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Home , Acylurea, Alsactide, Amadinone, Bezafibrate, Clinofibrate, Filenadol

US00779531 OB2

(12) United States Patent (10) Patent No.: US 7,795,310 B2 Lee et al. (45) Date of Patent: Sep. 14, 2010

(54) METHODS AND REAGENTS FOR THE WO WO 2005/025673 3, 2005 TREATMENT OF METABOLIC DISORDERS OTHER PUBLICATIONS (75) Inventors: Margaret S. Lee, Middleton, MA (US); Tenenbaum et al., “Peroxisome Proliferator-Activated Receptor Grant R. Zimmermann, Somerville, Ligand for Prevention of Type 2 Mellitus in MA (US); Alyce L. Finelli, Patients With Coronary Artery Disease'. Circulation, 2004, pp. 2197 Framingham, MA (US); Daniel Grau, 22O2.* Shen et al., “Effect of treatment in sulfonylurea-treated Cambridge, MA (US); Curtis Keith, patients with noninsulin-dependent diabetes mellitus'. The Journal Boston, MA (US); M. James Nichols, of Clinical Endocrinology & Metabolism, vol. 73, pp. 503-510, Boston, MA (US) 1991 (see enclosed abstract).* International Search Report from PCT/US2005/023030, mailed Dec. (73) Assignee: CombinatoRx, Inc., Cambridge, MA 1, 2005. (US) Lin et al., “Effect of Experimental Diabetes on Elimination Kinetics of in Rats.” Metab. Dispos. 17:147-152 (1989). (*) Notice: Subject to any disclaimer, the term of this Abstract only. patent is extended or adjusted under 35 Neogi et al., “Synthesis and Structure-Activity Relationship Studies U.S.C. 154(b) by 0 days. of Cinnamic Acid-Based Novel Antihyperglycemic Agents.” Bioorg. Med. Chem. 11:4059-4067 (21) Appl. No.: 11/171,566 (2003). Vessby et al., “Effects of Bezafibrate on the Serum Lipoprotein Lipid and Apollipoprotein Composition, Lipoprotein Removal (22) Filed: Jun. 30, 2005 Capacity and the Fatty Acid Composition of the Plasma Lipid Esters.” Atherosclerosis 37:257-269 (1980). Abstract only. (65) Prior Publication Data Windholz et al., eds., “Abstract No. 4866. The Merck Index. 10" ed., US 2006/OO69 161A1 Mar. 30, 2006 p. 723-724. Merck and Company, Inc.: Rahway, NJ (1983). Sone et al., “-Related Hypoglycemia in a Patient Receiving Related U.S. Application Data Sufonylurea” Annals of Internal Medicine 134:344, 2001. Yuan et al., “Reversal of Obesity- and Diet-Induced Insulin Resis (60) Provisional application No. 60/584,380, filed on Jun. tance with Salicylates or Targeted Disruption of Ikk?’ Science 30, 2004, provisional application No. 60/649,329, 293: 1673-1677, 2001. filed on Feb. 2, 2005. Search Report from European Application No. 05768.186 mailed on Jun. 2, 2008. Chen et al., “The Pharmacological Interactions of (51) Int. Cl. Antihyperglycemic Agents (1)”,Journal of Chinese Physician 29:47, A6 IK3I/I9 (2006.01) 2001. (52) U.S. Cl...... 514/568; 514/571; 514/866 Communication from the State Intellectual Property Office of the (58) Field of Classification Search ...... 514/568, People's Republic of China for Application No. 2005800289684 514/571,866 with English translation (issued Jul. 17, 2009), (pp. 1-16). See application file for complete search history. Yang et al., “The Comparison of Treatment of Type 2 Diabetes Mellitus Complicated with Hyperlipemia with Bezafibrate and (56) References Cited Sodium', Shanghai Medical Journal 21:478-480, 1998. U.S. PATENT DOCUMENTS * cited by examiner 6, 194.454 B1 2/2001 Dow ...... 514,522 Primary Examiner Kevin Weddington 6,500,823 B1* 12/2002 Literati Nagy et al. ... 514/231.5 (74) Attorney, Agent, or Firm—Clark & Elbing LLP 6,576,256 B2 6/2003 Liang et al. 6.593,347 B2 * 7/2003 Bandarage et al...... 514,327 (57) ABSTRACT 2002/0016293 A1 2/2002 Ratain et al...... 514/9 The invention features compositions, methods, and kits for FOREIGN PATENT DOCUMENTS the treatment of metabolic disorders such as diabetes and WO WOO3,059294 T 2003 obesity. WO WOO3,082283 10, 2003 WO WO 2004/052362 6, 2004 6 Claims, 3 Drawing Sheets U.S. Patent Sep. 14, 2010 Sheet 1 of 3 US 7,795,310 B2

FIG. 1

U.S. Patent Sep. 14, 2010 Sheet 2 of 3 US 7,795,310 B2

FIG 2

300

250

2 O O

150

100

50 ChoW HF Pio 6 DF Bez Bez/DF U.S. Patent Sep. 14, 2010 Sheet 3 of 3 US 7,795,310 B2

FIG. 3

60

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2 O

Chow HF Pio 6 DF Bez BezIDF US 7,795,310 B2 1. 2 METHODS AND REAGENTS FOR THE Given that the strategies currently available for the man TREATMENT OF METABOLIC DSORDERS agement of diabetes are suboptimal, there is a compelling need for treatments that are more effective and are not asso CROSS-REFERENCE TO RELATED ciated with such debilitating side-effects. APPLICATIONS SUMMARY OF THE INVENTION This application claims benefit from U.S. Ser. No. 60/584, 380, filed Jun. 30, 2004, and U.S. Ser. No. 60/649,329, filed The present invention features compositions, methods, and Feb. 2, 2005, each of which is hereby incorporated by refer kits for treating, preventing, and reducing metabolic disor CCC. 10 ders. This invention is particularly useful for treating patients having or at risk of having any condition that is characterized BACKGROUND OF THE INVENTION by a state of hyperglycemia, which may be caused, for example, by an alteration in the insulin signaling pathway The invention relates to the treatment, prevention, and (e.g., a reduction in insulin production, resistance to insulin, reduction of metabolic disorders, such as diabetes and obe 15 or both). Exemplary disorders amenable to treatment accord sity. ing to this invention are obesity, diabetes (e.g., type 1 diabe As the levels of rise postprandially, insulin is tes, type 2 diabetes, maturity-onset diabetes of the young secreted and stimulates cells of the peripheral tissues (skeletal (MODY), and gestational diabetes), satiety, endocrine defi muscles and fat) to actively take up glucose from the blood as ciencies of aging, and any of their associated complications a source of energy. Loss of glucose homeostasis as a result of (e.g., Syndrome X, diabetic retinopathy, diabetic nephropa faulty insulin secretion or action typically results in metabolic thy, diabetic neuropathy, peripheral vascular disease, hyper disorders such as diabetes, which may be co-triggered or lipidemia, hypertension, atherosclerosis, and coronary heart further exacerbated by obesity. Because these conditions are disease). often fatal, strategies to restore adequate glucose clearance In a first aspect, the invention features a composition that from the bloodstream are required. 25 includes (a) bezafibrate or an analog thereof, and (b) Although diabetes may arise secondary to any condition diflunisal or an analog thereof, wherein the bezafibrate and that causes extensive damage to the (e.g., pancreati diflunisal are present in amounts that, when administered to a tis, tumors, administration of certain such as corticos patient, are sufficient to treat, prevent, or reduce a metabolic teroids or pentamidine, iron overload (e.g., hemochromato disorder (e.g., diabetes or obesity). Exemplary combinations sis), acquired or genetic endocrinopathies, and Surgical 30 are bezafibrate and diflunisal; bezafibrate and bismuth Sub excision), the most common forms of diabetes typically arise salicylate; bezafibrate and nimosulide; bezafibrate and from primary disorders of the insulin signaling system. There , bezafibrate and ; bezafibrate and Sun are two major types of diabetes, namely type 1 diabetes (also dilac.; bezafibrate and ibuprofen; and diflunisal; known as insulin dependent diabetes (IDDM)) and type 2 clofibrate and bismuth subsalicylate; clofibrate and nimo diabetes (also known as insulin independent or non-insulin 35 sulide; clofibrate and oxaprozin; clofibrate and diclofenac: dependent diabetes (NIDDM)), which share common long clofibrate and Sundilac; clofibrate and ibuprofen; clofibrinc term complications in spite of their different pathogenic acid and diflunisal; clofibric acid and bismuth subsalicylate: mechanisms. clofibric acid and nimosulide; clofibric acid and oxaprozin, Type 1 diabetes, which accounts for approximately 10% of clofibric acid and diclofenac; clofibric acid and sundilac, all cases of primary diabetes, is an organ-specific autoim 40 clofibric acid and ibuprofen; and diflunisal; cli mune disease characterized by the extensive destruction of nofibrate and bismuth subsalicylate; clinofibrate and nimo the insulin-producing beta cells of the pancreas. The conse sulide; clinofibrate and oxaprozin; clinofibrate and quent reduction in insulin production inevitably leads to the diclofenac; clinofibrate and Sundilac; clinofibrate and ibupro deregulation of glucose metabolism. While the administra fen, gemfibrozil and diflunisal; gemfibrozil and bismuth sub tion of insulin provides significant benefits to patients suffer 45 salicylate; gemfibrozil and nimosulide; gemfibrozil and ing from this condition, the short serum half-life of insulin is oxaprozin, gemfibrozil and diclofenac; gemfibrozil and Sun a major impediment to the maintenance of normoglycemia. dilac, and gemfibrozil and ibuprofen. An alternative treatment is islet transplantation, but this strat In a second aspect, the invention features a composition egy has been associated with limited Success. that includes (a) bezafibrate or an analog thereof; and (b) Type 2 diabetes, which affects a larger proportion of the 50 cinnamic acid or an analog thereof, wherein the beZafibrate population, is characterized by a deregulation in the secretion and diflunisal are present in amounts that, when administered of insulin and/or a decreased response of peripheral tissues to to a patient, are sufficient to treat, prevent, or reduce a meta insulin, i.e., . While the pathogenesis of type bolic disorder. 2 diabetes remains unclear, epidemiologic studies suggest Exemplary bezafibrate analogs are binifibrate, , that this form of diabetes results from a collection of multiple 55 clinofibrate, clofibrate, clofibric acid, , , genetic defects or polymorphisms, each contributing its own or gemfibrozil. predisposing risks and modified by environmental factors, In either of the foregoing aspects, the composition may including excess weight, diet, inactivity, drugs, and excess include a third agent selected from the group consisting of consumption. Although various therapeutic treat Sulfonylureas, non-sulfonylurea secretagogues, insulin, insu ments are available for the management of type 2 diabetes, 60 lin analogs, glucagon-like , exendin-4 polypeptides, they are associated with various debilitating side effects. beta 3 adrenoceptoragonists, PPAR agonists, dipeptidyl pep Accordingly, patients diagnosed with or at risk of having type tidase IV inhibitors, biguanides, alpha-glucosidase inhibi 2 diabetes are often advised to adopt a healthier lifestyle, tors, immunomodulators, and -containing com including loss of weight, change in diet, exercise, and mod binations, angiotensin converting inhibitors, adeno erate alcohol intake. Such lifestyle changes, however, are not 65 sine A1 receptor agonists, adenosine A2 receptor agonists, Sufficient to reverse the vascular and organ damages caused aldosterone antagonists, alpha 1 adrenoceptor antagonists, by diabetes. alpha2-adrenoceptoragonists, alpha2-adrenoceptoragonists, US 7,795,310 B2 3 4 angiotensin receptor antagonists, antioxidants, ATPase wherein the bezafibrate and cinnamic acid are administered in inhibitors, atrial agonists, beta adrenoceptor antago amounts that together are sufficient to treat, prevent, or reduce nists, calcium channel agonists, calcium channel antagonists, a metabolic disorder. diuretics, dopamine D1 receptor agonists, endopeptidase In either of the foregoing aspects, the patient may also be inhibitors, endothelin receptor antagonists, guanylate cyclase administered a third agent selected from the group consisting stimulants, phosphodiesterase V inhibitors, protein kinase of Sulfonylureas, non-sulfonylurea secretagogues, insulin, inhibitors, Cdc2 kinase inhibitors, renin inhibitors, throm insulin analogs, glucagon-like peptides, exendin-4 polypep boxane synthase inhibitors, vasopeptidase inhibitors, vaso tides, beta 3 adrenoceptor agonists, PPAR agonists, dipepti pressin Iantagonists, Vasopressin2 antagonists, angiogenesis dyl peptidase IV inhibitors, biguanides, alpha-glucosidase inhibitors, advanced glycation end product inhibitors, bile 10 inhibitors, immunomodulators, statins and statin-containing acid binding agents, bile acid transport inhibitors, bone for combinations, angiotensin converting enzyme inhibitors, mation stimulants, apolipoprotein A1 agonists, DNA topoi adenosine A1 receptor agonists, adenosine A2 receptor ago somerase inhibitors, absorption inhibitors, cho nists, aldosterone antagonists, alpha 1 adrenoceptor antago lesterol antagonists, cholesteryl ester transfer protein nists, alpha 2 adrenoceptor agonists, alpha 2 adrenoceptor antagonists, cytokine synthesis inhibitors, DNA polymerase 15 agonists, angiotensin receptor antagonists, antioxidants, inhibitors, dopamine D2 receptoragonists, endothelin recep ATPase inhibitors, atrial peptide agonists, beta adrenoceptor tor antagonists, antagonists, insulin sensi antagonists, calcium channel agonists, calcium channel tizers, lipase inhibitors, lipid peroxidation inhibitors, lipopro antagonists, diuretics, dopamine D1 receptor agonists, tein A antagonists, microsomal transport protein inhibitors, endopeptidase inhibitors, endothelin receptor antagonists, microsomal triglyceride transfer protein inhibitors, nitric guanylate cyclase stimulants, phosphodiesterase Vinhibitors, oxide synthase inhibitors, oxidizing agents, phospholipase protein kinase inhibitors, Cdc2kinase inhibitors, renin inhibi A2 inhibitors, radical formation agonists, aggrega tors, synthase inhibitors, vasopeptidase inhibi tion antagonists, synthase stimulants, reverse tors, vasopressin I antagonists, vasopressin 2 antagonists, cholesterol transport activators, rho kinase inhibitors, selec angiogenesis inhibitors, advanced glycation end product tive estrogen receptor modulators, squalene epoxidase inhibi 25 inhibitors, bile acid binding agents, bile acid transport inhibi tors, squalene synthase inhibitors, antago tors, bone formation stimulants, apolipoprotein A1 agonists, nists, amylin agonists, receptor antagonists, DNA topoisomerase inhibitors, cholesterol absorption A agonists, corticotropin-releasing factor inhibitors, cholesterol antagonists, cholesteryl ester transfer agonists, dopamine uptake inhibitors, G protein-coupled protein antagonists, cytokine synthesis inhibitors, DNA poly receptor modulators, glutamate antagonists, glucagon-like 30 merase inhibitors, dopamine D2 receptor agonists, endothe peptide-1 agonists, insulin sensitizers, lipase inhibitors, lin receptor antagonists, growth hormone antagonists, insulin melanin-concentrating hormone receptor antagonists, nerve sensitizers, lipase inhibitors, lipid peroxidation inhibitors, growth factor agonists, neuropeptide Yagonists, neuropep lipoprotein A antagonists, microsomal transport protein tide Y antagonists, SNRIs, protein tyrosine phosphatase inhibitors, microsomal triglyceride transfer protein inhibi inhibitors, and serotonin 2C receptor agonists. 35 tors, synthase inhibitors, oxidizing agents, phos pholipase A2 inhibitors, radical formation agonists, platelet The compositions of the invention may be formulated for aggregation antagonists, prostaglandin Synthase stimulants, oral administration or systemic administration. reverse cholesterol transport activators, rho kinase inhibitors, The invention also features a method for treating, prevent selective estrogen receptor modulators, squalene epoxidase ing, or reducing a metabolic disorder in a patient in need 40 inhibitors, squalene synthase inhibitors, thromboxane A2 thereof by administering to the patient (i) bezafibrate or an antagonists, amylin agonists, cannabinoid receptor antago analog thereof, and (ii) diflunisal or an analog thereof, nists, cholecystokinin Aagonists, corticotropin-releasing fac wherein the bezafibrate and diflunisal are administered in tor agonists, dopamine uptake inhibitors, G protein-coupled amounts that together are sufficient to treat, prevent, or reduce receptor modulators, glutamate antagonists, glucagon-like a metabolic disorder. Exemplary combinations are bezafi 45 peptide-1 agonists, insulin sensitizers, lipase inhibitors, brate and diflunisal; bezafibrate and bismuth subsalicylate: melanin-concentrating hormone receptor antagonists, nerve bezafibrate and nimosulide; bezafibrate and oxaprozin; growth factor agonists, neuropeptide Yagonists, neuropep bezafibrate and diclofenac; bezafibrate and Sundilac, bezafi tide Y antagonists, SNRIs, protein tyrosine phosphatase brate and ibuprofen; clofibrate and diflunisal; clofibrate and inhibitors, and serotonin 2C receptor agonists. bismuth subsalicylate; clofibrate and nimosulide; clofibrate 50 and oxaprozin, clofibrate and diclofenac; clofibrate and Sun The two drugs may beformulated for oral administration or dilac; clofibrate and ibuprofen; clofibrinc acid and diflunisal; systemic administration. The first and second agents are clofibric acid and bismuth subsalicylate; clofibric acid and desirably administered within 10 days of each other, within 7 days of each other, within 24 hours of each other, or within 1 nimosulide; clofibric acid and oxaprozin, clofibric acid and hour of each other. diclofenac; clofibric acid and sundilac, clofibric acid and 55 ibuprofen; clinofibrate and diflunisal; clinofibrate and bis The invention also features a kit that includes (i) bezafi muth subsalicylate; clinofibrate and nimosulide; clinofibrate brate or an analog thereof, and (ii) instructions for adminis and Oxaprozin; clinofibrate and diclofenac; clinofibrate and tering beZafibrate and cinnamic acid oran analog thereof to a sundilac; clinofibrate and ibuprofen; gemfibrozil and patient having or at risk of having a metabolic disorder. diflunisal; gemfibrozil and bismuth Subsalicylate; gemfi 60 The invention also features a kit that includes (i) bezafi brozil and nimosulide; gemfibrozil and Oxaprozin, gemfi brate or an analog thereof, and (ii) instructions for adminis brozil and diclofenac; gemfibrozil and Sundilac, and gemfi tering beZafibrate and diflunisal or an analog thereof to a brozil and ibuprofen. patient having or at risk of having a metabolic disorder. The invention also features a method for treating, prevent The invention also features a kit that includes (i) diflunisal ing, or reducing a metabolic disorder in a patient in need 65 or an analog thereof, and (ii) instructions for administering thereof by administering to the patient (i) bezafibrate or an diflunisal and beZafibrate or an analog thereof to a patient analog thereof, and (ii) cinnamic acid or an analog thereof, having or at risk of having a metabolic disorder. US 7,795,310 B2 5 6 The invention also features a kit that includes (i) cinnamic aggregation antagonists, prostaglandin Synthase stimulants, acid oran analog thereof, and (ii) instructions for administer reverse cholesterol transport activators, rho kinase inhibitors, ing cinnamic acid and beZafibrate or an analog thereof to a selective estrogen receptor modulators, squalene epoxidase patient having or at risk of having a metabolic disorder. inhibitors, squalene synthase inhibitors, thromboxane A2 The invention also features a kit that includes (i) a compo antagonists, amylin agonists, cannabinoid receptor antago sition containing bezafibrate or an analog thereof and cin nists, cholecystokinin Aagonists, corticotropin-releasing fac namic acid or an analog thereof, and (ii) instructions for tor agonists, dopamine uptake inhibitors, G protein-coupled administering the composition to a patient having or at risk of receptor modulators, glutamate antagonists, glucagon-like having a metabolic disorder. peptide-1 agonists, insulin sensitizers, lipase inhibitors, The invention also features a kit that includes (i) a compo 10 melanin-concentrating hormone receptor antagonists, nerve sition containing beZafibrate or an analog thereof and growth factor agonists, neuropeptide Yagonists, neuropep diflunisal orananalog thereof, and (ii) instructions for admin tide Y antagonists, SNRIs, protein tyrosine phosphatase istering the composition to a patient having or at risk of inhibitors, and serotonin 2C receptor agonists. having a metabolic disorder. The compositions of the invention may be formulated for The invention also features a kit that includes (i) bezafi 15 oral administration or systemic administration. brate or an analog thereof; (ii) cinnamic acid or an analog The invention also features a method for treating, prevent thereof; and (iii) instructions for administering bezafibrate ing, or reducing a metabolic disorder in a patient in need and cinnamic acid to a patient having or at risk of having a thereof by administering to the patient (i) a PPAR agonist; and metabolic disorder. (ii) diflunisal oran analog thereof, wherein the PPARYagonist The invention also features a kit that includes (i) bezafi and diflunisal are administered in amounts that together are brate oran analog thereof (ii) diflunisal oran analog thereof; sufficient to treat, prevent, or reduce a metabolic disorder. and (iii) instructions for administering beZafibrate and The invention also features a method for treating, prevent diflunisal to a patient having or at risk of having a metabolic ing, or reducing a metabolic disorder in a patient in need disorder. thereof by administering to the patient (i) a PPAR agonist; and The invention also features a composition that includes (a) 25 (ii) cinnamic acid or an analog thereof, wherein the PPARY a PPAR agonist; and (b) diflunisal or an analog thereof, agonist and cinnamic acid are administered in amounts that wherein the PPAR agonist and diflunisal are present in together are sufficient to treat, prevent, or reduce a metabolic amounts that, when administered to a patient, are sufficient to disorder. treat, prevent, or reduce a metabolic disorder. The invention also features a kit that includes (i) a PPAR The invention also features a composition that includes (a) 30 agonist; and (ii) instructions for administering the PPAR ago a PPAR agonist; and (b) cinnamic acid or an analog thereof, nistand cinnamic acid oran analog thereof to a patient having wherein the PPAR agonist and diflunisal are present in or at risk of having a metabolic disorder. amounts that, when administered to a patient, are sufficient to The invention features a kit that includes (i) a PPAR ago treat, prevent, or reduce a metabolic disorder. nist; and (ii) instructions for administering the PPAR agonist In either of the foregoing aspects, the patient may also be 35 and diflunisal oran analog thereof to a patient having or at risk administered a third agent selected from the group consisting of having a metabolic disorder. of Sulfonylureas, non-sulfonylurea secretagogues, insulin, The invention features a kit that includes (i) diflunisal oran insulin analogs, glucagon-like peptides, exendin-4 polypep analog thereof, and (ii) instructions for administering tides, beta 3 adrenoceptor agonists, PPAR agonists, dipepti diflunisal and a PPAR agonist oran analog thereof to a patient dyl peptidase IV inhibitors, biguanides, alpha-glucosidase 40 having or at risk of having a metabolic disorder. inhibitors, immunomodulators, statins and statin-containing The invention also features a kit that includes (i) cinnamic combinations, angiotensin converting enzyme inhibitors, acid oran analog thereof, and (ii) instructions for administer adenosine A1 receptor agonists, adenosine A2 receptor ago ing cinnamic acid and a PPAR agonist oran analog thereof to nists, aldosterone antagonists, alpha 1 adrenoceptor antago a patient having or at risk of having a metabolic disorder. nists, alpha 2 adrenoceptor agonists, alpha 2 adrenoceptor 45 The invention also features a kit that includes (i) a compo agonists, angiotensin receptor antagonists, antioxidants, sition containing a PPAR agonist or an analog thereof and ATPase inhibitors, atrial peptide agonists, beta adrenoceptor cinnamic acid or an analog thereof, and (ii) instructions for antagonists, calcium channel agonists, calcium channel administering the composition to a patient having or at risk of antagonists, diuretics, dopamine D1 receptor agonists, having a metabolic disorder. endopeptidase inhibitors, endothelin receptor antagonists, 50 The invention also features a kit that includes (i) a compo guanylate cyclase stimulants, phosphodiesterase Vinhibitors, sition containing a PPAR agonist or an analog thereof and protein kinase inhibitors, Cdc2kinase inhibitors, renin inhibi diflunisal orananalog thereof, and (ii) instructions for admin tors, thromboxane synthase inhibitors, vasopeptidase inhibi istering the composition to a patient having or at risk of tors, vasopressin I antagonists, vasopressin 2 antagonists, having a metabolic disorder. angiogenesis inhibitors, advanced glycation end product 55 The invention also features a kit that includes (i) a PPAR inhibitors, bile acid binding agents, bile acid transport inhibi agonist or an analog thereof; (ii) cinnamic acid or an analog tors, bone formation stimulants, apolipoprotein A1 agonists, thereof; and (iii) instructions for administering the PPAR DNA topoisomerase inhibitors, cholesterol absorption agonist and cinnamic acid to a patient having or at risk of inhibitors, cholesterol antagonists, cholesteryl ester transfer having a metabolic disorder. protein antagonists, cytokine synthesis inhibitors, DNA poly 60 The invention also features a kit that includes (i) a PPAR merase inhibitors, dopamine D2 receptor agonists, endothe agonist or an analog thereof; (ii) diflunisal or an analog lin receptor antagonists, growth hormone antagonists, insulin thereof; and (iii) instructions for administering the PPAR sensitizers, lipase inhibitors, lipid peroxidation inhibitors, agonist and diflunisal to a patient having or at risk of having lipoprotein A antagonists, microsomal transport protein a metabolic disorder. inhibitors, microsomal triglyceride transfer protein inhibi 65 In any of the foregoing lits, the PPAR agonist is desirably tors, nitric oxide synthase inhibitors, oxidizing agents, phos a PPARY agonist (e.g., balaglitaZone, , pioglita pholipase A2 inhibitors, radical formation agonists, platelet Zone, , , , , US 7,795,310 B2 7 8 englitazone, , KRP-297, JTT-501, NC-2100, antagonists, diuretics, dopamine D1 receptor agonists, NIP-223, MCC-555, L-764486, CS-011, GI262570, endopeptidase inhibitors, endothelin receptor antagonists, GW.347845, or FK614). guanylate cyclase stimulants, phosphodiesterase Vinhibitors, The invention also features a method of treating a meta protein kinase inhibitors, Cdc2kinase inhibitors, renin inhibi bolic disorder by administering to a mammal (e.g., human) 5 tors, thromboxane synthase inhibitors, vasopeptidase inhibi one or more agents listed in Table 1 in an amount sufficient to tors, vasopressin I antagonists, vasopressin 2 antagonists, treat, prevent, or reduce the metabolic disorder. angiogenesis inhibitors, advanced glycation end product S. example, th As by E. N. inhibitors, bile acid binding agents, bile acid transport inhibi tered two agents listed in lable 1 within ays of each other tors, bone formation stimulants, apolipoprotein A1 agonists, in amounts that together- 0 are sufficient to treat, prevent, or 10 DNA topoisomerase inhibitors,- - - - - cholesterol absorption reduce the metabolic- 0 disorder. The two agents are desirably inhibitors,- - - - - cholesterol antagonists, cholesteryl ester transfer administered- 0 within 14 days of each other, more desirably protein antagonists, cytokine synthesis inhibitors,- - - - DNA poly within seven days of each other, and even more desirably inhibitors, d ine D2 t ist doth within twenty-four hours of each other, or even simulta- E. 1ditors, dopamine iG or agonists, enao i. neously (i.e., concomitantly). If desired, either one of the two 15 "eceptor antagonists, growt normone antagonists, insulin agents may be administered in low dosage. sensitizers, lipase inhibitors, lipid peroxidation inhibitors, Optionally, the mammal being treated may receive an addi- lipoprotein A antagonists, microsomal transport protein tional therapeutic regimen. Ifatherapeutic agent is employed inhibitors, microsomal triglyceride transfer protein inhibi as the additional therapeutic regimen, the agent or agents tors, nitric oxide synthase inhibitors, oxidizing agents, phos from Table 1 and the additional agent are present in amounts 20 pholipase A2 inhibitors, radical formation agonists, platelet that, when administered to a mammal, are together sufficient aggregation antagonists, prostaglandin synthase stimulants, to treat, prevent, or reduce a metabolic disorder. The addi- reverse cholesterol transport activators, rho kinase inhibitors, tional agent may be selected from the group consisting of selective estrogen receptor modulators, squalene epoxidase Sulfonylureas, non-sulfonylurea secretagogues, insulin, insu- inhibitorS, squalene synthase inhibitors, thromboxane A2 lin analogs, glucagon-like peptides, exendin-4 polypeptides, 25 antagonists, amylin agonists, cannabinoid receptor antago beta 3 adrenoceptoragonists, PPAR agonists, dipeptidyl pep- nists, cholecystokinin Aagonists, corticotropin-releasing fac tidase IV inhibitors, biguanides, alpha-glucosidase inhibi- tor agonists, dopamine uptake inhibitors, G protein-coupled tors, immunomodulators, statins and Statin-containing com- receptor modulators, glutamate antagonists, glucagon-like binations, angiotensin converting enzyme inhibitors, peptide-1 agonists, insulin sensitizers, lipase inhibitors, adenosine A1 receptor agonists, adenosine A2 receptor ago- 30 melanin-concentrating hormone receptor antagonists, nerve nists, aldosterone antagonists, alpha 1 adrenoceptor antago- growth factor agonists, neuropeptide Yagonists, neuropep nists, alpha 2 adrenoceptor agonists, alpha 2 adrenoceptor tide, Y antagonists, SNRIs, protein tyrosine phosphatase agonists, angiotensin receptor antagonists, antioxidants, inhibitors, and serotonin 2C receptor agonists. ATPase inhibitors, atrial peptide agonists, beta adrenoceptor If desired, more than one therapeutic agent may be used antagonists, calcium channel agonists, calcium channel with any of the agents listed in Table 1.

