WO2010121014A1 - Methods and compositions for treating type 2 diabetes and the metabolic syndrome - Google Patents

Abstract

The present invention relates generally to pharmaceutical compositions comprising combinations of metformin and L-4F, as well as to other compounds and their combinations, and to methods of treating and preventing type 2 diabetes, the metabolic syndrome and its individual components, and related disorders using the pharmaceutical compositions of the invention.

Description

METHODS AND COMPOSITIONS FOR TREATING TYPE 2 DIABETES AND THE

METABOLIC SYNDROME

FIELD OF INVENTION

[0001] The present invention relates generally to pharmaceutical compositions comprising combinations of metformin and L-4F, as well as to other compounds and their combinations, and to methods of treating and preventing type 2 diabetes, the metabolic syndrome and its individual components, and related disorders using the pharmaceutical compositions of the invention.

BACKGROUND OF THE INVENTION

[0002] Diabetes mellitus (DM) comprises a group of common metabolic disorders that share the phenotype of hyperglycemia. Several distinct types of DM exist and are caused by a complex interaction of genetics, environmental factors, and life-style choices. Depending on the etiology of the DM, factors that contribute to hyperglycemia may include reduced insulin secretion, decreased glucose usage, and increased glucose production. The metabolic dysregulation associated with DM causes secondary pathophysiological changes in multiple organ systems that impose a tremendous burden on the individual with DM and on the health care system. In the United States, DM is the leading cause of end-stage renal disease, nontraumatic lower extremity amputations, and adult blindness.

[0003] Type 2 DM is a heterogeneous group of disorders usually characterized by varying degrees of insulin resistance, impaired insulin secretion, and increased glucose production. Distinct genetic and metabolic defects give rise to the common phenotype of hyperglycemia in type 2 DM. Type 2 DM is characterized by three pathophysiologic abnormalities: impaired insulin secretion, peripheral insulin resistance, and excessive hepatic glucose production. Obesity is very commonly associated with type 2 DM, and insulin resistance associated with obesity augments the genetically determined insulin resistance of type 2 DM.

[0004] Insulin resistance is caused by the decreased ability of insulin to act effectively on peripheral target tissues, in particular, muscle and liver, and is a major feature of type 2 DM. Resistance to insulin action impairs glucose utilization by insulin-sensitive tissues and increases hepatic glucose output; both effects contribute to the hyperglycemia of diabetes. In type 2 DM, insulin secretion initially increases in response to insulin resistance in order to maintain normal glucose tolerance. Initially, the insulin secretory defect is mild, but eventually the defect progresses to a state of grossly inadequate insulin secretion.

[0005] During periods of fasting, the liver maintains plasma glucose through glyconeogenesis, glyconeolysis, and gluconeogenesis using substrates derived from skeletal muscle and fat. In type 2 DM, insulin resistance in the liver results from the failure of hyperinsulinemia to suppress gluconeogenesis, which results in fasting hyperglycemia and decreased glucose storage by the liver in the postprandial state. Increased hepatic glucose production occurs early in the course of diabetes, though likely after the onset of insulin secretory abnormalities and insulin resistance in skeletal muscle.

[0006] Insulin resistance is associated not only with type 2 diabetes, but also with hyperlipidemia, obesity, hypertension, and heart disease, among other conditions. This cluster of symptoms is known also as the metabolic syndrome, or "Syndrome X." Type 2 DM leads to many clinical complications, ranging from vascular disorders, eye disorders such as retinopathy, glaucoma and cataracts, nephropathy, diabetic neuropathy to a variety of infections and other conditions. In addition, several serious acute diabetic complications such as hyperosmolar nonketotic acidosis, diabetic ketoacidosis and lactic acidosis can occur, often with lethal consequences, particularly in the elderly.

