COMPOSITION COMPRISING A GLP-1 RECEPTOR AGONIST AND ACAT INHIBITOR (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 382,287, filed November 3, 2022, which is incorporated herein by reference.

Abstract

The present invention provides pharmaceutical compositions containing one or more ACAT inhibitors and one or more GLP-1RAs, and methods for controlling weight, comprising co-administering one or more ACAT inhibitors and one or more GLP-1RAs to a subject in need thereof.

Description

[Technical Field]

[0001] The present invention relates to compositions comprising one or more GLP-1 receptor agonists and one or more ACAT inhibitors, and methods for controlling weight by co-administering one or more GLP-1 receptor agonists and one or more ACAT inhibitors. [Background technology]

[0002] Obesity is characterized by an increase in fat mass due to an increase in the size and number of adipocytes. Enlarged adipocytes increase the uptake of free cholesterol, leading to its accumulation in lipid droplets in proportion to the increased triglyceride content. References: B.R.Krause, A.D.Hartman, "Adipose Tissue and Cholesterol Metabolism," J. Lipid Res. 25 (1984) 97-110; and F.T.Doole, T.Kumarage, R.Ashkar, M.F.Brown, "Lipid Membrane Stiffening by Cholesterol," J. Membr. Biol. 255 (2022) 385-405 (10.1007 / s00232-022-00263-9), are incorporated herein by reference. Acyl-coenzyme A:cholesterol acyltransferase (ACAT), which converts free cholesterol to cholesteryl esters (CEs) using adenosine triphosphate and coenzyme A, plays a key role in intracellular cholesterol storage. The article S. Mukherjee, G. Kunitake, and R.B. Alfin-Slater, "Esterification of Cholesterol and Palmitic Acid by Rat Liver Homogenates," J. Biol. Chem. 230 (1958) 91-96, is incorporated herein by reference. Increased ACAT1 expression has been shown to be associated with increased adiposity and adipogenesis in vitro. References Y. Zhu, C.Y. Chen, J. Li, J.X. Cheng, M. Jang, and K.H. Kim, "In vitro study of the contribution of ACAT to lipid droplet formation during adipogenesis," J Lipid Res 59 (2018) 820-829 (10.1194 / jlr.M081745), and Y. Xu, X. Du, N. Turner, A.J. Brown, and H. Yang, "Enhanced acyl-CoA:cholesterol acyltransferase activity increases lipid droplet surface cholesterol levels and reduces adipocyte function," J Biol Chem 294 (2019) 19306-19321, are incorporated herein by reference. Inhibition of ACAT activity has been reported to suppress the formation and expansion of lipid droplets during adipogenesis in vitro.The literature, Y. Zhu, C.Y. Chen, J. Li, J.X. Cheng, M. Jang, and K.H. Kim, "In vitro study on the contribution of ACAT to lipid droplet formation during adipogenesis," J Lipid Res 59 (2018) 820-829, is incorporated herein by reference. Furthermore, it has recently been reported that ACAT inhibitors induce significant weight loss in diet-induced obese (DIO) mice while simultaneously suppressing food intake, while reducing blood levels of markers associated with obesity and insulin resistance. The literature, Y. Zhu, S.Q. Kim, Y. Zhang, Q. Liu, and K.H. Kim, "Pharmacological inhibition of acyl-coenzyme A:cholesterol acyltransferase attenuates obesity and insulin resistance in diet-induced obese mice by regulating food intake," Metabolism 123 (2021) 154861, is incorporated herein by reference. It has been reported that ACAT inhibitors reduce body weight and food intake. US Patent Application No. 16,461,597 is incorporated herein by reference.

[0003] Glucagon-like peptide-1 receptor agonists (GLP-1RAs), including semaglutide and liraglutide, have been reported to reduce body weight and treat type 2 diabetes. References: Diabetes Ther. 2020 Sep;11(9):1965-1982; U.S. Patent Nos. 8,129,343 and 9,993,430 are incorporated herein by reference. A series of clinical trials titled "Semaglutide Therapeutic Efficacy in Obese Patients (STEP)" demonstrated that weekly subcutaneous injections of 2.4 mg of semaglutide effectively reduced body weight by 14.9% to 17.4% after 68 weeks. References: T.A. W. Adden, T.S. Bailey, L.K. Billings, M. Davies, J.P. Frias, A. Koroleva, I. Lingvay, P.M.O. Neil, D.M. Rubino, D.S. Skovgaard, S.R. Ballenstein, W.T. Garvey, "Effects of subcutaneous semaglutide versus placebo as an adjunct to intensive behavioral therapy in overweight or obese adults: the STEP 3 randomized clinical trial," JAMA 325(2021)1403-1413(10.1001 / jama.2021,1831), and reference D. Rubino, N. Abrahamsson, M. Davies, D. Hesse, FL Greenway, C. Jensen, I. Lingvay, O. Mosenzon, J. Rosenstock, M. Rubio, G. Rudofsky, S. Tadayon, T. A. Wadden, D. Dicker, "Continued weekly subcutaneous semaglutide versus placebo for maintaining weight loss in overweight or obese adults: the STEP 4 randomized clinical trial," JAMA 325(2021)1414-1425(10.1001 / jama.2021.3224), which is incorporated herein by reference. The success of semaglutide in treating obesity has resulted in double-digit weight loss rates and led to its clinical approval. However, concerns have arisen regarding dose-dependent gastrointestinal disturbances associated with semaglutide and rapid weight regain after treatment cessation. These concerns have prompted a search for new semaglutide therapies that may offer greater efficacy and improved tolerability.

