at 10 µm thickness. Sections were then fixed in 4% paraformaldehyde (PFA), followed by 60% isopropanol for 5 minutes. Aortic valve sections were then stained in an oil-Red O working solution (3:2 dilution with distilled water from stock solution of 0.5 mg / µl in 100% isopropanol). Valve plaque area was quantified in a blinded fashion using ImageJ as percentage of plaque area in the aortic lumen, as previously described. Corresponding sections of aortic value were also stained with 1:50 mouse anti-CD68 antibody. The primary antibody was detected with a 1:100 secondary antibody, donkey anti-rat IgG labeled with Alexa Fluor 555, followed by DAPI nucleus stain. Stained sections were then imaged on Leica Thunder DM6 B Microsystems inverted fluorescent microscope. The percentage of positively stained CD68 area relative to total aortic lumen was quantified using ImageJ software.

[0133] Similarly, the entire aorta from the aortic arch to the infrarenal aortic bifurcation weremicrodissected and resected en bloc at the time of sacrifice. Harvested aortic specimens were fixed in 4% PFA for 24 hours. The tissue was then effaced and stained using oil-Red O, and then imaged with Lecia S9i Microsystem microscope. The area of plaque that positively stained with oil-Red O was taken relative to the aortic valve segment area using ImageJ in a blinded fashion.

[0134] Immunofluorescence FASN staining

[0135] Differentiated macrophages were seeded onto fibronectin-coated coverslips in 24-wellplates at a density of 240,000 cells per well. The cells were treated with either PTM (20 µM) or DMSO under various human serum conditions: high cFAS / low LDL, low cFAS / low LDL, low cFAS / medium LDL, and low cFAS / low LDL for 48 hours. After treatment, the cells were rinsed three times with PBS and fixed with 4 % paraformaldehyde for 10 minutes at room temperature. Following fixation, the cells were washed with PBS and permeabilized with a 0.5% Triton X-100 solution for 15 minutes at room temperature. The cells were then blocked with 5% goat serum albumin for 1 hour at room temperature, followed by incubation with FAS- Alexa®488 (sc-48357; 1:100 dilution in PBS with 1% goat serum) for 18 hours at 4 °C. Thenext day, the cells were washed three times with PBS. Coverslips were mounted on slides using DAPI-containing mounting media. Imaging was performed using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope, and analysis was conducted with ImageJ software.

[0136] Tissue histology

[0137] Murine liver and white adipose tissue were immediately harvested at the time ofsacrifice and were embedded in OCT. Tissue was sectioned at 10 µm thickness and fixed in 4 % PFA and stained by H&E. Sections were also immuno-stained with 2% donkey blocking agent for 1 hour at room temperature, and then with 1:100 primary mouse monoclonal FAS antibody, or 1:50 mouse anti-CD68 antibody. The primary antibody was detected with a 1:100 secondary antibody donkey anti-rat IgG labeled with Alexa Fluor 555, followed by DAPI nucleus stain. Imaging assessments were performed using a Leica Thunder DM6 B Microsystems inverted fluorescent microscope, and staining was quantified using ImageJ software integrated density toolkit.

[0138] Statical analysis

[0139] Statistical correlations between continuous variables such as serum cFAS, tissue FAScontent or activity were evaluated with linear regression. Non-parametric two-tailed Man- Whitney tests were used to evaluate the differences between inter- and intra-group analysis. Endpoints obtained over a time course were evaluated using two-way ANOVA with multiple comparisons. All analyses were performed using GraphPad Prism (Prism 9.1 software, GraphPad Software Inc.). We considered p<0.05 to be statistically significant. All graphical data are presented as mean ± SEM.

[0140] Results

[0141] Serum cFAS induces macrophage foam cell formation

[0142] Macrophage cytoplasmic lipid droplet accumulation and foam cell formation is ahallmark of atheroprogression. We evaluated whether macrophages conditioned with native human serum either containing high cFAS or LDL can impact foam cell formation in vitro (Fig. 1A). Interestingly, we observed a significant correlation between serum cFAS content in the conditioned media and the percentage of macrophages that formed foam cells (Fig.1B; R2=0.44, p=0.049). On the other hand, no correlation was observed between serum LDL content and the percentage of macrophage foam cell formation (Fig.1B; R2=0.05, p=0.59).

[0143] Macrophages exposed to serum with varying cFAS and LDL levels showed differentialfoam cell formation (Fig.1C). Notably, high cFAS / low LDL serum (25.84 ng / μL) induced significantly more foam cells than low cFAS / high LDL conditions (p=0.008), evidenced by increased intracellular lipid droplets. Treatment with the FAS inhibitor PTM (20 µM) significantly decreased macrophage-derived foam cell formation when cells were conditioned with serum containing high cFAS (>17 ng / µL) and low LDL (<90 mg / dL; Fig.1D; p<0.01). Furthermore, intracellular FAS activity in macrophages that were conditioned with serum containing high cFAS was higher than macrophages conditioned with serum containing high LDL (Fig.1E; p<0.05). Treatment with PTM resulted in a marked reduction of intracellular FAS activity in treated macrophages (Fig.1E; p≤0.01), suggesting that cFAS is a potent factor in foam cell induction. Additionally, FAS inhibitors TVB-2640 (100 nM) and GSK2194069 (100 nM) significantly reduced foam cell formation in differentiated macrophages conditioned with high cFAS / low LDL serum (16.24 ng / μL), compared to untreated cells (p=0.003 and p<0.0001, respectively) (Fig.1F). The use of 5% FBS as a negative control confirmed that foam cell formation is specifically induced by FAS activity.

[0144] Intracellular FAS accumulation in macrophages under high cFAS / low LDL

[0145] To distinguish between the effects of exogenous and endogenous FAS, we performedFAS staining on macrophages under various conditions. Immunofluorescence analysis of FAS (FAS-Alexa®488) revealed distinct distribution patterns in macrophages exposed to different cFAS / LDL conditions, with and without PTM treatment (Fig.2A). Quantification of FAS fluorescence intensity showed significant variations across treatment groups (Fig.2B). Macrophages exposed to high cFAS / low LDL serum exhibited markedly increased FAS fluorescence intensity compared to low cFAS serum conditions (p<0.0001, n=10) in both PTM- treated and untreated groups. Notably, PTM treatment did not attenuate this effect (p>0.05, n=10). ELISA analysis of FAS content in macrophages corroborated these findings (Fig.2C). Differentiated macrophages conditioned with high cFAS / low LDL showed no significant difference in FAS content between PTM-treated and untreated cells. Cell viability assays confirmed that PTM did not significantly affect cell survival at the concentrations used 48 hours post treatment (p>0.05, n=3) (Fig.2D).

[0146] Knockdown and inhibition of FAS alters serum and tissue lipidomics

[0147] Fasn+ / +Cre-Apoe- / - and Fasnfl / flCreApoe- / - were maintained on a high-fat diet for 16weeks. A group of Fasn+ / +Cre+Apoe- / -mice also received PTM throughout this period, andserum, liver, and adipose tissue were collected (Fig.3A). Following 16 weeks of a high-fat diet, Fasnfl / flCre+Apoe- / -mice had significantly less weight gain compared to Fasn+ / +Cre-Apoe- / -mice (51% vs 54% increase in weight; p<0.05; Fig.3B). On the other hand, Fasn+ / +Cre-Apoe- / -mice treated with PTM had no significant change in body weight compared to untreated Fasn+ / +Cre-Apoe- / -mice (Fig.3B).

[0148] Compared to Fasn+ / +Cre-Apoe- / - mice, Fasnfl / flCre+Apoe- / - mice treated with andwithout PTM, demonstrated no significant differences in liver and adipose TGs at 16 weeks (Fig.3D & 3E). On the other hand, free fatty acids (FFAs) were notably decreased in the liver(Fig. 3F; ∆37%), and significantly elevated in adipose tissue of Fasn+ / +Cre-Apoe- / - mice thatreceived PTM treatment (Fig.3G; p<0.01).

[0149] After initiation of high-fat diet, Fasnfl / flCre+Apoe- / - mice demonstrated a differentpattern of FAS content and activity in the hepatic and adipose tissue. As expected, Fasnfl / flCre+Apoe- / -mice demonstrated a significant decrease in FAS content and activity in hepatic tissue (Fig.3G & 3H; p<0.05). Interestingly, Fasnfl / flCre+Apoe- / -mice demonstrated a significant increase in FAS content in white adipose (Fig.3I; p<0.005). PTM treatment did not impact FAS content in the liver but led to a significant decrease in FAS content and activity in white adipose (Fig.3I & 3J; p<0.001). Similarly, there was moderate, but not statistically significant, reduction in FAS activity in hepatic tissue of PTM-treated Fasn+ / +Cre-Apoe- / -mice (Fig.3G; p=0.14).

[0150] Conditional liver FAS knockdown and inhibition impacts serum cFAS and tissueFAS content and activity

[0151] All mice groups demonstrated universal hypercholesteremia with total cholesterol>1,400 mg / dL at 16 weeks (Fig.4D). We observed a significant decrease in serum cFAS content in Fasnfl / flCre+Apoe- / -mice prior to initiation of high-fat diet regimen (Fig.4A; p<0.05). Similarly, PTM treatment significantly reduced cFAS content in Fasnfl / flCre- Apoe- / -mice (Fig.4A; p<0.05). Interestingly, serum cFAS activity was reduced in Fasnfl / flCre+Apoe- / -mice, and PTM-treated Fasn+ / +Cre-Apoe- / -mice (Fig.4B). This reduction was more significant after 9 and 16 weeks with a high-fat diet (Fig.4C – 3D; p<0.05).