[0007] Many different therapeutic approaches targeting several different systems and relevant pathways have been used over the last few decades for the treatment and prevention of type 2 DM, insulin resistance, and its related diseases. While some of these therapeutics have had some success, there is a need for additional therapies that are efficacious in preventing and treating type 2 DM, and its symptoms, as well as the conditions leading up to type 2 DM and the conditions associated with type 2 DM and the metabolic syndrome. SUMMARY OF INVENTION

[0008] The present invention encompasses methods of treatment for (including management of, amelioration of symptoms of, and preventing the progression of) and prevention of type 2 DM, the metabolic syndrome, and related conditions and symptoms using certain combination therapies, as well as the pharmaceutical compositions comprising these combination therapies. Subjects are mammalian, and preferably are human.

[0009] Combinations of metformin and the apolipoprotein Al mimetic, L-4F, are particularly effective for preventing and treating type 2 DM, in addition to the individual components of the metabolic syndrome and its related conditions and symptoms, and also for certain disorders related to the brain.

[0010] In one aspect, the invention encompasses methods of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with therapeutically effective amount of metformin.

[0011] In one embodiment, L-4F is administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day.

[0012] In one embodiment, metformin is administered at a dose from about 1500 mg/day to about 2500 mg/day.

[0013] In another embodiment, L-4F is administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day and metformin is administered at a dose from about 1500 mg/day to about 2000 mg/day.

[0014] In yet another embodiment, L-4F and metformin are administered at a ratio of L-4F to metformin of about 1: about 1000.

[0015] In one embodiment, L-4F and metformin are administered orally.

[0016] Another aspect of the invention encompasses methods of treating or preventing insulin resistance, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin. [0017] In yet another aspect of the invention methods are provided of improving glucose tolerance, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0018] Another aspect of the invention encompasses methods of improving insulin sensitivity, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0019] Another aspect of the invention encompasses methods of treating or preventing the metabolic syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0020] Yet another aspect of the invention encompasses methods of treating or preventing hypertension, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0021] Yet another aspect of the invention encompasses methods of treating or preventing cardiovascular disease, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0022] Yet another aspect of the invention encompasses methods of treating or preventing diseases associated with inflammation, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0023] Another aspect of the invention encompasses methods of treating or preventing obesity, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0024] Another aspect of the invention encompasses methods of reducing or maintaining body weight, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin. [0025] Another aspect of the invention encompasses methods of reducing food intake, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0026] Methods are also provided for treating or preventing hyperlipidemia, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0027] Methods are also provided for preventing coronary artery disease, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0028] Methods are also provided for treating or preventing atherosclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0029] The invention also encompasses methods of treating or preventing Alzheimer's Disease with a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0030] In addition, the invention encompasses methods of treating or preventing Attention- Deficit Hyperactivity Disorder, preferably in adults, with a therapeutically effective amount of L- 4F in combination with a therapeutically effective amount of metformin.

[0031] Methods are also provided for treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that suppresses hepatic glucose production.

[0032] Another aspect of the invention encompasses treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that enhances peripheral glucose uptake. [0033] Another aspect of the invention encompasses methods of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of metformin in combination with a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al .

[0034] Another aspect of the invention encompasses methods of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al in combination with a therapeutically effective amount of a compound that suppresses hepatic glucose production.

[0035] Yet another aspects of the invention encompasses method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al in combination with a therapeutically effective amount of a compound that enhances peripheral glucose uptake.

[0036] Yet another aspects of the invention encompasses methods of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a biguanide compound.

[0037] In another embodiment, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0038] In one embodiment, the pharmaceutical composition comprises metformin administered at a dose from about 1500 mg/day to about 2500 mg/day.

[0039] In another embodiment, the pharmaceutical composition comprises metformin administered at a dose of about 2000 mg/day.

[0040] In another embodiment, the pharmaceutical composition comprises L-4F administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day. [0041] In another embodiment, the pharmaceutical composition comprises L-4F and metformin at a ratio of L-4F to metformin of about 1 : about 1000.

[0042] In another embodiment, the pharmaceutical composition comprising metformin and L-4F may also comprise one or more of a thiazolidenodione compound, a DPP-IV inhibitor, an incretin mimetic, an insulin secretagogue, an additional biguanide, and an additional apolipoprotein Al mimetic, among other therapies.

[0043] Also contemplated by the invention are methods of treating or preventing type 2 diabetes, obesity, or the metabolic syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F.

