Methods and compositions for inducing brown adipogenesis

Abstract

The present disclosure relates to compositions, methods and kits for the treatment of metabolic diseases such as diabetes and obesity.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 022,640, filed May 11, 2020, the entire contents of which are incorporated by reference, including all figures, amino acid or nucleic acid sequences.

[0002] The Sequence Listing of the present application is labeled "Seq-List.txt," was created on May 9, 2020, and is 2 KB. The entire contents of the Sequence Listing are incorporated herein by reference in their entirety.

[0003] Technical Field The present disclosure relates to compositions and methods related to enhancing brown adipocytes and / or brown adipocyte mass in conditions such as type 2 diabetes, obesity, insulin resistance, and dyslipidemia. Specifically, the present disclosure identifies and describes compounds that increase the differentiation of brown adipose tissue (BAT) progenitor cells isolated from skeletal muscle (CD34+ cells) into brown adipocytes. Additionally, the present disclosure identifies and describes compounds that interact with gene products involved in regulating brown adipocyte differentiation and / or mass. Furthermore, the present disclosure provides methods for the identification and therapeutic use of compounds for the prevention and treatment of type 2 diabetes, obesity, insulin resistance, and dyslipidemia. The present disclosure is useful for the study, prevention, and treatment of various metabolic diseases, such as obesity, type 2 diabetes, insulin resistance, and dyslipidemia. [Background technology]

[0004] The obesity epidemic is closely linked to the increasing prevalence of diabetes, hypertension, coronary heart disease, cancer, and other diseases. The role of white adipose tissue ("BAT") is to store lipids and is associated with obesity. The role of brown adipose tissue ("BAT") is, in effect, the opposite: it is specialized for lipid combustion and dissipation of energy as heat. Indeed, brown adipocytes contain numerous mitochondria (where cellular combustion occurs) and uniquely express uncoupling protein-1 ("UCP1"). UCP1 acts as an uncoupler of oxidative phosphorylation, leading to energy dissipation as heat. The sympathetic nervous system stimulates mitochondrial biogenesis, UCP1 expression, and activity. BAT-associated thermogenesis in rodents increases during cold exposure (e.g., to prevent hypothermia) or as a result of overeating, burning excess absorbed fat, and preventing weight gain. BAT also improves insulin sensitivity by altering susceptibility to weight gain and consuming large amounts of glucose. It therefore plays an important role in maintaining body temperature, energy balance and glucose metabolism.

[0005] Experiments using transgenic animals support the potential anti-obesity properties of BAT. For example, genetic ablation of BAT has been reported to cause obesity, whereas genetic increase in the amount and / or function of BAT (and / or UCP1 expression) has been reported to promote a lean and healthy phenotype. Specifically, mice with large amounts of BAT weigh less and are more insulin sensitive than control mice. Recently, ectopic BAT depots have been demonstrated in mouse muscle, which has been shown to provide a genetic mechanism for protection from weight gain and metabolic syndrome.

[0006] UCP1 has been reported to play a role in the regulation of energy balance in rodents, and although UCP1-expressing BAT is present in newborns, it has long been thought that physiologically relevant UCP1 expression was absent in adults. Indeed, UCP1-expressing BAT was thought to disappear during early life, and adults were thought to lack BAT. However, recently, numerous studies have demonstrated that BAT is indeed maintained in most adult humans, albeit at levels significantly lower than those in newborns and children.

[0007] Therefore, there is a need to carefully identify and study ways to provide more BAT and / or stimulate UCP1 expression in the adult human body for the study, prevention, and treatment of various metabolic diseases such as obesity, type 2 diabetes, insulin resistance, dyslipidemia, and type 1 diabetes. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] PCT International Patent Application No. PCT / US2009 / 003217 [Patent Document 2] PCT / US2012 / 064366 [Patent Document 3] PCT International Patent Application No. PCT / US2015 / 017392 [Patent Document 4] U.S. Patent No. 6,537,806 [Non-patent literature]

[0009] [Non-Patent Document 1] M. Klingenspor, Cold-induced recruitment of brown adipose tissue thermogenesis., Exp Physiol. 88 (2003) 141-148. [1] [Non-patent document 2] B. Cannon, J. Nedergaard, The biochemistry of an inefficient tissue: brown adipose tissue, Essays Biochem. 20 (1985) 110–164. [2]

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[18] [Non-Patent Document 18] L. Lehr, K. Canola, C. Asensio, M. Jimenez, F. Kuehne, J.P. Giacobino, P. Muzzin, The control of UCP1 is dissociated from that of PGC-I alpha or of mitochondriogenesis as revealed by a study using beta-less mouse brown adipocytes in culture, FEBS Lett. 580 (2006) 4661-4666.

[19] [Non-Patent Document 19] O. Champigny, B. R. Holloway, D. Ricquier, Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist, Mol Cell Endocrinol. 86 (1992) 73-82.

[20] [Non-Patent Document 20] M. Jimenez, C. Yvon, L. Lehr, B. Leger, P. Keller, A. Russell, F. Kuhne, P. Flandin, JP Giacobino, P. Muzzin, Eur J Biochem. 269 ​​(2002) 2878–2884.

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[30] [Non-Patent Document 26] Yoneshiro T, Aita S, Matsushita M, Kayahara T, Kameya T, et al. (2013) Recruited brown adipose tissue as an antiobesity agent in humans. J Clin Invest.

[31] [Non-Patent Document 27] Vijgen GH, Sparks LM, Bouvy ND, Schaart G, Hoeks J, et al. (2013) Increased Oxygen Consumption in Human Adipose Tissue From the "Brown Adipose Tissue" Region. J Clin Endocrinol Metab.

[32] [Non-Patent Document 28] Vijgen GH, van Marken Lichtenbelt WD (2013) Brown adipose tissue: clinical impact of a re-discovered thermogenic organ. Front Biosci (Elite Ed) E5: 823-833.

[33] [Non-Patent Document 29] van der Lans AA, Hoeks J, Brans B, Vijgen GH, Visser MG, et al. (2013) Cold acclimation recruits human brown fat and increases nonshivering thermogenesis. The Journal of Clinical Investigation 123: 3395-3403.

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[38] [Non-Patent Document 34] Cannon B, Nedergaard J. Nonshivering thermogenesis and its adequate measurement in metabolic studies, J. Exp. Biol. 214 (2011), 242-253.

[39] Summary of the Invention

[0010] The present disclosure provides compositions for mobilizing brown adipocytes in vitro and in vivo from BAT progenitor cells found in human skeletal muscle. These agents, or combinations thereof, can be used to promote the differentiation of BAT progenitor cells into brown adipocytes and / or to induce the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells in vitro, in vivo, or both. Furthermore, these agents can be used to treat metabolic diseases, including obesity, excess body fat, overweight, diabetes, hyperglycemia, insulin resistance, hyperlipidemia, and other conditions in patients.

[0011] The present disclosure is based in part on the knowledge that various mechanisms are involved in the differentiation of BAT progenitor cells, and the hypothesis that by screening various compounds, it is possible to identify those that effectively recruit brown adipocytes. In particular, various compounds disclosed herein have been found to significantly induce the differentiation of BAT progenitor cells isolated from human skeletal muscle into mature, functional brown adipocytes. Treating BAT progenitor cells with one or more of these various compounds induces the commitment of these cells to brown adipocyte differentiation.

[0012] In addition, two different compounds used together were found to have an additive or synergistic effect on the differentiation of progenitor cells into brown adipocytes, an effect that was greater than the effect obtained with either compound alone.

[0013] In some cases, treatment with one or more compounds for 3 days prior to introduction of the adipogenic medium results in brown adipocyte differentiation. In other cases, treatment with one or more compounds for 3 days concurrent with introduction of the adipogenic medium results in brown adipocyte differentiation.

[0014] Because brown adipose tissue (BAT) is specialized for energy expenditure, the methods described herein are useful for treating obesity and related diseases such as diabetes. The methods can also be used to reduce fat stores in subjects, including food animals, for example, to improve the quality of meat derived therefrom.

[0015]

[0010] Accordingly, in one aspect, the disclosure features a method of treating a subject, e.g., a method of reducing fat stores or body weight in a subject, such as a human. The method includes administering a compound or combination of compounds disclosed herein to the subject. In a further aspect, the disclosure features a method of administering a population of compound-activated BAT progenitor cells, wherein the population of compound-activated progenitor cells undergoes brown adipogenesis. The method optionally includes identifying a subject in need of fat store or body weight reduction. In a further aspect, the disclosure includes a method of enhancing insulin sensitivity in a subject, e.g., a subject who is insulin resistant. The method includes administering a compound or a population of compound-activated BAT progenitor cells to the subject, wherein the population of compound-activated BAT progenitor cells undergoes brown adipogenesis. The method optionally includes identifying a subject in need of enhanced insulin sensitivity.

[0016] In another aspect, the disclosure features a method of modulating brown adipose tissue function or expression, e.g., promoting BAT adipogenesis, in a subject. The method includes administering to the subject a compound or population of compound-activated BAT progenitor cells, where the population of compound-activated progenitor cells undergoes brown adipogenesis.

[0017] As used herein, "compound activated" means that BAT progenitor cells have been treated with a compound described herein. The cells may be autologous, allogeneic, or xenogeneic.

[0018] In some embodiments, the methods described herein involve transplanting a population of compound-activated BAT progenitor cells into a subject. The compound-activated cells can be directly implanted or administered in a scaffold, matrix, or other implantable device to which the cells adhere (e.g., carriers made from collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharides, fibrin, gelatin, self-assembling small peptides, and combinations thereof). Generally, the methods involve transplanting a population of compound-activated BAT progenitor cells containing a sufficient number of cells to promote an increase in brown adipocyte mass in the subject, e.g., to increase the amount of brown adipocytes in the subject by at least 1%, e.g., 2%, 5%, 7%, 10%, 15%, 20%, 25%, or more.

[0019] In some embodiments, the method includes assessing the level of BAT adipogenesis in a subject by contacting BAT progenitor cells isolated from the subject with one or more compounds disclosed herein. BAT differentiation can be assessed by measuring BAT markers, such as uncoupling proteins (UCPs), specifically UCP-I, expression, BAT morphology (e.g., visually, specifically, using microscopic examination of cells), or BAT thermodynamics, such as cytochrome oxidase activity, Na+-K+-ATPase enzyme activity, or other enzymes involved in BAT thermogenesis.