[0152] FAS conditional knockdown or pharmacological inhibition reducesatheroprogression.

[0153] We next evaluated arterial atheroprogression in Fasnfl / flCre+Apoe- / - mice and PTM-treated Fasn+ / +Cre-Apoe- / -mice that were maintained on a high fat diet for 16 weeks and aortasharvested for analysis (Fig.5A). Compared to Fasn+ / +Cre-Apoe- / -, Fasnfl / flCre+Apoe- / -mice demonstrated a significant reduction in total aortic atherosclerotic plaque formation (Fig.5A & B; p<0.01). This difference was evident in all aortic segments including the aortic arch, thoracic aorta, and infrarenal aorta (Fig.5CD - E; p<0.05). Similarly, PTM-treated Fasn+ / +Cre- Apoe- / -mice also demonstrated significantly reduced total aortic (Fig.5A & B; p<0.05), as well as reduced plaque in the aortic arch, thoracicand infrarenal aortic segments (Fig.5C- E; p<0.05).

[0154] Atherosclerotic plaque formation at the aortic valve roots were also evaluated after 16weeks of a high-fat diet regimen and with and without PTM treatment. Fasnfl / flCre+Apoe- / -mice and PTM-treated Fasn+ / +Cre-Apoe- / -mice demonstrated significantly reduced aortic valve root plaque formation (Fig.6A & B; p<0.01). Similarly, Fasnfl / flCre+Apoe- / -mice and PTM- treated Fasn+ / +Cre-Apoe- / -mice demonstrated reduced CD68+ macrophages in the aortic valve wall, while Fasn+ / +Cre-Apoe- / -mice had higher CD68 content in the valve wall atheroma (Fig. 6B; p<0.05).

[0155] Conditional knockdown or inhibition of FAS alters tissue inflammation

[0156] As expected, hepatic tissue of Fasnfl / flCre+Apoe- / - mice had diminished FASimmunostaining (Fig.7A & B; p<0.05), and reduced CD68+ macrophage content (Fig.7A & C; p<0.05). PTM-treated Fasn+ / +Cre-Apoe- / -mice also demonstrated a tendency towards reduction in liver FAS (Fig.7A & B; p=0.08), and significantly reduced CD68 content (Fig. 7A & C; p<0.001).

[0157] Interestingly, in white adipose tissue, liver Fasnfl / flCre+Apoe- / - mice demonstratedincreased adipocyte area (Fig.7A & D; p<0.05) and increased FAS content (Fig.7A & E; p<0.0001), but no change in CD68 content (Fig.7A & F). In PTM-treated Fasn+ / +Cre-Apoe- / -mice, there was a significant decrease in adipocyte area (Fig.7A & D; p<0.001), a strongly significant decrease in FAS content (Fig.7A & E; p<0.05), and a significant decrease in CD68 content (Fig.7A & G; p<0.05).

[0158] Discussion

[0159] Our study evaluates the role of tissue FAS and serum cFAS on atheroprogression andtissue inflammation. We observed a significant increase in macrophage foam cell formation when conditioned with serum containing higher cFAS content. On the other hand, treatment with PTM significantly blunted foam cell formation. Similarly, in vivo, conditional knockdown of FAS in the liver, or treatment with PTM, greatly reduced aortic atherosclerotic plaquevolume and macrophage content in aortic plaque regions. It was also observed that FAS targeting impacted liver-adipose tissue crosstalk. Remarkably, although Fasnfl / flCre+Apoe- / -mice exhibited hypercholesteremia while maintained on a 42% high-fat diet, they developed minimal aortic atherosclerotic plaque. Overall, these findings highlight the indispensable roles that tissue FAS and serum cFAS contribute to atheroprogression.

[0160] Dyslipidemia is a known risk factor for atheroprogression and cardiovascular disease.Individuals with familial hyperlipidemia are born with dramatically elevated serum LDL cholesterol, develop early atherosclerotic disease onset, and are at higher risk of cardiovascular complications if not intensively treated. Lipid-lowering medications, such as statins (co- enzyme A reductase inhibitors), fibrates, and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors, are first-line in the management of hyperlipidemia, and aim to reduce serum circulating LDL or TG content thereby reducing the risk of cardiovascular events such as myocardial infarction (MI), stroke, and major lower extremity amputations resulting from peripheral arterial occlusive disease. However, despite reduction of LDL with statins (20-50%) and PCSK9 inhibitors (50-65%), cardiovascular events are still only reduced by 30-60% in patients who are treated with medications within these drug categories. This leaves most individuals with significant residual risk of major cardiovascular events and an unclear management strategy to reduce cardiovascular morbidity and mortality. These persistent clinical gaps have contributed to a growing suspicion that in addition to traditional lipid mediators like LDL and TGs, there are likely additional key contributors to atheroprogression that are yet to be identified and therapeutically targeted.

[0161] While individuals with high serum LDL levels (>190 mg / dL) are known to have ahigher incidence of MI and stroke, this is indeed not always the case. For example, in The Multi-Ethnic Study of Atherosclerosis (MESA), which evaluated >23,000 over a 16-year period, high serum LDL was observed to not be a risk factor for the incidence of atherosclerotic cardiovascular disease in individuals who had a zero coronary artery calcium (CAC) score on CT angiography. Similarly, in a study of >136,000 patients who were hospitalized for an acute MI, it was observed that nearly 75% of patients had serum LDL levels that would indicate they were not at high risk of cardiovascular events. These studies highlight that beyond LDL cholesterol there are additional serum and / or tissue lipid mediators that can influence whether a patient is either at higher or lower risk for atherosclerotic disease progression.

[0162] Fatty acids are essential lipids that serve as functional components for TGs,phospholipids, and cholesterol esters. These lipid mediators impact a diverse array of cellular and tissue processes, including cell membrane structure and integrity, as well as serving as biological energy storage units during catabolism. On the other hand, dysregulation of fatty acid synthesis contributes to deleterious conditions such as obesity, non-alcoholic fatty liver disease, and type 2 diabetes. In macrophages, fatty acids play key roles in cholesterol uptake, esterification, and lipid efflux. However, dysregulation of fatty acid synthesis is known to impact macrophage function, polarization, and phenotypic transformation, Abrogation of fatty acid synthesis inhibits macrophage cholesterol efflux and foam cell formation. This is of particular importance since foam cell accumulation in the arterial intima has been linked to arterial wall atheroma progression and plaque vulnerability. It was demonstrated that on one end serum cFAS plays an important role in macrophage foam cell formation, and on the other end both serum cFAS and endogenous liver FAS play an important role in aortic atherosclerosis.

[0163] Additionally, it was observed that cFAS co-immunoprecipitated with ApoB in LDLcholesterol serum fractions. These findings previously led us to conclude that cFAS is produced by the liver and is released into the blood stream bound to ApoB in lipoproteins such as LDL. Given the relative concentrations of LDL and cFAS in human serum it is evident that cFAS concentrations are at least an order magnitude less than LDL. Meaning, while cFAS may serve as cargo attached to LDL particles in the serum, not all LDL particles will be saturated with cFAS and vice versa. This is presumably why we observed that human serum had variable content of cFAS and LDL. In the biobanked samples, there were samples that had higher cFAS content (>17 ng / µL), and others that were essentially undetectable cFAS. Naturally, we also observed serum samples that had very high LDL (>180 mg / dL), while others that had low LDL (<90 mg / dL). Since it was previously reported that there was no correlation between cFAS and LDL content in human serum, the impact of human serum samples with either high and low cFAS or LDLw was intentionally evaluated. It was observed that macrophages conditioned with serum containing high LDL, but low cFAS, did not lead to foam cell formation.

[0164] The mechanistic process that facilitates cFAS impact on foam cell formation iscurrently unknown. However, prior work demonstrates that endogenous FAS in macrophages is essential for the retention of plasma membrane cholesterol, cellular adhesion, and migration, as well as recruitment into adipose tissue that facilitates chronic tissue inflammation induced bynutrient dense diets. Macrophage-specific FAS deficiency exhibited a reduced inflammatory response, characterized by decreased expression of pro-inflammatory cytoskines (TNF-α, IL- 1β). Notably, these FAS-deficient macrophages exhibited upregulation of Abca1, that promotes the removal of excess cholesterol, potentially contributing to reduce from cell formation and atheroprogression. In this example, it was observed that pharmacological inhibition of FAS with PTM in mice maintained on 42% high-fat diet dramatically reduced macrophage infiltration in both hepatic and white adipose tissue (Fig.7C & 7F). While conditional knockdown of Fasn in liver tissue also reduced hepatic macrophage infiltration, it did not have as robust of a phenotype in white adipose tissue. Moreover, compared to conditional knockdown of Fasn, treatment with PTM had a more dramatic reduction of macrophages in the liver (127% difference) and white adipose (96% difference) tissue, suggesting that its inhibition of serum cFAS was likely playing a major role in these findings. EXAMPLE 2

[0165] The goal of this Example is to evaluate the impact of De novo lipogenesis (DNL)signaling and diabetes on peripheral atheroprogression. The primary objective of this experiment is to determine whether CEPT1, tissue FAS, and serum cFAS play crucial roles in peripheral arterial atheroprogression in the setting of diabetes. We have observed that CEPT1 and cFAS can fuel both cell-intrinsic and cell-extrinsic DNL signaling (Figure 9). Dysregulated DNL signaling in diabetes can lead to abnormal lipid accumulation and inflammation, which are key contributors to atheroprogression.