TABLE 1 5-10-Dihydro-5-10- Calcium Chloride S- Indocyanine Green Mono-Butyl Maleate Succinylcholine Dimethylphenazine Hydroxytryptophan (e.g., DL form) Candesartan (e.g., Dopamine (e.g., indomethacin Sulfaguanidine cilexetil salt) hydrochloride) Acenocoumarol Captopril Doxapram (e.g., nulin Sulfamethizole hydrochloride salt) Acetaminophen Doxycycline Wetase Nitroxoline Sulfasomidine Carbinoxamine (e.g., (e.g., opanoic Acid Norepinephrine (e.g., maleate salt) Succinate salt) bitartrate salt) Acetohexamide Dyclonine (e.g., ophenoxic Acid Olanzapine Tadalafil hydrochloride salt) Acetohydroxamic Cefamandole (e.g., Enalaprilat opromide Oleandomycin Tannic Acid Acid nafate salt) Acetrizoate (e.g., Cefpodoxime Proxetil Epinephrine (e.g., Soproterenol (e.g., (e.g., Sodium salt) bitartrate salt) Sulfate salt) citrate salt) Acetylcholine (e.g., Ciclopirox Mesylates Isotretinoin Oxaprozin Terbutaline (e.g., chloride salt) Sulfate salt) Acetyldigitoxin Cinnamic Acid Ethopropazine (e.g., Ketoconazole Oxybutynin (e.g., Tetrahydrozoline hydrochloride salt) chloride salt) (e.g., hydrochloride salt) Alachlor Clenbuterol (e.g., Etoposide Ketotifen (e.g., fumarate Oxymetazoline (e.g., Tinidazole hydrochloride salt) salt) hydrochloride salt) Albuterol Clioquinol Eucalyptol Lamivudine Pergolide (e.g., Tioconazole (Salbutamol) (e.g., meSylate salt) Sulfate salt) Alprenolol Copper bis-3,5- Evans Blue Lead Tolazoline (e.g., diisopropylsalicylate Dimethyldithiocarbamate hydrochloride salt) Amantadine (e.g., Cupric Chloride Exemestane Leflunomide Phenindione hydrochloride salt) Ametryn Flumixin (e.g., Levocabastine (e.g., Tretinoin meglumine salt) hydrochloride salt) Ammonium Diacerein Fumaric Acid Levonordefrin Triamterene Chloride Monoethyl Ester US 7,795,310 B2 9 10

TABLE 1-continued Arbutin Diallyl Maleate Gemfibrozil Loxapine (e.g., Phenylephrine (e.g., Triflupromazine hydrochloride salt) hydrochloride salt) (e.g., hydrochloride salt) Artemether Diatrizoate (e.g., Manganese Sulfate Phenylpropanolamine Tulobuterol (e.g., Sodium salt) (e.g. hydrochloride hydrochloride salt) salt) Aspartic Acid Diclofenac (e.g., GeranylAcetate Mebhydroline (e.g., 1,5- Vincamine Hydroxamate Sodium salt) Naphthalenedisulfonate salt) Atrazine Dicloxacillin (e.g., Glutamic Acid Meclofenoxate Pilocarpine (e.g., Warfarin Sodium salt) Hydrochloride hydrochloride salt) Azathioprine Diflunisal Guaiacol Prazosin (e.g., (e.g., hydrochloride salt) hydrochloride salt) Bamethan Digitoxin Guanfacine (e.g., (e.g., Zaprinast hydrochloride salt) hydrochloride salt) Benzbromarone Hexylcaine (e.g., Melphalan Prostaglandin (e.g., hydrochloride salt) prostaglandin E) Bezafibrate Dimethyl Fumarate Homatropine Mercaptoethanol Pyrilamine (e.g., Methylbromide maleate salt) Bismuth Diphemanil Hydroxocobalamin Metaproterenol (e.g., Quinacrine Subsalicylate Methylsulfate hemisulfate salt) Bopindolol Hydroxypropyl Methacholine (e.g., Ritodrine (e.g., (e.g., hydrochloride Cellulose chloride or salt) hydrochloride salt) salt) Brimonidine Diphenidol (e.g., budilast Methodilazine RO-20-1724 hydrochloride salt) Brompheniramine Diplivefrin (e.g., buprofen Methyl Linoleate Rolipram (e.g., maleate salt) hydrochloride salt) Calcitriol Dirithromycin mipramine (e.g., Methylergonovine (e.g., Spectinomycin hydrochloride salt) maleate salt)

If the mammal is administered more than one agent, the 30 immunosuppressant, immunomodulator, angiotensin con different agents may be admixed together in a single formu Verting enzyme (ACE) inhibitor, angiotensin II receptor lation. When administered in separate formulations, the blocker, and antioxidant, as well as instructions for adminis agents may be administered simultaneously or within 14 tering these two agents together to a patient having or at risk days, 7 days, or 1 day of each other. These agents may or may of having a metabolic disorder. not be administered by the same route of administration (e.g., 35 The invention also features a method of identifying a com oral, intravenous, intramuscular, ophthalmic, topical, dermal, bination of agents useful for the treatment, prevention, or Sub-cutaneous, and rectal). Optionally, the additional thera reduction of a metabolic disorder, involving the steps of: (a) peutic regimen may involve a lifestyle change, including the contacting cells with an agent listed in Table 1 and a candidate adoption of a low-fat diet or low-sodium diet, stress manage compound; and (b) determining whether this combination of ment, physical exercise, reduction in alcohol intake, or reduc 40 agents reduces glucose levels relative to cells contacted only tion in Smoking. with the agent from Table 1 but not contacted with the can In a further aspect, the present invention features a kit that didate compound. A reduction in glucose levels identifies the includes any one of the agents listed in Table 1 and instruc combination as being useful for the treatment, prevention, or tions for its administration to a patient having or at risk of reduction of a metabolic disorder. Glucose levels may be 45 reduced, for example, by altering insulin signaling (therefore having a metabolic disorder. Optionally, the kit contains two, increasing glucose uptake into cells and Subsequent storage three, four, or more than four agents from Table 1 that may or or metabolism, for example), altering glucose transporter may not be admixed in the same formulation. This kit may activity (e.g., increasing GLUT4 expression, translocation, or also contain instructions for administering this agent with a intrinsic activity), increasing the amount of insulin-sensitive second agent listed in Table 1. 50 tissue (e.g., by increasing adipocyte or muscle cell differen The invention also features a kit that includes (a) one, two, tiation), or altering genetranscription in adipocytes or muscle three, or more agents listed in Table 1 and (b) one or more of cells (e.g., altered secretion of factors from adipocytes and the following agents: a Sulfonylurea, non-sulfonylurea secre expression of metabolic pathway genes). tagogue, insulin, insulin analog, glucagon-like peptide, exen For example, the screening method for identifying a useful din-4, YM178, FK614, dipeptidyl peptidase IV inhibitor, ss therapeutic combination involves the steps of: (a) contacting biguanide, thiazalidinedione, alpha-glucosidase inhibitor, cells with an agent listed in Table 1 and a candidate com immunosuppressant, immunomodulator, angiotensin con pound; and (b) determining whether this combination of Verting enzyme (ACE) inhibitor, angiotensin II receptor agents alters insulin signaling Such that glucose levels are blocker, or antioxidant. The kit also includes instructions for reduced relative to cells contacted with the agent from Table administering these agents to a patient having or at risk of 60 1 but not contacted with the candidate compound. An alter having a metabolic disorder. ation in insulin signaling that reduces glucose levels identifies Alternatively, the kit of the invention may contain one, two, the combination as being useful for the treatment, prevention, three, or more agent listed in Table 1 or any one of the or reduction of a metabolic disorder. following agents: a Sulfonylurea, non-Sulfonylurea secreta Mammalian (e.g., human) cells may be employed in any of gogue, insulin, insulin analog, glucagon-like peptide, exen- 65 the screening methods described herein. Particularly useful din-4, YM178, FK614, dipeptidyl peptidase IV inhibitor, cells are muscle cells, intestinal cells, adipocytes, cells, biguanide, thiazalidinedione, alpha-glucosidase inhibitor, and pancreatic cells. Optionally, these cells have an alteration US 7,795,310 B2 11 12 in insulin signaling activity Such that glucose levels are Analogs of any of the compounds listed in Table 1 may be increased. Such a reduction in glucose levels may result from used in any of the methods, kits, and compositions of the an increase in insulin production, an increase in insulin secre tion, an increase in glucose uptake by peripheral tissues, a invention. Such analogs include any agent from the same reduction in hepatic glucose production, or a reduction in the chemical class, mechanistic class, or therapeutic class as absorption of carbohydrates from the intestines. those listed in Table 2.

TABLE 2 chemical class mechanistic class therapeutic class 5-10-dihydro-5-10 phenothiazine dimethylphenazine 5-hydroxytryptophan (e.g., intermediate metabolite of It in anti- dl form) the production of serotonin acemetacin indomethacin cyclooxegynase inhibitor anti-inflammatory agents derivative non-steroidal acenocoumarol heterocyclic coumarin anticoagulant thromboembolism atrial fibrillation pulmonary embolism acetaminophen Sodium Sulfate blocks NSAID (prostaglandin formation) acetazolamide sulfa-based carbonic anhydrase inhibitor anti-epileptic acetohexamide Sulfonylureas stimulate beta cell insulin oral hypoglycemic drugs Secretion acetohydroxamic acid specific inhibitor of urease antibacterial acetrizoate (e.g., sodium acetrizoic acid iodinated radiographic contrast salt) medium used in hysterosalpingography acetylcholine (e.g., mixed cholinergic agonist neurotransmitter chloride salt) acetyldigitoxin Sodium potassium atpase inotropic congestive heart failure alachlor acetamide herbicide albuterol (salbutamol) ethanolamine beta agonist bronchodilator sulfate alprenolol proponalamine andrenergic beta antagonist antihypertensive, anti-anginal, and anti-arrhythmic agent amantadine (e.g., antiparkinson hydrochloride salt) ametryn triazine herbicide ammonium chloride ammonia derivative diuretics, expectorants arbutin tyrosinase inhibitor dermatological artemether artemisinins anti-malarial aspartic acid hydroxamate asperigine antineoplastic agents antiviral agents Atrazine triazine selective triazine herbicide herbicide azathioprine heterocyclic immunosuppressive bamethan vasodilator benzbromarone benzofuran acts by increasing uric acid antigout clearance bezafibrate butyrate lowers cholesterol and antilipemic, cholesterol lowering bismuth Subsalicylate bismuth salt increases intracellular Ca2+, anti-diarrheal MAP-kinase activity, and cell proliferation in normal human gastric mucous epithelial cells bopindolol beta antagonist — antihypertensive brimonidine alpha-adrenergic receptor agonist anti-glaucoma brompheniramine maleate H1 antagonist anti-allergic agent calcitriol vitamin Danalog calcium channel agonists dermatological calcium chloride dietary Supplyment candesartan (e.g., cilexetil benzimidazole TL-type angiotensin II receptor antihypertensive salt) antagonist captopril mercaptain angiotensin-converting enzyme antihypertensive inhibitors carbamazepine iminostilbene derivative carbinoxamine (e.g., histamine H1 antagonist antihistamine maleate salt) carisoprodol carbamic acid muscle relaxent cefamandole (e.g., nafate cephalosporins cell wall synthesis inhibitor antibiotic salt) cefpodoxime proxetil cephalosporins antibiotic ciclopirox hydroxypyridone fungicide topical antifungal cinnamic acid cinnamate flavoring, coloring US 7,795,310 B2 13 14

TABLE 2-continued chemical class mechanistic class therapeutic class clenbuterol hydrochloride beta-2- beta agonist anticatabolic Symphatomimetic clioquinol 8-hydroxyquinoline antifungal, amoebicides derivative copper bis-3,5- salicyclic acid antineoplastic, hypoglycemic iisopropylsalicylate agent cupric chloride mineral antifungal exibuprofen single-enantiomer form inhibits prostaglandin synthesis NSAID of ibuprofen iacerein anthraquinones NSAID iallyl maleate mallic acid plastics additive iatrizoate (e.g., sodium X-ray contrast medium S alt) iclofenac sodium benzeneacetic acid NSAID icloxacillin (e.g., sodium penicillin derivative? inhibits bacterial cell wall antibiotic alt) beta-lactam synthesis iflunisal salicylate derivative cyclooxygenase inhibitors NSAID igitoxin cardiac glycoside inhibits Na+/K+ ATPase cell inotropic cardiovascular membrane transport complex agent, congestive heart failure imenhydrinate diphenhydramine and histamine H1 antagonists vertigo agent chlorotheophylline imethyl fumarate fumarate anti-psoriatic iphemanil methylsulfate synthetic quaternary anti-muscarinic agent ammonium compound iphenhydramine (e.g., ethanolamine histamine H1 receptor antagonist antihistamine hydrochloride salt) iphenidol (e.g., muscarinic antagonist anti-nausea, vomiting, hydrochloride salt) dizziness ipivefrin hydrochloride prodrug of epinephrine bronchodialator anti-glaucoma irithromycin inhibits bacterial protein antibacterial synthesis at the 50S ribosome (macrollide) opamine (e.g., neurotransmitter antidepressant hydrochloride) Oxapram (e.g., respiratory stimulant acute hypercarnic respiratory hydrochloride salt) failure oxycycline tetracycline derivative protein-synthesis-inhibitor antibiotic oxylamine (e.g., Succinate butanedioic acid antihistamine-H1 serotonin allergic rhinitis alt) secretion-inhibitor yclonine (e.g., nerve sodium-permeability pain reliever hydrochloride salt) inhibitor enalaprilat oligopeptide ACE inhibitor antihypertensive epinephrine bitartrate hormone induces cyclic AMP stimulant, asthma ergoloid mesylates dihydroergotoxine adrenergic alpha-antagonists Alzheimer's disease ethopropazine (e.g., phenothiazine Antidyskinetic Parkinson's disease hydrochloride salt) etoposide glucoside topoisomerase II inhibitor antineoplastic agent, phytogenic eucalyptol cyclohexanols local anti-infective agent evans blue azo dye diagnostic for blood volume determination exeneStane androstadiene aromatase antagonist inhibitor antineoplastic agent meglumine clinixin derivative prostaglandin antagonist NSAID fumaric acid monoethyl fumarate anti-psoriatic ester gemfibrozil pentonoic acid lowers elevated serum lipids lipid lowering primarily by decreasing serum triglycerides genistein isoflavone angiogenesis blocker geranyl isoflavone antineoplastic agent glutamic acid amino acid amino acid hydrochloride guaiacol phenol disinfectant, expectorant guanfacine (e.g., acetamide C2-adrenoceptor agonist antihypertensive hydrochloride salt) hexylcaine (e.g., benzoic acid derivitive topical anesthetic hydrochloride salt) homatropine tropine ester of antispasmodic and inhibitor of methylbromide mandelic acid Secretions hydroxocobalamin vitamin B12 inhibits vitamin B12 deficiency synthetic vitamin B12 hydroxypropyl cellulose cellulose Mechanical topical artificial tear ibudilast pyridine phosphodiesterase iv inhibitor anti-asthma ibuprofen phenylpropionates cyclooxygenase inhibitor NSAID imipramine (e.g., dibenzazepine blocks neuptake at adrenergic antidepressant hydrochloride salt) nerve endings indocyanine green tricarbocyanine infrared absorbance diagnostic for angiography indomethacin indole cyclooxygenase inhibitor NSAID US 7,795,310 B2 15 16