[0004] This "Background" section introduces aspects that are helpful in facilitating understanding of the present disclosure. Accordingly, these statements should be read in this light and not understood as admissions of what is or is not prior art. Summary of the Invention

[0005] One embodiment of the present invention provides a method for controlling weight, comprising co-administering to a subject in need thereof a therapeutically effective amount of one or more ACAT inhibitors, or pharmaceutically acceptable salts thereof, and a therapeutically effective amount of one or more GLP-1RAs, or pharmaceutically acceptable salts thereof.

[0006] In some embodiments, the GLP-1RA may be selected from the group consisting of lixisenatide, liraglutide, exenatide, exenatide extended release, albiglutide, semaglutide, ITCA650, dulaglutide, tirzepatide, retatortide, orfogliplon, rotigliplon, efpeglenatide, and taspoglutide.

[0007] In some embodiments, the ACAT inhibitor is avasimibe (CI-1011), CI-976, CP113,818, pactimibe, NTE-122, F-1394, PD140296, PD128042, PD132301-2, octimibate, DuP128, 58-035, HL-004, SMP-500, CL-277,082, SKF-9 9085, CS-505, eflucimibe (F12511), E5324, FR145237, CL277,082, YM-17E, FR129169, K-604, pyrocarbonate, beauveriolide I, and a methanol extract of the root of Saurus sinensis containing sorcerneol B and manassantin B.

[0008] In some embodiments, the GLP-1RA and ACAT inhibitor may be formed into a formulation, and the formulation is administered.

[0009] In some embodiments, the GLP-1RA and ACAT inhibitor may be formulated separately and administered simultaneously or sequentially.

[0010] In some embodiments, the GLP-1RA may be selected from the group consisting of semaglutide, liraglutide, and tirzepatide, and the ACAT inhibitor may be avasimibe or a pharmaceutically acceptable salt thereof.

[0011] Another aspect of the present invention provides a composition comprising an ACAT inhibitor or a pharmaceutically acceptable salt thereof, a GLP-1RA or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.

[0012] In some embodiments, the GLP-1RA may be selected from the group consisting of lixisenatide, liraglutide, exenatide, exenatide extended release, albiglutide, semaglutide, ITCA650, dulaglutide, tirzepatide, retatortide, orfogliplon, rotigliplon, efpeglenatide, and taspoglutide.

[0013] In some embodiments, the ACAT inhibitor may be selected from the group consisting of avasimibe (CI-1011), CI-976, CP113,818, pactimibe, NTE-122, F-1394, PD140296, PD128042, PD132301-2, octimibate, DuP128, 58-035, HL-004, SMP-500, CL-277,082, SKF-99085, CS-505, eflucimibe (F12511), E5324, FR145237, CL277,082, YM-17E, FR129169, K-604, pyrocarbonate, beauveriolide I, and a methanol extract of the root of Saurus sinensis containing saucerneol B and manassantin B.

[0014] In some embodiments, the GLP-1RA may be selected from the group consisting of semaglutide, liraglutide, and tirzepatide, and the ACAT inhibitor may be avasimibe or a pharmaceutically acceptable salt thereof.

[0015] Each embodiment disclosed herein is contemplated as applying to each of the other disclosed embodiments, and therefore all combinations of the various elements described herein are within the scope of the invention. [Brief explanation of the drawings]

[0016] [Figure 1A] FIG. 1 shows the effect of the combination of semaglutide and avasimibe in morbidly obese male mice administered subcutaneously daily for 28 days. Figure 1 shows the study design in obese male C57BL / 6 mice receiving semaglutide (0.04 mg / kg body weight) or vehicle solution in the presence or absence of avasimibe (10 mg / kg body weight) for the indicated number of days. [Figure 1B] FIG. 1B shows the weight change in the study of FIG. 1A. [Figure 1C] FIG. 1B shows the % body weight change on day 28 in the study of FIG. 1A. [Figure 1D] FIG. 1B shows the change in food intake in the test of FIG. 1A. [Figure 1E] FIG. 1B shows relative organ weights in the study of FIG. 1A. [Figure 2A] Figure 1 shows the effect of the combination of semaglutide and avasimibe in moderately obese male mice after subcutaneous administration once every three days for 23 days. Figure 1 shows the study design in obese male C57BL / 6 mice receiving semaglutide (0.04 mg / kg body weight) or vehicle solution every three days, with or without avasimibe (20 mg / kg body weight). [Figure 2B] The arrows indicate the days of drug administration in the test shown in FIG. 2A. [Figure 2C] FIG. 2B shows the % weight change at sacrifice for the study in FIG. 2A. [Figure 2D]FIG. 2B shows daily food intake in the study of FIG. 2A. [Figure 2E] FIG. 2B shows cumulative food intake in the study of FIG. 2A. [Figure 2F] FIG. 2B shows the results of OGTT in the study of FIG. 2A. [Figure 2G] FIG. 2B shows the area under the curve for the test of FIG. 2A. [Figure 3A] Figure 1 shows the effect of the combination of semaglutide and avasimibe on fat mass and adipocyte size after subcutaneous administration every three days for 23 days in moderately obese male mice, illustrating body composition. [Figure 3B] FIG. 3B shows relative organ weights in the study of FIG. 3A. [Figure 3C] FIG. 3B shows the PCC between body weight and lean body mass in the study of FIG. 3A. [Figure 3D] FIG. 3B shows the PCC between body weight and fat mass in the study of FIG. 3A. [Figure 3E] FIG. 3B shows the PCC between IngWAT and fat mass in the study of FIG. 3A. [Figure 3F] FIG. 3B shows H&E staining of IngWAT (scale bar: 50 μm) in the study of FIG. 3A. [Figure 3G] FIG. 3B shows the size distribution of adipocytes in IngWAT in the study of FIG. 3A.