[0020] Generally, the subject is a mammal. In some embodiments, the subject is a human, e.g., an obese human. In some embodiments, the subject is a non-human mammal, e.g., a laboratory animal, a companion animal, or a food animal raised for food, e.g., a cow, pig, or sheep. In some embodiments, the method includes assessing the subject for one or more of body weight, white adipose tissue reserves, brown adipose tissue reserves, adipose tissue morphology, insulin levels, insulin metabolism, glucose levels, thermogenic capacity, and cold sensitivity. Assessment can be performed before, during, and / or after administration of the compound or compound-activated BAT progenitor cells. For example, assessment can be performed at least 1 day, 2 days, 4 days, 7 days, 14 days, 21 days, or 30 days before and / or after administration.

[0021] In some embodiments, the method includes one or more additional rounds of treatment with a compound or transplantation of compound-activated BAT progenitor cells, e.g., to increase brown adipocyte mass, e.g., to maintain or further reduce obesity in the subject.

[0022] In some embodiments, the disclosure features a composition that includes (a) bezafibrate or an analog thereof, and (b) oxaprozin or an analog thereof, wherein the bezafibrate and oxaprozin or an analog thereof are present in an amount sufficient to treat, prevent, or reduce a metabolic disease (e.g., obesity or diabetes) when administered to a patient.

[0023] In other embodiments, the disclosure features a composition including (a) bezafibrate or an analog thereof, and (b) zaltoprofen or an analog thereof, wherein the bezafibrate and zaltoprofen or an analog thereof are present in an amount sufficient to treat, prevent, or reduce a metabolic disease (e.g., obesity or diabetes) when administered to a patient.

[0024] In yet another embodiment, the disclosure features a composition including (a) bezafibrate or an analog thereof, and (b) ozagrel or an analog thereof, wherein the bezafibrate and ozagrel or an analog thereof are present in an amount sufficient to treat, prevent, or reduce a metabolic disease (e.g., obesity or diabetes) when administered to a patient.

[0025] The compositions of the present disclosure are formulated for local or systemic administration. When two or more agents are used, the therapeutic agents may be delivered separately or mixed into a single formulation. When the agents are present in different pharmaceutical compositions, different routes of administration can be used. Routes of administration for various embodiments include, but are not limited to, topical, transdermal, and systemic administration (such as intravenous, intramuscular, subcutaneous, inhalation, rectal, buccal, vaginal, intraperitoneal, intraarticular, ocular, or oral administration). As used herein, "systemic administration" refers to all non-transdermal routes of administration and specifically excludes topical and transdermal routes of administration. Desirably, the agents of the present disclosure and the additional therapeutic agent are administered at least 1, 2, 4, 6, 10, 12, 18, 24 hours, 3 days, 7 days, 10 days, or 14 days apart. The dosage and frequency of each component of the combination can be independently controlled. For example, one compound can be administered three times daily, and the second compound can be administered once daily. Combination therapy is given in on-and-off cycles that include rest periods so that the patient's body has a chance to recover from any unexpected side effects. The compounds may also be formulated together to deliver both compounds in a single administration. Optionally, either of the agents in the combination can be administered at a low or high dose, each of which is defined herein.

[0026] Generally, when administered to humans, the dosage of any of the drugs in the combination of the present disclosure varies depending on the nature of the drug and can be easily determined by one of ordinary skill in the art. Typically, the dosage is usually about 0.001 mg to 2000 mg per day, preferably about 1 mg to 1000 mg per day, and more preferably about 5 mg to 500 mg per day. Doses of up to 2000 mg per day may be necessary. Each drug in the combination is administered independently, one to four times daily for one day to one year, and may even be administered for the patient's survival. In many cases, chronic, long-term administration is indicated.

[0027] The therapeutic agents of the present disclosure are mixed with additional active or inactive ingredients, for example, in a conventional pharmaceutically acceptable carrier. Pharmaceutical carriers are any compatible, non-toxic substance suitable for administering the compositions of the present disclosure to mammals. Pharmaceutically acceptable carriers include, for example, water, saline, buffers, and other compounds described in the Merck Index, Merck & Co., Rahway, NJ. Sustained-release formulations or sustained-release devices may also be used for continuous administration.

[0028] When two or more drugs are used, each drug can be formulated in a variety of ways known in the art. Desirably, the drugs are formulated together for simultaneous or near-simultaneous administration. Such co-formulations can include two drugs formulated together in the same pill, capsule, liquid, etc. When referring to the formulation of such a combination, it should be understood that the formulation techniques used are also useful for the formulation of the individual drugs in the combination, as well as other combinations disclosed herein. By using different formulation strategies for different drugs, the pharmacokinetic profiles for each drug can be appropriately matched.

[0029] The disclosed methods are also used prophylactically in patients at high risk of developing obesity, diabetes, or conditions associated with obesity or diabetes, such as insulin resistance. Risk factors include, for example, family history of diabetes, obesity, or related conditions, quality of nutrition, level of physical activity, presence of molecular markers of obesity or diabetes, age, race, or gender. Patients suffering from other unrelated diseases are also predisposed to secondary obesity or diabetes.

[0030] The disclosure also features a method of treating, preventing, or reducing a metabolic disorder in a patient in need thereof by administering to the patient (i) bezafibrate or an analog thereof and (ii) oxaprozin or an analog thereof, wherein the bezafibrate and oxaprozin or an analog thereof are administered in amounts sufficient together to treat, prevent, or reduce the metabolic disorder.

[0031] The disclosure also features a method of treating, preventing, or reducing a metabolic disorder in a patient in need thereof by administering to the patient (i) bezafibrate or an analog thereof and (ii) zaltoprofen or an analog thereof, wherein the bezafibrate and zaltoprofen or an analog thereof are administered in amounts sufficient together to treat, prevent, or reduce the metabolic disorder.

[0032] The disclosure also features a method of treating, preventing, or reducing a metabolic disorder in a patient in need thereof by administering to the patient (i) bezafibrate or an analog thereof and (ii) ozagrel or an analog thereof, wherein the bezafibrate and ozagrel or an analog thereof are administered together in amounts sufficient to treat, prevent, or reduce the metabolic disorder.

[0033] Individually or separately formulated medications can be packaged together as a kit. Examples include, but are not limited to, kits containing two pills, a pill and a powder, a vial of suppositories and a liquid, two topical creams, etc. The kit can include any components that aid in administering unit doses to patients, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, inhalers, etc. Additionally, unit dose kits can include instructions for preparing and administering the compositions. Kits can be manufactured as single-use unit doses for a single patient, multiple uses for a specific patient (at fixed doses, or where individual compounds vary in potency as treatment progresses), or the kits contain multiple doses suitable for administration to multiple patients ("bulk packaging"). Kit components can be assembled into cartons, blister packs, bottles, tubes, etc.

[0034] The disclosure also features a kit including (i) bezafibrate or an analog thereof, and (ii) instructions for administering bezafibrate and oxaprozin or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0035] The disclosure also features a kit including (i) Oxaprozin or an analog thereof, and (ii) instructions for administering Oxaprozin and bezafibrate or an analog thereof to a patient having or at risk of having a metabolic disease.

[0036] The disclosure also features a kit including (i) bezafibrate or an analog thereof, and (ii) instructions for administering bezafibrate and zaltoprofen or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0037] The disclosure also features a kit including (i) zaltoprofen or an analog thereof, and (ii) instructions for administering zaltoprofen and bezafibrate or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0038] The disclosure also features a kit including (i) bezafibrate or an analog thereof, and (ii) instructions for administering bezafibrate and ozagrel or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0039] The disclosure also features a kit including (i) ozagrel or an analog thereof, and (ii) instructions for administering ozagrel and bezafibrate or an analog thereof to a patient having or at risk of having a metabolic disease.

[0040] The disclosure also features a kit including (i) a composition containing bezafibrate or an analog thereof and oxaprozin or an analog thereof, and (ii) instructions for administering the composition to a patient having or at risk of having a metabolic disorder.

[0041] The disclosure also features a kit including (i) a composition containing bezafibrate or an analog thereof and zaltoprofen or an analog thereof, and (ii) instructions for administering the composition to a patient having or at risk of having a metabolic disorder.

[0042] The disclosure also features a kit including (i) a composition containing bezafibrate or an analog thereof and ozagrel or an analog thereof, and (ii) instructions for administering the composition to a patient having or at risk of having a metabolic disorder.

[0043] The disclosure also features a kit including (i) bezafibrate or an analog thereof, (ii) oxaprozin or an analog thereof, and (iii) instructions for administering bezafibrate and oxaprozin or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0044] The disclosure also features a kit including (i) bezafibrate or an analog thereof, (ii) zaltoprofen or an analog thereof, and (iii) instructions for administering bezafibrate and zaltoprofen or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0045] The disclosure also features a kit including (i) bezafibrate or an analog thereof, (ii) ozagrel or an analog thereof, and (iii) instructions for administering bezafibrate and its ozagrel analog to a patient having or at risk of having a metabolic disorder.

[0046] The disclosure also features compositions that include (a) a PPAR agonist (activator), and (b) Oxaprozin, where the PPAR activator and Oxaprozin are present in amounts sufficient to treat, prevent, or reduce a metabolic disease when administered to a patient.

[0047] The present disclosure also features a composition comprising (a) a PPAR agonist, and (b) zaltoprofen or an analog thereof, wherein the PPAR agonist and zaltoprofen or an analog thereof are present in an amount sufficient to treat, prevent, or reduce a metabolic disease when administered to a patient.

[0048] The disclosure also features a composition including (a) a PPAR activator agonist and (b) ozagrel or an analog thereof, wherein the PPAR activator and ozagrel or an analog thereof are present in an amount sufficient to treat, prevent, or reduce a metabolic disease when administered to a patient.

[0049] The disclosure also features a method for treating, preventing, or reducing a metabolic disease in a patient in need thereof by administering to the patient (i) a PPAR agonist and (ii) Oxaprozin or an analog thereof, wherein the PPAR agonist and Oxaprozin or an analog thereof are administered together in amounts sufficient to treat, prevent, or reduce the metabolic disease.

[0050] The disclosure also features a method for treating, preventing, or reducing a metabolic disease in a patient in need thereof by administering to the patient (i) a PPAR agonist and (ii) zaltoprofen or an analog thereof, wherein the PPAR agonist and zaltoprofen or an analog thereof are administered together in amounts sufficient to treat, prevent, or reduce the metabolic disease.

[0051] The disclosure also features a method for treating, preventing, or reducing a metabolic disease in a patient in need thereof by administering to the patient (i) a PPAR agonist and (ii) ozagrel or an analog thereof, wherein the PPAR agonist and ozagrel or an analog thereof are administered in amounts sufficient together to treat, prevent, or reduce the metabolic disease.

[0052] The disclosure features a kit that includes (i) a PPAR agonist and (ii) instructions for administering the PPAR agonist and Oxaprozin or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0053] The disclosure features a kit including (i) Oxaprozin or an analog thereof, and (ii) instructions for administering Oxaprozin or an analog thereof and a PPAR agonist to a patient having or at risk of having a metabolic disease.