TABLE 2-continued l8le chemical class mechanistic class therapeutic class oligosaccharide diagnostic for kidney health; probiotic; dietary fiber invertase beta-fructofuranosidase hydrolysis of the terminal enzyme nonreducing beta fructofuranoside residues in beta fructofuranosides iopanoic acid radiopaque compound antihyperthyroid agent; X-ray contrast medium iophenoxic acid radiopaque compound X-ray contrast medium iopromide benzoic acid derivitive isoproterenol (e.g., Sulfate amine beta adrenergic agonist inotropic agent salt) isoproterenol sulfate analog of epinephrine sympathomimetic, direct-acting beta agonist isotretinoin acne Vulgaris ketoconazole azole antifungal ketotifen fumarate cycloheptathiophene H1 receptor antagonist asthma, rhinitis, skin allergies, and anaphylaxis. lamivudine deoxyribonucleoside reverse transcriptase inhibitor anti-HIV treatment lead dimethyldithiocarbamate carbamic acid rubber accelerator leflunomide oxazole inhibits dihydroorotate DMARD dehydrogenase levocabastine (e.g., antihistaminic, H1-receptor; antiallergic; opthalmic hydrochloride salt) ophthalmic levonordefrin norepinephrine stimulates alpha- and beta vasoconstrictor, local derivative adrenergic systems anesthetic for dentistry loxapine (e.g., tricyclic antipsychotic agent hydrochloride salt) dibenzoxazepine manganese Sulfate sulfate mebhydroline (e.g., 1,5- aralkylamine carboline histamine H1 antagonists antihistamine naphthalenedisulfonate salt) mebhydroline 1,5- naphthalenesulfonates naphthalenedisulfonate salt meclofenoxate aminoalcohol stimulant mefenamic acid anthranilic acid cyclooxygenase inhibitor NSAID meloxicam derivative cyclooxygenase inhibitor NSAID melphalan nitrogen mustard alkylating antineoplastic not antineoplastic compound completely understood mercaptoethanol Sulfhydryl compound reducing agent biochem reagent metaproterenol (e.g., ethanolamine beta-adrenergic agonist treatment of asthma and hemisulfate salt) bronchoSpams metaproterenol hemisulfate synthetic beta agonist bronchodilator Salt sympathomimetic amine methacholine (e.g., quarternary ammonium muscarinic agonist parasympathomimetic bromide salt) bronchoconstrictor agent methacholine (e.g., quarternary ammonium muscarinic agonist parasympathomimetic chloride salt) bronchoconstrictor agent methdilazine phenothiazine antihistamine h1 receptor agonist allergies, antipsychotic methyl linoleate antioxidant flavoring agent methylergonovine (e.g., ergot alkyloid homolog Oxytocic obstetrics, to induce labor maleate salt) mono-butyl maleate maleate ester fumarate sar anti-inflammatory nefopam oxazocine nimeSulide Sulfonanilide COX-2 inhibitor NSAID nitroxoline chelating agent bactericidal chelation antibacterial, antifungal norepinephrine bitartrate catecholamine alpha adrenergic agonist vasoconstrictor olanzapine antiemetic anti-nausea oleandomycin macrollide binds to 23s rrna or 50s subunit antibiotic inhibits translocation or trina, inhibits peptidyl transferase interfere wiformation of 50s unit inhibit protein synthesis orphenadrine (e.g., citrate ethylamine muscarinic antagonist drug-induced parkinsonism salt) oxaprozin propionic acid prostaglandin inhibitor NSAID derivative oxybutynin (e.g., chloride mandelic acid competitive antagonist of antispasmodic, urinary tract salt) acetylcholine at postganglionic muscarinic receptors, oxymetazoline (e.g., imidazole adrenergic alpha-agonists, direct vasoconstrictor to relieve hydrochloride salt) acting sympathomimetic nasal congestion pergolide (e.g., meSylate ergoline dopamine agonist Parkinson's disease salt) phenacemide urea deriviative anti-epileptic drug phenindione indandione derivative anti-vitamin Kagent anticoagulent phensuximide anticonvulsant US 7,795,310 B2 17 18

TABLE 2-continued chemical class mechanistic class therapeutic class

phenylbutaZone pyrazole cyclooxegenase inhibitor 8 kylosing spondylitis, rheumatoid arthritis phenylephrine (e.g., ethanolamine alpha-adrenergic agonist op hthalmic examinations hydrochloride salt) phenylpropanolamine propanolamine adrenergic alpha-agonists nasal vasoconstrictor and an (e.g., hydrochloride salt) appetite depressant. phenytoin anticonvulsant pilocarpine (e.g., lactone imdiazole acetylcholine agonist antiglaucoma agent hydrochloride salt) praZosin (e.g., quinazoline piperazine alpha-adrenergic receptor antihypertensive hydrochloride salt) antagonist promethazine (e.g., phenathiazine histamine H1 receptor antihistamine hydrochloride salt) antagonists prostaglandine carboxylic acid prostaglandin vasodilator pyrilamine (e.g., maleate histamine H1 receptor antihistamine salt) antagonists quinacrine phospholipase inhibitor anti-malarial ritodrine hydrochloride catecholamine beta agonist relaxant RO-20-1724 polyphenyl imidazole camp phosphodiesterase inhibitor NSAID rolipram polyphenylpyrrole phosphodiesterase IV inhibitor NSAID, antidepressant, anti parkinsonian, tranquilizer spectinomycin ibiotic bacterial infection Succinylcholine etylcholine agonist Sulfaguanidine derivative i-bacterial agent antibiotic sulfamethizole Sulfa drug ibiotic urinary-tract infections Sulfasomidine Sulfa drug ibiotic urinary-tract infections suprofen thiopene ketoacid Prostaglandin-antagonistinsaid adalafil PDE phosphodiesterase inhibitor vasodilator annic acid glycoside astringent elmisartan carboxylic acid ACE inhibitor antihypertensive (angio 2 receptor antagonist) erbutaline (e.g., Sulfate bronchodilator asthma salt) etrahydrozoline (e.g., naphthalene imidazole HMG-coA reductase conjunctival congestion hydrochloride salt) inhibitori sympathomimetics alpha inidazole imidazole antiprotozoal ioconazole imidazole phenolic antioxitant antifungal olaZoline (e.g., imidazole vasodilator peripheral vascular-disorders hydrochloride salt) olfenamic acid anthranillic acid NSAID retinoin trans-retinoic acid ysosomal labilization keratolytic triamterene triaminopteridine aldosterone receptor antagonist diuretic triflupromazine (e.g., phenathiazine dopamine receptor antagonist antipsychotic hydrochloride salt) tulobuterol hydrochloride benzyl alcohol beta agonist bronchiodilator Vincamine vasodialator tinnitus anti-ischemic warfarin coumarin derivative anti-vitamin kagent anticoagulant Xylazine (e.g., thaizine alpha-2-adrenergic agonist analgesic hydrochloride salt) Zaprinast cyclic amidine phosphodiesterase v inhibitor vasodilator