[0166] It is hypothesized that DNL signaling in diabetes accelerates peripheralatheroprogression through molecular signaling interactions between the liver, adipose tissue, and the biochemical actions of CEPT1 and cFAS. To test this hypothesis, we will utilize our established novel animal models that accurately mimic peripheral atheroprogression, in conjunction with existing murine cell-specific genetic knockdown lines with induced hyperlipidemia and diabetes (Figure 10). These models will allow us to dissect the specific contributions of CEPT1 and cFAS on atherosclerosis development in the context of diabetes. Comprehensive cellular profiling, histopathological examinations, molecular, and biochemical / metabolomic analyses will be conducted to elucidate the mechanistic links between CEPT1, tissue FAS, serum cFAS, and peripheral atheroprogression in the setting of diabetes. These studies aim to provide critical insights into the potential therapeutic targeting of DNL pathways to mitigate PAD complications in diabetes.

[0167] Model for peripheral arterial atheroprogression: It was observed that Apoe- / - LepRdb / dbmice maintained on a 42% fat Western diet develop the majority of the features attributed to metabolic syndrome and diabetes, elevated plasma cholesterol, increase in serum LDL, markedly elevated aortic atheroprogression, and increased atherosclerosis in peripheral ilio- femoral arteries (Figure 11). This makes Apoe- / -LepRdb / dbmice a highly valuable model to evaluate potential molecular mechanisms that can influence DNL-mediated atheroprogression in the peripheral arterial system.

[0168] CEPT1 in PAD: Infrageniculate tibial arterial segments were collected and adjacentskeletal muscle tissue from amputated limbs of individuals with and without diabetes. CEPT1 was found to be notably present in the human arterial intima endothelial layer and microvasculature in skeletal muscle. In patients with diabetes, Cept1 gene expression and CEPT1 protein content were significantly elevated in maximally diseased tibial arterial intima, alongside higher levels of CEPT1-derived alkyl-phosphatidylcholines and plasmenyl- phosphatidylethanolamines (Figure 12). These findings provide the basis for our proposed exploration of DNL signaling in the peripheral arteries, and whether it directly or indirectly impacts atheroprogression in diabetes.

[0169] Cept1 plays a vital role in atheroprogression: Although phospholipids play a vital rolein PPARα activation, lipoprotein particle formation, and EC signaling, never before has the role of DNL in atheroprogression been evaluated. Interestingly, we observed that conditional knockdown of Cept1 in the endothelium in Apoe- / -mice (EC Cept1fl / flApoe- / -) maintained on a 42% fat Western diet significantly reduced serum total cholesterol and triglycerides (data not shown), aortic atherosclerosis, and CD68+ tissue macrophage content (Figure 13). This important finding confirms that DNL cell-intrinsic mechanisms play a vital role in atheroprogression and macrophage recruitment.

[0170] Endothelial Cept1 overexpression promotes atheroprogression: The impact ofconditional transgenic overexpression of Cept1 in the endothelium (EC Cept1Ox / OxCre+) was also explored. Relative to control, Cept1 overexpression in the endothelium lead to a significant increase in endothelial cell expression of macrophage recruitment markers Akt3, Pecam1, and Cdh5 (Figure 14). EC Cept1Ox / Ox Cre+ EC Cept1Ox / OxCre+Apoe- / -maintained on a 42% fat Western diet for 16 weeks developed significantly more atherosclerosis compared to controls (Figure 15). The burden of disease was reversible with fenofibrate administration.As expected, we observed that fenofibrate (a PPARα agonist) did not have an impact on atherosclerotic plaque formation in Ppara- / -mice.

[0171] Plaque FAS and serum cFAS are elevated in patients with diabetes and CLTI: It wasobserved that FAS protein content is elevated in peripheral arterial plaque of patients with diabetes and CLTI (Figure 16). We also observed increased FAS-derived saturated fatty acids (SFAs) in tibial arterial plaques of patients with diabetes and CLTI. cFAS is elevated in the serum of patients with CLTI and severe diabetes. There is a significant positive correlation between serum cFAS and plaque FAS (Figure 15). These clinical observations provide a foundation for the DNL cell-extrinsic mechanisms.

[0172] cFAS is LDL-associated and originates from the liver: It was discovered that cFASalmost exclusively fractionates in fast-protein liquid chromatography (FPLC)-derived LDL cholesterol isolates (Figure 17). Co-immunoprecipitation pull-down assay demonstrated that cFAS was associated with ApoB (dominant protein in LDL particles). Gold-labeled cFAS is observed on LDL particles using electron microscopy (EM). Like individuals with diabetes, mice with a diabetes phenotype (LepRdb / db) had the highest level of serum cFAS (Figure 17). Interestingly, selective knockdown of Fasn in liver or skeletal muscle (SM) tissue demonstrated that the liver is the dominant source of serum cFAS (Figure 17).

[0173] Liver Fasn impacts atherosclerosis: Conditional liver-specific knockdown of Fasn inApoe- / -mice (Liver Fasnfl / flApoe- / -; Figure 18) have a dramatic reduction in atherosclerosis and CD68+ macrophage content in atherosclerotic tissue. Interestingly, Fasn+ / +Apoe- / -mice are hyperlipidemic (elevated total cholesterol and serum triglycerides) despite having reduced atherosclerosis (data not shown). Treatment of Fasn+ / + Apoe- / - Fasn+ / +Apoe- / -mice with FAS- inhibitor PTM reduces serum cholesterol levels and also significantly reduces atherosclerosis and tissue macrophage content (Figure 18). scRNA sequencing of whole aortas demonstrated reduced Vecam1 expression in vascular smooth muscle cells (VSMCs) in Liver Fasnfl / flApoe- / -mice (Figure 19). These findings demonstrate that targeted molecular and pharmacological inhibition of FAS can lead to significantly reduced atherosclerosis and tissue macrophage recruitment.

[0174] Table 2: DNL Molecular Markers

[0175] As outlined in Fig. 10, mice with conditional endothelial-specific overexpression orknockdown of Cept1, or liver-specific knockdown of Fasn, on a Apoe- / -LepRdb / dbbackground will be tested. Both male and female mice, and Cre- controls (control for conditional knockdown or overexpression) will be included. Additionally, controls that are not on an Apoe- / -background (these controls were already partially tested; see preliminary data in Figures 11, 12, 13, 18, & 19) will also be included. We have all the proposed murine lines generated (including Apoe- / -LepRdb / dbmice). Moreover, throughout the experimental period, we will actively monitor murine body weights weekly, average food consumption, serum glucose and lipid profiles, and behavior using techniques and methodologies in which we have established expertise.

[0176] Molecular analysis: To investigate the impact of cell-intrinsic DNL signalingmechanisms, we will focus our molecular analysis on well-established DNL signaling markers (Table 2). This includes sterol regulatory element-binding protein 1c (SREBP-1c; encoded bySrebf1 and Srebf2), carbohydrate-responsive element-binding protein (ChREBP; encoded by Mlxipl), acetyl-CoA carboxylase (ACC; encoded by Acaca and Acacb), AMP-activated protein kinase (AMPK; encoded by Prkaa1, Prkaa2, Prkab1, Prkab2, Prkag1, Prkag2, and Prkag3), PPARα (encoded by Ppara), and insulin-induced gene 1 (INSIG1; encoded by Insig1). These molecular signaling mechanisms will be robustly evaluated in at least 5 pooled central aortic and peripheral arterial specimens in each animal group (male and female separately) using RT- PCR, multiplex bead array assays, immunofluorescence (IF), ELISA, and Western blotting. This will facilitate both robust qualitative / quantitative comparisons between DNL activating and stabilizing molecular signals in animal groups and both sexes.

[0177] Spatial transcriptomics in arterial specimens of diabetic and nondiabetic mice (at least5 per group) will be performed to visualize the spatial distribution of DNL signaling markers (Table 2) across entire sections of diseased and non-diseased central and peripheral arterial tissue segments. The spatial transcriptomics findings will be integrated with immunostaining of PPARα, CEPT1, and pro-inflammatory versus reparative macrophages (Table 3) in directly adjacent arterial sections.

[0178] Furthermore, using scRNA sequencing we will resolve expression profiles of distinctcell populations (stromal-, myeloid-, and lymphoid-derived; as a follow-up for preliminary data shown in Figure 12), and determine to what extent Cept1, Ppara, and Acox1 / Cpt1a (PPARα downstream genes), correlate with DNL signaling (Table 2). These correlations will beexamined between diabetic and non-diabetic animals, mouse strains, and between male and female sexes. Our analysis will allow for a novel in-depth assessment of the impact of Cept1 on DNL signaling, macrophage recruitment, and peripheral atherosclerosis in diabetes.

[0179] Biochemical analysis: To further elucidate the role of DNL signaling in peripheralarterial atheroprogression, we will complement our studies with detailed lipidomic and metabolomic analyses. CEPT1, FAS, and PPARα activity assays will be performed as previously described. Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) will also be used to profile lipid composition of arterial, hepatic, and adipose tissue. Samples will be homogenized, and lipids extracted using a chloroform-methanol method as we previously described. The lipid extracts will then be separated by liquid chromatography and analyzed by MS to quantify triglycerides, phospholipids, and cholesterol esters. Lipid species will be identified and quantified using lipid-specific databases and using LipidMaps software. These analyses will help identify lipid species that are impacted by Cept1 and Fasn-mediated DNL signaling in the setting of diabetes.