[0017] In Figures 1A–1E, all values ​​are shown as mean ± SEM, n = 10 mice per group. Multiple comparisons were performed using one-way ANOVA with Tukey's post-hoc test (Figure 1C) or two-way ANOVA with Tukey's post-hoc test (Figures 1B and 1D). "*" p < 0.05, "**" p < 0.01, "***" p < 0.001, "****" p < 0.0005 indicate significant differences compared to the vehicle group. "^" p < 0.05, "^^" p < 0.01, "^^^" p < 0.001 indicate significant differences compared to the semaglutide group. Student's t-test was used for Figures 1B, 1D, and 1E after sacrifice 1. "§" p < 0.05, "§§" p < 0.01, "§§§§" p < 0.0005. EpiWAT is epididymal white adipose tissue, PRAT is perirenal adipose tissue, and RetroWAT is retroperitoneal white adipose tissue.

[0018] In Figures 2A-G, time 0 is immediately before glucose challenge. All values ​​are shown as mean ± SEM. n = 5-6 mice per group. Multiple comparisons were performed using one-way ANOVA with Tukey's post-hoc test (Figures 2C, 2E, 2F, and 2G) or two-way ANOVA with Tukey's post-hoc test (Figures 2B and 2D). *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0005 indicate significant differences compared to the vehicle group.

[0019] In Figures 3A-G, all values ​​are shown as mean ± SEM. Multiple comparisons were performed using one-way ANOVA with Tukey's post-hoc test (Figures 3A, 3B, and 3G). *p<0.05, **p<0.01, and ***p<0.001 indicate significant differences compared to the vehicle group. EpiWAT refers to epididymal white adipose tissue, IngWAT refers to inguinal white adipose tissue, and RetroWAT refers to retroperitoneal white adipose tissue. DETAILED DESCRIPTION OF THE INVENTION

[0020] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to illustrated embodiments and specific language will be used to describe them, nevertheless it will be understood that no limitation of the scope of the disclosure is thereby intended.

[0021] In this disclosure, the term "about" can allow for a degree of variation in a value or range, such as, for example, within 10%, within 5%, or within 1% of a stated value or the limits of a stated range.

[0022] In this disclosure, the term "substantially" can allow for a degree of variation in a value or range, such as, for example, within 90%, within 95%, or within 99% of a stated value or the limits of a stated range.

[0023] As used herein, the terms "a," "an," or "the" are used to include one or more numbers unless the context clearly indicates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise specified. Furthermore, it should be understood that the phrases and terms used herein, unless otherwise defined, are for descriptive purposes only and not for limiting purposes. The use of section headings is to aid in the reading and comprehension of this document and should not be construed as limiting. Furthermore, the information associated with a section heading may be found both within and outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this specification are incorporated by reference in their entirety herein, as if individually incorporated by reference. In the event of a conflict of usage between this specification and a document incorporated by reference, the usage of the incorporated document should be considered supplementary to the usage of this specification, with the usage of this specification controlling in the event of any irreconcilable conflict.

[0024] As used herein, the term "GLP-1RA" refers to a glucagon-like peptide 1 receptor agonist. Non-limiting examples of GLP-1RA include lixisenatide, liraglutide, exenatide, exenatide extended release, albiglutide, semaglutide, ITCA650, dulaglutide, tirzepatide, retatortide, orfogliplon, rotigliplon, efpeglenatide, and taspoglutide.

[0025] As used herein, the term "ACAT inhibitor" refers to a small or large molecule capable of inhibiting ACAT activity. Non-limiting examples of ACAT inhibitors include avasimibe (CI-1011), K-604, CI-976, CP113,818, pactimibe, NTE-122, F-1394, PD140296, PD128042, PD132301-2, octimibate, DuP128, 58-035, HL-004, SMP-500, CL-27 7,082, SKF-99085, CS-505, eflucimibe (F12511) and F12511 analogs (analogs 1, 2, 2c, and 3 or F26) (US2006 / 0135785), E5324, FR145237, CL277,082, YM-17E, FR129169, diethyl pyrocarbonate (Cho, et al. al. 2003, Biochem. Biophys. Res. Comm. 309:864-872), beauveriolide I and beauveriolide III (Oshiro et al. 2007, J. Antibiotics 60:43-51), beauveriolide analogues (258, 274, 280, 285, 301) (Tomoda & Doi 2008, Accounts Chem. Res. 41:32-39), compound 1A and its derivatives (1B, 1C, 1D) (Lada et al. 2004, J. Lipid Res. 45:378-386), and methanol extract of Saurus sinensis root containing sorcerneal B and manassantin B (Lee et al. al. 2004, Bioorg. Med. Chem. Lett. 14:3109-3112), and derivatives of anilide, ureido or diphenylimidazole compounds (PCT / US2014 / 054917).