By “treating, reducing, or preventing a metabolic disorder dition predisposing to obesity or diabetes, high blood levels of is meant ameliorating Such a condition before or after it has 50 triglycerides, high blood levels of cholesterol, presence of occurred. As compared with an equivalent untreated control, molecular markers (e.g., presence of autoantibodies), and age such reduction or degree of prevention is at least 5%, 10%, (over 45 years of age). An individual is considered obese 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured when their weight is 20% (25% in women) or more over the by any standard technique. maximum weight desirable for their height. An adult who is A patient who is being treated for a metabolic disorder is 55 more than 100 pounds overweight, is considered to be mor one who a medical practitioner has diagnosed as having Such bidly obese. Obesity is also defined as a body mass index a condition. Diagnosis may be performed by any Suitable (BMI) over 30 kg/m. means, such as those described herein. A patient in whom the development of diabetes or obesity is being prevented may or By “a metabolic disorder is meant any pathological con may not have received Such a diagnosis. One in the art will 60 dition resulting from an alteration in a patient's metabolism. understand that patients of the invention may have been Sub Such disorders include those resulting from an alteration in jected to standard tests or may have been identified, without glucose homeostasis resulting, for example, in hyperglyce examination, as one at high risk due to the presence of one or mia. According to this invention, an alteration in glucose more risk factors, such as family history, obesity, particular levels is typically an increase in glucose levels by at least 5%, ethnicity (e.g., African Americans and Hispanic Americans), 65 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even gestational diabetes or delivering a baby that weighs more 100% relative to such levels in a healthy individual. Meta than nine pounds, hypertension, having a pathological con bolic disorders include obesity and diabetes (e.g., diabetes US 7,795,310 B2 19 20 type I, diabetes type II, MODY, and gestational diabetes), an agent that reduces glucose levels and that is formulated for Satiety, and endocrine deficiencies of aging. administration by inhalation will differ from a low dosage of By “reducing glucose levels” is meant reducing the level of the same agent formulated for oral administration. glucose by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, By a “high dosage' is meant at least 5% (e.g., at least 10%, 80%, 90%, 95%, or 100% relative to an untreated control. 20%, 50%, 100%, 200%, or even 300%) more than the high Desirably, glucose levels are reduced to normoglycemic lev est standard recommended dosage of a particular compound els, i.e., between 150 to 60 mg/dL, between 140 to 70 mg/dL, for treatment of any human disease or condition. between 130 to 70 mg/dL, between 125 to 80 mg/dL, and Compounds useful in the invention include those described preferably between 120 to 80 mg/dL. Such reduction in glu herein in any of their pharmaceutically acceptable forms, cose levels may be obtained by increasing any one of the 10 including isomers such as diastereomers and enantiomers, biological activities associated with the clearance of glucose salts, esters, Solvates, and polymorphs thereof, as well as from the blood. Accordingly, an agent having the ability to racemic mixtures and pure isomers of the compounds reduce glucose levels may increase insulin production, secre described herein. tion, or action. Insulin action may be increased, for example, By "' is meant any naturally occurring or by increasing glucose uptake by peripheral tissues and/or by 15 synthetic compound characterized by a hydrogenated cyclo reducing hepatic glucose production. Alternatively, the agent pentanoperhydrophenanthrene ring system and having of the invention may reduce the absorption of carbohydrates immunosuppressive and/or anti-inflammatory activity. Natu from the intestines, alter glucose transporter activity (e.g., by rally occurring are generally produced by the increasing GLUT4 expression, intrinsic activity, or translo adrenal cortex. Synthetic corticosteroids may behalogenated. cation), increase the amount of insulin-sensitive tissue (e.g., Exemplary corticosteroids are provided herein. by increasing muscle celloradipocyte cell differentiation), or By “non-steroidal immunophilin-dependent immunosup alter gene transcription in adipocytes or muscle cells (e.g., pressant’ or “NsIDI is meant any non-steroidal agent that altered secretion of factors from adipocytes expression of decreases proinflammatory cytokine production or secretion, metabolic pathway genes). Desirably, the agent of the inven binds an immunophilin, or causes a down regulation of the tion increases more than one of the activities associated with 25 proinflammatory reaction. Ns|DIs include calcineurin inhibi the clearance of glucose. tors, such as cyclosporine, tacrolimus, ascomycin, pimecroli By "alter insulin signaling pathway such that glucose lev mus, as well as other agents (peptides, peptide fragments, els are reduced' is meant to alter (by increasing or reducing) chemically modified peptides, or peptide mimetics) that any one of the activities involved in insulin signaling Such that inhibit the phosphatase activity of calcineurin. Ns|DIs also the overall result is an increase in the clearance of glucose 30 include rapamycin (sirolimus) and everolimus, which bind to from plasma. For example, the agent of the invention alters an FK506-binding protein, FKBP-12, and block antigen-in the insulin signaling pathway causing an increase in insulin production, secretion, or action, an increase in glucose uptake duced proliferation of white blood cells and cytokine secre by peripheral tissues, a reduction in hepatic glucose produc tion. tion, or a reduction in the absorption of carbohydrates from 35 By "Small molecule immunomodulator” is meant a non the intestines. steroidal, non-NSIDI compound that decreases proinflamma By "patient' is meant any animal (e.g., a human), including tory cytokine production or secretion, causes a down regula horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, tion of the proinflammatory reaction, or otherwise modulates guinea pigs, rats, mice, lizards, Snakes, sheep, cattle, fish, and the immune system in an immunophilin-independent manner. birds. 40 In the generic descriptions of compounds of this invention, By “an amount sufficient' is meant the amount of a com the number of atoms of a particular type in a Substituent group pound, alone or in combination with another therapeutic regi is generally given as a range, e.g., an alkyl group containing men, required to treat, prevent, or reduce a metabolic disorder from 1 to 4 carbon atoms or C alkyl. Reference to such a Such as diabetes in a clinically relevant manner. A Sufficient range is intended to include specific references to groups amount of an active compound used to practice the present 45 having each of the integer number of atoms within the speci invention for therapeutic treatment of conditions caused by or fied range. For example, an alkyl group from 1 to 4 carbon contributing to diabetes varies depending upon the manner of atoms includes each of C, C, C, and C. A. C. het administration, the age, body weight, and general health of eroalkyl, for example, includes from 1 to 12 carbon atoms in the mammal or patient. Ultimately, the prescribers will decide addition to one or more heteroatoms. Other numbers of atoms the appropriate amount and dosage regimen. Additionally, an 50 and other types of atoms may be indicated in a similar man effective amount may be an amount of compound in the . combination of the invention that is safe and efficacious in the As used herein, the terms “alkyl and the prefix “alk- are treatment of a patient having a metabolic disorder Such as inclusive of both straight chain and branched chain groups diabetes over eachagent alone as determined and approved by and of cyclic groups, i.e., cycloalkyl. Cyclic groups can be a regulatory authority (such as the U.S. Food and Drug 55 monocyclic or polycyclic and preferably have from 3 to 6 ring Administration). carbon atoms, inclusive. Exemplary cyclic groups include By “more effective' is meant that a treatment exhibits cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. greater efficacy, or is less toxic, safer, more convenient, or less By "Ca alkyl is meant a branched or unbranched hydro expensive than another treatment with which it is being com carbon group having from 1 to 4 carbon atoms. AC alkyl pared. Efficacy may be measured by a skilled practitioner 60 group may be substituted or unsubstituted. Exemplary Sub using any standard method that is appropriate for a given stituents include alkoxy, aryloxy, Sulfhydryl, alkylthio. indication. arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino, By a "low dosage' is meant at least 5% less (e.g., at least aminoalkyl, disubstituted amino, quaternary amino, 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest hydroxyalkyl, carboxyalkyl, and carboxyl groups. Calkyls standard recommended dosage of a particular compound for 65 include, without limitation, methyl, ethyl, n-propyl, isopro mulated for a given route of administration for treatment of pyl, cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec any human disease or condition. For example, a low dosage of butyl, tert-butyl, and cyclobutyl. US 7,795,310 B2 21 22 By “halogen' is meant bromine, chlorine, iodine, or fluo cation), increase the level of insulin-sensitive tissue (e.g., by 1. increasing adipocyte or muscle cell differentiation), or alter By “alkoxy” is meant a chemical substituent of the formula gene transcription in adipocytes or muscle cells (e.g., altered —OR, wherein R is selected from C, alkyl, C-, alkenyl, secretion of factors from adipocytes and expression of meta C2-7 alkynyl, C2-sheterocyclyl, Cs-12aryl, C7-14 alkaryl, Cso bolic pathway genes). alkheterocyclyl, or C, heteroalkyl. In one example, we propose that the administration of Other features and advantages of the invention will be beZafibrate and cinnamic acid to a patient having a metabolic apparent from the detailed description and from the claims. disorder such as diabetes within 14 days of each other will treat, prevent, or reduce the metabolic disorder. In another BRIEF DESCRIPTION OF THE DRAWINGS 10 example, the administration of bezafibrate and diflunisal to the patient within 14 days of each other will also treat, pre FIG. 1 is a graph showing insulin sensitivity in male Spra vent, or reduce the metabolic disorder. The two agents are gue Dawley rats, as determined using the homeostasis model desirably administered within 10 days of each other, more assessment (HOMA). Insulin resistance was induced by four desirably within seven days of each other, and even more weeks of high fat feeding (60% of calories derived from fat). 15 desirably within twenty-four hours of each other, one hour of Drug treatment began one week after initiation of high fat each other, or even simultaneously (i.e., concomitantly). If diet. Drugs were administered daily, by oral gavage for a three desired, either one of the two agents may be administered in week period. Following the three weeks of treatment, animals low dosage. were fasted for five hours and anesthetized, and blood col In view of this discovery, the aforementioned combina lected from the inferior vena cava for determination of serum tions of drugs can be used in a variety of compositions, glucose and insulin levels. Insulin sensitivity was determined methods, and kits, as described herein. Additionally, if using HOMA. desired, structural or functional analogs may be used in place of one or more of the drugs in a combination. Such analogs are described below. fasting serum glucose X fasting serum insulin 25 HOA = We have also discovered compounds that, alone or in com 22.5 bination, are effective in the treatment, reduction, or preven tion of metabolic disorders such as diabetes and obesity. Accordingly, a mammal that has been diagnosed with or is at Pio- ; Bez-bezafibrate; DF-diflunisal; HF-high risk of having a metabolic disorder is administered one, two, fat. 30 three, or more agents from Table 1. Optionally, analogs of FIG. 2 is a graph showing blood glucose levels in male these agents may be employed. In the case of diabetes and Sprague Dawley rats. Insulin resistance was induced by four obesity, for example, such administration may reduce the weeks of high fat feeding (60% of calories derived from fat). levels of glucose, reduce levels of LDL-cholesterol, increase Drug treatment began one week after initiation of high fat the levels of HDL-cholesterol, resultina more favorable ratio diet. Drugs were administered daily, by oral gavage for a three 35 between LDL-cholesterol and HDL-cholesterol, reduce the week period. Following the three weeks of treatment, animals triglyceride values, or reduce serum levels of CRP (C-reactive were fasted for five hours and anesthetized, and blood col protein). The ability of the agent to cause the clearance of lected from the inferior vena cava. glucose may be attributed, for example, to its ability to FIG. 3 is a graph showing blood serum insulin levels in increase insulin production, secretion, or action (e.g., stimu male Sprague Dawley rats. Insulin resistance was induced by 40 lation of glucose uptake by peripheral tissues and/or reduc four weeks of high fat feeding (60% of calories derived from tion in hepatic glucose production), reduce the absorption of fat). Drug treatment began one week after initiation of high fat carbohydrates from the intestines, alter glucose transporter diet. Drugs were administered daily, by oral gavage for a three activity (e.g., by increasing GLUT4 expression, intrinsic week period. Following the three weeks of treatment, animals activity, or translocation), increase the level of insulin-sensi were fasted for five hours and anesthetized, and blood col 45 tive tissue (e.g., by increasing adipocyte or muscle cell dif lected from the inferior vena cava. ferentiation), or alter gene transcription in adipocytes or muscle cells (e.g., altered secretion of factors from adipocytes DETAILED DESCRIPTION and expression of metabolic pathway genes). Optionally, the mammal may also receive other therapeutic regimens. We have discovered compounds that the certain bezafi 50 In one particular example, the mammal being treated is brate-containing combinations have in vitro and in vivo administered two agents listed in Table 1 within 28 days of activities that Suggest that these combinations may be useful each other in amounts that together are sufficient to treat, for treating a patient that has been diagnosed with or is at risk prevent, or reduce the metabolic disorder. The two agents are of having a metabolic disorder. Optionally, analogs of these desirably administered within 14 days of each other, more agents may be employed. In the case of diabetes and obesity, 55 desirably within seven days of each other, and even more for example, such administration may reduce the levels of desirably within twenty-four hours of each other, or even glucose, reduce levels of LDL-cholesterol, increase the levels simultaneously (i.e., concomitantly). If desired, either one of of HDL-cholesterol, result in a more favorable ratio between the two agents may be administered in low dosage. LDL-cholesterol and HDL-cholesterol, reduce the triglycer ide values, or reduce serum levels of CRP (C-reactive pro 60 Diagnosis of Metabolic Disorders tein). The ability of the agent to cause the clearance of glucose The methods and compositions of the present invention are may be attributed, for example, to its ability to increase insu useful for treating any patient that has been diagnosed with or lin production, secretion, or action (e.g., stimulation of glu is at risk of having a metabolic disorder, such as diabetes. A cose uptake by peripheral tissues and/or reduction in hepatic patient in whom the development of a metabolic disorder glucose production), reduce the absorption of carbohydrates 65 (e.g., diabetes or obesity) is being prevented may or may not from the intestines, alter glucose transporter activity (e.g., by have received such a diagnosis. One in the art will understand increasing GLUT4 expression, intrinsic activity, or translo that patients of the invention may have been subjected to US 7,795,310 B2 23 24 standard tests or may have been identified, without examina tion, as one at high risk due to the presence of one or more risk factors. COOH. O Diagnosis of metabolic disorders may be performed using O any standard method known in the art, such as those described 5 CH3 herein. Methods for diagnosing diabetes are described, for HC N example, in U.S. Pat. No. 6,537.806, hereby incorporated by H reference. Diabetes may be diagnosed and monitored using, for example, urine tests (urinalysis) that measure glucose and C ketone levels (products of the breakdown of fat); tests that 10 measure the levels of glucose in blood; glucose tolerance tests; and assays that detect molecular markers characteristic The synthesis of bezafibrate is described in U.S. Pat. No. of a metabolic disorder in a biological sample (e.g., blood, 3,781,328. serum, or urine) collected from the mammal (e.g., measure Bezafibrate Analogs ments of Hemoglobin Alc (Hb Alc) levels in the case of dia 15 Bezafibrate analogs include binifibrate, ciprofibrate, cli betes). nofibrate, clofibrate, etofibrate, fenofibrate, and gemfibrozil. Patients may be diagnosed as being at risk or as having Other bezafibrate analogs are described by formula (I). diabetes if a random plasma glucose test (taken at any time of the day) indicates a value of 200 mg/dL or more, if a fasting plasma glucose test indicates a value of 126 mg/dL or more (I) (after 8 hours), or if an oral glucose tolerance test (OGTT) indicates a plasma glucose value of 200 mg/dL or more in a blood sample taken two hours after a person has consumed a drink containing 75 grams of glucose dissolved in water. The OGTT measures plasma glucose at timed intervals over a 25 2 **C. O 3-hour period. Desirably, the level of plasma glucose in a diabetic patient that has been treated according to the inven CO-Z. tion ranges between 160 to 60 mg/dL, between 150 to 70 mg/dL, between 140 to 70 mg/dL, between 135 to 80 mg/dL, R4 and preferably between 120 to 80 mg/dL. 30 Optionally, a hemoglobin Alc (Hb Alc) test, which assesses In this formula, each of R and R is, independently, hydro the average blood glucose levels during the previous two and gen, halogen, Calkyl, and Calkoxy, each of R and Rais, three months, may be employed. A person without diabetes independently, hydrogen and C alkyl, n is 1, 2, and 3, and typically has an HbAlc value that ranges between 4% and 6%. Z is hydroxyl and C alkyl. For every 1% increase in Hb Alc, blood glucose levels 35 Specific bezafibrate analogs of formula (I) are alpha-4-(2- increases by approximately 30 mg/dL and the risk of compli methoxy-5-chlorobenzoylaminoethyl)-phenoxy-isobutyric cations increases. Preferably, the Hb Alc value of a patient acid, alpha-4-(4-methylbenzoylaminomethyl)-phenoxy being treated according to the present invention is reduced to isobutyric acid, alpha-4-(2-methylbenzoylaminoethyl)-phe less than 9%, less than 7%, less than 6%, and most preferably noxy-isobutyric acid, and alpha-4-(4-chlorobenzoylamino to around 5%. Thus, the Hb Alc levels of the patient being 40 ethyl)-phenoxy-proprionic acid. treated are preferably lowered by 10%, 20%, 30%, 40%, 50%, or more relative to such levels prior to treatment. Bezafibrate analogs are also described in U.S. Pat. Nos. Gestational diabetes is typically diagnosed based on 3,262,850; 3,674,836; 3,716,583; 3,723,446, 3,948,973: plasma glucose values measured during the OGTT. Since 4,010,279; 4,026,896; 4,042,711; 4,058,552; 4,151,303: glucose levels are normally lower during pregnancy, the 45 4,153,728; 4,238,506; 4,318,923; and 4,409,240, each of threshold values for the diagnosis of diabetes in pregnancy which is hereby incorporated by reference. are lower than in the same person prior to pregnancy. If a PPARAgonists woman has two plasma glucose readings that meet or exceed PPARS (Peroxisome Proliferator-Activated receptors) are any of the following numbers, she has gestational diabetes: a ligand-activated transcription factors belonging to the nuclear fasting plasma glucose level of 95 mg/dL, a 1-hour level of 50 receptor superfamily. Three different PPARs have been iden 180 mg/dL, a 2-hour level of 155 mg/dL, or a 3-hour level of tified to date (PPARC, PPARB and PPARy) each displaying 140 mg/dL. distinct tissue distribution pattern. PPARs are activated by Ketone testing may also be employed to diagnose type 1 natural ligands such as fatty acids and (leukot diabetes. Because ketones build up in the blood when there is rienes, ) and by pharmacological agonists Such not enough insulin, they eventually accumulate in the urine. 55 as , binding to PPARC. and glitazones, binding to High levels of blood ketones may result in a serious condition PPARY. Upon ligand activation, PPARs regulate the tran called ketoacidosis. scription of several genes. Activated PPARs heterodimerize The use of any of the above tests or any other tests known with another nuclear receptor, the retinoid X receptor, and in the art may be used to monitor the efficacy of the present modify the transcription of target genes after binding to spe treatment. Since the measurements of hemoglobin Alc 60 cific peroxisome proliferator response elements (PPRE). (HbAlc) levels is an indication of average blood glucose Bezafibrate is a and thus a PPARC. agonist. Because during the previous two to three months, this test may be used of the interaction between PPARC. and PPARy, PPARYago to monitor a patient’s response to diabetes treatment. nists may nonetheless be used in place of bezafibrate in the compositions, methods, and kits of the invention. For Bezafilbrate 65 example, glitaZones (e.g., balaglitaZone, troglitaZone, piogli Bezafibrate (2-4-2-(4-chlorobenzoyl)amino-ethylphe taZone, ciglitaZone, englitaZone, rosiglitaZone, darglitaZone, noZy-2-methylpropanoic acid) has the following structure: englitazone, netoglitazone, KRP-297, JTT-501, NC-2100, US 7,795,310 B2 25 26 NIP-223, MCC-555, L-764486, and CS-011), also referred to L652343, L745337, L748731, L752860, L761066, L768277, as , can be used in combination with cin L776967, L783003, L784520, L791456, L804600, L818571, namic acid, diflunisal, or an analog thereof, for treating a LAS33815, LAS34475, , LM 4108, lobuprofen, patient having a metabolic disorder. Similarly, tyrosine-based , , mabuprofen, , PPARY modulators (e.g., GI262570; (S)-2-(2-benzoylphe 5 meclofenamate Sodium, mefenamic acid, meloxicam, mer nylamino)-3-4-2-(5-methyl-2-phenyl-2-oxazol-4-yl) captoethylguanidine, mesoporphyrin, metoxibutropate, ethoxyphenylpropionic acid, and GW347845 (Cobb et al., J , mofebutazone, , MX1094, nabume Med. Chem.41:5055-5069, 1998)) can also be substituted for tone, Sodium, naproxen-sodium/metoclopramide, bezafibrate, as can other PPARYagonists (e.g., 3-(2,4-dichlo NCX1101, NCX284, NCX285, NCX4016, NCX4215, robenzyl)-2-methyl-N-(pentylsulfonyl)-3H-benzimidazole 10 NCX530, , nitric oxide-based COX-2 inhibitors 5-carboxamide (FK614)). and NSAIDs (NitroMed), nitrofenac, nitroflurbiprofen, Other PPARagonists that may be used in lieu of bezafibrate nitronaproxen, NS398, ocimum sanctum oil, ONO3144, in the compositions, methods, and kits of the invention are orpanoxin, oxaprozin, OXindanac, oXpinac, /ibu AA-10090, AD-5075, AMG-131, ARH-049020, AVE-0847, profen, , P10294, P54, P8892, , AVE-8134, AY-31637, BAY-549801, bexaroteine, 15 parcetasal, , PD138387, PD145246, PD164387, BM-131246, BM-501050, CLX-0921, CLX-0940, DRF pelubiprofen, pemedolac, phenylbutaZone, piraZolac, piroxi 10945, DRF-4832, E-3030, fargilitazar, fenofibrate/met cam, beta-cyclodextrin, piroXicam pivalate, pir formin, GW-0072, GW-1929, GW-2570, GW-409544, profen, , , R-, R-, GW-409890, GW-501516, GW-5393, GW-590735, , RP66364, RU43526, RU54808, RWJ63556, GW-7282, GW-9578, KRP-101, KT-6207, L-764406, S19812, S2474, S33516, salicylsalicylic acid, satigrel, LF-200337, LG-101506, LR-90, LY-465608, LY-510929, SC236, SC57666, SC58125, SC58451, SFPP, SKF105809, LY-518674, MBX-102, MK-0767, , navegli SKF86002, , sudoxicam, sulfasalazine, tazar, NC-2100, NS-220, ONO-5129, oxeglitazar, PD-72953, , suprofen, SVT2016, T3788, TA60, talmetacin, R-1 19702, ragaglitazar, reglitazar, SB-219994, , talniflumate, taZofelone, tebufelone, , tenoxican, tep 641597, and TY-51501. 25 oxalin, , tilmacoxib, tilnoprofen arbamel, Statins tinoridine, tiopinac, tioxaprofen, tolfenamic acid, , Statins or statin-containing drug combinations may be , tropesin, TY10222, TY10246, TY10474, UR8962, used in lieu of bezafibrate in the compositions, methods, and ursolic acid, , WAY 120739, WY28342, kits of the invention. Exemplary statins and statin-containing WY41770, Ximoprofen, YS134, , Zidometacin, combinations are acitemate, amlodipinefatorvastatin, atorv 30 and . astatin, /, BAY102987, BAY X 2678, BB476, bervastatin, BMY21950, BMY22089, , Cinnamic Acid and Analogs Thereof colestolone, CP83101, crilvastatin, dalvastatin, DMP565, Cinnamic acid (3-phenyl-2-propenoic acid) has the struc /, , glenvastatin, L659699, ture CH-CH=CHCOOH. Cinnamic acid analogs are L669262, , nicotinic acid/, nicotinic 35 described by formula (II): acid/simvastatin, P882222, P882284, PD134965, PD135022, , , RP61969, S2468, SC37.111, SC45355, simvastatin, SQ33600, SR 12813, SR45023A, (II) U20685, and U88156. NSAIDS 40 N COOH. NSAIDs may also be used in lieu of bezafibrate in the (X), it combinations, methods, and kits of the invention. Suitable 2 R R2 NSAIDs include A183827, ABT963, , acemeta cin, acetyl , AHR10037, , alminopro fen, , amtolimetinguacil, apaZone, , atlip 45 In formula (II), R and Reach represent a hydrogenatom or rofen methyl ester, AU8001, , a C alkyl group; R and Reach representahydrogenatom flufenamate, bermoprofen, bezpiperylon, BF388, BF389, or may be combined together to forman additional chemical BIRL790, BMS347070, , bucloxic acid, buti bond; X represents a hydroxyl group, a halogen atom, a bufen, BW755C, C53, C73, C85, , CBS1108, cele straight or branched chain saturated or unsaturated Calkyl coxib, CHF2003, chlorobiphenyl, choline magnesium trisali 50 group, a straight or branched chain Saturated or unsaturated cylate, CHX108, , cinnoxicam, clidanac, Calkoxy group, a C acyloxy group, or a C. cycloalkyl CLX1205, COX-2 inhibitor (PharmaVU/Vanderbilt Univer group; n is Zero oran integer from 1 to 3, with the proviso that sity), CP331, CS502, CS706, D1367, darbufelone, dera when n is 2 or 3, each X may be the same or different and that coxib, dexibuprofen, dexibuprofen lysine, , when two X's are commonly the alkyl or alkoxy group, both DFP. DFU, diclofenac (e.g., diclofenac potassium, diclofenac 55 X's may be combined together to form a ring. sodium), diflunisal, DP155, DRF4367, E5110, E6087, elte Specific cinnamic acid analogs of formula (II) are hydro nac, ER34122, esflurbiprofen, , F025, cinnamic acid, 2-, 3- and 4-methylhydrocinnamic acid, 2-, 3 ethyl, , , fenclozic acid, fenclozine, feno and 4-ethylhydrocinnamic acid, 2-, 3- and 4-propylhydrocin profen, , feprazone, filenadol, flobufen, florifenine, namic acid, 2-, 3- and 4-hydroxyhydrocinnamic acid, 2-, 3 flosulide, flubichin methanesulfonate, , flu 60 and 4-methoxyhydrocinnamic acid, 2-, 3- and 4-ethoxyhy profen, , FPL62064, FR122047, FR123826, drocinnamic acid, 2-, 3- and 4-chlorohydrocinnamic acid, 2-, FR140423, FR188582, FS205397, furofenac, GR253035, 3- and 4-bromohydrocinnamic acid, 2-, 3- and 4-fluorohy GW406381, HAI 105, HAI106, HCT2035, HCT6015, drocinnamic acid, 2.4-, 2.5- and 3,4-dimethylhydrocinnamic HGP12, HN3392, HP977, HX0835. HYAL AT2101, acid, 2,4-diethylhydrocinnamic acid, 2.3-, 2,4-, 2.5-, 2,6-, ibufenac, ibuprofen, -beta-cyclodextrin, icoduli 65 3,4- and 3,5-dihydroxyhydrocimmamic acid, 2.3-, 2,4-, 2.5- num, IDEA070, iguratimod, imrecoxib, indomethacin, 2,6-, 3.4- and 3,5-dimethoxyhydrocinnamic acid, 2.3-, 2,4-, , IP751, isoxepac, , KCT64, ketoprofen, 2.5-, 2,6-, 3.4- and 3,5-diethoxyhydrocinnamic acid, 2.3-, US 7,795,310 B2 27 28 2.4- and 3,4-dipropoxyhydrocinnamic acid, 2-hydroxy-3- Methods of making diflunisal are described in U.S. Pat. No. methoxyhydrocinnamic acid, 3-hydroxy-4-methoxyhydro 3,714.226. cinnamic acid, 4-hydroxy-3-methoxyhydrocinnamic acid, Diflunisal Analogs 3-ethoxy-4-methoxyhydrocinnamic acid, 4-hydroxy-3- Diflunisal analogs are described by formula (III): methoxyhydrocinnamic acid, 2-ethoxy-3-methoxyhydrocin namic acid, 3-ethoxy-4-methoxyhydrocinnamic acid, 4-ethoxy-3-methoxyhydrocinnamic acid, 3-methoxy-2-pro (III) poxyhydrocinnamic acid, 3-methoxy-4-propoxyhydrocin COR namic acid, 3.4-methylenedioxyhydrocinnamic acid, 2.4-, 10 2,6- and 3,4-dichlorohydrocinnamic acid, 2.3.4-, 2,4,5- and OR 3,4,5-trimethoxyhydrocinnamic acid, 2-bromo-4-hydroxy 5-methoxyhydrocinnamic acid, 4-isopropylhydrocinnamic acid, 3- and 4-isopropoxyhydrocinnamic acid, 3- and 4-isobutoxyhydrocinnamic acid, 3- and 4-sec-butoxyhydro cinnamic acid, 3-methoxy-4-isopropoxyhydrocinnamic acid, 15 2-, 3- and 4-allyloxyhydrocinnamic acid, 2-, 3- and 4-meth wherein each X is, independently, a halogen atom; R is allyloxyhydrocinnamic acid, 3-methoxy-4-allyloxyhydro selected from the group consisting of hydroxy, phenoxy, cinnamic acid, 3-methoxy-4-methallyloxyhydrocinnamic di (C-C)alkylamino, di(C-C)alkylamino (C-C)alkoxy; acid, 2-, 3- and 4-acetoxyhydrocinnamic acid, 3,4-trimethyl R is selected from the group consisting of hydrogen and enehydrocinnamic acid, and C- and/or B-alkyl-substituted (C-C)alkanoyl (such as acetyl, propionyl and butyryl); and hydrocinnamic acids carrying Substituents the same as those R is selected from the group consisting of hydrogen and mentioned in the case of the above-mentioned hydrocinnamic methyl. acids; and aromatic unsaturated carboxylic acids such as 2-, Other diflunisal analogs are flufenisal, 2-hydroxy-5-(4'- 3- and 4-methylcinnamic acid, 2-, 3- and 4-ethylcinnamic 25 fluorophenyl)benzoic acid; 2-acetoxy-5-(4'-fluorophenyl) acid, 2-, 3- and 4-propylcinnamic acid, 2-, 3- and 4-hydroxy benzoic acid; 2-hydroxy-5-(2'-fluorophenyl)benzoic acid; cinnamic acid, 2-, 3- and 4-methoxycinnamic acid, 2-, 3- and 2-hydroxy-5-(3'-fluorophenyl)benzoic acid; 2-hydroxy-5- 4-ethoxycinnamic acid, 2-, 3- and 4-propoxycinnamic acid, pentafluorophenyl benzoic acid; 2-hydroxy-3-methyl-5-(4- 2-, 3- and 4-butoxycinnamic acid, 2-, 3- and 4-fluorocinnamic fluorophenyl)benzoic acid; 2-hydroxy-5-(4-chlorophenyl) acid, 2-, 3- and 4-chlorocinnamic acid, 2-, 3- and 4-bromo 30 benzoic acid; N,N-dimethyl-5-(4'-fluorophenyl) cinnamic acid, 2.4- and 2.5 and 3,4-dimethylcinnamic acid, ; B-diethylaminoethyl-5-(4'-fluorophenyl) 2,4-diethylcinnamic acid, 2.3-, 2,4-, 2.5-2,6-, 3,4- and 3.5- salicylate; phenyl-5-(4'-fluorophenyl)salicylate: aluminum dihydroxycinnamic acid, 2.3-, 2,4-, 2.5-, 2,6-, 3,4- and 3.5- 2-acetoxy-5-(4-fluorophenyl)-benzoate salt; aluminum-2- dimethoxycinnamic acid, 2.3-, 2,4-, 2.5-, 2,6-, 3.4- and 3.5- hydroxy-5-(4-fluorophenyl)-benzoate salt; choline-2- diethoxycinnamic acid, 2.3-, 2,4- and 3,4- 35 acetoxy-5-(4'-fluorophenyl)-benzoate salt; choline-2- dipropoxycinnamic acid, 2-hydroxy-3-methoxycinnamic hydroxy-5-(4-fluorophenyl)-benzoate salt; sodium-2- acid, 3-hydroxy-4-methoxycinnamic acid, 4-hydroxy-3- acetoxy-5-(4'-fluorophenyl)-benzoate salt, sodium-2- methoxycinnamic acid, 2-ethoxy-3-methoxycinnamic acid, hydroxy-5-(4-fluorophenyl)-benzoate salt; 2-hydroxy-5- 3-ethoxy-4-methoxycinnamic acid, 4-ethoxy-3-methoxycin (pentafluorophenyl)-benzoic acid; 2-acetoxy-5- namic acid, 3-methoxy-2-propoxycinnamic acid, 3-meth 40 (pentafluorophenyl)-benzoic acid; B-diethylaminoethyl-2- oxy-4-propoxycinnamic acid, 3.4-methylenedioxycinnamic hydroxy-5-(4-fluorophenyl)-benzoate: acid, 2.4-, 2.6- and 3,4-dichlorocinnamic acid, 2,3,4-, 2,4,5- B-diethylaminoethyl-2-acetoxy-5-(4-fluorophenyl)-ben and 3,4,5-trimethoxycinnamic acid, 2-bromo-4-hydroxy-5- Zoate: 2-hydroxy-5-(4'-fluorophenyl)-benzamide: 2-hy methoxycinnamic acid, 4-isopropylcinnamic acid, 3- and droxy-5-(4'-fluorophenyl)-3-methyl benzamide: 2-acetoxy 4-isopropoxycinnamic acid, 3- and 4-isobutoxycinnamic 45 5-(4-fluorophenyl)-benzamide: 2-acetoxy-5-(4'- acid, 3- and 4-sec-butoxycinnamic acid, 3-methoxy-4-iso fluorophenyl)-benzmorpholide: 2-hydroxy-5-(4-fluoro-2'- proxycinnamic acid, 2-, 3- and 4-allyloxycinnamic acid, 2-, methoxyphenyl)benzoic acid; 2-acetoxy-5-(4-fluoro-2'- 3- and 4-methallyloxycinnamic acid, 3-methoxy-4-allyloxy methoxyphenyl)benzoic acid; 2-hydroxy-5-(4-fluoro-2'- cinnamic acid, 3-methoxy-4-methallyloxycinnamic acid, 2-, methylphenyl)benzoic acid; 2-acetoxy-5-(4-fluoro-3'- 3- and 4-acetoxycinnamic acid, 3,4-trimethylenecinnamic 50 methylphenyl)benzoic acid; 2-hydroxy-3-allyl-5-(4'- acid, and C- and/or 3-alkylsubstituted cinnamic acids carry fluorophenyl)benzoic acid; 2-hydroxy-3-propyl-5-(4'- ing Substituents the same as those mentioned in the case of the fluorophenyl)benzoic acid; and the compounds described in above-mentioned cinnamic acids. U.S. Pat. Nos. 3,674,870, 3,681,445, 3,692,821, 3,714,226, 4,044,049, 4,542,158, and 6,593,365. Diflunisal 55 If desired, other NSAIDs can be used in place diflunisal. Diflunisal (2',4'-difluoro-4-hydroxy-11'-biphenyl-3- Suitable NSAIDs include A183827, ABT963, aceclofenac, carboxylic acid) is an NSAID having the structure: acemetacin, acetyl salicylic acid, AHR10037, alclofenac, , ampiroxicam, amtolimetin guacil, apaZone, aspirin, atliprofen methyl ester, AU8001, benoxaprofen, ben COOH 60 Zydamine flufenamate, bermoprofen, bezpiperylon, BF388, BF389, BIRL790, BMS347070, bromfenac, bucloxic acid, butibufen, BW755C, C53, C73, C85, carprofen, CBS1108, , CHF2003, chlorobiphenyl, choline magnesium trisalicylate, CHX108, cimicoxib, cinnoxicam, clidanac, F 65 CLX1205, COX-2 inhibitor (PharmaVU/Vanderbilt Univer sity), CP331, CS502, CS706, D1367, darbufelone, dera coxib, dexibuprofen, dexibuprofen lysine, dexketoprofen, US 7,795,310 B2 29 30 DFP. DFU, diclofenac (e.g., diclofenac potassium, diclofenac between 80 and 130 mg/dL, and preferably between 80 and sodium), diflunisal, DP155, DRF4367, E5110, E6087, elte 120 mg/dL prior to a meal and between 90 mg/dL to 160 nac, ER34122, esflurbiprofen, etoricoxib, F025, felbinac mg/dL, between 90 mg/dL to 150 mg/dL, and preferably ethyl, fenbufen, fenclofenac, fenclozic acid, fenclozine, feno between 90 to 140 mg/dL two hours after eating. A particu profen, fentiazac, feprazone, filenadol, flobufen, florifenine, larly desirable result of treatment is a reduction of any of the flosulide, flubichin methanesulfonate, flufenamic acid, flu symptoms associated with the metabolic disorder. profen, flurbiprofen, FPL62064, FR122047, FR123826, Analogs of any of the compounds listed in Table 1 may be FR140423, FR188582, FS205397, furofenac, GR253035, used in any of the methods, kits, and compositions of the GW406381, HAI 105, HAI106, HCT2035, HCT6015, invention. Analogs are known in the art (e.g., as described HGP12, HN3392, HP977, HX0835. HYAL AT2101, 10 herein). Acemetacin analogs are described in German patent ibufenac, ibuprofen, ibuproxam-beta-cyclodextrin, icoduli DE 2234651 and U.S. Pat. No. 3,910,952. Acenocoumarol num, IDEA070, iguratimod, imrecoxib, indomethacin, analogs are described in U.S. Pat. No. 2,648,682. Acetami indoprofen, IP751, isoxepac, isoxicam, KCT64, ketoprofen, nophen analogs are described in U.S. Pat. No. 2,998.450 and L652343, L745337, L748731, L752860, L761066, L768277, German patent DE 453577. Acetazolamide analogs are L776967, L783003, L784520, L791456, L804600, L818571, 15 described in U.S. Pat. No. 2,554,816. Acetohexamide analogs LAS33815, LAS34475, licofelone, LM 4108, lobuprofen, are described in GB 912789. Acetyldigitoxin analogs are lornoxicam, lumiracoxib, mabuprofen, meclofenamic acid, described in U.S. Pat. No. 2,776,963. Alachlor analogs are meclofenamate sodium, mefenamic acid, meloxicam, mer described in NL 6602564. Albuterol (Salbutamol) analogs are captoethylguanidine, mesoporphyrin, metoxibutropate, described in ZA6705591 and U.S. Pat. No. 3,644,353. Alpre miroprofen, mofebutazone, mofezolac, MX1094, nabume nolol analogs are described in NL 6605692 and NL 6612958. tone, naproxen Sodium, naproxen-sodium/metoclopramide, Ametryn analogs are described in CH 337019 and U.S. Pat. NCX1101, NCX284, NCX285, NCX4016, NCX4215, No. 3,558,622. Atrazine analogs are described in CH342784, NCX530, niflumic acid, nitric oxide-based COX-2 inhibitors CH 342785, HU 149189, and FR 1317812. Azathioprine and NSAIDs (NitroMed), nitrofenac, nitroflurbiprofen, analogs are described in U.S. Pat. No. 3,056,785 and ben nitronaproxen, NS398, ocimum sanctum oil, ONO3144, 25 Zbromarone analogs are described in BE 553621 and U.S. orpanoxin, oxaprozin, OXindanac, oXpinac, oxycodone/ibu Pat. No. 3,012,042. Bezafibrate analogs are described in DE profen, oxyphenbutazone, P10294, P54, P8892, pamicogrel, 2149070 and U.S. Pat. No. 3,781,328. Bopindolol analogs are parcetasal, parecoxib, PD138387, PD145246, PD164387, described in U.S. Pat. No. 4,340,541 and DE 2635209. Bri pelubiprofen, pemedolac, phenylbutaZone, piraZolac, piroxi monidine analogs are described in U.S. Pat. No. 3,890,319 cam, piroXicam beta-cyclodextrin, piroXicam pivalate, pir 30 and German patent DE 23091.60. Brompheniramine (e.g., profen, pranoprofen, resveratrol, R-ketoprofen, R-ketorolac, maleate salt) analogs are described in U.S. Pat. No. 2,567, rofecoxib, RP66364, RU43526, RU54808, RWJ63556, 245, U.S. Pat. No. 2,676,964, and U.S. Pat. No. 3,061,517. S19812, S2474, S33516, salicylsalicylic acid, satigrel, Candesartan (e.g., cilexetil salt) analogs are described in EP SC236, SC57666, SC58125, SC58451, SFPP, SKF 105809, 459136 and U.S. Pat. No. 5,196,444, captopril analogs are SKF86002, sodium salicylate, sudoxicam, sulfasalazine, 35 described in DE 2703828, U.S. Pat. No. 4,046,889, and U.S. sulindac, suprofen, SVT2016, T3788, TA60, talmetacin, Pat. No. 4,105,776, and carbamazepine analogs are described talniflumate, taZofelone, tebufelone, tenidap, tenoxican, tep in U.S. Pat. No. 2,948,718. Carbinoxamine (e.g., maleate oxalin, tiaprofenic acid, tilmacoxib, tilnoprofen arbamel, salt) analogs are described in U.S. Pat. No. 2,606,195, U.S. tinoridine, tiopinac, tioxaprofen, tolfenamic acid, tolmetin, Pat. No. 2,800,485, and GB 905993. Carisoprodol analogs triflusal, tropesin, TY10222, TY10246, TY10474, UR8962, 40 are described in U.S. Pat. No. 2,937,119 and cefamandole ursolic acid, valdecoxib, WAY 120739, WY28342, (e.g., nafate salt) analogs are described in German patents DE WY41770, Ximoprofen, YS134, Zaltoprofen, Zidometacin, 2018600 and DE 2312997, as well as U.S. Pat. No. 3,641,021 and Zomepirac. and U.S. Pat. No. 3,840.531. Cefpodoxime Proxetil analogs are described in EP 49118 and U.S. Pat. No. 4486,425. Additional Therapeutic Agents 45 Ciclopirox analogs are described in ZA 69 06039 and U.S. The present invention involves the administration of an Pat. No. 3,883,545. Clenbuterol analogs are described in ZA effective amount of one, two, three, four, or more agents listed 6705692 and U.S. Pat. No. 3,536,712. Diacerein analogs are in Table 1 to a mammal at risk of having or having a metabolic described in German patent DE 2711493, Japanese Patent JP disorder, thereby treating, preventing, and reducing Such a Kokai 83 225015, U.S. Pat. No. 4,244,968, and U.S. Pat. No. disorder. 