[0180] For metabolomic analysis, tissue samples will be processed to extract metabolitesusing a methanol-water extraction method. The extracts will be analyzed using LC-MS and GC-MS to profile key metabolites involved in glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid oxidation pathways. Targeted metabolomics will be performed using standards for known metabolites (phosphatidylcholine; PC, phosphatidylethanolamine; PE, lysophosphatidylcholine; LPC, sphingomyleins, ceramides (such as C16),28 choline, diacylglycerol (DAG), and cardiolipin. Untargeted metabolomics will allow the discovery of novel metabolic changes in phospholipid and fatty acid-derived metabolites. Data will be processed and analyzed using MetaboAnalyst. These analyses will enable us to identify metabolic pathways that are significantly altered in diabetic versus non-diabetic conditions, different mouse strains, and diseased versus non-diseased peripheral arterial segments.

[0181] Immune cell profiling in murine tissue of diabetic animals: We will pool arterial,hepatic, and adipose tissue from at least 5 animals in each animal group to facilitate tissue flow cytometry. Freshly collected tissue will be digested into a single cell suspension. Cells will be immunostained for CD45, CD64, Ly6C, GFP, and MHC-II. Isotype control antibodies will be used to validate the gating strategy. Cell populations will be defined using the following markers: CD45+CD11b+CD64+Ly6C+ for monocytes, CD45+CD11b+CD64+Ly6C- for macrophages, and CD45+CD11c+CD64+Ly6C+MHC2+ for dendritic cell. Similar analysis willalso be conducted on circulating blood monocytes, as well as selective markers for pro- inflammatory (CCR2+, CD38+), reparative (Arg1+, CD301b+) or transition (CXCR4+) macrophages and monocytes (Table 3). FloJo will be used to identify the specific cell types that are most associated with arterial atherosclerosis and are differentially present between different mouse lines and arterial segments (aortic arch, thoracic aorta, abdominal aorta, peripheral arterial iliofemoral segment (Fig.11).

[0182] Histopathological characterization: Atherosclerotic and non-atherosclerotic arterialsegments from murine aortic arch, thoracic aorta, abdominal aorta, and peripheral iliofemoral segment will be collected and analyzed with H&E, Verhoeff-Van Gieson (VVG), and ORO staining (Figs.11 & 13). ORO staining intensity and plaque volumes will be analyzed as previously described. Arterial wall disease severity will be characterized as mild, moderate, or severe based on American Heart Association (AHA) criteria, which includes the degree of VSMC loss, elastic layer disruption, cholesterol deposits as previously described. Differences between disease and non-diseased regions of central aortic and peripheral arterial tissue will be scored in a blinded fashion. Immune cell (pro-inflammatory, reparative, vs transition; Table 2) infiltration within the arterial wall will also be characterized using appropriate markers, and blinded observers will evaluate the staining intensity in diseased and non-diseased arterial segments as previously described. Across mouse groups, we will also analyze liver and white adipose tissue for immune cell infiltration (Table 3 & Figure.20).

[0183] We anticipate that altered endothelial Cept1 expression will significantly impactperipheral atheroprogression in the setting of diabetes. Specifically, we expect that endothelial- specific knockdown of Cept1 will lead to reduced inflammatory response and lower atherosclerotic plaque burden in the peripheral arteries of Apoe- / -LepRdb / dbmice. This reduction in atheroprogression will likely be associated with diminished expression of pro- inflammatory macrophage recruitment markers such as CD38+ and CCR2+, as well as decreased Arg1+ and CD301b macrophage content in the arterial walls. Conversely, endothelial overexpression of Cept1 is anticipated to exacerbate atherosclerosis, with increased endothelial cell activation and heightened inflammatory responses, resulting in more pronounced atheroprogression in the setting of diabetes. The interplay between CEPT1 and PPARα is expected to play a crucial role in these processes, with altered expression and / or activation of DNL cell-intrinsic signaling via SREBP-1c, ACC, and AMPK. Accordingly, through these biochemical pathways, we anticipate observing that Cept1 knockdown will lead to a significantdecrease in DAG, SFAs within phospholipids, and the tissue content of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (20:4) and docosahexaonoic acid (22:6), which play important roles in tissue inflammation and atheroprogression. Conversely, Cept1 overexpression may lead to increased C16 and LPC, and decreased plasmenyl-PE, thus potentially promoting atheroprogression in diabetes.

[0184] We also anticipate that liver-specific Fasn expression will be critically involved inperipheral atheroprogression in the setting of diabetes. We expect that conditional knockdown of Fasn in the liver will result in reduced hepatic DNL activity, leading to lower serum cFAS levels and reduced serum saturated fatty acids that are known contributors to atherosclerosis like palmitic acid (16:0), stearic acid (18:0), myristic acid (14:0), and lauric acid (12:0). This reduction in hepatic DNL activity and serum circulating factors is anticipated to correlate with decreased atherosclerotic plaque formation in the peripheral arteries and reduced macrophage infiltration in the arterial wall.

[0185] Data / Statistical analysis: Descriptive statistics will be used to summarize differences inhistology, immunostaining, immune cell content, and pathological properties between diabetic / non-diabetic, and male / female mice. Generalized linear mixed models will also be used to analyze the differences in DNL signaling among the various animal strains, diseased / non-diseased arterial segments, and central / peripheral arterial segments. Similar analysis will also be performed for liver and adipose tissue among the different animal strains. Differences in spatial transcriptomics, gene expression, tissue cytokine profiles, and DNL signaling activity between diseased / non-diseased arterial specimens, and between different animal strains, will be assessed using ANOVA or Kroskal-Wallis test as appropriate. Our preliminary studies suggest similar atheroprogression between male and female mice (unpublished). However, since sex is a biological variable that impacts PAD disease progression in diabetes, all rodent experiments will be performed using equal cohorts of both male and female animals. Based on our preliminary analysis (Figures 13, 15, 18, & 20), we anticipate that 20 mice per group and per sex (male / female) will achieve 80% power for both sexes to detect a two-sided Spearman correlation of 0.62 assuming 2-sided alpha = 0.05.

[0186] We observed a robust phenotype in atheroprogression when endothelial Cept1 isaltered (Figures 13 & 15). However, in the setting of diabetes, altered expression limited to only endothelial cells might not significantly impact peripheral atheroprogression due to compensatory mechanisms or other atherogenic pathways. If a modest impact is observed, wewill explore additional genetic manipulations, including knockdown or overexpression of Cept1 in VSMCs to assess if a combined approach enhances the impact on atheroprogression. Incomplete endothelial-specific modulation of Cept1 could also lead to variable results. We will validate gene modulation efficiency using quantitative PCR and Western blotting in isolated endothelial cells in all mouse lines. If necessary, we will optimize transgenic models or use alternative gene delivery methods like adeno-associated virus (AAV) vectors for robust Cept1 overexpression. We expect that liver-specific knockdown of Fasn will have a robust phenotype in the setting of diabetes. However, if this does not occur, we will investigate other key hepatic DNL enzymes involved in fatty acid synthesis, such as ACC, to determine their roles in peripheral atheroprogression. Incomplete knockdown of Fasn in the liver could lead to inconsistent or mild changes. We will confirm the efficiency of liver-specific Fasn knockdown using quantitative PCR and Western blotting in liver tissues to ensure robust results. EXAMPLE 3

[0187] The goal of this Example is to evaluate the impact of PPARα agonists and GLP-1receptor agonist on DNL signaling and peripheral arterial atherosclerosis.

[0188] Hypothesis: Fenofibrate, semaglutide (Ozempic®), and liraglutide (Victoza®) willmitigate peripheral arterial atheroprogression by modulating DNL signaling in diabetes.

[0189] Rationale: Emerging evidence highlights the significant role of PPARα agonists andGLP-1 receptor agonists in regulating lipid metabolism and inflammatory pathways, making them promising candidates for mitigating the impact of DNL signaling on atherosclerosis. The FIELD study (Fenofibrate Intervention and Event Lowering in Diabetes) demonstrated that fenofibrate, which is known to enhance fatty acid oxidation, reduce triglyceride levels, and improve endothelial function, also significantly reduced amputation rates in individuals with diabetes. This underscores the potential benefit of fenofibrate in managing complications associated with PAD. Our preliminary data shows fenofibrate can reduce the burden of atherosclerosis in mice with endothelial Cept1 overexpression (Figure 15). Similarly, GLP-1 receptor agonists such as semaglutide and liraglutide have shown substantial cardioprotective effects in major clinical trials. The SUSTAIN-6 trial revealed that semaglutide significantly reduced cardiovascular events in patients with diabetes. The mechanism for this is currently unknown. Our preliminary data indicates that semaglutide can reduce serum circulating cFAS activity (Figure 21), suggesting its role in influencing DNL signaling. By targeting DNLsignaling, established pharmacological agents such as fenofibrate, semaglutide, and liraglutide may offer dual benefits: improving metabolic control and reducing the progression of atherosclerosis, particularly in the peripheral arteries affected by PAD in patients with diabetes. It remains unknown whether these agents are directly or indirectly impacting the peripheral arterial system. By robustly studying this, we aim to elucidate critical mechanisms that can further optimize care and reduce the PAD burden in individuals with diabetes.