[0026] As used herein, the terms "treat," "treating," or "treatment" refer to a method of alleviating, alleviating, or ameliorating the symptoms of a disease or condition, preventing further symptoms, ameliorating or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or condition, arresting the onset of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, alleviating the conditions caused by the disease or condition, or prophylactically and / or therapeutically arresting the symptoms of a disease or condition.

[0027] As used herein, the term "subject" or "patient" includes mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0028] As used herein, the terms "administration" or "administering" a subject compound refers to providing a compound of the present invention and / or a prodrug thereof to a subject in need of treatment.

[0029] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the administration of a sufficient quantity of an active ingredient described herein to relieve to some extent one or more symptoms of the disease or condition being treated. This result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound as disclosed herein required to provide a clinically significant reduction in disease symptoms. An appropriate "effective" amount in any individual case may be determined using techniques such as dose escalation studies. By way of example only, therapeutically effective amounts of the compounds of the present invention range, for example, from about 0.01 mg / kg / day to about 1000 mg / kg / day, from about 0.1 mg / kg / day to about 500 mg / kg / day, or from about 0.1 mg (×2) / kg / day to about 500 mg (×2) / kg / day.

[0030] Furthermore, such compounds and compositions may be administered alone or in combination with one or more additional therapeutic agents.The administration methods of such compounds and compositions may include, but are not limited to, intravenous administration, inhalation, oral administration, rectal administration, parenteral administration, intravitreal administration, subcutaneous administration, intramuscular administration, intranasal administration, transdermal administration, topical administration, ophthalmic administration, buccal administration, intratracheal administration, bronchial administration, sublingual administration, or intraocular administration.The compounds provided herein can be administered in known pharmaceutical formulations, including tablets, capsules, or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, lotions, gels, ointments, or creams for topical administration, etc.In some embodiments, such pharmaceutical compositions are formulated as tablets, pills, capsules, liquids, inhalants, nasal spray solutions, suppositories, solutions, gels, emulsions, ointments, eye drops, or ear drops.

[0031] The therapeutically effective amount may vary depending on the disease being treated, the severity of the disease, the age and relative health of the subject, the potency of the compound being administered, the mode of administration, and the treatment desired. The required dosage will also vary depending on the mode of administration, the particular condition being treated, and the effect desired.

[0032] The compounds described herein include all stereoisomers, geometric isomers, tautomers, isotopes, and prodrugs of the depicted structures. The compounds described herein may exist in various forms, including crystalline, powdered, and amorphous forms of the compounds, pharmaceutically acceptable salts, e.g., polymorphs, pseudopolymorphs, solvates, hydrates, nonsolvated polymorphs (including anhydrates), conformational variants, and amorphous forms, and mixtures thereof.

[0033] As used herein, the term "pharmaceutically acceptable" refers to a material, such as a carrier or diluent, that does not abolish the biological activity or properties of the compounds described herein. Such a material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0034] As used herein, the term "pharmaceutically acceptable salt" refers to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and that does not abolish the biological activity and properties of the compounds described herein.

[0035] The pharmaceutically acceptable salt form may include pharmaceutically acceptable acidic / anionic salts or basic / cationic salts (see UK Journal of Pharmaceutical and Biosciences Vol. 2(4), 01-04, 2014, incorporated herein by reference). Pharmaceutically acceptable acidic / anionic salts include acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycolylsanilate, hexylresorcinolate, hydrobromide, hydrochloride, hydroxynaphthoate, Pharmaceutically acceptable basic / cationic salts include sodium, potassium, calcium, magnesium, diethanolamine, N-methyl-D-glucamine, L-lysine, L-arginine, ammonium, ethanolamine, piperazine, and triethanolamine salts.

[0036] Pharmaceutically acceptable acid addition salts of the compounds of the present invention can be prepared by methods known in the art and can be formed by reacting the free base form of the compound with a suitable inorganic or organic acid, including, but not limited to, hydrobromic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, succinic acid, maleic acid, formic acid, acetic acid, propionic acid, fumaric acid, citric acid, tartaric acid, lactic acid, benzoic acid, salicylic acid, glutamic acid, aspartic acid, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, ethanesulfonic acid, naphthalenesulfonic acids such as 2-naphthalenesulfonic acid, and hexanoic acid. Pharmaceutically acceptable acid addition salts may include or be, for example, hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, phosphate, succinate, maleate, formate, acetate, propionate, fumarate, citrate, tartrate, lactate, benzoate, carbonate, benzathine, chloroprocaine, choline, histidine, meglumine, procaine, triethylamine, besylate, decanoate, ethylenediamine, salicylate, glutamate, aspartate, p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate, naphthalenesulfonate (e.g., 2-naphthalenesulfonate), and hexanoate salts.

[0037] Pharmaceutically acceptable base addition salts of the compounds of the present invention can also be prepared by methods known in the art and can be formed by reacting the free base form of the compound with a suitable inorganic or organic base, including, but not limited to, hydroxides or other salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, tromethamine, glycolate, hydrabamine, methyl bromide, methyl nitrate, octanoate, oleate, and the like.

[0038] The free acid or free base forms of the compounds of the present invention may be prepared by methods known in the art (e.g., see L.D. Bigley, S.M. Berg, D.C. Monkhouse, "Encyclopedia of Pharmaceutical Technology," Eds., J. Swarbrick and J.C. Boylam, Vol. 13, Marcel Dekker, Inc., 1995, pp. 453-499, incorporated herein by reference, for further details). For example, a compound of the present invention in the form of an acid addition salt may be converted to the corresponding free base form by treatment with a suitable base (e.g., aqueous ammonium hydroxide, sodium hydroxide, etc.). A compound of the present invention in the form of a base addition salt may be converted to the corresponding free acid by treatment with a suitable acid (e.g., hydrochloric acid, etc.).