50 4.346,103. Diclofenac (e.g., sodium salt) analogs are In the case of diabetes, the agent of the invention increases described in U.S. Pat. No. 3,558,690 and NL 6604752. the clearance of glucose from the plasma by any mechanism Dicloxacillin (e.g., sodium salt) analogs are described in GB thereby reducing glucose levels to normoglycemic levels. For 978299 and U.S. Pat. No. 3,239,507. Diflunisal analogs are example, a combination of the invention may alter the insulin described in ZA 67 01021, FR 1522570, and U.S. Pat. No. signaling pathway, reduce the absorption of carbohydrates 55 3,714.226. Dimenhydrinate analogs are described in U.S. Pat. from the intestines, alter glucose transporter activity (e.g., by No. 2,499,058 and U.S. Pat. No. 2,534,813. Diphemanil increasing GLUT4 expression, intrinsic activity, or translo Methylsulfate analogs are described in U.S. Pat. No. 2,739, cation), increase the level of insulin-sensitive tissue (e.g., by 968. Diphenhydramine (e.g., hydrochloride salt) analogs are increasing adipocyte or muscle cell differentiation), or alter described in U.S. Pat. No. 2,421,714, U.S. Pat. No. 2,427,878, gene transcription in adipocytes or muscle cells (e.g., altered 60 and U.S. Pat. No. 2,397,799 as well as Japanese patent JP 64 secretion of factors from adipocytes expression of metabolic 243. Diphenidol (e.g., hydrochloride salt) analogs are pathway genes). Given that plasma glucose levels prior to described in U.S. Pat. No. 2.41 1,664 and GB 683950. Dipiv eating typically range between 80 and 120 mg/dL and that efrin (e.g., hydrochloride salt) analogs are described in Ger postprandial blood levels range between 100 to 140 mg/dL, manpatents DE 2152058 and DE 2343657 as well as U.S. Pat. the plasma glucose levels of the patient being treated accord 65 No. 4,085,270, U.S. Pat. No. 3,809,714, and U.S. Pat. No. ing to the invention is stabilized such that glucose levels range 3,839,584. Dirithromycin analogs are described in BE between 60 and 150 mg/dL, between 70 and 140 mg/dL, 840431 and U.S. Pat. No. 4,048,306. 5-Hydroxytryptophan US 7,795,310 B2 31 32 (e.g., DL form) analogs are described in CA 619472 and GB in U.S. Pat. No. 2,643.258. Phenylbutazone analogs are 845034. Doxapram (e.g., hydrochloride salt) analogs are described in U.S. Pat. No. 2,562,830 and GB 812449. Phe described in BE 613734 and U.S. Pat. No. 3,192.206. Doxy nylephrine analogs are described in U.S. Pat. No. 1,932.347 cycline analogs are described in U.S. Pat. No. 3,019,260 and and U.S. Pat. No. 1,954.389. Phenyloin analogs are described U.S. Pat. No. 3.200,149. Enalaprilat analogs are described in in U.S. Pat. No. 2,409,754. Prazosin (e.g., hydrochloride salt) EP 12401 and U.S. Pat. No. 4.374,829. Ethopropazine (e.g., analogs are described in GB 1156973, U.S. Pat. No. 3.511, hydrochloride salt) analogs are described in U.S. Pat. No. 836, and NL 7206067. Promethazine (e.g., hydrochloride 2,607,773. Etoposide analogs are described in U.S. Pat. No. salt) analogs are described in U.S. Pat. No. 2.530,451 and 3,524,844. Evans Blue analogs are described in German pat U.S. Pat. No. 2,607,773. Prostaglandin (e.g., Prostaglandin ents DE 35341, DE 38802, DE 3949, DE 57327, and DE 10 E) analogs are described in GB 851827, U.S. Pat. No. 3,598, 75469. Exemestane analogs are described in U.S. Pat. No. 858, NL 6505799, DE 2126127, U.S. Pat. No. 3,657,327, 4,808,616 and German patent DE 3622841. Flumixin (e.g., U.S. Pat. No. 3,069,322, and U.S. Pat. No. 3,598,858. Pyril meglumine salt) analogs are described in BE 679271, BE amine (e.g., maleate salt) analogs, are described in U.S. Pat. 812772, as well as U.S. Pat. No. 3,337,570 and U.S. Pat. No. No. 2.502,151. Quinacrine analogs are described in German 3,839,344. Gemfibrozil analogs are described in DE 1925423 15 patents DE 553072, DE 571499, U.S. Pat. No. 2,113,357, and U.S. Pat. No. 3,674,836 and U.S. Pat. No. 4,126,637. U.S. Pat. No. 1,782,727, and U.S. Pat. No. 1,889,704. Rito Indocyanine Green analogs are described in U.S. Pat. No. drine (e.g., hydrochloride salt) analogs are described in BE 2.895,955. Indomethacin analogs are described in U.S. Pat. 660244 and U.S. Pat. No. 3,410,944. Rolipram analogs are No. 3,161,654 and BE 679678. Iopanoic Acid analogs are described in BE 826923 and U.S. Pat. No. 4,193.926. Succi described in U.S. Pat. No. 2,705,726. Iophenoxic Acid ana nylcholine analogs are described in AT 171411. Sulfaguani logs are described in GB 726987. Iopromide analogs are dine analogs are described in U.S. Pat. No. 2,218,490, U.S. described in DE 2909439 and U.S. Pat. No. 4,364,921. Iso Pat. No. 2,229,784, U.S. Pat. No. 2,233,569, and GB551524. proterenol (e.g., Sulfate salt) analogs are described in German Sulfamethizole analogs are described in U.S. Pat. No. 2,447. patent DE 723278 and U.S. Pat. Nos. 2,308.232 and 2,715, 702. Suprofen analogs are described in U.S. Pat. No. 4,035, 141. Isotretinoin analogs are described in EP 111325 and U.S. 25 376. Telmisartan analogs are described in EP 502314. Terb Pat. No. 4,556,518. Ketoconazole analogs are described in utaline (e.g., sulfate salt) analogs are described in BE 704932 German patent DE 2804096, U.S. Pat. No. 4,144,346, and and U.S. Pat. No. 3,937,838. Tetrahydrozoline (e.g., hydro U.S. Pat. No. 4,223,036. Ketotifen (e.g., fumarate salt) ana chloride salt) analogs are described in U.S. Pat. No. 2,731,471 logs are described in German patent DE 21 11071 and U.S. and U.S. Pat. No. 2,842,478. Timidazole analogs are Pat. No. 3,682.930. Lamivudine analogs are described in PCT 30 described in U.S. Pat. No. 3,376,311. Tioconazole analogs are WO 91/17159. Leflunomide analogs are described in German described in BE 841309 and U.S. Pat. No. 4,062,966. Tola patent DE 2854439 and U.S. Pat. No. 4,284,786. Levocabas Zoline (e.g., hydrochloride salt) analogs are described in U.S. tine (e.g., hydrochloride salt) analogs are described in EP Pat. No. 2,161,938. Tolfenamic acid analogs are described in 34415 and U.S. Pat. No. 4.369,184. Loxapine (e.g., hydro NL 6600251 and U.S. Pat. No. 3,313,848. Triamterene ana chloride salt) analogs are described in NL 6406089 and U.S. 35 logs are described in U.S. Pat. No. 3,081,230. Triflupro Pat. No. 3,546,226 and U.S. Pat. No. 3,412,193. Mebhydro mazine (e.g., hydrochloride salt) analogs are described in GB line (e.g., 1.5-Naphthalenedisulfonate salt) analogs are 813861. Tulobuterol (e.g., hydrochloride salt) analogs are described in U.S. Pat. No. 2,786,059. Meclofenoxate analogs described in German patent DE 2244737. Vincamine analogs are described in FR M398. Mefenamic Acid analogs are are described in German patent DE 2115718 and U.S. Pat. described in BE 605302 and U.S. Pat. No. 3,138,636. Meloxi 40 No. 3,770,724. Warfarin analogs are described in U.S. Pat. cam analogs are described in U.S. Pat. No. 4,233,299. Mel No. 2,427,578, U.S. Pat. No. 2,765,321, U.S. Pat. No. 2,777, phalan analogs are described in U.S. Pat. No. 3,032.584 and 859, and U.S. Pat. No. 3.239,529. Xylazine (e.g., hydrochlo U.S. Pat. No. 3,032.585. Mercaptoethanol analogs are ride salt) analogs are described in BE 634552, DE 1173475, described in U.S. Pat. No. 2,402,665 and U.S. Pat. No. 3,394, and U.S. Pat. No. 3,235,550. All of these references are 192. Metaproterenol (e.g., hemisulfate salt) analogs are 45 hereby incorporated by reference. described in U.S. Pat. No. 3,341,594 and BE 611502. Metha If desired, the patient may also receive additional therapeu choline analogs are described in U.S. Pat. No. 2,040,146. tic regimens. For example, therapeutic agents may be admin Methdilazine analogs are described in U.S. Pat. No. 2,945, istered with the agent or agents described herein at concen 855. Methylergonovine (e.g., maleate salt) analogs are trations known to be effective for Such therapeutic agents. described in U.S. Pat. No. 2.265.207. Nateglinide analogs are 50 Particularly useful agents include those that reduce glucose described in EP 196222 and U.S. Pat. No. 4,816,484. Nefo levels or those used to treat, prevent, or reduce metabolic pam analogs are described in NL 6606390 and U.S. Pat. No. disorders. Such agents include those that alter the insulin 3,830,803. Nimesulide analogs are described in BE 801812 signaling pathway, such as agents that increase the insulin and U.S. Pat. No. 3,840,597. Norepinephrine (e.g., bitartrate Supply, reduce insulin resistance, increase the effectiveness of salt) analogs are described in U.S. Pat. No. 2,774,789. Olan 55 insulin, reduce hepatic glucose output, control blood glucose Zapine analogs are described in EP 454436 and U.S. Pat. No. and triglyceride levels, and reduce the absorption of carbo 5.229,382. Oleandomycin analogs are described in U.S. Pat. hydrates from the intestine. Exemplary agents are Sulfony No. 2,757,123 and U.S. Pat. No. 2,842,481. Orphenadrine lureas (e.g., acetohexamide, chlorpropamide, tolaZamide, (e.g., citrate salt) analogs are described in U.S. Pat. No. 2,567, , , glipizide, and glyburide), non-sul 351 and U.S. Pat. No. 2,991.225. Oxaprozin analogs are 60 fonylurea Secretagogues (e.g., nateglinide and repaglinide). described in FR 2001036, GB 1206403, and U.S. Pat. No. insulin, insulin analogs (e.g., insulin lispro, insulin aspart, 3.578,671. Oxybutynin (e.g., chloride salt) analogs are insulin glarginine, NPH, lente insulin, ultralente insulin, described in GB940540. Oxymetazoline (e.g., hydrochloride humulin, novolin), glucagon-like peptides (e.g., GLP-1), salt) analogs are described in German patent DE 1117588. exendin-4 (e.g., AAC2993), beta-3 adrenergic receptor ago Pergolide (e.g., mesylate salt) analogs are described in U.S. 65 nists (e.g., YM178), PPARgamma agonists (e.g., FK614), Pat. No. 4,166,182. Phenacemide analogs are described in dipeptidyl peptidase IV inhibitors, biguanides (e.g., met U.S. Pat. No. 2,887,513. Phensuximide analogs are described formin and metformin/glyburide), thiazalidinediones (e.g., US 7,795,310 B2 33 34 troglitaZone, pioglitaZone and rosiglitaZone), alpha-glucosi changes may be helpful to control glucose levels and include dase inhibitors (e.g., acarbose and miglitol), immunosuppres weight loss, physical exercise, diet control, reduction in alco sants or immunomodulators (e.g., glucocorticoids, cyclo hol intake, or reduction in Smoking. phosphamide, cyclosporine A, rapamycin, FK506 cytokines Corticosteroids such as IL-4 and -IL-10, OK-432, LZ-8, BCG, and CFA, A corticosteroid may be formulated in the composition of angiotensin converting enzyme (ACE) inhibitors (e.g., the invention or administered to the mammal being treated benazepril, captopril, cilaZapril, enalapril, enalaprilat, fosi according to the invention. Suitable corticosteroids include nopril, lisinopril, moexipril, perindopril, quinapril, ramipril, 11-alpha, 17-alpha, 21-trihydroxypregn-4-ene-3,20-dione; and trandolapril), angiotensin II receptor blockers (e.g., can 11-beta, 16-alpha, 17.21-tetrahydroxypregn-4-ene-3,20-di desartan, eprosartan, irbesarten, losartin, telmisartan, and Val 10 one; 11-beta, 16-alpha, 17.21-tetrahydroxypregn-1,4-diene Sartan), antioxidants (e.g., , Vitamin E. , 3.20-dione; 11-beta, 17-alpha, 21-trihydroxy-6-alpha-meth MDL29311 and U78518F), and combinations thereof. ylpregn-4-ene-3,20-dione; 11-dehydrocorticosterone: It may also be desirable to administer to the patient thera 11-deoxycortisol; 11-hydroxy-1,4-androstadiene-3,17-di peutic compounds, such as corticosteroids, NSAIDs (e.g., one; 11-ketotestosterone; 14-hydroxyandrost-4-ene-3,6,17 naproxen Sodium, diclofenac sodium, diclofenac potassium, 15 trione; 15,17-dihydroxyprogesterone; 16-methylhydrocorti aspirin, Sulindac, diflunisal, piroxicam, indomethacin, ibu sone; 17,21-dihydroxy-16-alpha-methylpregna-1,4,9(11)- profen, , choline magnesium trisalicylate, triene-3,20-dione; 17-alpha-hydroxypregn-4-ene-320 Sodium salicylate, Salicylsalicylic acid, , flurbi dione; 17-alpha-hydroxypregnenolone; 17-hydroxy-16-beta profen, ketoprofen, meclofenamate sodium, meloxicam, methyl-5-beta-pregn-9(11)-ene-320-dione; 17-hydroxy-4,6, oxaprozin, Sulindac, and tolmetin), COX-2 inhibitors (e.g., 8(14)-pregnatriene-320-dione; 17-hydroxypregna-4,9011)- rofecoxib, celecoxib, Valdecoxib, and lumiracoxib), diene-320-dione; 18-hydroxycorticosterone: DMARD, anti-cytokine agents or agents that modulate the 18-hydroxycortisone; 18-oxocortisol; 21-deoxyaldosterone: immune response to positively effect disease, Such as agents 21-deoxycortisone; 2-deoxyecdysone; 2-methylcortisone; that influence cell adhesion, or biologics (i.e., agents that 3-dehydroecdysone: 4-pregnene-17-alpha, 20-beta, 21-triol block the action of IL-6, IL-1, IL-2, IL-12, IL-15 or TNFC. 25 3,11-dione: 6,17.20-trihydroxypregn-4-ene-3-one: 6-alpha (e.g., etanercept, adelimumab, infliximab, or CDP-870). hydroxycortisol; 6-alpha-fluoroprednisolone, 6-alpha-meth If more than one agent is employed, therapeutic agents may ylprednisolone, 6-alpha-methylprednisolone 21-acetate, be delivered separately or may be admixed into a single 6-alpha-methylprednisolone 21-hemisuccinate sodium salt, formulation. When agents are present in different pharmaceu 6-beta-hydroxycortisol, 6-alpha, 9-alpha-difluoropredniso tical compositions, different routes of administration may be 30 lone 21-acetate 17-butyrate, 6-hydroxycorticosterone: 6-hy employed. Routes of administration for the various embodi droxydexamethasone; 6-hydroxyprednisolone; 9-fluorocor ments include, but are not limited to, topical, transdermal, and tisone; alclometasone dipropionate; aldosterone; : systemic administration (such as, intravenous, intramuscular, alphaderm; amadinone; amcinonide; ; andros Subcutaneous, inhalation, rectal, buccal, vaginal, intraperito tenedione; anecortave acetate; beclomethasone; beclometha neal, intraarticular, ophthalmic or oral administration). As 35 Sone dipropionate; beclomethasone dipropionate monohy used herein, “systemic administration” refers to all nonder drate; betamethasone 17-valerate; betamethasone sodium mal routes of administration, and specifically excludes topi acetate; betamethasone sodium phosphate; betamethasone cal and transdermal routes of administration. Desirably, the Valerate; bolasterone; budesonide; calusterone; chlormadi agent of the invention and additional therapeutic agents are none; chloroprednisone; chloroprednisone acetate; choles administered within at least 1, 2, 4, 6, 10, 12, 18, 24 hours, 3 40 terol; clobetasol; clobetasol propionate; clobetaSone; clocor days, 7 days, or 14 days apart. The dosage and frequency of tolone; clocortolone pivalate; ; cloprednol; administration of each component of the combination can be corticosterone; ; cortisol acetate; cortisol butyrate; controlled independently. For example, one compound may cortisol cypionate; cortisol octanoate; cortisol sodium phos be administered three times per day, while the second com phate; cortisol sodium Succinate; cortisol Valerate; cortisone; pound may be administered once per day. Combination 45 cortisone acetate; cortodoxone; daturaolone; deflazacort, therapy may be given in on-and-off cycles that include rest 21-deoxycortisol, ; ; periods so that the patient’s body has a chance to recover from deoxycorticosterone; deprodone; descinolone; desonide; any as yet unforeseen side effects. The compounds may also desoximethasone; dexafen; dexamethasone; dexamethasone be formulated together such that one administration delivers 21-acetate; dexamethasone acetate; dexamethasone sodium both compounds. Optionally, any of the agents of the combi 50 phosphate; dichlorisone; diflorasone; diflorasone diacetate; nation may be administered in a low dosage or in a high diflucortolone; dihydroelatericina; domoprednate: doxibeta dosage, each of which is defined herein. Sol, ecdysone; ecdysterone; endrysone; enoXolone; flucino The therapeutic agents of the invention may be admixed lone; fludrocortisone; fludrocortisone acetate; ; with additional active or inert ingredients, e.g., in conven flumethasone; flumethasone pivalate; flumoxonide; tional pharmaceutically acceptable carriers. A pharmaceuti 55 flunisolide; fluocinolone; fluocinolone acetonide; fluocinon cal carrier can be any compatible, non-toxic Substance Suit ide; 9-fluorocortisone; fluocortolone; fluorohydroxyandros able for the administration of the compositions of the present tenedione; ; ; flu invention to a mammal. Pharmaceutically acceptable carriers oxymesterone; fluprednidene; fluprednisolone: include, for example, water, Saline, buffers and other com flurandrenolide; fluticasone; fluticaSone propionate; forme pounds described for example in the Merck Index, Merck & 60 bolone; formestane; formocortal; gestonorone; glyderinine; Co., Rahway, N.J. Slow release formulation or a slow release halcinonide; hyrcanoside; halometaSone; halopredone; halo apparatus may be also be used for continuous administration. , hydrocortiosone cypionate; hydrocortisone; In addition to the administration of therapeutic agents, the hydrocortisone 21-butyrate; hydrocortisone aceponate; second therapeutic regimen may involve transplantation of hydrocortisone acetate; hydrocortisone buteprate; hydrocor insulin-producing cells, tissues, or organs (e.g., pancreatic 65 tisone butyrate; hydrocortisone cypionate; hydrocortisone cells, beta pancreatic cells, or pancreas) or a modification to hemisuccinate; hydrocortisone probutate; hydrocortisone the lifestyle of the patient being treated. Such lifestyle Sodium phosphate; hydrocortisone sodium Succinate; hydro US 7,795,310 B2 35 36 cortisone Valerate; hydroxyprogesterone; inokosterone; isof Non-Steroidal Immunophilin-Dependent Immunosup lupredone; isoflupredone acetate; isoprednidene; meclor pressants isone; mecortolon; ; ; The present invention may also involve the administration medry Sone; ; ; ; of a non-steroidal immunophilin-dependent immunosuppres meprednisone; methandrostenolone; methylprednisolone; sant (NSIDI). In healthy individuals the immune system uses methylprednisolone aceponate; methylprednisolone acetate; cellular effectors, such as B-cells and T-cells, to target infec methylprednisolone hemisuccinate; methylprednisolone tious microbes and abnormal cell types while leaving normal Sodium Succinate; methyltestosterone; ; cells intact. In individuals with an autoimmune disorder (e.g., ; mometasone furoate; mometasone furoate diabetes), activated T-cells damage healthy tissues. Cal 10 cineurin inhibitors (e.g., cyclosporines, tacrolimus, pime monohydrate; nisone; ; ; norvinis crolimus), and rapamycin target many types of immunoregu terone; oxymesterone; paramethasone; paramethasone latory cells, including T-cells, and Suppress the immune acetate; ponasterone; prednisolamate; prednisolone; pred response in autoimmune disorders. Immunosuppressants are nisolone 21-hemisuccinate; prednisolone acetate; predniso particularly useful if the mammal is also receiving an organ, lone farnesylate; prednisolone hemisuccinate; prednisolone 15 tissue, or cellular implant. Exemplary immunosuppressants 21 (beta-D-glucuronide); prednisolone metasulphobenzoate; are provided below. prednisolone sodium phosphate; prednisolone steaglate; Cyclosporines prednisolone tebutate; prednisolone tetrahydrophthalate: The cyclosporines are fungal metabolites that comprise a prednisone; prednival; prednylidene; ; procino class of cyclic oligopeptides that act as immunosuppressants. nide; tralonide; progesterone; ; rhapontister Cyclosporine A, and its deuterated analogue ISAtX247, is a one; rimexolone; roxibolone; rubrosterone; stizophyllin; hydrophobic cyclic polypeptide consisting of eleven amino tiXocortol; topterone; triamcinolone; triamcinolone acids. Cyclosporine A binds and forms a complex with the acetonide; 21-palmitate; triamcino intracellular receptor cyclophilin. The cyclosporine/cyclo lone diacetate; triamcinolone hexacetonide; ; philin complex binds to and inhibits calcineurin, a Ca"- turkesterone; and wortmannin. 25 calmodulin-dependent serine-threonine-specific protein Standard recommended dosages for corticosteroids are phosphatase. Calcineurin mediates signal transduction events provided, e.g., in the Merck Manual of Diagnosis & Therapy required for T-cell activation (reviewed in Schreiber et al., (17th Ed. MH Beers et al., Merck & Co.) and Physicians Cell 70:365-368, 1991). Cyclosporines and their functional Desk Reference 2003 (57" Ed. Medical Economics Staffet and structural analogs Suppress the T-cell-dependent immune al., Medical Economics Co., 2002). In one embodiment, the 30 response by inhibiting antigen-triggered signal transduction. dosage of corticosteroid administered is a dosage equivalent This inhibition decreases the expression of proinflammatory to a prednisolone dosage, as defined herein. For example, a cytokines, such as IL-2. low dosage of a corticosteroid may be considered as the Many cyclosporines (e.g., cyclosporine A, B, C, D, E, F, G, dosage equivalent to a low dosage of prednisolone. H. and I) are produced by fungi. Cyclosporine A is a com Other compounds that may be used as a substitute for or in 35 mercially available under the trade name NEORAL from addition to a corticosteroid in the methods, compositions, and Novartis. Cyclosporine A structural and functional analogs kits of the invention A-348441 (Karo Bio), adrenal cortex include cyclosporines having one or more fluorinated amino extract (GlaxoSmithKline), alsactide (Aventis), amebucort acids (described, e.g., in U.S. Pat. No. 5,227.467); cyclospo (Schering AG), amelometasone (Taisho), ATSA (Pfizer), rines having modified amino acids (described, e.g., in U.S. bitolterol (Elan), CBP-2011 (InKine Pharmaceutical), 40 Pat. Nos. 5,122,511 and 4.798,823); and deuterated cyclospo cebaracetam (Novartis) CGP-13774 (Kissei), ciclesonide rines, such as ISAtx247 (described in U.S. Patent Publication (Altana), ciclometasone (Aventis), clobetaSone butyrate No. 20020132763). Additional cyclosporine analogs are (GlaxoSmithKline), cloprednol (Hoffmann-La Roche), col described in U.S. Pat. Nos. 6,136,357, 4,384,996, 5,284,826, lismycin A (Kirin), cucurbitacin E (NIH), deflazacort (Aven and 5,709,797. Cyclosporine analogs include, but are not tis), deprodone propionate (SSP), dexamethasone acefurate 45 limited to, D-Sar (C-SMe) Val-DH-Cs (209-825), Allo-Thr (Schering-Plough), dexamethasone linoleate (GlaxoSmith 2-Cs, Norvaline-2-Cs. D-Ala (3-acetylamino)-8-Cs. Thr-2- Kline), dexamethasone Valerate (Abbott), difluprednate Cs, and D-MeSer-3-Cs, D-Ser (O-CHCH-OH)-8-Cs, (Pfizer), domoprednate (Hoffmann-La Roche), ebiratide and D-Ser-8-Cs, which are described in Cruz et al. (Antimi (Aventis), etiprednol dicloacetate (IVAX), fluazacort (Vicu crob. Agents Chemother. 44:143-149, 2000). ron), flumoxonide (Hoffmann-La Roche), fluocortin butyl 50 Cyclosporines are highly hydrophobic and readily precipi (Schering AG), fluocortolone monohydrate (Schering AG), tate in the presence of water (e.g., on contact with body GR-250495X (GlaxoSmithKline), halometasone (Novartis), fluids). Methods of providing cyclosporine formulations with halopredone (Dainippon), HYC-141 (Fidia), icomethasone improved are described in U.S. Pat. Nos. enbutate (Hovione), itrocinonide (AstraZeneca), L-6485 (Vi 4,388,307, 6,468,968, 5,051,402, 5,342,625, 5,977,066, and curon), Lipocort (Draxis Health), locicortone (Aventis), 55 6,022,852. Cyclosporine microemulsion compositions are meclorisone (Schering-Plough), naflocort (Bristol-Myers described in U.S. Pat. Nos. 5,866,159, 5,916,589, 5,962,014, Squibb), NCX-1015 (Nicox), NCX-1020 (Nicox), NCX 5,962,017, 6,007,840, and 6,024,978. 1022 (NicOx), nicocortonide (Yamanouchi), NIK-236 (Ni Cyclosporines can be administered either intravenously or kken Chemicals), NS-126 (SSP), Org-2766 (Akzo Nobel), orally, but oral administration is preferred. To counteract the Org-6632 (AkzoNobel), P16CM, propylmesterolone (Scher 60 hydrophobicity of cyclosporine A, an intravenous cyclospo ing AG), RGH-1113 (Gedeon Richter), rofileponide (Astra rine A is usually provided in an ethanol-polyoxyethylated Zeneca), rofileponide palmitate (AstraZeneca), RPR-106541 castor oil vehicle that must be diluted prior to administration. (Aventis), RU-26559 (Aventis), Sch-19457 (Schering Cyclosporine A may be provided, e.g., as a microemulsion in Plough), T25 (Matrix Therapeutics), TBI-PAB (Sigma-Tau), a 25 mg or 100 mg tablets, or in a 100 mg/ml oral solution ticabesone propionate (Hoffmann-La Roche), tifluadom 65 (NEORALTM). (Solvay), timobesone (Hoffmann-La Roche), TSC-5 Typically, patient dosage of an oral cyclosporine varies (Takeda), and ZK-73634 (Schering AG). according to the patient’s condition, but some standard rec US 7,795,310 B2 37 38 ommended dosages in prior art treatment regimens are pro While Suggested dosages will vary with a patient’s condi vided herein. Patients undergoing organ transplant (e.g., pan tion, standard recommended dosages used in prior art treat creatic islet transplantation) typically receive an initial dose ment regimens are provided below. Patients having a trans of oral cyclosporine A in amounts between 12 and 15 mg/kg/ planted organ typically receive doses of 0.1-0.2 mg/kg/day of day. Dosage is then gradually decreased by 5% per week until oraltacrolimus. Patients being treated for rheumatoid arthritis a 7-12 mg/kg/day maintenance dose is reached. For intrave typically receive 1-3 mg/day oral tacrolimus. For the treat nous administration, 2-6 mg/kg/day is preferred for most ment of psoriasis, 0.01-0.15 mg/kg/day of oral tacrolimus is patients. Often cyclosporines are administered in combina administered to a patient. Patients receiving oral tacrolimus tion with other immunosuppressive agents, such as glucocor capsules typically receive the first dose no Sooner than six 10 hours after transplant, or eight to twelve hours after intrave ticoids. Additional information is provided in Table 3. nous tacrolimus infusion was discontinued. Thus, Suggested tacrolimus dosages include 0.005-0.01 mg/kg/day, 0.01-0.03 TABLE 3 mg/kg/day, 0.03-0.05 mg/kg/day, 0.05-0.07 mg/kg/day, 0.07 NSIDIS 0.10 mg/kg/day, 0.10-0.25 mg/kg/day, or 0.25-0.5 mg/kg/ 15 day. Compound Transplant Tacrolimus is extensively metabolized by the mixed-func Cyclosporine A ~7-12 mg/kg/day tion oxidase system, in particular, by the cytochrome P-450 (NEORAL) system. The primary mechanism of metabolism is demethy Tacrolimus 0.1-0.2 mg/kg/day (oral) lation and hydroxylation. While various tacrolimus metabo Pimecrolimus 40-120 mg/day (oral) lites are likely to exhibit immunosuppressive biological activ ity, the 13-demethyl metabolite is reported to have the same Tacrolimus activity as tacrolimus. Pimecrolimus and Ascomycin Derivatives Tacrolimus (PROGRAF, Fujisawa), also known as FK506, Ascomycin is a close structural analog of FK506 and is a is an immunosuppressive agent that targets T-cell intracellu 25 potent immunosuppressant. It binds to FKBP-12 and Sup lar signal transduction pathways. Tacrolimus binds to an presses its proline rotamase activity. The ascomycin-FKBP intracellular protein FK506 binding protein (FKBP-12) that complex inhibits calcineurin, a type 2B phosphatase. is not structurally related to cyclophilin (Harding etal. Nature Pimecrolimus (also known as SDZASM-981) is an33-epi 341:758-7601, 1989: Siekienka et al. Nature 341:755-757, chloro derivative of the ascomycin. It is produced by the strain 1989; and Soltoff et al., J. Biol. Chem. 267: 17472-17477, 30 Streptomyces hygroscopicus var. ascomyceitus. Like tacroli 1992). The FKBP/FK506 complex binds to calcineurin and mus, pimecrolimus (ELIDELTM, Novartis) binds FKBP-12, inhibits calcineurin's phosphatase activity. This inhibition inhibits calcineurin phosphatase activity, and inhibits T-cell prevents the dephosphorylation and nuclear translocation of activation by blocking the transcription of early cytokines. In NFAT, a nuclear component that initiates gene transcription particular, pimecrolimus inhibits IL-2 production and the required for lymphokine (e.g., IL-2, gamma interferon) pro 35 release of other proinflammatory cytokines. duction and T-cell activation. Thus, tacrolimus inhibits T-cell Pimecrolimus structural and functional analogs are activation. described in U.S. Pat. No. 6,384,073. Pimecrolimus is par Tacrolimus is a macrollide antibiotic that is produced by ticularly useful for the treatment of atopic dermatitis. Pime Streptomyces tsukubaensis. It suppresses the immune system crolimus is currently available as a 1% cream. While indi and prolongs the Survival of transplanted organs. It is cur 40 vidual dosing will vary with the patient’s condition, some rently available in oral and injectable formulations. Tacroli standard recommended dosages are provided below. Patients mus capsules contain 0.5 mg, 1 mg, or 5 mg of anhydrous having an organ transplant can be administered 160-240 tacrolimus within a gelatin capsule shell. The injectable for mg/day of pimecrolimus. Thus, useful dosages of pimecroli mulation contains 5 mg anhydrous tacrolimus in castor oil mus range between 0.5-5 mg/day, between 5-10 mg/day, and alcohol that is diluted with 9% sodium chloride or 5% 45 between 10-30 mg/day, between 40-80 mg/day, between dextrose prior to injection. While oral administration is pre 80-120 mg/day, or even between 120-200 mg/day. ferred, patients unable to take oral capsules may receive Rapamycin injectable tacrolimus. The initial dose should be administered Rapamycin (Rapamune(R) Sirolimus, Wyeth) is a cyclic lac no sooner than six hours after transplant by continuous intra tone produced by Steptomyces hygroscopicus. Rapamycin is venous infusion. 50 an immunosuppressive agent that inhibits T-lymphocyte acti Tacrolimus and tacrolimus analogs are described by Vation and proliferation. Like cyclosporines, tacrolimus, and Tanaka et al., (J. Am. Chem. Soc., 109:5031, 1987), and in pimecrolimus, rapamycin forms a complex with the immu U.S. Pat. Nos. 4,894,366, 4,929,611, and 4,956,352. FK506 nophilin FKBP-12, but the rapamycin-FKBP-12 complex related compounds, including FR-900520, FR-900523, and does not inhibit calcineurin phosphatase activity. The rapa FR-900525, are described in U.S. Pat. No. 5.254.562; O-aryl, 55 mycin-immunophilin complex binds to and inhibits the mam O-alkyl, O-alkenyl, and O-alkynylmacrollides are described malian target of rapamycin (mTOR), a kinase that is required in U.S. Pat. Nos. 5,250,678, 532,248, 5,693,648; amino for cell cycle progression. Inhibition of mTOR kinase activity O-aryl macrollides are described in U.S. Pat. No. 5,262,533; blocks T-lymphocyte proliferation and lymphokine secretion. alkylidene macrollides are described in U.S. Pat. No. 5.284, Rapamycin structural and functional analogs include 840; N-heteroaryl, N-alkylheteroaryl, N-alkenylheteroaryl, 60 mono- and diacylated rapamycin derivatives (U.S. Pat. No. and N-alkynylheteroaryl macrollides are described in U.S. 4.316,885); rapamycin water-soluble prodrugs (U.S. Pat. No. Pat. No. 5,208,241; aminomacrollides and derivatives thereof 4,650,803); carboxylic acid esters (PCT Publication No. WO are described in U.S. Pat. No. 5,208,228; fluoromacrollides 92/05179); carbamates (U.S. Pat. No. 5,118,678); amide are described in U.S. Pat. No. 5,189,042; amino O-alkyl, esters (U.S. Pat. No. 5,118,678); biotinesters (U.S. Pat. No. O-alkenyl, and O-alkynylmacrollides are described in U.S. 65 5,504.091); fluorinated esters (U.S. Pat. No. 5,100,883); Pat. No. 5,162.334; and halomacrollides are described in U.S. acetals (U.S. Pat. No. 5,151,413); silyl ethers (U.S. Pat. No. Pat. No. 5,143,918. 