[0190] Experimental Design

[0191] Animal models and treatment protocols: Apoe- / - LepRdb / db mice (Figure 11) will beused, which exhibit hyperlipidemia, diabetes, and peripheral iliofemoral arterial disease. We will match mice by age, sex, and initial body weight. Mice will be divided into 4 groups (Fig. 22): Group 1, oral or subcutaneous daily vehicle injection. Group 2, oral fenofibrate (as previously performed, Fig.15). Group 3, subcutaneous semaglutide administration (3 to 15nM / kg / day weekly increase taper over a 6-week period and then 15nM / kg thereafter, as previously described, Fig.21). Group 4, subcutaneous liraglutide administration (100 to 300ug / kg / day weekly increase taper over a 3-week period and then 300ug / kg thereafter, as previously described). Weekly blood draws will be collected to isolate serum from all mice. Mouse food consumption, weight gain, and behavior will be monitored weekly throughout the experimental protocol as previously described.

[0192] Lipidomics and Metabolomics: Post-treatment, arterial, hepatic, and adipose tissueswill be collected, homogenized, and lipids extracted using a chloroform-methanol method. Lipid extracts will be analyzed using LC-MS and GC-MS to quantify triglycerides, phospholipids, cholesterol esters, and metabolites involved in key metabolic pathways (as outlined in Example above). This will identify specific lipid species and DNL signaling that are altered by fenofibrate, semaglutide, and liraglutide in the liver, adipose, or arteries.

[0193] Histopathology and Molecular Analysis: Atherosclerotic lesions will be assessed in theaortic arch, thoracic aorta, abdominal aorta, and peripheral arteries using H&E, VVG, and ORO staining. Plaque volume and composition will be quantified, and RT-PCR, Western blotting, ELISA, and immunofluorescence will be used to measure the expression of DNL markers (CEPT1, FAS, SREBP-1c, ChREBP, ACC, AMPK, PPARα, INSIG1; Table 1), as well as inflammatory markers (Table 3). Additionally, spatial transcriptomics will be used to visualize the distribution of DNL signaling markers across arterial tissue sections, and scRNA sequencing will identify cell-specific responses to treatments, providing a detailedunderstanding of molecular changes in both somatic (ECs, VSMCs, fibroblasts) and myeloid cell types (monocytes / macrophages). These multidimensional approaches will also help delineate whether relative to controls our proposed treatments are directly or indirectly impacting DNL in peripheral arterial structures.

[0194] Data Analysis: Descriptive statistics will be used to summarize imaging, immuneactivity, and pathological properties of the animal groups. Generalized linear mixed models will be used to analyze differences in the characteristics (DNL signaling, disease severity, and in blood / tissue lipid metabolites) between sexes, central / peripheral arterial, disease / non- diseased regions, and between animal Groups 1-4. Differences in cytokine profile, DNL enzyme activity, and tissue metabolites will be assessed using ANOVA or Kroskal-Wallis test as appropriate. Sex as a biological variable will be analyzed in all animal groups to facilitate comparisons between male and female mice. Power calculation. For vehicle and fenofibrate experiments (Groups 1 & 2), the impact on atherosclerosis we observed in our preliminary experiments (Figure 15) indicated that with 20 animals per group we would achieve 90% power to detect a Spearman correlation of 0.65 assuming 2-sided alpha = 0.05. For GLP-1 receptor agonist experiments (Groups 3 & 4), based on our preliminary findings (Figure 21) 20 animals per group will achieve 85% power. The sample size also allows 80% at 2-sided alpha = 0.05 to detect an effect size (Cohen’s d) of 0.91 for between-group differences based on preliminary data (Figures 15 & 21).

[0195] We anticipate that animals in Groups 2, 3, and 4 will have significantly reducedperipheral arterial atheroprogression, pro-inflammatory macrophage infiltration, and DNL signaling. We chose a tapered dose increase in GLP-1 receptor agonist regimens to avoid gastrointestinal side effects. However, if gastrointestinal side effects become an issue over the 16-week experimental protocol period, we will plan to use a longer dose escalation taper (over 5-6 weeks). Fenofibrate and semaglutide have been shown to reduce atherosclerosis in non- diabetic mice. If we find that the proposed agents do not substantially reduce the burden of disease in diabetic Apoe- / -LepRdb / db, then we will try to adjust the dose and / or frequency of the agents. If this also fails to procedure reliable results, we may instead consider the use of alternative agents like gemfibrozil (another PPARα agonist) or dulaglutide (Trulicity®; another GLP-1 receptor agonist). It is potentially possible that, despite our multi-omic analysis, we are unable to clearly elucidate the direct versus indirect impact of the proposed treatments on the peripheral arterial system. If this becomes an issue, we will pursue the outlined experimentsusing animal lines proposed in Example above (e.g., EC Cept1fl / flApoe- / -LepRdb / db, EC Cept1Ox / OxApoe- / -LepRdb / db, and Liver Fasnfl / flApoe- / -LepRdb / dbmice). EXAMPLE 4

[0196] The goal of this Example is to evaluate the therapeutic efficacy of cFAS-specificinhibitors on macrophage foam cell formation and peripheral arterial atherosclerosis.

[0197] Experimental & pharmacological rationale: It was demonstrated the efficacy of cFASinhibition using 1) a Fasn conditional knockout mouse model and 2) a FAS inhibitor, PTM (Figure 18). Our working model is that FAS is normally synthesized by the liver and in the setting of diabetes, cFAS is pathologically released from the liver via lipoproteins and transported to the peripheral arterial system where it catalyzes SFA production and contributes to atheroprogression. Additionally, human serum containing high cFAS (but low LDL) leads to robust macrophage foam cell formation (Figure 23), which is a well-established marker for atheroprogression. Interestingly, human serum containing high LDL, but low cFAS, does not lead to foam cell formation. These observations lead us to hypothesize that specific pharmacological targeting of cFAS may lead to inhibition of foam cell formation and reduce atheroprogression.

[0198] Although the invention has been described with reference to the disclosedembodiments, those skilled in the art will readily appreciate that the specific examples and studies detailed above are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention.

[0199] Detailed description of figures:

[0200] Fig 1. cFAS induces macrophage foam cell formation in vitro. (A) Schematicrepresentation of in vitro experiments used to evaluate the impact of cFAS or LDL on macrophage foam cell formation. (B) Correlation between the percentage of Oil Red O-positive foam cells and serum concentrations of cFAS and LDL.(C) Evaluation of foam cell formation in differentiated macrophages exposed to varying concentrations of cFAS and LDL levels. The percentage of foam cells was quantified for each condition. (D) Assessment of foam cell formation in differentiated macrophages under different conditions: high cFAS / low LDL and low cFAS / high LDL, with or without treatment with PTM (20 μM). (E)Effect of PTM treatment (20 μM) on FAS activity in differentiated macrophages under high cFAS / low LDL and low cFAS / high LDL conditions. (F) Differentiated macrophages conditioned with highcFAS / low LDL serum were treated with different FAS inhibitors: PTM (20 μM), TVB-2640 (100 nM), and GSK2194069 (100 nM). Untreated cells served as a positive control, and cells cultured in 5% FBS served as a negative control. Scale bar: 50μm. Arrows indicate lipid droplets within macrophages. Data are presented as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 compared to control (untreated) conditions.

[0201] Fig 2. Effect of conditioned serum on intracellular and extracellular FAS inmacrophages. (A) Immunofluorescence of FAS (FAS-Alexa®488) in macrophages under various cFAS / LDL conditions ± PTM treatment. (B) Quantification of FAS fluorescence intensity from (A) (n=10). (C) ELISA of FAS content in macrophages treated with high cFAS / low LDL ± PTM, compared to 5% FBS control (n=3). (D) Percentage of cell viability after 48 hours exposure to various PTM concentrations (n=3). Scale bar, 50 μm. Data are mean ± SEM (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.

[0202] Fig 3. FAS inhibition affects murine lipid homeostasis. (A) Schematic representationof in vivo murine experiments using Fasn+ / +Cre-Apoe- / - (1) and Fasnfl / flCre+Apoe- / - (2) that were maintained on a high- fat diet for 16 weeks. A group of Fasn+ / +Cre+ Apoe- / - mice also received PTM (3) throughout this period. Liver, and white adipose tissue were collected from all mouse groups. (B) Impact of Fasn knockdown of on murine body weight (n = 5 per group) over 16-week period. (C) Total serum cholesterol for each murine group (n = 3 per group). (D & E) Liver and white adipose tissue triglyceride content (n = 3 per group). (F & G) Liver and white adipose tissue non-esterified free fatty acid content (n = 3). * p<0.05, ** p<0.01

[0203] Fig 4. Conditional liver-specific knockdown of FAS and PTM treatment impacts serumcFAS content and activity. Serum specimens from Fasn+ / +Cre- Apoe- / - mice, Fasnfl / fl Cre+ Apoe- / - mice, and Fasn+ / +Cre- Apoe- / - mice treated with PTM, were analyzed for cFAS content (A) and cFAS activity (B – D; n = 5 per mouse group). (E & F) The supernatant of digested liver was evaluated for cFAS content and activity (n = 5). (G & H) The supernatant of digested white adipose tissue was evaluated for cFAS content and activity (n = 5). * p<0.05, ** p<0.01, *** p<0.001

[0204] Fig 5. Targeting FAS reduces aortic atherosclerotic plaque burden. (A) Schematic of invivo murine experiments using Fasn+ / +Cre- Apoe- / - mice, Fasnfl / fl Cre+ Apoe- / - mice, and Fasn+ / +Cre- Apoe- / - mice treated with PTM, that were maintained on a high-fat diet for 16 weeks. (B) Representative enfacements of aortic specimens from different mouse groups thatwere stained with Oil Red O. Plaque areas are visualized in red. (C) Total aortic plaque assessment in Fasn+ / +Cre-Apoe- / - (n = 10), Fasnfl / fl Cre+Apoe- / - (n = 10), and Fasn+ / +Cre- Apoe- / - that were treated with PTM (n = 8). (D) Plaque burden in the aortic arch segment, (E) thoracic aorta, and (F) infrarenal aorta. * p<0.05, ** p<0.01, *** p<0.001

[0205] Fig 6. Targeting FAS reduced aortic root plaque burden. (A) Hearts were isolated fromFasn+ / +Cre- Apoe- / - mice, Fasnfl / fl Cre+ Apoe- / - mice, and Fasn+ / +Cre- Apoe- / - mice treated with PTM, that were maintained on a high-fat diet for 16 weeks. Aortic valve leaflets were sectioned at 10μm and stained with Oil Red O. Plaque area is visualized in red. (B) Plaque lesion area percentage was evaluated in each mouse group (n = 3). (C) Aortic valve leaflet sections were also stained with the macrophage marker CD68 (green) and DAPI nuclear stain (blue). * Indicates lumen. (D) Integrated density was analyzed to evaluate CD68 content in aortic valve sections of each mouse group (n = 3).