[0039] Embodiments of the present disclosure include prodrug forms of any of the compounds described herein. Any convenient prodrug form of the subject compound can be prepared according to the strategies and methods described, for example, by Rautio et al., "Prodrugs: Design and Clinical Applications," Nature Reviews Drug Discovery 7, 255-270 (February 2008).

[0040] Prodrug derivatives of the compounds of the invention may be prepared by methods known to those skilled in the art (see, for example, Saulnier et al., Bioorg Med. Chem. Letters, 1994, 4, 1985, which is incorporated herein by reference, for further details). Protected derivatives of the compounds of the invention may be prepared by means known to those skilled in the art. A detailed description of the techniques applicable to the creation of protecting groups and their removal can be found in T.W. Greene, "Protecting Groups in Organic Chemistry," 3rd edition, John Wiley and Sons, Inc., 1999, which is incorporated herein by reference, and in "Design of Prodrugs", ed. 11. Bundgaard, Protecting Groups in Organic Chemistry, Elsevier, 1985.

[0041] The compounds of the present disclosure may be prepared as stereoisomers. If the compounds possess at least one chiral center, they may exist as enantiomers. If the compounds possess two or more chiral centers, they may exist as diastereomers. The compounds of the present invention may be prepared as racemic mixtures. Alternatively, the compounds of the present invention may be prepared as individual enantiomers or diastereomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of diastereoisomeric compounds, separating the diastereomers, and recovering the optically pure enantiomer. Separation of enantiomers may be accomplished using covalent diastereomeric derivatives of the compounds of the present invention or dissociable complexes (e.g., crystalline diastereomeric salts). Diastereomers have different physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and can be readily separated by exploiting these dissimilarities. Diastereomers can be separated by chromatography or separation / resolution techniques based on differential solubility. The optically pure enantiomers are then recovered along with the resolving agent by any practical means that does not result in racemization. A more detailed description of the techniques applied to the separation of stereoisomers of compounds from racemic mixtures can be found in the book Jean Jacques, Andre Collet and Samuel H. Wilen, "Enantiomers, Racemates and Separations", John Wiley and Sons, Inc., 1981, which is incorporated herein by reference.

[0042] The compounds of the present invention may be prepared as solvates (e.g., hydrates). The term "solvate" refers to a complex of variable stoichiometry formed by a solute (e.g., a compound of the present invention or a pharmaceutically acceptable salt thereof) and a solvent. For the purposes of the present invention, such solvents do not interfere with the biological activity of the solute. Non-limiting examples of suitable solvents include water, acetone, methanol, ethanol, and acetic acid. Preferably, the solvent used is a pharmaceutically acceptable solvent.

[0043] Additionally, the compounds of the present invention may be prepared in crystalline form, which may exist as polymorphs.

[0044] In view of the close relationship between the compounds of the present invention and their other forms, it should be noted that whenever a compound is referred to in this context herein, the corresponding salts, diastereomers, enantiomers, racemates, crystals, polymorphs, prodrugs, hydrates, or solvates are also intended, where possible or appropriate under the particular circumstances.

[0045] Another aspect of the present invention provides a composition for controlling body weight, comprising a therapeutically effective amount of one or more ACAT inhibitors and a therapeutically effective amount of one or more GLP-1RAs.

[0046] As used herein, the term "composition" is intended to encompass products that contain a therapeutically effective amount of a compound, salt, diastereomer, enantiomer, racemate, hydrate, solvate, or pharmaceutical combination thereof, as well as other products that are derived directly or indirectly from the claimed compound, salt, diastereomer, enantiomer, racemate, hydrate, solvate, or pharmaceutical combination thereof.

[0047] As used herein, the term "pharmaceutical composition" refers to a mixture of a therapeutically active ingredient (ingredient) with one or more other ingredients, which may be chemically or biologically active or inactive. Such ingredients include, but are not limited to, carriers, stabilizers, diluents, dispersing agents, suspending agents, thickeners, excipients, and adjuvants.

[0048] As used herein, the term "pharmaceutical combination" refers to a product resulting from the mixing or combining of two or more therapeutically active ingredients.

[0049] As used herein, the term "acceptable" with respect to a formulation, composition, or ingredient means that it has no lasting adverse effects on the general health of the subject being treated.

[0050] As used herein, the term "carrier" refers to a chemical or biological material that can facilitate the incorporation of one or more therapeutically active ingredients into cells or tissues.

[0051] Suitable excipients may include, for example, water, paraffin (e.g., petroleum fractions), vegetable oils (e.g., peanut oil or sesame oil), pharmaceutically acceptable organic solvents such as monofunctional or polyfunctional alcohols (e.g., ethanol or glycerol), natural mineral powders (e.g., kaolin, clay, talc, chalk), synthetic mineral powders (e.g., highly dispersed silicic acid and silicates), sugars (e.g., cane sugar, lactose, glucose), emulsifiers (e.g., lignin, spent sulfite liquor, methylcellulose, starch, and polyvinylpyrrolidone), and lubricants (e.g., magnesium stearate, talc, stearic acid, sodium lauryl sulfate, and the like).