[0054] The present disclosure features a kit including (i) a PPAR agonist and (ii) instructions for administering the PPAR agonist and zaltoprofen or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0055] The present disclosure features a kit including (i) zaltoprofen or an analog thereof, and (ii) instructions for administering zaltoprofen or an analog thereof and a PPAR agonist to a patient having or at risk of having a metabolic disorder.

[0056] The disclosure features a kit that includes (i) a PPAR agonist and (ii) instructions for administering the PPAR agonist and ozagrel or an analog thereof to a patient having or at risk of having a metabolic disorder.

[0057] The present disclosure features a kit including (i) ozagrel or an analog thereof, and (ii) instructions for administering ozagrel or an analog thereof and a PPAR agonist to a patient having or at risk of having a metabolic disorder.

[0058] The disclosure also features a kit including (i) a composition containing a PPAR agonist and Oxaprozin or an analog thereof, and (ii) instructions for administering the composition to a patient having or at risk of having a metabolic disease.

[0059] The disclosure also features a kit including (i) a composition containing a PPAR agonist and zaltoprofen or an analog thereof, and (ii) instructions for administering the composition to a patient having or at risk of having a metabolic disorder.

[0060] The disclosure also features a kit including (i) a composition containing a PPAR agonist and ozagrel or an analog thereof, and (ii) instructions for administering the composition to a patient having or at risk of having a metabolic disease.

[0061] The disclosure also features a kit including (i) a PPAR agonist, (ii) Oxaprozin or an analog thereof, and (iii) instructions for administering the PPAR agonist and Oxaprozin or an analog thereof to a patient having or at risk of having a metabolic disease.

[0062] The present disclosure also features a kit including (i) a PPAR agonist, (ii) zaltoprofen or an analog thereof, and (iii) instructions for administering the PPAR agonist and zaltoprofen or an analog thereof to a patient having or at risk of having a metabolic disease.

[0063] The disclosure also features a kit including (i) a PPAR agonist, (ii) ozagrel or an analog thereof, and (iii) instructions for administering the PPAR agonist and ozagrel or an analog thereof to a patient having or at risk of having a metabolic disease. [Brief explanation of the drawings]