[0206] Fig 7. FAS inhibition reduces tissue FAS and inflammation response. (A) Liver andwhite adipose tissue were collected from Fasn+ / +Cre- Apoe- / - mice, Fasnfl / fl Cre+ Apoe- / - mice, and Fasn+ / +Cre- Apoe- / - mice treated with PTM, that were maintained on a high-fat diet for 16 weeks. Tissues were then sectioned and stained with H&E, and immunostained for FAS and CD68. (B) Quantification of liver FAS staining (n = 5), and (C) liver CD68 staining (n = 5). (D) Average adipocyte vacuole area (n = 5), (E) FAS staining (n = 5), and (F) CD68 staining (n = 5).

[0207] Fig 8. Patient with diabetes and severe end-stage PAD and CLTI. A) Lower leg x-rayshows severe plaque extending in the lower leg arteries (arrows). B) angiogram shows severe multivessel arterial occlusive disease (arrows). C) Calcaneal eschar consistent with severe tissue loss.

[0208] Fig 9. Proposed mechanisms linking de novo lipogenesis (DNL) to PAD. Prior studiesdemonstrate molecular signals that can stabilize or promote DNL signaling (via cytokine, nutrient, hormonal, and transcriptional factors). We demonstrated that DNL can lead to cell- extrinsic (cFAS and C16) and cell-intrinsic (PPARα and CEPT1) mechanisms associated with atheroprogression. This renewal application proposes to evaluate these mechanisms using a complement of histomorphic, genomic, and metabolomic studies. The proposed studies will use established murine models, unique reagents, novel models evaluating the peripheral arterial system, as well as conventional and proprietary targeted pharmacological interventions (GLP1 receptor agonist semaglutide, PPARα agonist fenofibrate, and selective cFAS inhibitors derivedfrom GSK2194069). AMP-activated protein (AMPK), insulin-Induced Gene 1 (Insig1), sterol regulatory element-binding protein-1c (SREBP-1c), acetyl-CoA carboxylase (ACC).

[0209] Fig 10. Experimental design.

[0210] Fig 11. Novel ilio-femoral peripheral arterial atherosclerosis model in diabetic micemaintained on a Western diet. A) CEPT1 content is increased in the peripheral arterial segments of adult Apoe- / - LepRdb / db mice maintained on a Western diet. n=3 male mice; *p<0.05. B) Aorta (1), iliac (2), and femoral (3) peripheral arterial whole mount segments dissected from a 21-week-old C57BL6 adult wildtype (LepR+ / +) mouse maintained on at least a 12-week course of 42% fat-content Western diet. Peripheral arterial segment whole mount Oil Red-O (ORO) staining reveals minimal lipid-rich plaque regions. C) Aorto-iliac-femoral whole mount and ORO-stained arterial segment of a 21-week-old Apoe- / - LepRdb / db mouse maintained on a 16-week course of Western diet demonstrates obvious peripheral arterial occlusive plaques (white arrows) that are lipid rich (yellow arrows).

[0211] Fig 12. CEPT1 relative to PAD severity. A) CEPT1 co-localizes with CD31 in theintima endothelial layer of the aorta and in microvascular structures of skeletal muscle (inserts 1.5x magnification). B) Min and Max diseased tibial arterial segments from a diabetic patient with CLTI undergoing amputation. C) RT-PCR shows higher Cept1 expression in Max diseased arterial intima of diabetic patients (n=8) compared to non-diabetic (n=15). D) Pooled Western blot shows higher CEPT1 in Max arterial intima segments of a diabetic patient. E) Immunohistochemistry shows higher CEPT1 in Max diseased arterial segments in both diabetic (n=4) and non-diabetic patients (n=4). F) Mass spectrometry reveals higher aPC content in Max diseased segments of diabetic patients (n=14). G) Higher pPE content in Max diseased segments of diabetic patients (n=14). No differences in non-CEPT1 derived phospholipids (PS, PG, PI; data not shown). No sex differences observed. *p<0.05.

[0212] Fig 13. Conditional knockdown of Cept1 in the endothelium decreased atherosclerosis.A) Male and female, 6-week-old mice (Groups 1 & 2; n=6 male and n=6 female per group) were maintained on a 42% high-fat diet for 16 weeks. Aorta was collected at the conclusion of the feeding protocol for analysis. B & C) Mice with conditional Cept1 in the endothelium (2: EC Cept1fl / fl Cre+ Apoe- / -) demonstrated significantly reduced plaque in the aortic arch and carotids. D & E) EC Cept1fl / fl Cre+ Apoe- / - demonstrated reduced aortic valve plaque. F) Quantification of CD68 staining intensity in aortic valves. No differences were observed between male and female mice (data not shown). *p<0.05.

[0213] Fig 14. Conditional overexpression of Cept1 in the endothelium leads to increasedmacrophage recruitment genes. Aortas were isolated from 10-week-old EC Cept1Ox / Ox Cre+ and EC Cept1Ox / Ox Cre- mice (n=3 males per group). Single-cell (sc) RNA sequencing was performed using the 10X genomics platform. A) TSNE map shows the clustering of transcriptomes among cell types between the two groups. Within endothelial cell cluster 2 (EC2), there is significantly increased expression of Cept1 (B), Akt3 (C), Pecam1 (D), and Cdh5 (E). ***p<0.001.

[0214] Fig 15. Conditional overexpression of Cept1 in the endothelium leads toatheroprogression. A) Male and female, 6-week-old mice (Groups 1-6; n=5 male and n=5 female per group) were maintained on a 42% high-fat diet for 16 weeks, with or without fenofibrate (FFB; 0.2mg / g / day) in their chow. Aorta was collected at the end of the feeding protocol. B & C) Mice with conditional Cept1 in the endothelium (3: EC Cept1Ox / Ox Cre+ Apoe- / -) that did not receive fenofibrate had the highest aortic plaque volume. Plaque volume was significantly reduced with fenofibrate treatment (4), and with Ppara knockdown (5 & 6). Fenofibrate did not significantly impact plaque volume in Ppara- / - mice. No differences were observed between male and female (data not shown). *p<0.05.

[0215] Fig 16. Plaque FAS and serum cFAS are elevated in patients with diabetes and CLTI.A) Higher FAS content in lower extremity plaques of diabetic (n=10; 6M & 4F) versus non- diabetic patients (n=13; 8M & 5F) (ELISA, relative units). B) Higher SFA to PUFA ratio in plaques of diabetic (n=10; 4M & 6F) versus non-diabetic patients (n=12; 6M & 6F) (mass spectrometry). C) Higher serum cFAS in CLTI patients (n=29; 16M & 13F) compared to moderate PAD (n=30; 18M & 12F) and no PAD (n=22; 11M & 11F) (ELISA). D) Higher serum cFAS in patients with severe (DCSI>5, n=15; 8M & 7F) versus mild (DCSI<5, n=14; 8M & 6F) diabetes-related complications and no diabetes (n=41; 22M & 19F). E) Positive correlation between serum cFAS and plaque FAS content (n=23; 12M & 11F). *p<0.05

[0216] Fig 17. Serum cFAS is LDL associated and elevated in setting of diabetes. A) cFAS islargely present in LDL serum cholesterol fractions isolated by FPLC (n=13). B) FAS and ApoB co-IP from LDL cholesterol fractions isolated from one diabetic patient (1) and one non- diabetic patient (2). C) Representative EM image demonstrating gold labeled cFAS (black oval) associated with LDL particle (light gray ovals). D) Representative blot demonstrating decreased serum cFAS in Liver Fasnfl / fl mice. E) LepRdb / db mice have the higher serum cFAScompared to control (+ / +), and Liver Fasnfl / fl mice have lower cFAS compared to SM Fasnfl / fl mice (with selective knockdown of Fasn in skeletal muscle; n=6 per group). **p<0.01.