[0052] Any suitable pharmaceutically acceptable carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, and adjuvants known to those skilled in the art for use in pharmaceutical compositions may be selected and used in the compositions described herein. The compositions described herein may be in the form of a solid, liquid, or gas (aerosol). For example, they may be in the form of tablets (coated tablets) made from Kollidon or shellac, gum arabic, talc, titanium dioxide, or sugar, capsules (gelatin), solutions (aqueous or aqueous ethanolic solutions), syrups containing active substances, emulsions, or inhalable powders (mixtures of various sugars, such as lactose or glucose, salts, and these excipients), and aerosols (inhalable solutions with or without propellants). The compositions described herein may also be formulated for sustained or delayed release.

[0053] Other embodiments and uses will be apparent to those skilled in the art in light of the present disclosure. The following examples are provided merely as illustrations of various embodiments and should not be construed as limiting the invention in any way. [Example]

[0054] Example 1: Materials and Methods 1. Animal care and drug administration Cohort 1: The first set of animal experiments was conducted at the ChemOn Inc. nonclinical laboratory, and the experimental procedures were approved by the Animal Care and Use Committee (serial number: 2021-09-007). Eight-week-old male C57BL / 6 mice were purchased from Orient Bio Inc., Korea. After 2 weeks of acclimation, they were fed a high-fat diet (HFD) (catalog number: D12492, Research Diets Inc.) for 12 weeks to allow DIO to develop. These mice were then randomly assigned to a control group, avasimibe group, semaglutide group, or combination group. Avasimibe solution was prepared to a final concentration of 10 mg / kg body weight as described in Y. Zhu, S. Q. Kim, Y. Zhang, Q. Liu, and K. H. Kim, "Pharmacological inhibition of acyl-coenzyme A: cholesterol acyltransferase attenuates obesity and insulin resistance in diet-induced obese mice by modulating food intake," Metabolism 123 (2021) 154861. Semaglutide stock solution, prepared in DMSO (1 mg / ml), was diluted with PBS to a final concentration of 0.04 mg / kg body weight. Mice were injected subcutaneously with this drug solution or vehicle solution at a volume of 10 μl / g body weight daily for the indicated days.

[0055] Cohort 2: The second set of animal experiments was conducted in accordance with a protocol approved by the Purdue University Animal Care and Use Committee (Protocol Number: 11129000347). Male C57BL / 6 DIO mice were purchased from the Jackson Laboratory. After 8 weeks of HFD treatment, mice were randomly assigned to the same groups as in Cohort 1: control, avasimibe, semaglutide, and combination treatment. Avasimibe (20 mg / kg body weight) and semaglutide (0.04 mg / kg body weight) were prepared as in Cohort 1, and the drugs were injected subcutaneously every 3 days for 23 days. Mice were housed in a humidity- and temperature-controlled facility under a 12-hour / 12-hour light / dark cycle with free access to food and water.

[0056] 2.Body composition and blood analysis Immediately before sacrificing the second cohort of mice, changes in body composition (e.g., fat mass, lean body mass, free water, and total water) were assessed using EchoMRI (Echo Medical Systems). Blood samples were collected after a 3-h fast and stored frozen until analysis. Aspartate transaminase (AST), alanine aminotransferase (ALT), glucose, triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were also measured (AU680 Chemistry Analyzer, Beckman Coulter). Serum leptin (catalog number: ab100718, Abcam) and insulin (catalog number: ab277390, Abcam) were determined by enzyme-linked immunosorbent assay kit, and cholesterol / CE (catalog number: ab65359, Abcam) was determined by enzymatic assay according to the manufacturer's instructions.

[0057] 3. Oral glucose tolerance test (OGTT) Mice were fasted for 6 h (07:00–13:00) before oral administration of D-glucose (2 g / kg BW). Tail blood samples were collected before glucose loading (0 min) and at 15, 30, 60, 90, and 120 min after glucose loading, and blood glucose levels were measured using a CONTOUR® NEXT glucometer (Ascensia Diabetes Care, Parsippany, NJ, USA).

[0058] 4. Tissue Harvesting and Histological Measurement of Adipocyte Size Upon animal sacrifice, the adipose tissue was washed with saline, fixed in 10% neutral formalin buffer, and embedded in paraffin. The tissue was then cut into 4 μm sections, stained with hematoxylin and eosin (H&E), and cell size (~100 cells / mouse) was measured using ImageJ software (NIH).

[0059] 5.Statistical analysis Data are presented as mean ± SEM, and analyses were performed using GraphPad Prism 9.4.1 (GraphPad). Comparisons between experimental and control groups were performed by one-way ANOVA (Figures 1C, 2C, 2E, 2F, 2G, 3A, 3B, 3G) or two-way ANOVA (Figures 1B, 1D, 2B, 2D), followed by Tukey's post-hoc test. Differences were considered significant at p ≤ 0.05. For comparisons between two groups, Student's t-tests were performed in some of Figures 1B and 1E.