5,120,842); bicyclic derivatives (U.S. Pat. No. 5,120,725); US 7,795,310 B2 39 40 rapamycin dimers (U.S. Pat. No. 5,120,727); O-aryl, O-alkyl, groups for amines include amides, such as formamides, O-alkyenyl and O-alkynyl derivatives (U.S. Pat. No. 5.258, acetamides, trifluoroacetamides, Sulfonamides, trifluo 389); and deuterated rapamycin (U.S. Pat. No. 6,503.921). romethanesulfonyl amides, trimethylsilylethanesulfona Additional rapamycin analogs are described in U.S. Pat. Nos. mides, and tert-butylsulfonyl amides. Examples of com 5,202,332 and 5,169,851. monly used protecting groups for carboxyls include esters, Everolimus (40-O-(2-hydroxyethyl)rapamycin; CERTI such as methyl, ethyl, tert-butyl, 9-fluorenylmethyl 2-(trim CANTM; Novartis) is an immunosuppressive macrollide that is ethylsilyl)ethoxy methyl, benzyl, diphenylmethyl, O-ni structurally related to rapamycin, and has been found to be trobenzyl, ortho-esters, and halo-esters. Examples of com particularly effective at preventing acute rejection of organ monly used protecting groups for alcohols include ethers, transplant when given in combination with cyclosporin A. 10 Such as methyl, methoxymethyl, methoxyethoxymethyl, Rapamycin is currently available for oral administration in methylthiomethyl, benzyloxymethyl, tetrahydropyranyl, liquid and tablet formulations. RAPAMUNETM liquid con ethoxyethyl, benzyl, 2-napthylmethyl, O-nitrobenzyl, P-ni tains 1 mg/mL rapamycin that is diluted in water or orange trobenzyl, P-methoxybenzyl, 9-phenylxanthyl, trityl (includ juice prior to administration. Tablets containing 1 or 2 mg of ing methoxytrityls), and silyl ethers. Examples of commonly rapamycin are also available. Rapamycin is preferably given 15 used protecting groups for sulfhydryls include many of the once daily as soon as possible after transplantation. It is same protecting groups used for hydroxyls. In addition, Sulf absorbed rapidly and completely after oral administration. hydryls can be protected in a reduced form (e.g., as disulfides) Typically, patient dosage of rapamycin varies according to the oran oxidized form (e.g., as Sulfonic acids, Sulfonic esters, or patient’s condition, but some standard recommended dosages Sulfonic amides). Protecting groups can be chosen Such that are provided below. The initial loading dose for rapamycin is selective conditions (e.g., acidic conditions, basic conditions, 6 mg. Subsequent maintenance doses of 2 mg/day are typical. catalysis by a nucleophile, catalysis by a lewis acid, or hydro Alternatively, a loading dose of 3 mg, 5 mg, 10 mg, 15 mg, 20 genation) are required to remove each, exclusive of other mg, or 25 mg can be used with a 1 mg, 3 mg, 5 mg, 7 mg, or protecting groups in a molecule. The conditions required for 10 mg per day maintenance dose. In patients weighing less the addition of protecting groups to amine, alcohol, Sulfhy than 40 kg, rapamycin dosages are typically adjusted based 25 dryl, and carboxyl functionalities and the conditions required on body surface area: generally a 3 mg/m/day loading dose for their removal are provided in detail in T. W. Green and P. and a 1-mg/m/day maintenance dose is used. G. M. Wuts, Protective Groups in Organic Synthesis (2" Ed.), John Wiley & Sons, 1991 and P. J. Kocienski, Protecting Conjugates Groups, Georg Thieme Verlag, 1994. Additional synthetic If desired, the drugs used in any of the combinations 30 details are provided below. described herein may be covalently attached to one another to form a conjugate of formula XXX. Linkers The linker component of the invention is, at its simplest, a (A)-(L)-(B) (XXX) bond between drug (A) and drug (B), but typically provides a Informula XXX. (A) is a drug listed on Table 1 covalently 35 linear, cyclic, or branched molecular skeleton having pendant tethered via a linker (L) to (B), a sulfonylurea, a non-sulfo groups covalently linking drug (A) to drug (B). nylurea secretagogue, insulin, an insulin analog, glucagon Thus, linking of drug (A) to drug (B) is achieved by cova like peptide, exendin-4, YM178, FK614, a dipeptidyl pepti lent means, involving bond formation with one or more func dase IV inhibitor, biguanide, thiazalidinedione, an alpha tional groups located on drug (A) and drug (B). Examples of glucosidase inhibitor, an immunosuppressant, an 40 chemically reactive functional groups which may be immunomodulator, an angiotensin converting enzyme (ACE) employed for this purpose include, without limitation, amino, inhibitor, an angiotensin II receptor blocker, an antioxidant, hydroxyl, sulfhydryl, carboxyl, carbonyl, carbohydrate or a second drug listed on Table 1. groups, vicinal diols, thioethers, 2-aminoalcohols, 2-ami Conjugates of the invention can be administered to a Sub nothiols, guanidinyl, imidazolyl, and phenolic groups. ject by any route and for the treatment of any disease 45 The covalent linking of drug (A) and drug (B) may be described herein. effected using a linker which contains reactive moieties The conjugates of the invention can be prodrugs, releasing capable of reaction with Such functional groups present in drug (A) and drug (B) upon, for example, cleavage of the drug (A) and drug (B). For example, an amine group of drug conjugate by intracellular and extracellular (e.g., (A) may react with a carboxyl group of the linker, or an amidases, esterases, and phosphatases). The conjugates of the 50 activated derivative thereof, resulting in the formation of an invention can also be designed to largely remain intact in amide linking the two. Vivo, resisting cleavage by intracellular and extracellular Examples of moieties capable of reaction with sulfhydryl enzymes. The degradation of the conjugate in Vivo can be groups include C-haloacetyl compounds of the type controlled by the design of linker (L) and the covalent bonds XCHCO— (where X=Br, C1 or I), which show particular formed with drug (A) and drug (B) during the synthesis of the 55 reactivity for sulfhydryl groups, but which can also be used to conjugate. modify imidazolyl, thioether, phenol, and amino groups as Conjugates can be prepared using techniques familiar to described by Gurd, Methods Enzymol. 11:532 (1967). N-Ma those skilled in the art. For example, the conjugates can be leimide derivatives are also considered selective towards Sulf prepared using the methods disclosed in G. Hermanson, Bio hydryl groups, but may additionally be useful in coupling to conjugate Techniques, Academic Press, Inc., 1996. The Syn 60 amino groups under certain conditions. Reagents such as thesis of conjugates may involve the selective protection and 2-iminothiolane (Traut et al., Biochemistry 12:3266 (1973)), deprotection of alcohols, amines, ketones, sulfhydryls or car which introduce athiol group through conversion of an amino boxyl functional groups of drug (A), the linker, and/or drug group, may be considered as Sulfhydryl reagents if linking (B). For example, commonly used protecting groups for occurs through the formation of disulphide bridges. amines include carbamates, such as tert-butyl, benzyl, 2.2.2- 65 Examples of reactive moieties capable of reaction with trichloroethyl, 2-trimethylsilylethyl, 9-fluorenylmethyl, amino groups include, for example, alkylating and acylating allyl, and m-nitrophenyl. Other commonly used protecting agents. Representative alkylating agents include: US 7,795,310 B2 41 42 (i) C.-haloacetyl compounds, which show specificity ing reagents such as carbodiimides, which react through towards amino groups in the absence of reactive thiol groups O-acylurea formation followed by amide bond formation, and are of the type XCH-CO— (where X=Cl, Br or I), for may also be employed. example, as described by Wong Biochemistry 24:5337 It will be appreciated that functional groups in drug (A) (1979); and/or drug (B) may, if desired, be converted to other func (ii) N-maleimide derivatives, which may react with amino tional groups prior to reaction, for example, to confer addi groups either through a Michael type reaction or through tional reactivity or selectivity. Examples of methods useful acylation by addition to the ring carbonyl group, for example, for this purpose include conversion of amines to carboxyls as described by Smyth et al., J. Am. Chem. Soc. 82:4600 using reagents such as dicarboxylic anhydrides; conversion (1960) and Biochem. J. 91:589 (1964); 10 of amines to thiols using reagents such as N-acetylhomocys (iii) aryl halides Such as reactive nitrohaloaromatic com teine thiolactone, S-acetylmercaptosuccinic anhydride, pounds; 2-iminothiolane, or thiol-containing Succinimidyl deriva (iv) alkyl halides, as described, for example, by McKenzie tives; conversion of thiols to carboxyls using reagents such as et al., J. Protein Chem. 7:581 (1988); C-haloacetates; conversion of thiols to amines using reagents (v) aldehydes and ketones capable of Schiffs base forma 15 Such as ethylenimine or 2-bromoethylamine; conversion of tion with amino groups, the adducts formed usually being carboxyls to amines using reagents such as carbodiimides stabilized through reduction to give a stable amine; followed by diamines; and conversion of alcohols to thiols (vi) epoxide derivatives such as epichlorohydrin and bisox using reagents such as tosyl chloride followed by transesteri iranes, which may react with amino, Sulfhydryl, or phenolic fication with thioacetate and hydrolysis to the thiol with hydroxyl groups; Sodium acetate. (vii) chlorine-containing derivatives of S-triazines, which So-called Zero-length linkers, involving direct covalent are very reactive towards nucleophiles Such as amino, Sufhy joining of a reactive chemical group of drug (A) with a reac dryl, and hydroxyl groups; tive chemical group of drug (B) without introducing addi tional linking material may, if desired, be used in accordance (viii) aziridines based on S-triazine compounds detailed 25 with the invention. above, e.g., as described by Ross, J. Adv. Cancer Res. 2:1 Most commonly, however, the linker will include two or (1954), which react with nucleophiles such as amino groups more reactive moieties, as described above, connected by a by ring opening: spacer element. The presence of such a spacer permits bifunc (ix) squaric acid diethyl esters as described by Tietze, tional linkers to react with specific functional groups within Chem. Ber: 124:1215 (1991); and 30 drug (A) and drug (B), resulting in a covalent linkage between (X) C-haloalkyl ethers, which are more reactive alkylating the two. The reactive moieties in a linker may be the same agents than normal alkyl halides because of the activation (homobifunctional linker) or different (heterobifunctional caused by the ether oxygenatom, as described by Bennecheet linker, or, where several dissimilar reactive moieties are al., Eur: J. Med. Chem. 28:463 (1993). present, heteromultifunctional linker), providing a diversity Representative amino-reactive acylating agents include: 35 of potential reagents that may bring about covalent attach (i) isocyanates and isothiocyanates, particularly aromatic ment between drug (A) and drug (B). derivatives, which form stable urea and thiourea derivatives Spacer elements in the linker typically consist of linear or respectively; branched chains and may include a Coalkyl, Coalkenyl, (ii) sulfonyl chlorides, which have been described by C2-lo alkynyl, C2-a heterocyclyl, C-2 aryl, C7-14 alkaryl, Herzig et al., Biopolymers 2:349 (1964); 40 Coalkheterocyclyl, or Coheteroalkyl. (iii) acid halides: In some instances, the linker is described by formula (iv) active esters such as nitrophenylesters or N-hydrox (XXXI): ySuccinimidyl esters; (V) acid anhydrides such as mixed, symmetrical, or N-car (XXXI) boxyanhydrides: 45 Informula (XXXI), G' is a bond between drug (A) and the (vi) other useful reagents for amide bond formation, for linker: G is a bond between the linker and drug (B): Z, Z. example, as described by M. Bodansky, Principles of Peptide Z, and Z' each, independently, is selected from O, S, and Synthesis, Springer-Verlag, 1984; NR, R is hydrogen, Calkyl, Calkenyl, Calkynyl, (vii) acylazides, e.g. wherein the azide group is generated C2-6 heterocyclyl, C-2 aryl, C7-14 alkaryl, Cao alkhetero 50 cyclyl, or C, heteroalkyl:Y' and Y are each, independently, from a preformed hydrazide derivative using sodium nitrite, selected from carbonyl, thiocarbonyl, Sulphonyl, orphospho as described by Wetz et al., Anal. Biochem, 58:347 (1974): ryl: o, p, S, t, u, and V are each, independently, 0 or 1; and Rio and is a Co alkyl, Coalkenyl, C2-io alkynyl, C2-6 heterocy (viii) imidoesters, which form stable amidines on reaction clyl, C-2 aryl, C7-14 alkaryl, Cao alkheterocyclyl, or Co with amino groups, for example, as described by Hunter and 55 heteroalkyl, or a chemical bond linking G'-(Z)-(Y)- Ludwig, J. Am. Chem. Soc. 84:3491 (1962). (Z)- to -(Z)-(Y°)-(Z)-G’. Examples of homobifunc Aldehydes and ketones may be reacted with amines to form tional linkers useful in the preparation of conjugates of the Schiffs bases, which may advantageously be stabilized invention include, without limitation, diamines and diols through reductive amination. Alkoxylamino moieties readily selected from ethylenediamine, propylenediamine and hex react with ketones and aldehydes to produce stable alkoxam 60 amethylenediamine, ethylene glycol, diethylene glycol, pro ines, for example, as described by Webb et al., in Bioconju pylene glycol, 1,4-butanediol, 1.6-hexanediol, cyclohex gate Chem. 1:96 (1990). anediol, and polycaprolactone diol. Examples of reactive moieties capable of reaction with carboxyl groups include diazo compounds such as diaZoac Formulation etate esters and diazoacetamides, which react with high speci 65 Any of the agents employed according to the present inven ficity to generate ester groups, for example, as described by tion may be contained in any appropriate amount in any Herriot, Adv. Protein Chem. 3:169 (1947). Carboxyl modify Suitable carrier Substance, and is generally present in an US 7,795,310 B2 43 44 amount of 1-95% by weight of the total weight of the com assays generally known in the art, examples of which are position. The composition may be provided in a dosage form described herein. For example, candidate compounds may be that is suitable for the oral, parenteral (e.g., intravenously, tested, alone or in combination (e.g., with an agent that intramuscularly), rectal, cutaneous, nasal, vaginal, inhalant, reduces glucose levels, such as those described herein) and skin (patch), or ocular administration route. Thus, the com applied to adipocytes or muscle cells in the presence of insu position may be in the form of, e.g., tablets, capsules, pills, lin and glucose. After a suitable time, these cells are examined powders, granulates, Suspensions, emulsions, solutions, gels for glucose uptake. An increase in glucose uptake identifies a including hydrogels, pastes, ointments, creams, plasters, candidate compound or combination of agents as an effective drenches, osmotic delivery devices, Suppositories, enemas, agent to treat, prevent, or reduce a metabolic disorder. injectables, implants, sprays, or aerosols. The pharmaceutical 10 The agents of the invention are also useful tools in eluci compositions may be formulated according to conventional dating mechanistic information about the biological path pharmaceutical practice (see, e.g., Remington: The Science ways involved in glucose transport and glucose utilization. and Practice of Pharmacy, 20th edition, 2000, ed. A. R. Such information can lead to the development of new com Gennaro, Lippincott Williams & Wilkins, Philadelphia, and binations or single agents for treating, preventing, or reducing Encyclopedia of Pharmaceutical Technology, eds. J. Swar 15 diabetes. Methods known in the art to determine biological brick and J. C. Boylan, 1988-1999, Marcel Dekker, New pathways can be used to determine the pathway, or network of York). pathways affected by contacting cells (e.g., adipocytes, If more than one agent is employed, each agent may be muscle cells, or any cells that utilizes glucose as a source of formulated in a variety of ways that are known in the art. energy) in the presence of insulin and glucose or by contact Desirably, the agents are formulated together for the simul ing pancreatic cells (any cell that has the ability to produce taneous or near simultaneous administration of the agents. insulin) with the compounds of the invention. Such methods Such co-formulated compositions can include the two agents can include, analyzing cellular constituents that are expressed formulated together in the same pill, capsule, liquid, etc. It is or repressed after contact with the compounds of the inven to be understood that, when referring to the formulation of tion as compared to untreated, positive or negative control Such combinations, the formulation technology employed is 25 compounds, and/or new single agents and combinations, or also useful for the formulation of the individual agents of the analyzing some other metabolic activity of the cell Such as combination, as well as other combinations of the invention. enzyme activity, nutrient uptake, and proliferation. Cellular By using different formulation strategies for different agents, components analyzed can include gene transcripts, and pro the pharmacokinetic profiles for each agent can be suitably tein expression. Suitable methods can include standard bio matched. 30 chemistry techniques, radiolabeling the compounds of the The individually or separately formulated agents can be invention (e.g., ''C or H labeling), and observing the com packaged together as a kit. Non-limiting examples include pounds binding to proteins, e.g. using 2D gels, gene expres kits that contain, e.g., two pills, a pill and a powder, a Sup sion profiling. Once identified. Such compounds can be used pository and a liquid in a vial, two topical creams, etc. The kit in in vivo models (e.g., NOD mice) to further validate the tool can include optional components that aid in the administra 35 or develop new agents or strategies to treat, prevent, or reduce tion of the unit dose to patients, such as vials for reconstitut metabolic disorders. ing powder forms, syringes for injection, customized IV As indicated above, the methods of this invention may also delivery systems, inhalers, etc. Additionally, the unit dose kit be used prophylactically, in patients who are an increased risk can contain instructions for preparation and administration of of developing diabetes or a condition associated with diabe the compositions. The kit may be manufactured as a single 40 tes. Risk factors include for example, family history of dia use unit dose for one patient, multiple uses for a particular betes or obesity conditions, quality of nutrition, level of patient (at a constant dose or in which the individual com physical activity, presence of molecular markers of diabetes, pounds may vary in potency as therapy progresses); or the kit age, race, or sex. Patients affected with other non-related may contain multiple doses Suitable for administration to disorders may also be predisposed to secondary diabetes. multiple patients (“bulk packaging”). The kit components 45 may be assembled in cartons, blisterpacks, bottles, tubes, and Exemplary Candidate Compounds the like. Peptide Moieties Peptides, peptide mimetics, and peptide fragments Dosages (whether natural, synthetic or chemically modified) are suit Generally, when administered to a human, the dosage of 50 able for use in practicing the invention. Exemplary inhibitors any of the agents of the combination of the invention will include compounds that reduce the amount of target protein depend on the nature of the agent, and can readily be deter or RNA levels (e.g., antisense compounds, dsRNA, mined by one skilled in the art. Typically, Such dosage is ribozymes) and compounds that compete with endogenous normally about 0.001 mg to 2000 mg per day, desirably about mitotic kinesins or protein tyrosine phosphatases for binding 1 mg to 1000 mg per day, and more desirably about 5 mg to 55 partners (e.g., dominant negative proteins or polynucleotides 500 mg per day. Dosages up to 200 mg per day may be encoding the same). necessary. Antisense Compounds Administration of each drug in the combination can, inde pendently, be one to four times daily for one day to one year, The biological activity of any enzyme that increases blood and may even be for the life of the patient. Chronic, long-term glucose concentration can be reduced through the use of an 60 antisense compound directed to RNA encoding the target administration will be indicated in many cases. protein. Antisense compounds that reduce expression of Sig Additional Applications naling molecules can be identified using standard techniques. If desired, the compounds of the invention may be For example, accessible regions of the target the mRNA of the employed in mechanistic assays to determine whether other target enzyme can be predicted using an RNA secondary combinations, or single agents, are as effective as the combi 65 structure folding program such as MFOLD (M. Zuker, D. H. nation in treating, reducing, or preventing metabolic disor Mathews & D. H. Turner, Algorithms and Thermodynamics ders (e.g., diabetes or any of its associated conditions) using for RNA Secondary Structure Prediction: A Practical Guide. US 7,795,310 B2 45 46 In: RNA Biochemistry and Biotechnology, J. Barciszewski & Preparation of Gelatinized Plates B. F. C. Clark, eds., NATO ASI Series, Kluwer Academic Cell culture plates were gelatinized as follows. Gelatin (1% Publishers, (1999)). Sub-optimal folds with a free energy value within 5% of the predicted most stable fold of the w/v in distilled water) was autoclaved and stored at room mRNA are predicted using a window of 200 bases within temperature. The bottom of each cell culture well was cov which a residue can find a complimentary base to form a base ered uniformly in the gelatin Solution, ensuring that no pair bond. Open regions that do not form a base pair are bubbles were formed. This solution was removed leaving Summed together with each Suboptimal fold and areas that are behind a thin film of gelatin. These plates were left to dry predicted as open are considered more accessible to the bind under the tissue culture hood. Plates were next washed with ing to antisense nucleobase oligomers. Other methods for 10 PBS, after which a 0.5% glutaric dialdehyde solution (glut antisense design are described, for example, in U.S. Pat. No. araldehyde in distilled water) was added to the cell culture 6,472,521, Antisense Nucleic Acid Drug Dev. 1997 7:439 wells. After ten minutes, wells were washed twice with 444, Nucleic Acids Research 28:2597-2604, 2000, and DMEM containing pen-strep. Each washing step should last Nucleic Acids Research 31:4989-4994, 2003. for approximately five minutes. RNA Interference 15 Cell Culture The biological activity of a signaling molecule can be 3T3-L1 pre-adipocyte cells were split approximately every reduced through the use of RNA interference (RNAi), employing, e.g., a double stranded RNA (dsRNA) or small 2-3 days or upon reaching a confluence of approximately interfering RNA (siRNA) directed to the signaling molecule 60%. Overconfluency may affect the ability of these cells to in question (see, e.g., Miyamoto et al., Prog. Cell Cycle Res. differentiate into adipocytes. 5:349-360, 2003: U.S. Patent Application Publication No. Other Reagents 20030157030). Methods for designing such interfering D-(+)-glucose ("cold glucose, not radiolabeled) was RNAs are known in the art. For example, software for design added to DPBS mix to a final concentration of 10 mM. ing interfering RNA is available from Oligoengine (Seattle, Lysis buffer, a mixture of a base (e.g., sodium hydroxide at Wash.). 25 Dominant Negative Proteins a final concentration of 0.5N) and a detergent (e.g., sodium One skilled in the art would know how to make dominant dodecyl sulphate (SDS) diluted to a final concentration of negative proteins to the signaling molecules to be targeted. 0.1% w/v) was freshly prepared each time (within one to two Such dominant negative proteins are described, for example, hours of use). Prior to use, lysis buffer was warmed up to a in Gupta et al., J. Exp. Med., 186:473–478, 1997: Maegawa et 30 temperature exceeding that of room temperature for a period al., J. Biol. Chem. 274:30236-30243, 1999; Woodford-Tho of approximately 30 minutes to avoid precipitation of the mas et al., J. Cell Biol. 1 17:401–414, 1992). buffer. Determination of Glucose Uptake Example 1 Pre-adipocyte 3T3-L1 cells were plated at a density of 35 approximately 5000 cells/well (in black NUNC 96 well An Increase in Insulin-Stimulated Glucose Uptake In plate). These cells were differentiated into adipocytes in two Vitro separate steps. Initially, cells were cultured in differentiation medium-1 (DM1) (day 1 of adipocyte differentation) for a Differentiated mouse adipocytes were employed to iden period of two to three days. DM1 prevents proliferation and tify combinations of agents that have the ability to increase 40 glucose uptake upon insulin stimulation, as detected by Scin induces the expression of adipocyte-specific genes. Cells tillation counting of radiolabelled glucose (using, for were next cultured in differentiation medium-2 (DM2) for 3 example, the Perkin Elmer 1450 Microbeta JET reader). to 4 days, after which the culture media was replaced by fresh These assays were conducted as follows. DM2. The glucose uptake assay was performed at day 9-15 of Materials and Methods 45 differentiation. Prees Media Two days prior to the experiment (at day 7-13 of differen Complete media, also referred to as “Prees' media, was tiation), DM2 was removed and replaced with fresh Prees prepared as follows. Dulbecco's Modified Eagle's Medium media. Candidate compounds were added at this time, allow (DMEM) was supplemented with L-glutamine, penicillin-G ing an incubation period of approximately 48 hours. On the and streptomycin (pen/strep), and heat-inactivated fetal 50 day of the experiment, cells (now at day 9 to 15 of differen bovine serum (FBS) (heat inactivated at 65° C. for 30 min tiation) were serum starved for three hours in DPBS, magne utes). Because serum can affect the growth, adherence, and sium sulfate (0.8 mM), and Hepes (10 mM) at pH -7. After differentiation of cells, any new lot of serum was first tested this incubation period, fresh DPBS containing insulin (10 prior to use. Media was equilibrated in the incubator (5% nM) was added to the adipocytes. Fresh DPBS without any CO) until the pH was within the proper range (-7), as indi 55 cated by the red/orange color of the indicator dye. If the media insulin were placed on cells that served as a negative control. became pink (indicating a high pH), we discarded the media Following an incubation period of 25 minutes at 37° C. as basic conditions can affect cells and denature the insulin radioactive glucose (labeled with 'C, at a final concentration used in the differentiation medium-1 (DM1) and the differ of 0.04 mM, ~0.26 uCi C-glucose in each well) was added entiation medium-2 (DM2). 60 to the media for a period of 15 minutes at room temperature. Differentiation Medias Media was next removed and cells were washed thoroughly Differentiation media-1 (DM1) was prepared by supple and lysed. Upon lysis, cells form a small, cloudy mass, menting DMEM with 10% FBS, L-glutamine, pen/strep, detached from the well bottom. 10% glacial acetic acid was IBMX (375uM), insulin (120 nM), and dexamethasone (188 added to each well to neutralize the lysis reaction. Scintilla nM). Differentiation media-2 (DM2) was prepared by supple 65 tion fluid was next added to the wells and the incorporation of menting DMEM with 10% FBS, L-glutamine, pen/strep, and glucose was determined by measuring the amount of radio insulin (120 nM). activity in each well using the MicroBeta plate reader. US 7,795,310 B2 48 The data are shown in Tables 4 and 5, below. Materials and Methods Prees Media TABLE 4 Complete media, also referred to as “Prees' media, was prepared as follows. Dulbecco's Modified Eagle's Medium Diflunisal (LIM 5 (DMEM) was supplemented with L-glutamine, penicillin-G 36 18 9 O and streptomycin (pen/strep), and heat-inactivated fetal bovine serum (FBS) (heat inactivated at 65° C. for 30 min Bezafibrate 36 1.69 1.74 1.74 1.57 (IM) 18 1.67 1.62 1.57 1.37 utes). Because serum can affect the growth, adherence, and 9 1.65 1.42 1.27 1.16 differentiation of cells, any new lot of serum was first tested O 1.52 1.19 1.12 O.99 10 prior to use. Media was equilibrated in the incubator (5% CO) until the pH was within the proper range (-7), as indi cated by the red/orange color of the indicator dye. If the media The average fold increase in insulin-stimulated glucose became pink (indicating a high pH), we discarded the media uptake compared to vehicle treated control was determined. The average of four biological replicates is shown. At all as basic conditions can affect cells and denature the insulin 15 used in the differentiation medium-1 (DM1) and the differ combination doses there is an increase in glucose uptake as entiation medium-2 (DM2). compared to the single agents. Differentiation Medias Differentiation media-1 (DM1) was prepared by supple TABLE 5 menting DMEM with 10% FBS, L-glutamine, pen/strep, IBMX (375uM), insulin (120 nM), and dexamethasone (188 Bezafibrate (IM 2O nM). Differentiation media-2 (DM2) was prepared by supple 36 18 9 O menting DMEM with 10% FBS, L-glutamine, pen/strep, and Cinnamic 12 146 1.36 123 1.10 insulin (120 nM). acid (IM) 6 1.49 136 1.17 1.03 Preparation of Gelatinized Plates 3 130 126 1.10 O.98 25 Cell culture plates were gelatinized as follows. Gelatin (1% O 1.15 1.21 O.94 O.88 w/v in distilled water) was autoclaved and stored at room temperature. The bottom of each cell culture well was cov The average fold increase in insulin-stimulated glucose ered uniformly in the gelatin Solution, ensuring that no uptake compared to vehicle treated control was determined. bubbles were formed. This solution was removed leaving The average of three biological replicates is shown. At all 30 behind a thin film of gelatin. These plates were left to dry combination doses there is an increase in glucose uptake as under the tissue culture hood. Plates were next washed with compared to the single agents. PBS, after which a 0.5% glutaric dialdehyde solution (glut araldehyde in distilled water) was added to the cell culture Example 2 wells. After ten minutes, wells were washed twice with 35 DMEM containing pen-strep. Each washing step should last The Combination of Bezafibrate and Diflunisal for approximately five minutes. Reduce Insulin Sensitivity in a Rat Model Cell Culture 3T3-L1 pre-adipocyte cells were split approximately every Insulin resistance was induced in male Sprague Dawley 2-3 days or upon reaching a confluence of approximately rats by four weeks of high fat feeding (60% of calories derived 40 60%. Overconfluency may affect the ability of these cells to from fat). Drug treatment began one week after initiation of differentiate into adipocytes. high fat diet. Drugs were administered daily, by oral gavage Other Reagents for a three week period. D-(+)-glucose ("cold glucose, not radiolabeled) was Following the three weeks of treatment, animals were added to DPBS mix to a final concentration of 10 mM. fasted for five hours and anesthetized, and blood collected 45 Lysis buffer, a mixture of a base (e.g., sodium hydroxide at from the inferior Vena cava for determination of serum glu a final concentration of 0.5N) and a detergent (e.g., sodium cose and insulin levels. Insulin sensitivity was determined dodecyl sulphate (SDS) diluted to a final concentration of using the homeostasis model assessment (HOMA). 0.1% w/v) was freshly prepared each time (within one to two hours of use). Prior to use, lysis buffer was warmed up to a 50 temperature exceeding that of room temperature for a period fasting serum glucose X fasting serum insulin of approximately 30 minutes to avoid precipitation of the HOA = 22.5 buffer. Determination of Glucose Uptake Pre-adipocyte 3T3-L1 cells were plated at a density of The results are shown in FIGS. 1-3. 55 approximately 5000 cells/well (in black NUNC 96 well plate). These cells were differentiated into adipocytes in two Example 3 separate steps. Initially, cells were cultured in differentiation medium-1 (DM1) (day 1 of adipocyte differentation) for a Screening Assays Identify Additional Agents that period of two to three days. DM1 prevents proliferation and Increase Glucose Uptake by Adipocytes 60 induces the expression of adipocyte-specific genes. Cells were next cultured in differentiation medium-2 (DM2) for 3 Differentiated mouse adipocytes were employed to iden to 4 days, after which the culture media was replaced by fresh tify combinations of agents that have the ability to increase DM2. The glucose uptake assay was performed at day 9-15 of glucose uptake upon insulin stimulation, as detected by Scin differentiation. tillation counting of radiolabelled glucose (using, for 65 Two days prior to the experiment (at day 7-13 of differen example, the Perkin Elmer 1450 Microbeta JET reader). tiation), DM2 was removed and replaced with fresh Prees These assays were conducted as follows. media. Candidate compounds were added at this time, allow US 7,795,310 B2 49 50 ing an incubation period of approximately 48 hours. On the pounds that exhibited glucose uptake activity are shown in day of the experiment, cells (now at day 9 to 15 of differen Table 4. Table 4 shows glucose uptake activity (denoted as F tiation) were serum starved for three hours in DPBS, magne (fold over base) with the standard deviation (sE)) of each of sium sulfate (0.8 mM), and Hepes (10 mM) at pH -7. After the compounds at various concentrations. S/N denotes the this incubation period, fresh DPBS containing insulin (10 signal over noise ratio. nM) was added to the adipocytes. Fresh DPBS without any In another set of experiments, human skeletal myoblasts insulin were placed on cells that served as a negative control. obtained by the conditional immortalization of cells derived Following an incubation period of 25 minutes at 37° C. from a non-diabetic subject, were used to screen the effect of radioactive glucose (labeled with ''C, at a final concentration a multitude of compounds on glycogen synthesis. For each of 0.04 mM, -0.26 uCi C-glucose in each well) was added 10 compound, two doses were tested in triplicate. The vehicle to the media for a period of 15 minutes at room temperature. was used as a negative control and insulin was used as a Media was next removed and cells were washed thoroughly positive control. Prior to treatment, cells were serum-starved and lysed. Upon lysis, cells form a small, cloudy mass, for 12 to 18 hours in Hams F10 medium. Cells were next detached from the well bottom. 10% glacial acetic acid was incubated either with the test compounds or control for a added to each well to neutralize the lysis reaction. Scintilla- 15 period of two hours in serum-free media containing radiola tion fluid was next added to the wells and the incorporation of beled glucose, after which, glycogen synthesis was mea glucose was determined by measuring the amount of radio sured. The results of these experiments are shown in Table 4 activity in each well using the MicroBeta plate reader. Com (denoted as XlsyZ).