[0217] Fig 18. Conditional hepatic knockdown of Fasn and systemic pharmacologicaltargeting of FAS leads to decreased atherosclerosis. A) Male and female, 6-week-old mice (Groups 1-3; n=5 male and n=5 female per group) were maintained on a 42% high-fat diet for 16 weeks, with feeding chow containing or not containing platensimycin (PTM; 0.1mg / g / day). Aorta was collected at the conclusion of the feeding protocol for analysis. B, C) Mice with conditional Fasn in the liver (2: Liver Fasnfl / fl Apoe- / -) and mice that received PTM (3: Fasn+ / + Apoe- / - + PTM) demonstrated significantly reduced plaque in the aortic arch, branch arteries, and aortic valves. There was reduced macrophage CD68 staining in the aortic valves of Groups 2 and 3 mice. D) Quantification of aortic valve plaque. E) Quantification of CD68 staining intensity in aortic valves. No differences were observed between male and female mice (data not shown). *p<0.05

[0218] Fig 19. Conditional hepatic knockdown of Fasn leads to reduced Vecam1 expression inVSMCs. A) Uniform Manifold Approximation and Projection (UMAP) plot of scRNA sequencing data from cell clusters isolated from pooled aortic specimens from Fasn+ / + Apoe- / - and Liver Fasn+ / + Apoe- / - mice (n=3 males per group). The color intensity represents the expression level, with a gradient from beige (low expression) to dark purple (high expression). The blue dashed circle highlights a cluster of VSMCs with distinctly high Vecam1 expression in Fasn+ / + Apoe- / - mice. B) The blue dashed circle corresponds to the same region and indicates reduced VSMCs that express Vecam1 in Liver Fasn+ / + Apoe- / - mice.

[0219] Fig 20. Conditional hepatic knockdown of Fasn reduced inflammation in liver andadipose tissue. A) Male and female, 6-week-old mice (Groups 1-3; n=5 male and n=5 female per group) were maintained on a 42% high-fat diet for 16 weeks, with feeding chow containing or not containing platensimycin (PTM; 0.1mg / g / day). Liver and adipose tissue were collected at the conclusion of the feeding protocol for immunohistochemistry with H&E, FAS, and CD68. B) Quantification of FAS staining in liver. C) Quantification of CD68 staining in liver. D) Quantification of FAS staining in white adipose tissue. E) Quantification of CD68 staining in white adipose. No differences were observed between male and female mice (data not shown). *p<0.05, **p<0.01, ***p<0.001

[0220] Fig 21. Semaglutide reduced hepatic steatosis, hepatic Fasn expression, and serumcFAS activity. A) C57BL6 mice maintained on a 42% fat Western diet were maintained ondaily semaglutide injection course and livers harvested after 6 weeks. Gross examination demonstrates reduced fatty liver and lipid droplet deposits on H&E and ORO staining (black arrows). B) Quantification of ORO lipid droplet area (n=12 male mice). C) RT-PCR of Fasn in liver tissue from treated and untreated mice (n=3 male mice). D) Serum cFAS activity in treated and untreated mice (n=3 male mice). *p<0.05, **p<0.01, ***p<0.001

[0221] Fig 22. Experimental outline.

[0222] Fig 23. Foam cell and cFAS activity assays. A) High cFAS containing human seruminduces macrophage foam cell formation (ORO+ red staining indicating lipid droplet formation). B) PTM significantly reduces cFAS-induced foam cell formation, and C) In vitro cFAS activity.

[0223] Pharmacological inhibition of FAS is a topic of multiple prior investigations,particularly since FAS is elevated in malignant tissue, and serum cFAS is also elevated in individuals with certain metastatic tumors. Indeed, there are currently FAS inhibitors that are undergoing efficacy testing in phase II human clinical trials and are demonstrating promise. PTM is a commonly used FAS inhibitor that is naturally derived from Streptomyces platensis bacteria. It selectively and competitively binds to both bacterial and mammalian FAS and forms stable complexes with FAS subunits. In Db / Db mice, PTM inhibits de novo fatty acid synthesis and enhances glucose oxidation. Consistent with findings in Db / Db mice, we observed that PTM treatment of Fasn+ / +Cre- Apoe- / -mice supported normal weight gain. Prior studies suggest that this phenotype is observed due to improved hepatic glucose uptake and glycolysis. However, our study demonstrates that PTM clearly also impacts white adipose FAS content and activity, as well as adipocyte lipid storage (expressed as adipocyte area). This may reflect a compensatory mechanism and tissue-specific metabolic adaptation where adipose tissue increases FFAs uptake and storage in response to systemic FAS inhibition, potentially through upregulation of fatty acid transporters and altered lipolysis regulation. Importantly, this observation suggests that adipose tissue may serve as a lipid buffer, compensating for decreased FAS content and activity in the liver by increasing its capacity to store lipids. The remarkable crosstalk between liver and adipose tissue was not only limited to mice treated with PTM, but this was also observed in Fasnfl / flCre- Apoe- / -mice, which after 16 weeks of a high- fat diet regimen demonstrated significantly elevated FAS content. These findings underscore the importance of considering tissue-specific metabolic adaptations when developing therapies targeting lipid metabolism. Liver and adipose signaling in relation to fatty acid synthesis andmacronutrient metabolism has been reported extensively and is a highly orchestrated process. In humans, dietary nutrients, and de novo lipid synthesis (in these organ tissue) is thought to influence obesity and fatty liver disease. Our findings suggest that cFAS may in part be a vehicle of communication between liver and white adipose tissue.

[0224] In conclusion, we report that FAS targeting through conditional liver knockdown andtargeted pharmacological inhibition, reduces tissue FAS and serum cFAS activity in macrophages, liver, and white adipose tissue. This leads to a significant reduction in atherosclerosis after 16 weeks of a high-fat diet regimen. Additionally, knockdown or inhibition of FAS reduces tissue infiltration in arterial plaque, liver, and adipose tissue. These findings highlight the utility of targeting tissue FAS or serum cFAS for the management of atheroprogression. DEFINITIONS

[0225] All publications, patents and patent applications cited herein, whether supra or infra,are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0226] The term “composition” refers to a mixture of materials which comprise thecomposition, as well as reaction products and decomposition products formed from the materials of the composition.

[0227] The terms “comprising,” “including,” “having” and their derivatives, are not intendedto exclude the presence of any additional component, step, or procedure, whether or not the same is specifically disclosed. In order to avoid any doubt, all compositions claimed through use of the term “comprising” may include any additional additive, adjuvant, or compound, whether polymeric or otherwise, unless stated to the contrary.

[0228] The term “or,” unless stated otherwise, refers to the listed members individually aswell as in any combination. Use of the singular includes use of the plural and vice versa.

[0229] The terms “a,” “an,” “the” and similar referents used in the context of describing theinventive features (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Thus, for example, reference to a “starch” can include one, two or more starches.

[0230] Numbers, percentages, ratios, or other values stated herein may include that value, andalso other values that are about or approximately the stated value, as would be appreciated by one of ordinary skill in the art. A stated value should therefore be interpreted broadly enough to encompass values that are at least close enough to the stated value to perform a desired function or achieve a desired result, and / or values that round to the stated value. The stated values include at least the variation to be expected in a typical manufacturing process, and may include values that are within 25%, 15%, 10%, within 5%, within 1%, etc. of a stated value.

[0231] The term “effective amount” refers to the amount of a therapy (e.g., an active agentsuch as a FAS inhibitor described herein) which is sufficient to accomplish a stated purpose or otherwise achieve the effect for which it is administered. An effective amount can be sufficient to reduce and / or ameliorate the progression, development, recurrence, severity and / or duration of a given disease, disorder or condition and / or a symptom related thereto. An effective amount can be a “therapeutically effective amount” which refers to an amount sufficient to provide a therapeutic benefit such as, for example, the reduction or amelioration of the advancement or progression of a given disease, disorder or condition, reduction or amelioration of the recurrence, development or onset of a given disease, disorder or condition, and / or to improve or enhance the prophylactic or therapeutic effect(s) of another therapy. A therapeutically effective amount of a composition described herein can enhance the therapeutic efficacy of a therapeutic agent.

[0232] The “FAS inhibitor” described herein is referred to as the “FAS inhibitor” or the “FASinhibitor compound.”

[0233] The “GLP-1 receptor agonist” described herein is also referred to as the “GLP-1receptor agonist compound.”

[0234] The “PPAR agonist” described herein is also referred to as the “PPAR agonistcompound.”

[0235] The terms “treating” or “treatment” refer to any indicia of success or amelioration ofthe progression, severity, and / or duration of a disease, pathology or condition, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a patient's physical or mental well-being.

[0236] The term “administering” refers to the act of delivering a composition or atherapeutically effective amount of an active agent and / or compound as described herein into a subject by routes such as oral, mucosal, topical, suppository, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration. Parenteral administration includes intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Administration generally occurs after the onset of the disease, disorder, or condition, or its symptoms but, in certain instances, can occur before the onset of the disease, disorder, or condition, or its symptoms (e.g., administration for patients prone to such a disease, disorder, or condition).

[0237] The term “regimen” refers to a protocol for dosing and timing the administration ofone or more compositions or active agents comprising an FAS inhibitor compound described herein, for treating a disease, disorder, or condition described herein. A regimen can include periods of active administration and periods of rest as known in the art. Active administration periods include administration of compositions described herein and the duration of time of efficacy of such compositions. Rest periods of regimens described herein include a period of time in which no compound is actively administered, and in certain instances, includes time periods where the efficacy of such compounds can be minimal. Combination of active administration and rest in regimens described herein can increase the efficacy and / or duration of administration of the compositions described herein.

[0238] The term “patient” or “subject” refers to a mammal, such as a human, bovine, rat,mouse, dog, monkey, ape, goat, sheep, cow, or deer. Generally, a patient as described herein is human.

[0239] The terms “inhibition,” “inhibit,” “inhibiting” refer to a reduction in the activity orexpression of a polypeptide and / or an enzyme (e.g., FAS) or reduction or amelioration of a disease, disorder, or condition or a symptom thereof. Inhibiting as used here can include partially or totally blocking stimulation, decreasing, preventing, or delaying activation, or inactivating, desensitizing, or down-regulating protein or enzyme activity. An “inhibitor,” as used herein is a means that carries out the “inhibition” and / or “inhibits.”