[0060] Example 2: Daily Administration—Body Weight and Food Intake of DIO Mice Cohort 1 mice were used to investigate the effects of combined administration of semaglutide and low-dose avasimibe compared with semaglutide injection alone in morbidly obese mice (Figure 1A). On day 1 of the experiment, the average mouse weight was recorded at 48.84 ± 2.26 g (Figure 1B). After 4 weeks of treatment, the weights of mice in the semaglutide and combination groups reached a plateau and were significantly lower than those of mice maintained on a normal diet, so the mice were sacrificed (data not shown). Mice in the vehicle control and avasimibe groups were treated for an additional 16 days before sacrifice (Figure 1A). Over the first 4 weeks, the average weight of vehicle-treated animals increased by 1.2% compared to their starting weight. In contrast, the mean weight changes in mice treated with avasimibe, semaglutide, or the combination of both drugs were -6.29%, -21.66%, and -30.47% of their starting weight, respectively (Figure 1C). Notably, administration of a low dose of avasimibe (10 mg / kg BW) alone resulted in only a modest weight loss compared to our previous findings, in which a 24% weight loss was achieved by daily intraperitoneal administration of 20 mg / kg BW avasimibe for 14 days (see references Y. Zhu, S. Q. KIM, Y. Zhang, Q. Liu, K. H. KIM, 2014, incorporated herein by reference). "Pharmacological inhibition of acyl-coenzyme A:cholesterol acyltransferase attenuates obesity and insulin resistance in diet-induced obese mice by modulating food intake," Metabolism 123 (2021) 154861. Consequently, in subsequent studies using a second cohort of mice, the dose of avasimibe was increased to 20 mg / kg BW. The weight loss observed after semaglutide and combination therapy was attributed to a rapid suppression of food intake compared with mice treated with vehicle or avasimibe alone (Figure 1D). However, as the treatment regimen continued, food intake gradually returned to baseline levels, and the effect of semaglutide diminished around day 26. Furthermore, organ weight measurements confirmed that all fat depots sampled were reduced in mice treated with the combination therapy compared with those treated with semaglutide (Figure 1E). Although kidney weights showed no significant differences, liver and brain weights were significantly increased in the combination group compared with the semaglutide group (Figure 1E). To investigate the potential toxic effects of combination therapy on the liver, plasma ALT and AST levels were measured. Both ALT and AST levels were higher in the combination group compared with the semaglutide group and were similar to those seen in the vehicle control group (Table 1). These results suggest that repeated subcutaneous administration of semaglutide and avasimibe resulted in greater weight loss, primarily due to a reduction in fat mass in DIO mice.

[0061] [Table 1]

[0062] Values ​​are mean ± SEM of n=10 mice / group (ALT, alanine aminotransferase; AST, aspartate transaminase; CE, cholesteryl ester; HDL, high-density lipoprotein; LDL, low-density lipoprotein; NS, not significant; TG, triglycerides). For comparisons between two groups sacrificed on the same day, values ​​were analyzed using Student's t-test. * indicates a significant difference between the vehicle and avasimib groups sacrificed on day 43, and § indicates a significant difference between the semaglutide and combination groups.

[0063] Example 3: Cholesterol and leptin levels in daily dosing-DIO mice To assess whether avasimib could enhance the plasma lipoprotein profile and major lipid components associated with obesity, plasma samples from mice in Cohort 1 were analyzed. Daily subcutaneous administration of avasimib at 10 mg / kg BW for 6 weeks did not significantly reduce cholesterol or CE levels compared with vehicle administration (Table 1). However, 4 weeks of combination treatment significantly reduced both free cholesterol and CE levels compared with semaglutide alone (Table 1). Because mice naturally lack CE transfer protein and store most of their cholesterol in HDL, the reduction in circulating cholesterol levels in the combination group was also reflected in HDL levels. (References Z. Kaabia, J. Poirier, M. Moughaizel, A. Aguesse, S. Billon-Crossouard, F. Fall, M. Durand, E. Dagher, M. Krempf, M. Croyal, "Plasma lipid analysis reveals strong similarities in lipid fingerprints of humans, hamsters, and mice compared with other animal species," Sci Rep. 8 (2018) 15893.) Interestingly, avasimib did not improve triglyceride (TG) levels; rather, they increased in the combination group compared with the semaglutide group, reaching levels comparable to those in the vehicle control group (Table 1). Furthermore, circulating leptin levels were significantly lower in the combination group compared with the semaglutide group, indicating a reduction in the fat fraction in adipose tissue. In this study, the avasimib group showed lower glucose levels compared with the vehicle group, and the combination group showed lower glucose levels compared with the semaglutide group, but the difference in insulin levels did not reach statistical significance (Table 1). In summary, repeated subcutaneous administration of semaglutide and avasimib in combination proved effective in lowering circulating cholesterol and glucose levels without improving TG levels. The reduction in fat mass with combination administration was further supported by the observation that plasma leptin levels were lower in DIO mice.

[0064] Example 4: Every 3 Days Dosing - Body Weight and Food Intake of DIO Mice To examine the effects of combined semaglutide and avasimibe administration in DIO mice and mimic the once-weekly semaglutide regimen in humans, we utilized Cohort 2 mice (Figure 2A). On day 1, the average mouse weight was recorded at 37.37 ± 3.58 g. During the treatment period, mice in the vehicle group continued to gain weight, reaching an average weight of 41.58 ± 4.74 g, a 10.07% increase from their starting weight (Figure 2B). In contrast, mice receiving subcutaneous administration of avasimibe alone or in combination with semaglutide every three days successfully prevented weight gain induced by the HF diet. Specifically, the average weight loss in mice treated with avasimibe, semaglutide, and the combination of both drugs was 0.82%, 8.56%, and 14.89% of their starting weight, respectively (Figure 2C). Notably, each administration of semaglutide initially reduced food intake, which then recovered within 48 hours. The compensatory effect on food intake became more pronounced by the seventh administration, and by day 22, food intake in the semaglutide and combination groups exceeded that of the vehicle group (Figure 2D). However, cumulative food intake analysis revealed that mice in the combination group ate less food than mice in the vehicle group throughout the entire experimental period (Figure 2E). Next, to verify the suggested improvement in glucose metabolism by avasimibe, an OGTT was performed (Figures 2F and 2G). Subcutaneous administration of avasimibe alone every 3 days did not enhance glucose tolerance or amplify the hypoglycemic effect of semaglutide. These results suggest that the combination of semaglutide and avasimibe resulted in a greater percentage of weight loss compared to the effect observed with semaglutide alone in moderately obese mice, which showed little improvement in glucose tolerance.