TABLE 6 XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ Name CRXF CRX SF CRX S.N CRX uM F(L) sE(L) S/N (L) uM(L) F(H) sF(H) S/N (H) uM(H) Tetrahydrozoline 1.0225 0.0755 13.54 39S477 1.2564 O.O39 32.22 4.3938 1.6 O.1451 11.03 43.9375 Hydrochloride SOPROTERENOL 15553 0.0975 15.95 39.494 6714 O.O815, 20.51 3.2331 1.5225 0.1165 13.07 32.3311 SULFATE Acetyldigitoxin O.8077 O.1165 6.93 11.153S 1.3016 O.0434 29.99 12.39.16 12593 0.047 26.79 1.2392 NOREPINEPHRINE 1.7559 O.2784 6.31 27.797 3758 0.0924 4.89 3.0882 14353 0.1497 9.59 30.882S BITARTRATE Dipivefrin 1.8856 0.113 16.69 23.2042 1.2562 O.O375 33.50 2.6553 2.6154 0.3631 7.20 26.5532 Hydrochloride Brimonidine 1.4251 O.2O61 6.91 31.4307 1.6094 O.O966 6.66 3.492 1.2967 O.O784 16.54 349195 Epinephrine Bitaritrate 1.651 1 0.1809 9.13 9.43O4 1511 O.1387 O.89 3.2103 1.7681 0.1006 17.58 32.1032 Terbutaline Sulfate 1.2758 O.O854 14.94 33.7985 1.3013 O.O271 48.02 3.755 14059 0.1136 12.38 37.55O1 Homatropine 1.0613 O.O74 14.34 25.0363 134O1 O.O401 33.42 2.781S 1.3333 0.1324 10.07 27.8153 Methylbromide Loxapine O.9649 O.122 7.91 18.8457 14364 O.1353 O.62 2.0938 1.2281 0.0377 32.58 20.9376 Hydrochloride Ethopropazine O.9375 0.1223 7.67 28.8937 1.395 O.09 5.50 3.2101 O.84O6 O.O212 39.65 32.1009 Hydrochloride Candesartan Cilexetil .33 O.1416 9.39 15.929S 1.7284 O. 1165 4.84 1.7698 0.6667 O.O223 29.90 17.6977 nulin O249 0.0918 1116 4576 1.3377 0.1023 3.08 16194 1.OO89 O.0339 29.76 O.1619 Metaproterenol 7249 0.2108 8.18 16.8778 1.4052 O.O959 4.65 1.7812 1.1261 O.O292 38.57 17.81.16 Hemisulfate Salt Wetase 2175 0.1785 6.82 843 3974. O.108 2.94 2.02 O.8561 O.O3OS 28.07 O.2O2 Methylergonovine O.S26S O.2648 1.99 20.748S 1.2906 0.08.23 S.68 2.3052 1.05 O.O271 38.75 23.0516 Maleate Prostaglandin E S723 O.1088 4.45 26.0518 1.7037. O.16S O.33 2.8944 1 O. 149 6.71 28.94.36 Guanfacine O.6974 O.2239 3.11 31861 6 14264 O.11 2.97 3.5398 1.1364 O.O349 32.56 35.3982 Hydrochloride Methacholine Chloride 152 O.114 O.11 47.8331 1.3086 OO698 8.75 5.3143 1.0156 0.0467 21.75 S3.1426 Digitoxin O.88O1 O.O449 9.60 11.8851 1.3846 O.1713 8.08 13.2043 1.146 (O.0486 23.58 1.3204 GeranylAcetate O.9527 OO694 3.73 45.8528 1.3523 O.1396 9.69 S.O942 O.9 O.O642 14.02 SO9424 Evans Blue O.90O8 O. 115 7.83 9.6492 13913 O.1889 737 10.72O2 1.0519 O.O4O6 25.91 1.072 Xylazine O978 O.O81 3.55 51.1733 1.34O1 O.OS37 24.96 S.6854 1.0857 O.OS21 20.84 56.8536 Hydrochloride Oleandomycin 1OO2 O.O811 3.57 11.567S 1.6271 O.2313 7.03 12.8515 O.9744 0.0403 24.18 1.2852 Prazosin 2437. O.1464 8. SO 21.4358 1.3333 O-1184 11.26 2.3815 O.7021 O.O264 26.59 23.8152 Hydrochloride Tinidazole O177 O.0646 5.75 36.3967 1.2177 0.1579 7.71 4.0437 1.0588 0.066 16.04 40.4367 Enalaprilat O.992 O.1543 6.43 26.0636 1.2739 O.O814 15.65 2.8957 0.8333 O.O692 12.04 28.9567 Tannic Acid O.8674 O.O779 1.13 5.2909 12949 O.1048 12.36 5.8782 1.0522 O.O2.19 48.05 O.S878 Azathioprine 7493 0.22OS 7.93 37.0961 12275 O.O948 12.95 3.6062 1.0938 O.O352 31.07 36.062 Ritodrine SO11 O.1595 9.41 27.7977 1.2121 O.O44S 27.24 3.0883 1.039 O.OS63 18.45 3O8833 Hydrochloride Guaiacol O722 O.062 7.29 72.5294 1.2105 O.08O1 15.11 S.6529 O.9873 0.028 35.26 56.5291 Eucalyptol 1325 O.1543 7.34 4948.67 2047 O.1552 7.76 549.797 1.038 0.0387 26.82 S497.97 Promethazine O.9391 O.1273 7.38 28.0489 12585 0.18 6.99 3.1162 O.S797 0.0416 13.94 31.1624 Hydrochloride METHANTHELINE .2479 0.1282 9.73 23.0738 1.219 O.1438 8.48 2.563S 1.1136 O.O3O1 37.OO 25.6349 BROMIDE METHDILAZINE O.9361 O.1008 9.29 31.6248 1.2127 O.O957 12.67 3.5135 0.75 O.O258 29.07 35.1351 Glutamic Acid O.964 O2332 4.13 43.24.63 12292 O.O384 32.01 4.8047 O.953S 0.0658 14.49 48.0466 Hydrochloride US 7,795,310 B2 51 52