[0064] [Figure 1] The effect of the maximum effective concentration of rosiglitazone (1 μM), BMP7 (6 nM), or the combination of both drugs (incubated with brown adipocyte precursor cells on days -3 to 0) on PPARγ2 mRNA expression is shown. p = 0.0001 for rosiglitazone vs. (vehicle), p = 0.0025 for BMP7 vs. (vehicle), p = 0.0001 for rosiglitazone + BMP7 vs. (vehicle), p = 0.0024 for rosiglitazone + BMP7 vs. rosiglitazone, and p = 0.0001 for rosiglitazone + BMP7 vs. BMP7 (unpaired t-test, two-tailed). [Figure 2] The effect of the maximum effective concentration of rosiglitazone (1 μM), BMP7 (6 nM), or the combination of both drugs (incubated with brown adipocyte precursor cells on days -3 to 0) on UCP1 mRNA expression is shown. p=0.021 for rosiglitazone-(vehicle), p=0.018 for BMP7 vs-(vehicle), p=0.0006 for rosiglitazone+BMP7 vs-(vehicle), p=0.045 for rosiglitazone+BMP7 vs rosiglitazone, p=0.004 for rosiglitazone+BMP7 vs BMP7. [Figure 3A] Fluorescence microscopy showing immunohistochemistry (IHC) assay results for UCP1 protein expression (FITC, green) and cell nuclei count (DAPI, blue). After exposure to rosiglitazone (1 μM), CD34+ cells differentiated in minimal differentiation medium (MDM) for 8 days significantly differentiate into brown adipocytes that express high levels of UCP1. [Figure 3B] Fluorescence microscopy showing immunohistochemistry (IHC) assay results for UCP1 protein expression (FITC, green) and cell nuclei number (DAPI, blue). Cells not exposed to rosiglitazone show very low levels of differentiation and UCP1 expression. [Figure 3C] Fluorescence microscopy showing immunohistochemistry (IHC) assay results for UCP1 protein expression (FITC, green) and cell nuclei count (DAPI, blue). Cells maintained in growth medium (EGM-2) do not differentiate or express UCP1. [Figure 4] BODIPY500 / 510C1, C12 fluorescence signals are shown after brown adipocyte precursor cells were induced to differentiate in culture for 9 days under various conditions (rosiglitazone and BMP7 were used from day -3 to day 0). [Figure 5] Fluorescence micrographs showing BODIPY assay results for intracellular lipid droplets (BODIPY500 / 510C1, C12, green). CD34+ cells were exposed to rosiglitazone (1 μM) for 3 days (days -3 to 0) and then differentiated in minimal differentiation medium (MDM) for 8 days. [Figure 6A] Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with vehicle, rosiglitazone. CD34+ cells were picked 6 days after the end of treatment and switched to MDM medium. [Figure 6B] Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with BMP7, rosiglitazone and BMP7 in combination. CD34+ cells were picked 6 days after the end of treatment and switched to MDM medium. [Figure 6C] Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with diflunisal and probenecid. CD34+ cells were picked 6 days after the end of treatment and switched to MDM medium. [Figure 6D] Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with tianeptine and zaltoprofen is shown. CD34+ cells were harvested 6 days after the end of treatment and switched to MDM medium. [Figure 6E] Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with ozagrel and gliquidone. CD34+ cells were harvested 6 days after the end of treatment and switched to MDM medium. [Figure 6F]Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with bezafibrate and alprostadil. CD34+ cells were harvested 6 days after the end of treatment and switched to MDM medium. [Figure 6G] Light microscopy of CD34+ cells and brown adipocyte differentiation after treatment with Oxaprozin, which promotes brown adipogenesis. CD34+ cells were picked 6 days after the end of treatment and switched to MDM medium. [Figure 7] Figure 1 shows the effect of glimepiride (0.1-10 μM), gliquidone (0.1-10 μM), or oxaprozin (1-50 μM) incubated with brown adipocyte precursor cells on days -3 to 0 on PPARγ2 mRNA expression. Rosiglitazone (1 μM), BMP7 (6 nM), or the combination of both drugs are used as a reference. [Figure 8] Figure 1 shows the effect of glimepiride (0.1-10 μM), gliquidone (0.1-10 μM), or oxaprozin (1-50 μM) incubated with brown adipocyte precursor cells on days -3 to 0 on UCP1 mRNA expression. Rosiglitazone (1 μM), BMP7 (6 nM), or the combination of both drugs are used as a reference. [Figure 9] 1 shows the effect of probenecid (50 μM), tianeptine (50 μM), alprostadil (10 μM), ozagrel (50 μM), zaltoprofen (50 μM), gliquidone (10 μM), or bezafibrate (50 μM) (incubated with brown adipocyte precursor cells on days −3 to 0) on PPARγ2 mRNA expression. [Figure 10] The effect of probenecid (50 μM), tianeptine (50 μM), alprostadil (10 μM), ozagrel (50 μM), zaltoprofen (50 μM), gliquidone (10 μM), or bezafibrate (50 μM) (incubated with brown adipocyte precursor cells on days −3 to 0) on UCP1 mRNA expression is shown. [Figure 11]Dose response of a compound (indomethacin) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. [Figure 12] Dose response of a compound (bezafibrate) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. [Figure 13]Dose response of a compound (glimepiride) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. [Figure 14] Dose response of a compound (ozagrel) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. [Figure 15]Dose response of a compound (diflunisal) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. [Figure 16] Dose response of a compound (alprostadil) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. [Figure 17]Dose response of a compound (tianeptine) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, after switching to MDM medium. Concentrations are in μM. [Figure 18] Dose response of a compound (probenecid) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) switched to MDM medium at the end of cell differentiation, approximately 6-12 days after the end of compound treatment. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, switched to MDM medium. Concentrations are in μM. [Figure 19]Dose response of a compound (gliquidone) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) switched to MDM medium at the end of cell differentiation, approximately 6-12 days after the end of compound treatment. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, switched to MDM medium. Concentrations are in μM. [Figure 20] Dose response of a compound (Oxaprozin) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, when cells were switched to MDM medium. Concentrations are in μM. [Figure 21]Dose response of a compound (zaltoprofen) that promotes brown adipogenesis (recruitment of CD34+ cells into brown adipocytes) in culture is shown. A. Adipocyte score determined by light microscopy of multilocular lipid-containing cells (per well) switched to MDM medium at the end of cell differentiation, approximately 6-12 days after the end of compound treatment. Concentrations are in μM. B. PPARγ2 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, switched to MDM medium. Concentrations are in μM. Y-axis units are percent of vehicle-treated cells. C. UCP1 mRNA expression (per well) at the end of cell differentiation, approximately 6-12 days after the end of compound treatment, switched to MDM medium. Concentrations are in μM. [Figure 22] 1 shows the effect of bezafibrate (115 mg / kg body weight, once daily by oral gavage) on body weight and body fat in DIO mice. [Figure 23] 1 shows the effect of bezafibrate (115 mg / kg BW, once daily by oral gavage for 3 weeks) on glucose tolerance in DIO mice. [Figure 24] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage) and diflunisal (100 mg / kg BW) on body weight and body fat in DIO mice. [Figure 25] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage for 3 weeks) and diflunisal (100 mg / kg BW) on glucose tolerance in DIO mice. [Figure 26] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage) and probenecid (100 mg / kg BW) on body weight and body fat in DIO mice. [Figure 27] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage for 3 weeks) and probenecid (100 mg / kg BW) on glucose tolerance in DIO mice. [Figure 28]1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage) and tianeptine (10 mg / kg BW) on body weight and body fat in DIO mice. [Figure 29] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage for 3 weeks) and tianeptine (10 mg / kg BW) on glucose tolerance in DIO mice. [Figure 30] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage) and glimepiride (0.6 mg / kg BW) on body weight and body fat in DIO mice. [Figure 31] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW, once daily by oral gavage for 3 weeks) and glimepiride (0.6 mg / kg BW) on glucose tolerance in DIO mice. [Figure 32] 1 shows the effect of a combination of zaltoprofen (50 mg / kg BW administered orally by gavage once daily) and glimepiride (0.6 mg / kg BW) on body weight and body fat in DIO mice. [Figure 33] 1 shows the effect of a combination of zaltoprofen (50 mg / kg BW, once daily by oral gavage for 3 weeks) and glimepiride (0.6 mg / kg BW) on glucose tolerance in DIO mice. [Figure 34] 1 shows the effect of a combination of probenecid (100 mg / kg body weight, once daily by oral gavage for 3 weeks) and ozagrel (30 mg / kg body weight) on glucose tolerance in DIO mice. [Figure 35] 1 shows the effect of a combination of probenecid (100 mg / kg body weight, once daily by oral gavage for 3 weeks) and tianeptine (10 mg / kg body weight) on glucose tolerance in DIO mice. [Figure 36] 1 shows the effect of a combination of tianeptine (10 mg / kg BW, once daily by oral gavage for 3 weeks) and glimepiride (0.6 mg / kg BW) on glucose tolerance in DIO mice. [Figure 37]Figure 1 shows the effect of bezafibrate (115 mg / kg body weight, once daily by oral gavage for 28 days) alone or in combination with diflunisal (100 mg / kg body weight), probenecid (100 mg / kg body weight), tianeptine (10 mg / kg body weight), or glimepiride (0.6 mg / kg body weight) on perigonadal (epididymal) fat depot size in DIO mice. [Figure 38] Figure 1 shows the effect of bezafibrate (115 mg / kg body weight, once daily by oral gavage for 28 days), alone or in combination with diflunisal (100 mg / kg body weight), probenecid (100 mg / kg body weight), tianeptine (10 mg / kg body weight), or glimepiride (0.6 mg / kg body weight), on plasma leptin levels in DIO mice. [Figure 39] Figure 1 shows the effect of bezafibrate (115 mg / kg body weight, once daily by oral gavage for 28 days) alone or in combination with diflunisal (100 mg / kg body weight), probenecid (100 mg / kg body weight), tianeptine (10 mg / kg body weight), or glimepiride (0.6 mg / kg body weight), or in combination with probenecid (100 mg / kg body weight) and ozagrel (30 mg / kg body weight) or diflunisal (100 mg / kg body weight), or in combination with zaltoprofen (50 mg / kg body weight) and glimepiride (0.6 mg / kg body weight) on plasma triglyceride levels in DIO mice. [Figure 40] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on inguinal subcutaneous fat mass (WATing) in DIO mice. [Figure 41] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on plasma leptin levels in DIO mice. [Figure 42] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body weight in DIO mice. [Figure 43] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body fat content in DIO mice. [Figure 44] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on inguinal subcutaneous fat mass (WATing) in DIO mice. [Figure 45] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on gonadal visceral adipose mass (WATing) in DIO mice. [Figure 46] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma leptin levels in DIO mice. [Figure 47] Baseline (pre-dose) levels of fed plasma insulin in DIO mice used to administer each agent are shown. [Figure 48] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on fed plasma insulin levels in DIO mice. [Figure 49] 1 shows the effect of a combination of rosiglitazone (3 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body weight in DIO mice. [Figure 50] 1 shows the effect of a combination of rosiglitazone (3 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body fat content in DIO mice. [Figure 51]1 shows the effect of a combination of rosiglitazone (3 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on inguinal subcutaneous fat mass (WATing) in DIO mice. [Figure 52] 1 shows the effect of a combination of rosiglitazone (3 mg / kg BW, once daily by oral gavage) and oxaprozin (50 mg / kg BW, once daily by oral gavage) on gonadal visceral fat pad mass (WATing) in DIO mice. [Figure 53] 1 shows the effect of a combination of rosiglitazone (3 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma leptin levels in DIO mice. [Figure 54] Baseline (pre-dose) levels of fed plasma insulin in DIO mice used to administer each agent are shown. [Figure 55] 1 shows the effect of a combination of rosiglitazone (3 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on fed plasma insulin levels in DIO mice. [Figure 56] 1 shows the effect of a combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on the body weight of DIO mice. [Figure 57] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on the body weight of DIO mice. [Figure 58] 1 shows the effect of combined administration of bezafibrate (115 mg / kg body weight, orally administered once daily) and zaltoprofen (25 mg / kg body weight, orally administered once daily) on the body weight of DIO mice. [Figure 59]1 shows the effect of a combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body weight in DIO mice. [Figure 60] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body weight in DIO mice. [Figure 61] 1 shows the effect of a combination of bezafibrate (30 mg / kg body weight, once daily by oral gavage) and zaltoprofen (25 mg / kg body weight, once daily by oral gavage) on body fat (measured by MRI) in DIO mice. [Figure 62] 1 shows the effect of a combination of bezafibrate (60 mg / kg body weight, once daily by oral gavage) and zaltoprofen (25 mg / kg body weight, once daily by oral gavage) on body fat (measured by MRI) in DIO mice. [Figure 63] 1 shows the effect of a combination of bezafibrate (115 mg / kg body weight, once daily by oral gavage) and zaltoprofen (25 mg / kg body weight, once daily by oral gavage) on body fat (measured by MRI) in DIO mice. [Figure 64] 1 shows the effect of a combination of bezafibrate (30 mg / kg body weight, once daily by oral gavage) and oxaprozin (50 mg / kg body weight, once daily by oral gavage) on body fat (measured by MRI) in DIO mice. [Figure 65] Figure 1 shows the effect of combined administration of bezafibrate (60 mg / kg body weight, once daily by oral gavage) and oxaprozin (50 mg / kg body weight, once daily by oral gavage) on body fat (measured by magnetic resonance imaging, MRI) in DIO mice. Bezafibrate (60) + oxaprozin (50) synergistically reduced body fat mass (p=0.048). [Figure 66] 1 shows the effect of a combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on plasma leptin levels in DIO mice. [Figure 67] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on plasma leptin levels in DIO mice. [Figure 68] 1 shows the effect of a combination of bezafibrate (115 mg / kg body weight, orally administered once daily) and zaltoprofen (25 mg / kg body weight, orally administered once daily) on plasma leptin levels in DIO mice. [Figure 69] 1 shows the effect of a combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma leptin concentrations in DIO mice. [Figure 70] Figure 1 shows the effect of the combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma leptin levels in DIO mice. The combination dramatically reduced leptin relative to vehicle, and the two drugs acted synergistically (p=0.001). [Figure 71] 1 shows the effect of a combination of bezafibrate (30 mg / kg BW by oral inhalation once daily) and zaltoprofen (25 mg / kg BW by oral inhalation once daily) on blood glucose levels in DIO mice. [Figure 72] 1 shows the effect of a combination of bezafibrate (60 mg / kg body weight / day, once daily by oral gavage) and zaltoprofen (25 mg / kg body weight / day, once daily by oral gavage) on blood glucose levels in DIO mice. [Figure 73] 1 shows the effect of a combination of bezafibrate (115 mg / kg body weight, once daily by oral gavage) and zaltoprofen (25 mg / kg body weight, once daily by oral gavage) on blood glucose levels in DIO mice. [Figure 74] 1 shows the effect of a combination of bezafibrate (30 mg / kg body weight, once daily by oral gavage) and oxaprozin (50 mg / kg body weight, once daily by oral gavage) on blood glucose levels in DIO mice. [Figure 75] 1 shows the effect of a combination of bezafibrate (60 mg / kg body weight, once daily by oral gavage) and oxaprozin (50 mg / kg body weight, once daily by oral gavage) on blood glucose levels in DIO mice. [Figure 76] 1 shows the effect of a combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on plasma insulin levels in DIO mice. [Figure 77] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on plasma insulin levels in DIO mice. [Figure 78] 1 shows the effect of a combination of bezafibrate (115 mg / kg body weight, orally administered once daily) and zaltoprofen (25 mg / kg body weight, orally administered once daily) on plasma insulin levels in DIO mice. [Figure 79] 1 shows the effect of a combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma insulin levels in DIO mice. [Figure 80] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma insulin levels in DIO mice. [Figure 81] Figure 1 shows the effect of the combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on insulin sensitivity in DIO mice, as determined using the homeostasis model assessment of insulin resistance (HOMA-IR): HOMA-IR = (fasting or fed plasma glucose [mM] × fasting or fed plasma insulin [microIU / ml]) / 22.5. [Figure 82]1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on insulin sensitivity in DIO mice as determined using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). [Figure 83] 1 shows the effect of a combination of bezafibrate (115 mg / kg BW once daily by oral gavage) and zaltoprofen (25 mg / kg BW once daily by oral gavage) on insulin sensitivity in DIO mice, as determined using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). [Figure 84] Figure 1 shows the effect of the combination of bezafibrate (30 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on insulin sensitivity in DIO mice as determined using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). [Figure 85] Figure 1 shows the effect of the combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on insulin sensitivity in DIO mice as determined using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). [Figure 86] 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body weight in DIO mice. [Figure 87] Effect of the combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on body fat (measured by medical resonance imaging, MRI) in DIO mice. Baseline p=0.2316, endpoint p=0.0249. [Figure 88]1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma leptin concentrations in DIO mice. Endpoint p<0.0001. [Figure 89] Effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on blood glucose levels in DIO mice. Baseline p=0.8019, endpoint p=0.0123. [Figure 90] Figure 1 shows the effect of a combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on plasma insulin levels in DIO mice. Baseline p=0.7627, endpoint p<0.0001. [Figure 91] Figure 1 shows the effect of the combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on insulin sensitivity in DIO mice as determined using the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Baseline p=0.6413, endpoint p<0.0001. [Figure 92] Effect of the combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on energy expenditure [kcal / h] assessed over 24 hours post-dose. p=0.0075. [Figure 93] 1 shows the effect of the combination of bezafibrate (60 mg / kg BW once daily by oral gavage) and oxaprozin (50 mg / kg BW once daily by oral gavage) on energy expenditure [kcal / hr] assessed 1 hour before and 2 hours after norepinephrine infusion, and post-dose. [Figure 94]Effect of the combination of bezafibrate (60 mg / kg BW once daily by gavage) and oxaprozin (50 mg / kg BW once daily by gavage) on energy expenditure [kcal / h] assessed post-dose, 2 hours after norepinephrine infusion. Post-dose p=0.0112. DETAILED DESCRIPTION OF THE INVENTION

[0065] According to Patent Documents 1 and 2, the presence of cells in various tissues that can differentiate into brown adipocytes has been previously identified. A population of such cells, called BAT progenitor cells, has been found to exist in skeletal muscle. Patent Document 3 provides an assay that induces expression of the UCP1 gene and enables the identification of agents (e.g., compounds, proteins, biological agents, etc.) that promote the differentiation of BAT progenitor cells into brown adipocytes in vitro, promote the differentiation of BAT progenitor cells into brown adipocytes in vivo, or promote a combination of these activities.