[0240] Antibodies described herein can be polyclonal or monoclonal.

[0241] The pharmaceutically acceptable salts of the compounds described herein include, forexample, a salt with an alkali metal such as lithium, sodium, potassium, etc.; a salt with analkaline earth metal such as calcium, magnesium, etc.; a salt with zinc or aluminum; a salt with an organic base such as ammonium, choline, diethanolamine, lysine, ethylenediamine, t- butylamine, t-octylamine, tris(hydroxymethyl)aminomethane, N-methyl glucosamine, triethanolamine and dehydroabietylamine; a salt with an inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; or a salt with an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, etc.; or a salt with an acidic amino acid such as aspartic acid, glutamic acid, etc. EXEMPLARY CLAUSES

[0242] Clause 1. A method of reducing atheroprogression in a subject in need thereofcomprising: administering to the subject a Fatty Acid Synthase (FAS) inhibitor or a pharmaceutically acceptable salt thereof.

[0243] Clause 2. The method of clause 1, wherein the FAS inhibitor is a natural FAS inhibitoror a synthetic FAS inhibitor.

[0244] Clause 3. The method of clause 1, wherein the FAS inhibitor is selected from a smallmolecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

[0245] Clause 4. The method of clause 1, wherein the FAS inhibitor inhibits tissue FAS andserum circulating Fatty Acid Synthase (cFAS).

[0246] Clause 5. The method of clause 1, wherein the FAS inhibitor reduces livertriglycerides and Free Fatty Acids (FFAs).

[0247] Clause 6. The method of clause 3, wherein the FAS inhibitor is selected from GSK2194069, GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat, G28UCM, Fasnall, imidazopyridine, epigallocatechin-3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, and 910249-49-9.

[0248] Clause 7. The method of clause 1, wherein about 100 mg / kg / day of the FAS inhibitoris administered to the subject.

[0249] Clause 8. The method of clause 1, wherein about 50 to 200 mg of the FAS inhibitor isadministered.

[0250] Clause 9. The method of clause 1, wherein the subject is a human patient.

[0251] Clause 10. The method of clause 1, wherein the FAS inhibitor is administered incombination with an additional therapeutic agent.

[0252] Clause 11. A method of regulating a disease governed by Fatty Acid Synthase (FAS)in a subject in need thereof comprising administering to the subject a a Fatty Acid Synthase (FAS) inhibitor; wherein the FAS inhibitor inhibits tissue FAS and serum circulating FAS (cFAS); and wherein the FAS inhibitor reduces liver triglycerides and Free Fatty Acids (FFAs).

[0253] Clause 12. The method of clause 11, wherein the disease is selected from cancer, viralinfections, nonalcoholic fatty liver disease and a metabolic disorder.

[0254] Clause 13. The method of clause 12, wherein the metabolic disorder is diabetes.

[0255] Clause 14. The method of clause 12, wherein the metabolic disorder is atherosclerosis.

[0256] Clause 15. The method of clause 11, wherein the FAS inhibitor is selected from asmall molecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

[0257] Clause 16. The method of clause 15, wherein the FAS inhibitor is selected from GSK2194069, GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat, G28UCM, Fasnall, imidazopyridine, epigallocatechin-3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, and 910249-49-9.

[0258] Clause 17. The method of clause 11, wherein about 100 mg / kg / day of the FASinhibitor is administered.

[0259] Clause 18. The method of clause 11, wherein about 50 to 200 mg of the FAS inhibitoris administered.

[0260] Clause 19. The method of clause 11, wherein the subject is a human patient.

[0261] Clause 20. The method of clause 11, wherein the FAS inhibitor is administered incombination with an additional therapeutic agent.

[0262] Clause 21. A method of treating peripheral artery disease in a subject in need thereofcomprising: administering to the subject a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

[0263] Clause 22. The method of clause 21, wherein the GLP-1 receptor agonist is selectedfrom a small molecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

[0264] Clause 23. The method of clause 21, wherein the GLP-1 receptor agonist is a GLP-1analog.

[0265] Clause 24. The method of clause 23, wherein the GLP-1 receptor agonist is selectedfrom dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, and tirzepatide.

[0266] Clause 25. The method of clause 21, wherein the GLP-1 receptor agonist isadministered in combination with an additional therapeutic agent.

[0267] Clause 26. The method of clause 25, wherein the additional therapeutic agent ismetformin, thiazolidinediones, sulfonylureas, a dipeptidyl peptidase 4 inhibitor, or sodium glucose co-transporter.

[0268] Clause 27. A method of treating peripheral artery disease in a subject in need thereofcomprising administering to the subject a PPAR agonist or a pharmaceutically acceptable salt thereof.

[0269] Clause 28. The method of clause 27, wherein the PPAR agonist is selected from asmall molecule compound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

[0270] Clause 29. The method of clause 27, wherein the PPAR agonist is a PPARα agonist ora PPARγ agonist.

[0271] Clause 30. The method of clause 27, wherein the PPAR agonist is clofibrate,gemfibrozil, ciprofibrate, bezafibrate, fenofibrate, thiazolidinediones, pioglitazone or rosiglitazone.

Claims

CLAIMS What is claimed is:

1. A method of reducing atheroprogression in a subject in need thereof comprising:administering to the subject a Fatty Acid Synthase (FAS) inhibitor or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the FAS inhibitor is a natural FAS inhibitor or asynthetic FAS inhibitor.

3. The method of claim 1, wherein the FAS inhibitor is selected from a small moleculecompound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

4. The method of claim 1, wherein the FAS inhibitor inhibits tissue FAS and serumcirculating Fatty Acid Synthase (cFAS).

5. The method of claim 1, wherein the FAS inhibitor reduces liver triglycerides and FreeFatty Acids (FFAs).

6. The method of claim 3, wherein the FAS inhibitor is selected from GSK 2194069,GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat, G28UCM, Fasnall, imidazopyridine, epigallocatechin- 3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, and 910249-49-9.

7. The method of claim 1, wherein about 100 mg / kg / day of the FAS inhibitor is administered tothe subject.

8. The method of claim 1, wherein about 50 to 200 mg of the FAS inhibitor is administered.

9. The method of claim 1, wherein the subject is a human patient.

10. The method of claim 1, wherein the FAS inhibitor is administered in combination with anadditional therapeutic agent.

11. A method of regulating a disease governed by Fatty Acid Synthase (FAS) in a subject in needthereof comprising administering to the subject a a Fatty Acid Synthase (FAS) inhibitor; wherein theFAS inhibitor inhibits tissue FAS and serum circulating FAS (cFAS); and wherein the FAS inhibitor reduces liver triglycerides and Free Fatty Acids (FFAs).

12. The method of claim 11, wherein the disease is selected from cancer, viral infections,nonalcoholic fatty liver disease and a metabolic disorder.

13. The method of claim 12, wherein the metabolic disorder is diabetes.

14. The method of claim 12, wherein the metabolic disorder is atherosclerosis.

15. The method of claim 11, wherein the FAS inhibitor is selected from a small moleculecompound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

16. The method of claim 15, wherein the FAS inhibitor is selected from GSK 2194069,GSK837149A, TVB-2640, TVB-3166, TVB-3567, TVB-3664, IPI-9119, BI 99179, FT-113, platensimycin, C75, cerulenin, orlistat, G28UCM, Fasnall, imidazopyridine, epigallocatechin- 3-gallate, omeprazole, 1375105-96-6, 1309805-49-9, 1808260-84-5, and 910249-49-9.

17. The method of claim 11, wherein about 100 mg / kg / day of the FAS inhibitor is administered.

18. The method of claim 11, wherein about 50 to 200 mg of the FAS inhibitor is administered.

19. The method of claim 11, wherein the subject is a human patient.

20. The method of claim 11, wherein the FAS inhibitor is administered in combination with anadditional therapeutic agent.

21. A method of treating peripheral artery disease in a subject in need thereof comprising:administering to the subject a GLP-1 receptor agonist or a pharmaceutically acceptable salt thereof.

22. The method of claim 21, wherein the GLP-1 receptor agonist is selected from a small moleculecompound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

23. The method of claim 21, wherein the GLP-1 receptor agonist is a GLP-1 analog.

24. The method of claim 23, wherein the GLP-1 receptor agonist is selected from dulaglutide,exenatide, liraglutide, lixisenatide, semaglutide, and tirzepatide.

25. The method of claim 21, wherein the GLP-1 receptor agonist is administered in combinationwith an additional therapeutic agent.

26. The method of claim 25, wherein the additional therapeutic agent is metformin,thiazolidinediones, sulfonylureas, a dipeptidyl peptidase 4 inhibitor, or sodium glucose co-transporter.

27. A method of treating peripheral artery disease in a subject in need thereof comprisingadministering to the subject a PPAR agonist or a pharmaceutically acceptable salt thereof.

28. The method of claim 27, wherein the PPAR agonist is selected from a small moleculecompound, a nucleic acid, a peptide, a protein, an antibody, a peptibody, a diabody, a minibody, a single-chain variable fragment (ScFv), and a fragment or variant thereof.

29. The method of claim 27, wherein the PPAR agonist is a PPARα agonist or a PPARγ agonist.

30. The method of claim 27, wherein the PPAR agonist is clofibrate, gemfibrozil, ciprofibrate,bezafibrate, fenofibrate, thiazolidinediones, pioglitazone or rosiglitazone.

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