TABLE 6-continued

Name CRXF CRX SF CRX SN CRX uM

Ergoloid Mesylates ..1518 O.1728 6.67 21.2S36 2045 53.06 1.S158 1.109S 38.52 15.1584 QUINACRINE OOS8 0.1727 S.82 2O.SS61 2378 26.45 2.2838 1046 12.41 22.8378 Meclofenoxate 1681 O. 1143 10.22 34.8777 2103 22.09 3.8749 1.0625 37.02 38.7492 GENISTEIN O4522 O.0432 10.47 33.312 2013 27.43 3.701 0.7857 22.13 37.OO96 Pergolide Mesylate 1326 O.1253 9.04 21.9213 2698 9.54 2.4355 1.039 24.45 24.3546 Triflupromazine 0.9717 O.1634 5.95 23.1445 2443 18.14 2.571.4 O.2479 12.27 25.7136 Hydrochloride Levocabastine 2727 O. 1069 11.91 19.6956 2127 O.O791 15.33 2.1882 O.9667 O.O687 14.07 21.8818 Hydrochloride Hydroxypropyl O957 20.63 1.9082 2583 O.1044 12.05 2.12 107 O.0519 21.33 O.212 Cellulose Oxybutynin Chloride O483 O.1851 S.66 23.0734 O.0483 22.46 2.5634 5652 O.1325 11.81 25.6345 DOXAPRAM 1028 O.O713 15.47 21.6.188 O.O375 24.24 24O18 4615 O.2139 6.83 24.0185 HYDROCHLORIDE Pyrilamine Maleate 1294 12.11 24.66 35.27 2.7397 3778 15:29 27.3973 LEAD 524 3.76 21.9875 17.53 2.4428 2527 32.04 244281 DIETHYLDITHIO CARBAMATE Dimenhydrinate 1102 O.O697 15.93 19.7254 0.9571 26.15 2.1915 5385 O.1913 8.04 21.9149 Bamethan 1895 O.1516 7.85 17.7718 O.9106 22.10 19744 3846 O.1387 9.98 19.7445 Dyclonine 1238 O.1232 9.12 29.5519 O.9339 27.96 3.2832 9231 O.2533 7.59 32.8322 Hydrochloride Dopamine 1203 0.1517 7.38 47.4731 O909 0.0756 14.43 S.2743 O.2348 8.52 52.7426 Hydrochloride Spectinomycin O474 O.1049 9.98 25.0888 1245 46.09 2.7874 2881 O.O819 15.73 27.8737 Acetohexamide O591 O.1317 8.04 27.7463 O179 12.47 3.0826 3939 0.1572 8.87 30.8261 DL-5- O187 O.O972 10.48 40.876 1353 17.60 4.5413 2903 O.O608 21.22 45.4133 Hydroxytryptophan PioglitaZone 2.1107 O.2432 8.68 7.6519 2016 2.5967 8586 170.51 2.SSO4 Hydrochloride Doxylamine Succinate 1094 O.OS46 20.32 24.SS84 26.28 2.7284 .7692 7.23 27.2844 Phensuximide 1431 O.O842 13.58 48.5249 36.23 5.3911 .3333 13.29 S3.9112 Melphalan 398 O.1619 8.63 27.6151 13.57 3.068 2982 40.07 30.68O3 Phenylpropanolamine .077 0.1579 6.82 49.8718 40.14 S.S408 4222 13.96 SS4O76 Hydrochloride Acetylcholine Chloride O512 O. 1161 9.OS 50.5279 O.O689 16.84 S. 6136 3305 20.89 5.7788 Carbamazepine O 7464 O.1508 4.95 41.9001 O.0482 22.28 4.6SS1 4211 12.97 46.SS1 Doxycycline O.447 O. 1142 3.91 19.0911 O.0288 36.73 2.1.21 .7692 5.59 21.2102 Phenylephrine .2537 O.O89 14.09 44.187 O.0482 24.46 4.9092 6061 1S.O2 49.0918 Hydrochloride Diclofenac Sodium 3966 O.O673 20.75 30.3169 32.82 3.458 4615 7.43 34.58O3 Acetaminophen O21 O.1744 5.85 60.1251 24.25 6.6799 6154 11.46 66.7989 Carbinoxamine 0.7798 O.1566 4.98 21.8995 18.66 2433 6842 1342 24.3303 Maleate Diphenhydramine O.931 O.1519 6.13 31.1546 29.35 3.4613 9231 4.19 34.6127 Hydrochloride Oxymetazoline O. 1035 8.66 35.4664 18.25 3.4704 3623 O.1591 8.56 34.703S Hydrochloride mipramine 7.04 28.403 11.9S 3.1556 3218 9.07 31.5557 Hydrochloride Phenacemide O.0733 3.99 53.54O7 40.49 5.9484 .3333 3.43 59.4837 Carisoprodol O.O708 5.97 35.9621 27.48 3.99S4 3846 9.14 39.9539 Pilocarpine O.1514 5.79 37.8869 1423 4.2092 2889 4.60 42.0924 Hydrochloride Acetrizoate Sodium 0.0957 3.15 15.7065 20.46 1.745 3158 O.115 17.4499 Hydrosocobalamin O.215 5.45 6.752 31.44 7.5O15 2416 O.1306 0.75O1 Calcium Chloride O.2489 2.96 83.8859 24.29 9.3197 2121 O.1806 93.1973 Cupric Chloride O.O795 7.18 53.31.91 32.65 S.9238 25 O. 1173 59.2375 Vincamine O.O952 8.43 25.3976 33.08 2.8.217 2619 O.0733 28.2167 Acetohydroxamic Acid O.083 2.56 130.639 23.03 14.514 .3333 O.1459 145.14 Dirithromycin O.O698 5.33 11.8561 32.87 1.3172 2533 O.0543 13.1721 Sulfamethizole O.O786 3.68 33.9657 38.63 3.7736 O.O806 37.7358 Ammonium Chloride O.1503 S.91 173.288 1910 19.2523 21 OS O.0912 192S23 Tolazoline O.0701 3.79 47.5899 29.68 52872 2469 O.0997 52.8724 Hydrochloride Diphenidol O. 1118 9.12 26.2819 O.9781 27.40 2.91.99 3846 O.3815 3.63 29.1992 Hydrochloride Bismuth Subsalicylate O.1409 1.99 24.8575 1.0089 23.57 3.1759 21 OS O.1328 9.12 31.7592 PHENYTOIN O.1697 6.93 35.6754 O.9796 28.39 3.963S 2174 O.O874 13.93 39.63.54 Dicloxacillin Sodium O.1082 9.28 18.6968 1.0511 15.46 2.0772 3846 O.1962 7.06 20.7721 Indocyanine Green O.1506 5.86 12.O364 1.0603 13.42 1.3372 O.O405 29.63 13.3724 HEXYLCAINE O.08O2 3.00 30.86O2 1.1545 17.41 3.4286 2121 O.O947 12.8O 34.2857 HYDROCHLORIDE AMANTADINE O.0917 6.74 47.9536 O.8699 13.06 5.3276 2308 O. 1149 10.71 53.2765 HYDROCHLORIDE Orphenadrine Citrate O. 1018 9.58 O.97.04 14.21 2.2318 2174 O.0661 1842 22.3.185 US 7,795,310 B2

TABLE 6-continued XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ XlsyZ Name CRx F CRx sf CRx S/N CRx uM F(L) sR(L) S/N (L) uM(L) F(H) sR(H) S/N (H) uM(H) Diflunisal S694 O.O744 21.09 36.8314 O.9421 O.O31S 29.91 4.0767 1.23O8 O.1346 9.14 40.7674 DIPEHEMANIL 2O78 O.O672 17.97 24.5269 O.9708 O.0573 16.94 2.7249 1.23O8 O.2763 4.45 27.2494 METHYLSULFATE Acetazolamide O.8994. O.1774 S.O7 41.3183 1.0268 O.O421. 24.39 4.59OS 1.3077 O.2004 6.53 45.9046 Sulfasomidine 1803 0.0729 16.19 32.6659 1.016S O.O312 32.58 3.6292 1.3077 O.1693 7.72 36.2918 Diatrizoate Sodium O.9858 0.0493 20.OO 16.2778 O.9562 O.O343 27.88 1.808S 1.3077 O.15 8.72 18-0846 Cefamandole Nafate 23S3 0.073S 16.81 17.914 1.016S O.O397 25.60 19902 1.25 0.109 11.47 19.9024 Arbutin O786 0.1077 10.01 36.6911 1.056 O.O489 21.60 4.0764 1.2O4S O.O495 24.33 40.7639 opromide O.8499 0.0662. 12.84 11.720S 0.9852 O.O316 31.18 13022 1.25 O.O82S 15.15 13.0215 Phenylbutazone 2098. O.O872 13.87 29.4777 O.967S O.O642 15.07 3.275 2444 O.O894 13.92 32.7497 SUCCINYLCHOLINE 1.04O9 O.112S 9.2iS 23.7879 0.9577 O.O383 25.01 2.6428 1.2231 O.09S 12.87 26.4284 Captopril O662 0.3 2.21 44.321 10804 O.OSO3 21.48 4.9241 2632 O.109 11.59 49.24O7 ohenoxic Acid O641 O.O986 10.79 16.3681 1.OSO4 O.O382 27.50 1.818S 1.2119 O.O796 15.22 1.8.185 OLANZAPINE OSO6 0.2278 4.61 28.8121 10681 0.0595 17.9S 3.2O1 .2 O.OSO7 23.67 32.0102 Etoposide O37 0.1142 9.08 15.5979 0.7273 O.O491. 14.81 17329 12468 O.O929 13.42 17.3293 Nefopam O.88S9 0.1159 7.64 31.68O2 1.1617 O.O329 35.31 3.5.197 12368 O.OS69 21.74 35.1967 Suprofen 4298 O.2O3 7.04 34.5789 1.0962 O.O233 47.OS 3.8417 1.22O3 O.O788 1549 38.4172 opanoic Acid 252 0.06O1 20.83 187617 O.8708 (O.O323 26.96 2.0844 12318 O.O871. 14.14 20.8443

Other Embodiments All publications, patent applications, and patents men- 25 R3 R4 (II) tioned in this specification are herein incorporated by refer- 3 CCC. N COOH Various modifications and variations of the described (X) R. R. method and system of the invention will be apparent to those 21 R2 skilled in the art without departing from the scope and spirit of 30 the invention. Although the invention has been described in connection with specific desired embodiments, it should be wherein R and R are each, independently, a hydrogen understood that the invention as claimed should not be unduly atom or a C alkyl group; R and Reach represent a limited to such specific embodiments. Indeed, various modi hydrogenatom or may be combined together to forman fications of the described modes for carrying out the invention 35 additional chemical bond; n is Zero or an integer from 1 that are obvious to those skilled in the fields of medicine, to 3; each X is, independently, a hydroxyl group, a immunology, pharmacology, endocrinology, or related fields halogen atom, a straight or branched chain Saturated or are intended to be within the scope of the invention. unsaturated C alkyl group, a straight or branched chain Saturated or unsaturated Calkoxy group, a Ca 40 acyloxy group, or a C cycloalkyl group; when n is 2 or The invention claimed is: 3 and two X's are commonly C alkyl or C. alkoxy, 1. A composition consisting of active and inertingredients, both X's may be combined together to form a ring. wherein the sole active ingredients are: 2. A composition consisting of active and inactive ingredi ents, wherein the sole active ingredients are bezafibrate and (a) a fibrate selected from the group consisting of bezafi 45 brate, binifibrate, ciprofibrate, clinofibrate, clofibrate, diflunisal. etofibrate, fenofibrate, and gemfibrozil; and 3. A composition consisting of active and inactive ingredi (b) diflunisal, a diflunisal analog, cinnamic acid, or a cin ents, wherein the sole active ingredients are bezafibrate and namic acid analog, wherein said diflunisal analog has the cinnamic acid. structure of formula (III): 50 4. A composition consisting of active and inactive ingredi ents, wherein the sole active ingredients are: (a) bezafibrate or a bezafibrate analog having a structure of (III) formula (I): COR 55 (I) M OR O xx(1-5) / C R3 N N (i.V A 60 R-t H wherein each X is, independently, a halogen atom; R is A-4 O hydroxy, phenoxy, di(C-C)alkylamino, di(C-C)alky R lamino, or (C-C)alkoxy; R is hydrogen or (C-C)al R3 CO-Z kanoyl; and R is hydrogen or methyl: 65 R4 wherein said cinammic acid analog has the structure of formula (II): US 7,795,310 B2 55 56 wherein R and R2 are each, independently, hydrogen, halo wherein R and R2 are each, independently, a hydrogenatom gen, Calkyl, or Calkoxy; R and Ra are each, indepen or a C alkyl group; R and Reach represent a hydrogen dently, hydrogen or Calkyl, n is 1, 2, or 3; and Z is hydroxyl or C. alkyl, and atom or may be combined together to form an additional (b) diflunisal, a diflunisal analog, cinnamic acid, or a cin chemical bond; n is Zero or an integer from 1 to 3; each X is, namic acid analog; wherein said diflunisal analog has independently, a hydroxyl group, a halogenatom, a straight or the structure of formula (III): branched chain Saturated or unsaturated C alkyl group, a (III) straight or branched chain Saturated or unsaturated Ca COR alkoxy group, a C14 acyloxy group, or a C- cycloalkyl 10 group; when n is 2 or 3 and two X's are commonly Calkyl OR or Calkoxy, both X's may be combined together to form a ring. 5. The composition of claim 4, wherein said bezafibrate R3 15 analog is selected from the group consisting of alpha-4-(2- methoxy-5-chlorobenzoylaminoethyl)-phenoxy-isobutyric wherein each X is, independently, a halogen atom; R is hydroxy, phenoxy, di(C-C)alkylamino, di(C-C)alky acid, alpha-4-(4-methylbenzoylaminomethyl)-phenoxy lamino, or (C-C)alkoxy; R is hydrogen or (C-C)al isobutyric acid, alpha-4-(2-methylbenzoylaminoethyl)-phe kanoyl; and R is hydrogen or methyl; 2 O noxyisobutyric acid, and alpha-4-(4-chlorobenzoylamino wherein said cinnamic acid analog has the structure of ethyl)-phenoxy-proprionic acid. formula (II): 6. The composition of claim 4, wherein said bezafibrate or (II) beZafibrate analog and said diflunisal, diflunisal analog, cin 25 namic acid, or cinnamic acid analog are present in amounts N COOH that, when administered to a patient, are sufficient to treat or R R2 reduce type II diabetes. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 7,795.310 B2 Page 1 of 3 APPLICATIONNO. : 11/171566 DATED : September 14, 2010 INVENTOR(S) : Lee et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On Cover page, under OTHER PUBLICATIONS, in Yuan et al., replace “Science with --Science--. Under OTHER PUBLICATIONS, in Chen et al., replace “(1).Journal with --(1), Journal--. Column 2, Line 36, replace “clofibrinc' with --clofibric-, Lines 64-65, replace “adenosine with -adenosine--. Column 3, Line 52, replace “clofibrinc' with --clofibric-. Column 6, Line 65, replace “lits with --lists--. Column 9, Line 63, replace “or more agent with --or more agents--. Column 12, Table 2, under mechanistic class, replace “andrenergic beta antagonist with -adrenergic beta antagonist--. Table 2, under therapeutic class, replace “dietary supplyment with -dietary supplement--. Table 2, under therapeutic class, replace “sedative/muscle relaxent with --Sedative/muscle relaxant--.

Column 13, Table 2, under chemical class, replace “mallic acid with --malic acid--. Column 17, under chemical class, replace “imdiazole with --imidazole--, Table 2, under chemical class, replace “thaizine' with--thiazine--.

Signed and Sealed this Tenth Day of April, 2012

David J. Kappos Director of the United States Patent and Trademark Office CERTIFICATE OF CORRECTION (continued) Page 2 of 3 U.S. Pat. No. 7,795,310 B2 Column 18, Table 2, under mechanistic class, replace “phenolic antioxitant with --phenolic antioxidant--. Table 2, under mechanistic class, replace “vasodialator with --vasodilator--. Line 55, replace “overweight, is considered with --overweight is considered--. Column 21, Lines 45-46, replace “and blood collected with -and blood was collected-: Lines 50-54, replace “We have discovered compounds that the certain bezafibrate-containing combinations have in vitro and in vivo activities that suggest that these combinations may be useful for treating a patient that has been diagnosed with or is at risk of having a metabolic disorder. with --Wehave discovered that combinations of certain bezafibrate-containing compounds have in vitro and in vivo activities suggesting that these combinations may be useful for treating a patient who has been diagnosed with, or is at risk of having a metabolic disorder.--. Column 24, Line 51, replace “each displaying with --each displaying a--, Line 55, replace “fibrates, binding to PPARO and glitazones, binding to with --fibrates, binding to PPARO, and glitazones, binding to--. Column 27, Line 31, replace "2.5 and 2,4-dimethylcinnamic acid with --2,5- and 2,4-dimethylcinnamic acid--. Column 28, Line 55, replace “in place diflunisal with -in place of diflunisal--. Column 29, Line 10, replace “HX0835. HYAL with-HX0835, HYAL.--. Column 32, Line 4, replace “Phenyloin with --Phenytoin--.

Column 33, Line 33, replace “such as, intravenous with --such as: intravenous--. Column 37, Lines 28-29, replace “Harding et al. Nature 341: 758-7601 with --Harding et al. Nature 341: 758-760-: Line 57, replace “532,248 with --5,532,248--. Column 38, Line 38, replace “is an' with --is a--. CERTIFICATE OF CORRECTION (continued) Page 3 of 3 U.S. Pat. No. 7,795,310 B2 Column 39, Line 8, replace “rapamycin, and has been with --rapamycin and has been--. Line 43, replace “listed on Table 1.'' with --listed in Table 1.--. Column 44, Line 22, replace “can include, analyzing with --can include analyzing-- Line 29, replace “transcripts, and with--transcripts and-: Line 40, replace “include for example, with -include, for example.--. Column 45, Line 1, replace “In: RNA with --RNA-. Column 46, Line 39, replace “adipocyte differentation with -adipocyte differentiation--. Column 47, Line 45, replace “blood collected with -blood was collected--. Column 48, Line 58, replace “adipocyte differentation with -adipocyte differentiation--. Column 50, Line 8, replace “subject, were with --subject were--. Column 53, Line 63-64, replace “di(C1-C4)alkylamino, di(C1-C4)alkylamino with --di(C1-C4) alkylamino-:

Column 53, Line 66, replace “cinammic acid with --cinnamic acid--.