[0066] Provided herein, in some embodiments, are combinations of two or more effective brown adipocyte mobilizing agents that have additive or synergistic effects, thereby providing greater efficacy than a single agent or reducing toxicity associated with a single agent by allowing for the use of lower doses, providing superior product candidates for treating metabolic diseases such as obesity, type 2 diabetes, insulin resistance, and dyslipidemia. For example, the combination of rosiglitazone and BMP7 results in greater in vitro PPARγ2 and UCP1 expression than either agent alone. Multidrug combinations, including combinations of two compounds that exhibit additive or synergistic activity on metabolic parameters important in metabolic diseases, were identified by testing candidate brown adipocyte mobilizing agents in an animal model of obesity and type 2 diabetes (DIO mice).

[0067] The present disclosure provides drug combinations that promote the differentiation of BAT progenitor cells into brown adipocytes both in vitro and in vivo. The combination of effective brown adipocyte mobilizing agents can provide greater efficacy than either compound alone. The combination of rosiglitazone and BMP7 results in greater PPARγ2 and UCP1 expression than either compound alone.

[0068] Thus, in some embodiments, compositions comprising: a cyclooxygenase inhibitor such as zaltoprofen or oxaprosin, a thromboxane synthetase inhibitor such as ozagrel, or a pan-PPAR (α, δ, γ) ligand such as bezafibrate, bezafibrate in combination with oxaprozin, or bezafibrate in combination with azagrel, can be used to promote differentiation of BAT progenitor cells into brown adipocytes and / or induce expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells in vitro, in vivo, or both.

[0069] In still other embodiments, additional agents or combinations that can be used to promote differentiation of BAT precursor cells into brown adipocytes and / or induce expression of UCP1 include a combination of rosiglitazone or pioglitazone with oxaprozin or zaltoprofen or ozagrel, a combination of bezafibrate with diflunisal or probenecid or tianeptine or glimepiride, a combination of zaltoprofen and glimepiride or probenecid, a combination of probenecid with tianeptine or ozagrel or diflunisal, or a combination of tianeptine and glimepiride.

[0070] In some embodiments, treatment of a subject, including a human, with the compositions described herein results in increased UCP1 mRNA or protein production in the subject's skeletal muscle. For example, treatment of a subject with rosiglitazone, in some embodiments, induces the development or differentiation of brown adipocytes in skeletal muscle, enhances UCP1 gene expression in existing brown adipocytes in or near skeletal muscle (between muscle fibers, on the surface of skeletal muscle tissue, and / or adjacent to skeletal muscle tissue), or both. In some embodiments, the development or differentiation of brown adipocytes in skeletal muscle is induced in a subject suffering from a metabolic disease. Brown adipocytes provide a glucose sink with high mitochondrial respiration, cellular respiration, and fatty acid oxidation rates, and dissipate energy as heat (uncoupled oxidative phosphorylation). This can increase a subject's metabolic rate and induce weight loss. Inducing the development or differentiation of brown adipocytes also results in improved insulin sensitivity, blood glucose homeostasis, and cardiovascular disease risk factors. Brown adipocytes further secrete factors that contribute to achieving a healthy energy balance and low body fat levels, increasing insulin sensitivity and improving blood glucose homeostasis, and cardiovascular health.

[0071] Thus, in some embodiments, the agents disclosed herein or combinations thereof can be used to treat subjects, including humans. In some aspects, these agents promote the differentiation of BAT progenitor cells into brown adipocytes. In other aspects, these agents induce the expression of UCP1, FABP4(aP2), PPARγ2, mtTFA, PGC-lα, and / or COX IV in BAT progenitor cells in vitro, in vivo, or both.

[0072] In some embodiments, the metabolic disease treated is obesity, overweight, type II diabetes, insulin resistance, hyperinsulinemia, hyperglycemia, prediabetes, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovary syndrome, acanthosis nigricans, hyperphagia, endocrine disorders, triglyceride storage diseases, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.

[0073] In other embodiments, the compositions are used to activate isolated BAT progenitor cells, which are then used to treat a subject, including a human.

[0074] The assay previously disclosed by U.S. Patent Application Publication No. 2009 / 0129999 can be used to identify additive or synergistic effects of two or more compounds promoting the differentiation of BAT progenitor cells into brown adipocytes. However, because some compounds individually induce nearly complete (100% or close to 100%) brown adipocyte differentiation, it is difficult to detect significant differences between the combination of two compounds and the individual compounds. Therefore, in vivo studies in mice with obesity, insulin resistance, and impaired glucose tolerance were used to complement cell culture assays to identify combinations of two compounds that produce additive or synergistic effects on any of the metabolic parameters of interest: body weight, body fat content, adiposity, plasma leptin concentration, blood glucose concentration, plasma glucose concentration, plasma insulin concentration, HOMA-IR (Homeostasis Model Assessment of Insulin Tolerance), glucose tolerance, or plasma triglyceride concentration.

[0075] The combinations tested in animals contain compounds with different known or suspected molecular mechanisms of action. For example, bezafibrate, a fibrate used to treat dyslipidemia, acts through a different molecular target (PPAR) and pathway than Oxaprozin. Oxaprozin is an NSAID that acts as an inhibitor of cyclooxygenase-1 and -2 (prostaglandin G / H synthase-1 and -2). However, Oxaprozin likely acts as described on body weight, body fat, etc. through a molecular target other than cyclooxygenase.

[0076] The present inventors have found that certain combinations of bezafibrate-containing compounds have in vitro and in vivo activity.It is suggested that these combinations are useful for treating patients diagnosed with or at risk of metabolic disease.In the case of obesity and diabetes, for example, such administration reduces body weight / fat and / or blood glucose levels.

[0077] In one example, the inventors propose that the mutual administration of bezafibrate and oxaprozin within 14 days to a patient with a metabolic disorder such as obesity or diabetes will treat, prevent, or reduce the metabolic disorder.

[0078] In another example, mutual administration of bezafibrate and zaltoprofen to a patient within 14 days also treats, prevents, or reduces metabolic disorders.

[0079] In another example, mutual administration of bezafibrate and ozagrel to a patient within 14 days also treats, prevents, or reduces metabolic disorders.

[0080] The two agents are preferably administered within 10 days of each other, more preferably within 7 days, and even more preferably within 24 hours, within 1 hour, or simultaneously (i.e., concomitantly). If desired, either one of the two agents can be administered at a lower dose.

[0081] In light of this finding, the aforementioned drug combinations can be used in various compositions, methods, and kits, as described herein.

[0082] "Treating, reducing, or preventing a metabolic disorder" means ameliorating such a condition before or after it occurs. The degree of such reduction or prevention, as measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an equivalent untreated control.

[0083] A patient being treated for a metabolic disease is one who has been diagnosed by a physician as having such a condition. Diagnosis may be performed by any appropriate means, such as those described herein. Patients being prevented from developing diabetes or obesity may or may not have received such a diagnosis. One of ordinary skill in the art will understand that patients of the present disclosure may have undergone standard testing or may have been identified as high-risk patients without testing due to the presence of one or more risk factors, such as family history, obesity, certain ethnicities (e.g., African American, Hispanic American), gestational diabetes, or delivery of an infant weighing more than 9 pounds, high blood pressure with a pathological condition predisposing to obesity or diabetes, high blood levels of triglycerides, high blood levels of cholesterol, the presence of molecular markers (e.g., the presence of autoantibodies), and age (over 45 years old). A person is considered obese if their weight is 20% or more of the maximum desirable weight for their height (25% for women). Adults who are more than 100 pounds overweight are considered morbidly obese. Obesity is also defined as a body mass index (BMI) greater than 30 kg / m2.

[0084] "Metabolic disease" refers to any pathological condition resulting from an alteration in a patient's metabolism. Such diseases include, for example, diseases resulting from alterations in glucose homeostasis resulting in hyperglycemia. According to the present disclosure, the alteration in glucose levels is typically an increase in glucose levels of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% compared to such levels in a healthy individual. Metabolic diseases include obesity and diabetes (e.g., type 1 diabetes, type 2 diabetes, MODY, and gestational diabetes), dyslipidemia, and age-related endocrine deficiencies.

[0085] "Lowering glucose levels" means lowering glucose levels by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to untreated controls. Desirably, glucose levels are lowered to normoglycemic levels, i.e., 150-60 mg / dL, 140-70 mg / dL, 130-70 mg / dL, 125-80 mg / dL, and preferably 120-80 mg / dL.

[0086] "Patient" means any animal (e.g., a human), including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cows, fish, and birds.

[0087] By "sufficient amount" is meant the amount of compound, alone or in combination with other therapeutic regimens, needed to treat, prevent, or reduce a metabolic disease, such as diabetes, in a clinically relevant manner. The amount of active compound sufficient to be used in practicing the present disclosure for the therapeutic treatment of a metabolic disease will vary depending on the method of administration, the age, weight, and general health of the mammal or patient. Ultimately, the prescriber will determine the appropriate amount and dosing regimen. Furthermore, an effective amount is the amount of compound in the combination of the present disclosure that is safe and effective in treating patients with a metabolic disease, such as diabetes, more than each agent alone, as determined and approved by regulatory authorities (such as the U.S. Food and Drug Administration).

[0088] By "more effective" is meant that the treatment is more effective, less toxic, safer, more convenient, or less expensive than other treatments to which it is being compared. Effectiveness is measured by one of skill in the art using any standard method appropriate for a given indication.

[0089] Compounds useful in the present disclosure include the compounds described herein in any pharmaceutically acceptable form, including isomers such as diastereomers and enantiomers, salts, esters, solvates, and polymorphs, as well as racemic mixtures and pure isomers of the compounds described herein.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials are described herein for use in this disclosure; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0091] Diagnosis of metabolic diseases The disclosed methods and compositions are useful for treating patients who have been diagnosed with or are at risk for a metabolic disease, such as obesity or diabetes. The patient in whom the development of a metabolic disease (e.g., obesity or diabetes) is being prevented may or may not have been diagnosed with such a disease. Those skilled in the art will understand that the disclosed patients may have been subjected to standard testing, or may have been identified without testing as high-risk patients due to the presence of one or more risk factors.

[0092] Body mass index 30 kg / m 2 If so, the individual is considered obese. Adults with a BMI of over 40 are considered morbidly obese.

[0093] Diagnosis of other metabolic diseases can be performed using any standard method known in the art, such as those described herein. Methods for diagnosing diabetes are described, for example, in U.S. Patent No. 6,277,629, which is incorporated herein by reference. Diabetes is diagnosed and monitored using, for example, urine tests (urinalysis) to measure glucose and ketone levels (products of fat breakdown), tests to measure blood glucose levels, glucose tolerance tests, and assays to detect molecular markers characteristic of metabolic diseases in biological samples (e.g., blood, serum, or urine) collected from a mammal (e.g., measuring hemoglobin A1c (Hb A1c) levels in the case of diabetes).