[0065] Example 5: Fat mass and adipocyte size in DIO mice treated every 3 days Next, we tested whether the enhanced weight loss observed in the combination group was mediated by avasimib-induced reduction in fat mass. Only the combination group showed a significant reduction in fat mass compared to the vehicle group (Figure 3A). However, when examining the weights of different adipose tissues, no significant differences were observed between groups due to the high variability (Figure 3B). Furthermore, we found that fat mass correlated more strongly with body weight than lean mass (Figure 3C and Figure 3D). Among various adipose tissues, the weight of inguinal adipose tissue best represented total fat mass (Figure 3E). Because hypertrophic adipocytes are known to exhibit impaired insulin response and secrete proinflammatory cytokines [16-18], we analyzed digital images of H&E-stained inguinal fat layers (Figure 3F and Figure 3G). The avasimib group showed a significantly higher proportion of small adipocytes (5,001-10,000 μm²) and a lower proportion of large adipocytes (20,001-100,000 μm²) compared with the vehicle group. The semaglutide group showed a lower proportion of large adipocytes (40,001-100,000 μm²) compared with the vehicle group. However, no significant changes in adipocyte size distribution were observed in the combination group. Taken together, these findings suggest that combination treatment reduced fat mass in DIO mice, in part due to a reduction in adipocyte size by avasimib.

[0066] Those skilled in the art will recognize that numerous variations can be made to the specific embodiments described above. The embodiments should not be limited to the specific limitations set forth. Other embodiments are possible.

[0067] The scope of the method and apparatus is intended to be defined by the following claims. However, it should be understood that the present disclosure may be practiced otherwise than as specifically described and illustrated without departing from its spirit or scope. Those skilled in the art should understand that various alternatives to the embodiments described herein may be employed in implementing the following claims without departing from the spirit and scope thereof as defined by the claims.

Claims

1. A method for controlling body weight, comprising co-administering to a subject in need thereof a therapeutically effective amount of one or more ACAT inhibitors or pharmaceutically acceptable salts thereof and a therapeutically effective amount of one or more GLP-1RAs or pharmaceutically acceptable salts thereof.

2. 2. The method of claim 1, wherein the GLP-1RA is selected from the group consisting of lixisenatide, liraglutide, exenatide, exenatide extended release, albiglutide, semaglutide, ITCA650, dulaglutide, tirzepatide, retatortide, orfogliplon, rotigliplon, efpeglenatide, and taspoglutide.

3. The ACAT inhibitor is selected from the group consisting of avasimibe (CI-1011), CI-976, CP113,818, pactimibe, NTE-122, F-1394, PD140296, PD128042, PD132301-2, octimibate, DuP128, 58-035, HL-004, SMP-500, CL-277,082, SKF-99085, and CS-5 05, eflucimibe (F12511), E5324, FR145237, CL277,082, YM-17E, FR129169, K-604, pyrocarbonate, beauveriolide I, and a methanol extract of the root of Saurus sinensis containing sorcerneol B and manassantin B.

4. 10. The method of claim 1, wherein the GLP-1RA and the ACAT inhibitor are formulated and the formulation is administered.

5. 10. The method of claim 1, wherein the GLP-1RA and the ACAT inhibitor are formulated separately and administered simultaneously or sequentially.

6. 2. The method of claim 1, wherein the GLP-1RA is selected from the group consisting of semaglutide, liraglutide, and tirzepatide, and the ACAT inhibitor is avasimibe or a pharmaceutically acceptable salt thereof.

7. an ACAT inhibitor or a pharmaceutically acceptable salt thereof; GLP-1RA or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable excipient or carrier.

8. 8. The pharmaceutical composition of claim 7, wherein the GLP-1RA is selected from the group consisting of lixisenatide, liraglutide, exenatide, exenatide extended-release, albiglutide, semaglutide, ITCA650, dulaglutide, tirzepatide, retatortide, orfogliplon, rotigliplon, efpeglenatide, and taspoglutide.

9. The ACAT inhibitor is selected from the group consisting of avasimibe (CI-1011), CI-976, CP113,818, pactimibe, NTE-122, F-1394, PD140296, PD128042, PD132301-2, octimibate, DuP128, 58-035, HL-004, SMP-500, CL-277,082, SKF-99085, and CS-505. , eflucimibe (F12511), E5324, FR145237, CL277,082, YM-17E, FR129169, K-604, pyrocarbonate, beauveriolide I, and a methanol extract of the root of Saurus sinensis containing saucerneol B and manassantin B.

10. 8. The pharmaceutical composition of claim 7, wherein the GLP-1RA is selected from the group consisting of semaglutide, liraglutide, and tirzepatide, and the ACAT inhibitor is avasimibe or a pharmaceutically acceptable salt thereof.

Images