[0094] A patient is diagnosed as at risk for or with diabetes if a random plasma glucose test (taken any time during the day) shows a value of 200 mg / dL or greater, if a fasting plasma glucose test shows a value of 126 mg / dL or greater (after 8 hours), or if an oral glucose tolerance test (OGTT) shows a plasma glucose value of 200 mg / dL or greater in a blood sample taken 2 hours after ingesting a beverage containing 75 grams of glucose dissolved in water. The OGTT measures plasma glucose at time intervals over a 3-hour period. Desirably, plasma glucose levels in diabetic patients treated according to the present disclosure range from 160-60 mg / dL, 150-70 mg / dL, 140-70 mg / dL, 135-80 mg / dL, and preferably 120-80 mg / dL.

[0095] Optionally, a hemoglobin A1c (Hb A1c) test can be used to assess average blood glucose levels over the past two and three months. Non-diabetic individuals typically have Hb A1c values ​​in the 4% to 6% range. For every 1% increase in Hb A1c, blood glucose levels increase by approximately 30 mg / dL, increasing the risk of complications. Preferably, patients treated according to the present disclosure have Hb A1c values ​​reduced to less than 9%, less than 7%, or less than 6%, and most preferably, to about 5%. Thus, treated patients' Hb A1c levels are preferably reduced by 10%, 20%, 30%, 40%, 50%, or more compared to pre-treatment levels.

[0096] Gestational diabetes is typically diagnosed based on plasma glucose levels measured during an OGTT. Because blood glucose levels are usually low during pregnancy, the threshold for diagnosing diabetes during pregnancy is lower than for the same person before pregnancy. A woman is considered to have gestational diabetes if she has two plasma glucose measurements that meet or exceed any of the following numbers: a fasting plasma glucose level of 95 mg / dL, a 1-hour level of 180 mg / dL, a 2-hour level of 155 mg / dL, or a 3-hour level of 140 mg / dL.

[0097] The effectiveness of treatment can be monitored using the above tests or any other test known in the art. Measurement of hemoglobin A1c (HbA1c) levels is an indicator of average blood glucose over the past 2-3 months, and so this test is used to monitor a patient's response to diabetes treatment.

[0098] Bezafibrate (2-(4-{2-[(4-chlorobenzoyl)amino]ethyl}phenoxy)-2-methylpropanoic acid) has the following structure: [ka]

[0099] Oxaprozin (3-(4,5-diphenyloxazol-2-yl)propionic acid) has the following structure: [ka]

[0100] Zaltoprofen (2-(6-oxo-5H-benzo[b][1]benzothiepin-3-yl)propanoic acid) has the following structure: [ka]

[0101] Ozagrel ((2E)-3-{4-[(1H-imidazol-1-yl)methyl]phenyl}prop-2-enoic acid) has the following structure: [ka]

[0102] Example Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings.

[0103] Example 1: Screening for voltage modulation circuits of human UCP1 mRNA by quantification using TaqMan real-time PCR CD34+ cells can be used as a tool to induce cell differentiation into brown adipocytes or to identify drugs (small molecule compounds, proteins, biologics, etc.) that modulate UCP1 expression. For example, RT-PCR-based approaches can be used to measure UCP1 mRNA levels affected by specific drugs.

[0104] This allows the identification of agents that can enhance the differentiation of CD34+ cells into brown adipocytes and / or the expression of UCP1 by enhancing transcription of the UCP1 gene and / or by stabilizing UCP1 transcripts.

[0105] For example, PPARγ ligands such as rosiglitazone can be used to promote the differentiation of CD34+ progenitor cells into brown adipocytes (Figures 1-10). Another example is the use of the recombinant protein human BMP-7 (Figures 1-2, 4, 6-9).

[0106] A previously described robust method was used for the detection of CD34+ cell differentiation into brown adipocytes by simultaneously quantifying mRNA species corresponding to the brown adipocyte marker UCP1, the adipocyte marker PPARγ2, and the “housekeeping” gene cyclophilin A, which were used as internal controls.

[0107] This method allows for the analysis of a large number of samples and identifies agents that enhance the differentiation of CD34+ cells into brown adipocytes. Upon differentiation into brown adipocytes, CD34+ cells express very high levels of UCP1 and PPARγ2 mRNA for a given level of cyclophilin A. UCP1 and PPARγ2 mRNA levels normalized to cyclophilin A mRNA levels provide an indication of the level of differentiation of CD34+ cells into brown adipocytes, regardless of the total number of cells in the sample.

[0108] Therefore, quantification of UCP1, PPARγ2, and cyclophilin A mRNA by multiplexed TaqMan real-time PCR was used to quantify the differentiation of CD34+ cells into brown adipocytes.

[0109] The applicant hypothesized that some previously approved drugs are active in mobilizing brown adipocytes (inducing the differentiation of brown adipocyte precursor cells into brown adipocytes) and therefore may be beneficial for treating obesity and diabetes. Furthermore, two drugs that mobilize brown adipocytes via different molecular mechanisms could produce greater effects on brown adipocyte mobilization than the individual drugs and enhance in vivo efficacy on parameters of metabolic health.

[0110] Materials and Methods Screening for brown adipocyte mobilizing agents UCP1 is a key protein in brown adipocytes involved in uncoupled respiration and is highly specific for brown adipocyte differentiation. It is expressed only by fully differentiated brown adipocytes, not by CD34+ progenitor cells. We used a real-time, semi-quantitative RT-PCR-based assay for the detection of UCP1 expression to screen for brown adipocyte mobilizing agents. We multiplexed this assay for message normalization to cell number to simultaneously detect UCP1 (brown fat-specific), PPARγ2 (adipocyte-specific; CD34+ cells do not become white, but only brown, confirming brown differentiation / maturation in cells), and cyclophilin A. This assay has the added advantage of allowing light microscopic visualization of the distinct morphological changes that accompany brown adipocyte differentiation. This serves as further confirmation of differentiation. The system responded predictably to several positive controls, including PPARγ activators such as rosiglitazone, pioglitazone, and ciglitazone, and the protein BMP7, with an appropriate dose-response behavior.

[0111] Combining compounds demonstrates the impact of the combination. We have shown that in CD34+ cells, both rosiglitazone and BMP7 robustly increase brown adipocyte mobilization. Given that they act via different mechanisms, they were tested together at their most effective individual concentrations to determine whether they promote further mobilization beyond that seen with either compound. Indeed, the two compounds together were significantly more effective than either compound alone (Figures 1, 2, 6: rosiglitazone + BMP7 vs. rosiglitazone and BMP7), suggesting that the two compounds have different mechanisms and, more generally, that multidrug combinations may be more effective than individual compounds for brown adipocyte mobilization. The potential for combining active compounds for greater efficacy was demonstrated. Alternatively, the additive (or synergistic) effects of compound combinations could allow for the use of lower doses of one or both drugs, reducing the side effects associated with their use.

[0112] cell culture 10,000 cells / cm 2 Cells were seeded in 48-well tissue culture plates (Chemglass #CLS-3500-048) and cultured in endothelial cell growth medium-2 (EGM2) (BulletKit growth medium, Lonza #CC-3162) at 37°C until confluent (1-4 days) and differentiated (6-12 days). After 1-4 days in EGM2 medium, cells were incubated with test agents (or positive controls) for 2-3 days (day -3 or day -2 to day 0). Rosiglitazone (1 μM) and rhBMP7 (6.3 nM) were used as reference agents (positive controls). Then, on day 0, the culture medium (containing the test agents or positive controls) was removed, minimal adipogenic medium (MDM) was added, and the cells were allowed to differentiate for 6-12 days. MDM medium is a modification of the by-product medium described by Rodriguez et al.

[21] and contains DMEM / Ham's F-12 50 / 50 mix (3.151 g / L, 17.5 mM D-glucose, 3.651 g / L glutamine) (Cellgro #10-090-CV), 5 μg / ml (0.86 μM) insulin, 1 μM dexamethasone, 100 μM 3-isobutyl-1-methylxanthine, 0.2 nM 3,3',5-triiodo-L-thyronine, 10 μg / ml (127 nM) transferrin, and 1% penicillin-streptomycin. Adipocyte scores were determined by light microscopy and defined as the number of cells per well containing multilocular lipid droplets at the end of cell differentiation, approximately 6–12 days after the end of compound treatment, and switched to MDM medium.

[0113] Quantitation of UCP1 and PPARγ2 mRNA by quantitative reverse transcription and real-time PCR Total RNA was prepared from cells using the PureLink RNA isolation kit (Invitrogen #12183-016). Alternatively, cells were simply lysed by freezing (-80°C). First-strand cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and random primers.

[0114] Quantitative real-time PCR was performed using an Applied Biosystems StepOnePlus® instrument, TaqMan Gene Expression Master Mix (Applied Biosystems #4369016), and custom TaqMan gene expression probes and primers for human uncoupling protein-1 "UCP1" (GenBank NM_021833) and human peptidylprolyl isomerase A "cyclophilin A" (GenBank NM_021130). Custom TaqMan gene expression reagents were also developed for the simultaneous measurement of peroxisome proliferator-activated receptor gamma, transcript variant 2 (PPARγ2) (GenBank NM_015869) in a multiplexed format (with UCP1 and cyclophilin A). UCP1 FAM-MGB probe: TCA AGG GGT TGG TAC CTT CC (SEQ ID NO: 1), sense primer: CAC TAA CGA AGG ACC AAC GG (SEQ ID NO: 2), antisense primer: TTC CAG GAT CCA AGT CGC AA (SEQ ID NO: 3), cyclophilin A NED-MGB probe: ACT GCC AAG ACT GAG TGG TT (SEQ ID NO: 4), sense primer: CAA ATG CTG GAC CCA ACA CA (SEQ ID NO: 5), antisense primer: TCA CTT TGC CAA ACA CCA CA (SEQ ID NO: 6), PPARγ2 VIC-MGB probe: TCA CAA GAA ATG ACC ATG GTT G (SEQ ID NO: 7), sense primer: AGC GAT TCC TTC ACT GAT ACA C (SEQ ID NO: 8), and antisense primer: CCA GAA TGG CAT CTC TGT GT (SEQ ID NO: 9).

[0115] Cyclophilin A was used as a control to account for variations due to the efficiency of reverse transcription. Arbitrary units were determined by normalizing target mRNA levels to cyclophilin A mRNA levels (based on Ct).

[0116] Cell morphology images Images of cells were taken using a handheld digital camera (Nikon Coolpix 950) and an inverted microscope (Nikon TMS) used for cell culture observation. Images were optimized using the Paint.net version 4.0 function for auto-leveling brightness and contrast.

[0117] result We incubated CD34+ brown adipocyte precursors with compounds (10 μM) from a collection of 1018 FDA-approved drugs (FDA-approved Drug Screening Library Collection, Selleckchem, Houston, TX). Compounds found to be active in the initial screening (10 μM) were retested at concentrations ranging from 1 nM to 50 μM to confirm dose-dependence.

[0118] Using this method, the following drugs were identified or confirmed to promote the differentiation of brown adipocyte progenitors into brown adipocytes, as indicated by the expression of UCP1 and PPARγ2 in vitro: prostaglandin E1 (PGE1) analogs, such as alprostadil; pan-PPAR (α, δ, γ) ligands, such as benzofibrates (bezafibrate); cyclooxygenase inhibitors, such as diflunisal, zaltoprofen, indomethacin, acemetacin, diclofenac, mefenamic acid, niflumic acid, meclofenamate, or oxaprozin; thromboxane synthetase inhibitors, such as ozagrel; sulfonylureas, such as glimepiride or gliquidone; or probenecid, tianeptine, and epalrestat (Figures 6C-6G, 7-21).

[0119] These agents (with the possible exceptions of bezafibrate and indomethacin) were not predicted to induce brown adipocyte precursor differentiation, and their biological activity as brown adipocyte mobilizing agents in vitro could not be predicted based on their known molecular targets or approved indications.

[0120] Unless otherwise noted, all analytical or molecular biology grade organic and inorganic chemicals were purchased from Cayman Chemical (including rosiglitazone, #71742), R&D Systems (including recombinant human BMP7 (rhBMP7), 100 μg / ml, 6.3 μM, #354-BP-010), Sigma Chemical Co. (St Louis, MI), and Life Technologies (Grand Island, NY). The 1018 FDA-approved drug screening library collection was purchased from Selleck Chemicals / Selleckchem (Houston, TX).

[0121] Using this method, the following agents were identified or confirmed to promote the differentiation of BAT progenitor cells into brown adipocytes and / or induce the expression of UCP1, FABP4 (aP2), PPARγ2, mtTFA, PGC-1α, and / or COX IV in BAT progenitor cells in vitro, in vivo, or both: prostaglandin E1 (PGE1) analogs such as alprostadil, pan-PPAR (α, δ, γ) ligands such as benzofibrates (bezafibrate), cyclooxygenase inhibitors such as diflunisal, zaltoprofen, indomethacin, acemetacin, diclofenac, mefenamic acid, niflumic acid, meclofenamate, or oxaprozin, thromboxane synthetase inhibitors such as ozagrel, sulfonylureas such as glimepiride or gliquidone, or probenecid, tianeptine, or epalrestat.

[0122] Example 2: Quantification of UCP1 protein by fluorescent immunohistochemistry (IHC) The differentiation of brown adipocyte precursors into brown adipocytes can be detected through quantification of UCP1 protein by immunohistochemistry.

[0123] CD34+ cells were cultured and differentiated into brown adipocytes using adipogenic differentiation medium lacking 1 μM rosiglitazone (Minimal Differentiation Medium, MDM) or containing 1 μM rosiglitazone (Reference Differentiation Medium, RDM). After 15 days, differentiated cells were fixed with 4% paraformaldehyde in PBS pH 7.4 and incubated with UCP1 antibody (Abcam ab23841) and Alexafluor 488 goat anti-rabbit antibody to quantify relative UCP1 levels (green) according to standard protocols. Prior to fixation, nuclei were labeled with 5 μM DAPI (blue) for 10 min. Each treatment condition was evaluated in triplicate in a 96-well plate, corresponding to approximately 360–480 cells in total for each data point. Using the InCell 1000 Developer Toolbox software, an automated cell detection script was developed to measure UCP1 signal intensity using nuclear and cytoplasmic detection algorithms. As readout, the total intensity of the intracellular UCP1 signal was used and normalized to cell number.

[0124] In some embodiments, drugs or combinations thereof identified using this technology include famotidine, tiapride hydrochloride, guanfacine hydrochloride, reserpine, minoxidil, spiperone, diflunisal, syrosingopine, probenecid, metformin, thiethylperazine, colchicine, and felodipine.

[0125] Example 3: Detection of brown adipocyte differentiation using BODIPY BODIPY fluorescent dye-labeled neutral lipids are incorporated into cytoplasmic lipid droplets, allowing for analysis of cellular fatty acid uptake and adipocyte differentiation by fluorescent cell imaging. Cells are incubated with C1-BODIPY® 500 / 510 C12 (Molecular Probes #D-3823) for 3-6 hours and then imaged with a microplate-based, high-throughput, high-content, brightfield and fluorescent cell imager and analyzer (Cyntellect Celigo® or GE Healthcare IN Cell Analyzer).

[0126] Example 4: The combination of bezafibrate and oxaprozin induces weight loss, increased insulin sensitivity, and improved blood glucose homeostasis in a mouse model of obesity and diabetes. Agents that promote the differentiation of brown adipocyte precursor cells into brown adipocytes, i.e., agents that mobilize brown adipocytes or brown adipose tissue in vivo, are predicted to result in improvements in any of the following parameters of metabolic health in obese individuals or animals: reductions in body weight, body fat content, plasma levels of leptin, glucose, and insulin, and the index of insulin resistance, HOMA-IR (HOMA-IR = (plasma insulin [microIU / ml] × plasma glucose [mM]) / 22.5).

[0127] We investigated the effects of agents found to mobilize brown adipocytes in vitro on parameters of metabolic health in obese, prediabetic mice. To clarify possible combinatorial effects between compounds, we also investigated the effects of combining two agents that mobilize brown adipocytes in vitro and that are known or thought to affect different molecular targets and intracellular signaling pathways.

[0128] Materials and Methods Animal experiments Obesity and insulin resistance, early stages of the development of type 2 diabetes (or prediabetes), were induced in C57Bl / 6 mice by feeding them a high-fat diet (Research diet, Cat# D12492, 60% fat kcal) for 12 weeks, starting at 6 weeks of age. Mice were maintained at 22-23°C with copious amounts of bedding, and animals were maintained at their near-thermoneutral environmental temperature on a 12-h / 12-h light / dark cycle for the entire treatment period, starting 2 weeks before the treatment period.

[0129] Mice were administered once daily by oral gavage (200 μl per mouse) first with vehicle (PBS + 0.5% CMC + 0.1% Tween-80) for 3 days to allow for acclimation to the study, and then either vehicle alone or test material dissolved in vehicle for 28–55 days (4–8 weeks).

[0130] Body weight was recorded every 3 days, body composition (fat and lean mass using EchoMRI) was assessed at the end of the study (University of Cincinnati Mouse Metabolic Phenotyping Center), and blood glucose levels (measured by glucometer) were monitored 3–5 days before (baseline) and at the end of the treatment period.

[0131] At the end of the dosing period, mice were fasted for 6 hours, euthanized by CO2, blood was collected, and plasma was isolated and stored at -20°C. Plasma glucose, insulin, and leptin levels were assessed 3–5 days before the dosing period (baseline) and at the end of the period (mice were fasted for 6 hours before all plasma collection) (University of Cincinnati Mouse Metabolic Phenotyping Center). Insulin sensitivity was determined using the homeostasis model assessment of insulin resistance (HOMA-IR)

[38] . HOMA-IR = (plasma insulin [microIU / ml] × plasma glucose [mM]) / 22.5.

[0132] Mice were maintained on a high-fat diet (60% calories from fat) throughout the experiment, and were fasted overnight on the last night of the experiment in preparation for a glucose tolerance test.

[0133] Animal experiments using indirect calorimetry (bezafibrate (60) + oxaprozin (50)) Diet-induced obese mice: C57Bl / 6 male mice were fed a high-fat diet (Research Diet, Cat# D12492, 60% fat kcal) for 12 weeks starting at 6 weeks of age. Mice were maintained at 22-23°C with copious amounts of bedding, and animals were maintained at their thermoneutral environmental temperature on a 12-12 hour light / dark cycle for the entire treatment period, starting 2 weeks prior to the treatment period.

[0134] Mice were administered either vehicle alone (PBS + 0.5% CMC + 0.1% Tween-80) or a combination of 60 mg / kg bezafibrate dissolved in vehicle + 50 mg / kg oxaprozin once daily by oral gavage (200 μl per mouse) for 24 days.

[0135] Body weight was recorded daily and body composition (fat and lean mass by EchoMRI) was assessed at baseline (pre-treatment) and at the end of the treatment period (University of Michigan Mouse Metabolic Phenotyping Center).

[0136] At the end of the dosing period, mice were fasted for 6 hours, euthanized by CO2, blood was collected, and plasma was isolated and stored at -20° C. Plasma glucose, insulin, and leptin levels were assessed at baseline and at the end of the dosing period (mice were fasted for 6 hours before all plasma collection) (University of Michigan Mouse Metabolic Phenotyping Center).

[0137] After 24 days of treatment, mice were transferred to a calorimetry chamber (one mouse per cage) and maintained at 30°C, a temperature close to thermoneutrality for mice, for 3 days. During the 3 days in the calorimetry chamber (TSE Systems Phenomaster), the following parameters were measured: energy expenditure by indirect calorimetry (using oxygen consumption rate (VO2), carbon dioxide production (VCO2), and respiratory quotient (RQ)), food intake, locomotor activity, ambulatory activity, and distance traveled (beam break).

[0138] The first day in the calorimetry chamber was used as an acclimation period (measured parameters were not used in data analysis). Values ​​obtained on the second day (over 24 h) in the calorimetry chamber were used as unstimulated / resting values. On the third day in the calorimetry chamber, mice were measured for whole-body nonshivering thermogenesis, a measure of whole-body brown adipose tissue volume, using a single injection of 1 mg / kg norepinephrine

[39] .

[0139] statistical analysis Data from cell culture experiments are shown as mean ± SEM. Significance was assessed using paired or unpaired Student's t-tests using the online GraphPad QuickCalcs t-test calculator (GraphPad Software, San Diego, CA). Significance was set at p<0.05.

[0140] Data from in vivo mouse experiments are shown as mean ± SEM. Significance was assessed using paired or unpaired Student's t-test, two-way ANOVA, or one-way ANOVA with Bonferroni or Dunnett's multiple comparison test using GraphPad Prism version 7 or 8 (GraphPad Software, San Diego, CA). Significance was set at p<0.05; *: p<0.05 vs. vehicle; **: p<0.01 vs. vehicle; ***: p<0.001 vs. vehicle.

[0141] result In the first experiment reported here, we examined the effects of 56 days of administration of bezafibrate (60 mg / kg), oxaprozin (50 mg / kg), and a combination of bezafibrate (60) plus oxaprozin (50) on metabolic health parameters in DIO mice.

[0142] These data were generated using bezafibrate at approximately half the recommended dosage level in humans receiving treatment for hyperlipidemia and allometrically scaled for mice according to current FDA guidelines. Oxaprozin was used in this animal study at approximately one-quarter of the scaled recommended human dose.

[0143] Treatment of DIO mice with bezafibrate (60 mg / kg) or oxaprozin (50 mg / kg) for 56 days significantly reduced body weight (Figure 60), fat mass (Figure 65), plasma leptin (Figure 70), blood glucose (Figure 75), plasma insulin (Figure 80), and insulin resistance index (HOMA-IR) (Figure 85) compared to vehicle treatment.

[0144] In addition, we found that the combination of bezafibrate (60 mg / kg) and oxaprozin (50 mg / kg) further reduced all of these metabolic parameters. In fact, the effect of the combination on weight loss (Figure 60) and body fat (Figure 65) was statistically synergistic (i.e., greater than additive).

[0145] We found that bezafibrate plus oxaprozin resulted in a significant reduction in body weight in DIO mice over 56 days (p = 2.6 x 10-10). The observed effect was synergistic (greater than the sum of the effects of the two individual drugs versus vehicle (p = 0.043)). All P values ​​in the figures for this 56-day study of bezafibrate (60) plus oxaprozin were determined based on two-tailed Z-tests with standard errors estimated by bootstrap. To assess synergy, we tested whether the percent change from baseline with the combination was greater than the sum of the percent change from baseline with the two individual drugs. For measurements at the end of the study, logarithmic transformation was used for Z-tests of between-group differences. These data were generated using bezafibrate at approximately half the dosage level recommended in humans receiving treatment for hyperlipidemia and were allometrically scaled for mice according to current FDA guidelines. Oxaprozin was used in this animal study at approximately one-quarter of the scaled recommended human dose.

[0146] Bezafibrate is known to reduce plasma levels of triglycerides and LDL cholesterol through activation of PPARα in the liver and is used to treat dyslipidemia and increased cardiovascular risk. Oxaprozin is a nonsteroidal anti-inflammatory (NSAID) drug that inhibits cyclooxygenase 1 and 2 and is used to treat rheumatoid arthritis.

[0147] Based on this, the synergistic effects of the combination of bezafibrate and oxaprozin on body weight, body fat content, and plasma leptin levels could not be predicted based on the known effects of these individual drugs. The known molecular targets of these two drugs may mediate their brown adipocyte recruitment (and metabolic) effects. Alternatively, one or both of these drugs may affect brown adipocyte recruitment and metabolic health through other molecular targets.

[0148] Using this general approach, certain existing drug combinations have been identified or confirmed to induce weight loss, increased insulin sensitivity, and improved blood glucose homeostasis in mouse models of obesity and diabetes, including bezafibrate in combination with oxaprozin or zaltoprofen or ozagrel or diflunisal or probenecid or tianeptine or glimepiride, rosiglitazone or pioglitazone in combination with oxaprozin or zaltoprofen or ozagrel, zaltoprofen in combination with glimepiride or probenecid, tianeptine or ozagrel or probenecid in combination with ozagrel, and tianeptine in combination with glimepiride.

[0149] In a second experiment, the effects of the combination of bezafibrate (60 mg / kg) and oxaprozin (50 mg / kg) on ​​parameters of metabolic health and energy expenditure (metabolic rate) at rest and after maximal sympathetic stimulation with norepinephrine were examined in DIO mice over 24 days of treatment. The objective of this experiment was to evaluate the energy expenditure and whole-body thermogenic capacity of mice after 24 days of treatment with bezafibrate (60) and oxaprozin (50). The objective was also to assess whether mice treated with bezafibrate (60) and oxaprozin (50) showed evidence of increased brown fat mass (and volume) compared to mice given vehicle.

[0150] Treatment of DIO mice with bezafibrate (60 mg / kg) + oxaprozin (50 mg / kg) for 24 days significantly reduced body weight (Figure 86), fat mass (Figure 87), plasma leptin (Figure 88), blood glucose (Figure 89), plasma insulin (Figure 90), and insulin resistance index (HOMA-IR) (Figure 91) compared to vehicle treatment.

[0151] Additionally, energy expenditure assessment by indirect calorimetry showed that mice treated with the combination of bezafibrate (60) + oxaprozin (50) had significantly higher energy expenditure (over 24 hours, unstimulated, Figure 92).

[0152] Furthermore, the thermogenic (energy expenditure) response to norepinephrine injection was significantly higher in mice treated with the combination of bezafibrate (60) and oxaprozin (50) versus vehicle-treated mice (Figures 93-94), demonstrating an increase in the thermogenic capacity, i.e., brown adipose tissue capacity / mass, of the animals.

[0153] There were no significant differences between the mouse groups in food intake, locomotor activity, ambulatory activity, and distance traveled.

[0154] These data clearly demonstrate that the combination of bezafibrate (60) plus oxaprozin (50) induces an increase in mobilized, thermogenic brown / beige adipose tissue in DIO mice, resulting in improved energy expenditure (metabolic rate) and improved parameters of metabolic health (reduced body weight, fat mass, plasma leptin, glucose levels, and insulin levels, an index of insulin resistance, HOMA-IR).

[0155] The section headings and subheadings used herein are for organizational purposes only and are not to be construed as limiting the described subject matter in any way. Moreover, while the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. Rather, the present teachings encompass various variations, modifications, and equivalents, as will be appreciated by those skilled in the art.

[0156] Other embodiments Various aspects of the present disclosure may be used alone, in combination, or in various configurations not specifically discussed in the foregoing embodiments, and therefore the application is not limited to the details and arrangements of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

[0157] The use of ordinal terms such as "first," "second," "third," etc. in a claim to modify a claim element does not, in itself, imply that the priority, precedence, or order of the claim element is more important than the elements of other claims or the chronological order in which the method actions are performed, but is merely used as a label (but for the use of ordinal terms) to distinguish one claim element having a certain name from another element having the same name to distinguish the claim elements.

[0158] Also, the phraseology and terminology used herein are not to be construed as limiting. The use of "including," "comprising," "having," "containing," "including," and variations thereof are intended to include the items listed thereafter, equivalents, and other additional items.

[0159] The present disclosure provides, inter alia, novel compositions capable of mobilizing brown adipocytes in vitro and in vivo. Although specific embodiments of the present disclosure have been discussed, the above specification is illustrative and not limiting. Upon reviewing this specification, many variations of the present disclosure will become apparent to those skilled in the art. The full scope of the present disclosure should be determined by reference to the claims, their full scope of equivalents, the specification, and variations thereof.

[0160] Incorporated by reference All publications, patents, and patent applications referenced herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference.

Claims

1. A pharmaceutical composition comprising bezafibrate as a first active ingredient, a second active ingredient selected from the group consisting of oxaprozin and zaltoprofen, and a pharmaceutically acceptable carrier.

2. 10. The pharmaceutical composition of claim 1, comprising bezafibrate and oxaprozin.

3. 3. The pharmaceutical composition of claim 2, comprising: (a) bezafibrate in the range of 25% to 75% of the clinically approved dose of BEZALIP® SR (bezafibrate extended-release); and (b) a therapeutically effective amount of oxaprozin in the range of 25% to 100% of the clinically approved dose of DAYPRO® (oxaprozin).

4. 4. The pharmaceutical composition of claim 3, wherein the therapeutically effective amount of bezafibrate is in the range of 100 mg to 300 mg, and the therapeutically effective amount of oxaprozin is in the range of 300 mg to 1200 mg.

5. 3. The pharmaceutical composition of claim 2, comprising: (a) a therapeutically effective amount of bezafibrate in the range of 25% to 100% of the clinically approved dose of BEZALIP® SR; and (b) a therapeutically effective amount of oxaprozin in the range of 25% to 75% of the clinically approved dose of DAYPRO®.

6. 6. The pharmaceutical composition of claim 5, wherein the therapeutically effective amount of bezafibrate is in the range of 100 mg to 400 mg or 5 mg to 500 mg, and the therapeutically effective amount of oxaprozin is in the range of 300 mg to 900 mg or 5 mg to 500 mg.

7. The pharmaceutical composition of claim 1 , comprising bezafibrate and zaltoprofen.

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the first and second active ingredients are provided in therapeutically effective amounts sufficient to treat or reduce obesity when administered to a subject.

9. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the first and second active ingredients are provided in therapeutically effective amounts sufficient to treat or reduce type II diabetes when administered to a subject.

10. 8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the first and second active ingredients are provided in therapeutically effective amounts capable of inducing expression of UCP1, FABP4 (aP2), PPARγ2, mtTFAs, PGC-1α and / or COX IV in human skeletal muscle BAT progenitor cells in vitro, in vivo or both.

11. The composition comprises: (a) increased heat production in brown adipose tissue and / or skeletal muscle tissue; (b) increasing insulin sensitivity in skeletal muscle, white adipose tissue, or liver; (c) increased glucose tolerance; (d) an increase in basal respiration, maximum respiration rate, or uncoupled respiration; (e) increased metabolic rate; (f) reduction of hepatosteatosis; (g) weight loss; (h) reduction in body fat mass; (i) a decrease in plasma leptin levels; (j) a decrease in blood glucose; (k) decreased plasma insulin concentration; (l) reducing insulin resistance; 11. The pharmaceutical composition according to any one of claims 1 to 10, for modulating biological activity in a subject, the pharmaceutical composition having one or more biological activities selected from the group consisting of:

12. The pharmaceutical composition according to any one of claims 1 to 7, for use in a subject in need of modulation of a metabolic response.

13. The pharmaceutical composition according to any one of claims 1 to 7, for use in a subject in need of prevention or treatment of a metabolic disease.

14. The pharmaceutical composition of claim 13, wherein the metabolic disease to be prevented or treated is one or more of obesity, overweight, type II diabetes, insulin resistance, hyperinsulinemia, hyperglycemia, prediabetes, hypertension, hyperlipidemia, hepatosteatosis, fatty liver, non-alcoholic fatty liver disease, hyperuricemia, polycystic ovary syndrome, acanthosis nigricans, bulimia, endocrine disorders, triglyceride storage diseases, Bardet-Biedl syndrome, Laurence-Moon syndrome, Prader-Willi syndrome, neurodegenerative diseases, and Alzheimer's disease.

15. A pharmaceutical composition according to claim 13 or 14, comprising a therapeutically effective amount of bezafibrate in the range of 100 mg to 400 mg, 100 mg to 300 mg, or 5 mg to 500 mg, and a therapeutically effective amount of oxaprozin in the range of 300 mg to 900 mg, 300 mg to 1200 mg, or 5 mg to 500 mg.

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