[0044] In one embodiment, L-4F is administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day.

[0045] The invention also encompasses methods of treating or preventing insulin resistance, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F.

[0046] The invention also encompasses methods of improving insulin sensitivity, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F.

[0047] The invention also encompasses methods of improving glucose tolerance, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F.

[0048] Another aspect of the invention encompasses pharmaceutical compositions comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that suppresses hepatic glucose production.

[0049] Another aspect of the invention encompasses pharmaceutical compositions comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that enhances peripheral glucose uptake.

[0050] Yet another aspect of the invention encompasses pharmaceutical compositions comprising a therapeutically effective amount of metformin in combination with a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al .

BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIGS. 1A-1D: (A) Body weight of vehicle-treated or L-4F-treated obese mice. The mice were weighed at the times shown on the x-axis given as the age of the mice in weeks. The data are the weights in grams as mean ± SEM (average of two independent experiments); n=8 for vehicle treated and n=10 for L-4F treatment, L-4F discontinued. L-4F recommenced *p<0.05 obese-vehicle treated vs. obese-L-4F treated mice. (B) food intake in vehicle treated or L-4F treated obese mice during the first two weeks of treatment. (C) Representative photographs of mice after 6 weeks of treatment. (D) The weight of subcutaneous and visceral fat after L-4F treatment, *p<0.05 vs. vehicle treated obese animals.

[0052] FIGS. 2A-2B: (A) Glucose tolerance and insulin sensitivity after treatment with vehicle or vehicle containing L-4F. After six weeks of treatment with vehicle or vehicle containing L-4F obese mice were injected intraperitoneally with 2 gm/kg of glucose and plasma glucose levels were determined as described in Methods for the intraperitoneal glucose tolerance test (IPGTT). *p<0.05 vs. vehicle treated obese mice, 0 minutes; *p<0.001 vs. L-4F treated obese mice, 0 minutes; **p<0.05 vs. L4F treated obese mice, 0 minutes. (B) After six weeks of treatment with vehicle or vehicle containing L-4F obese mice were injected intraperitoneally with 2.0 U/kg of insulin and plasma glucose levels were determined as described in Methods for the intraperitoneal insulin tolerance test (IPITT). The results are expressed as mean ± SEM; n=4. p<0.001 vs. L-4F -treated obese mice, 0 minutes.

[0053] FIGS. 3A-3B: Plasma glucose and insulin levels in vehicle-treated obese mice and L- 4F-treated obese mice after six weeks of treatment. (A) Glucose levels in vehicle -treated obese and L-4F-treated obese mice after six weeks of treatment, *p<0.002 vehicle-treated obese vs. L- 4F-treated obese mice. (B) Insulin levels in vehicle- treated obese mice and L-4F-treated obese mice after six weeks of treatment, *p<0.036 vehicle -treated obese vs. L-4F-treated obese mice. Glucose and Insulin levels were measured before sacrifice as described in Methods. Data are shown as the mean ± SEM; n=5 for vehicle -treated and n=7 for L-4F-treated obese mice. [0054] FIGS. 4A-4C: (A) Adiponectin levels in lean and obese mice. Vehicle or vehicle containing L-4F were administered daily for 6 weeks (as described in the Examples) and serum samples were obtained immediately prior to sacrifice. The results are expressed as μg/ml serum. Levels of significance: *p<0.027 vehicle treated lean vs. vehicle treated obese mice; *p<0.04 L- 4F treated obese vs. vehicle treated obese mice. (B) and (C): Serum IL- lβ and IL-6 levels in lean and obese mice. Vehicle or vehicle containing L-4F were administered as described in the Examples. Serum samples were obtained immediately prior to sacrifice. The results for lean vehicle-treated or obese-L-4F treated vs. obese- vehicle treated mice for IL-I β (FIG. 4B) and for IL-6 (FIG. 4C) are shown as the mean ± SEM. Levels of significance: FIG. 2B: *p<0.02 vehicle treated obese vs. vehicle treated lean mice; *p<0.05 L-4F-treated obese vs. vehicle-treated obese mice. FIG 4C: *p<0.05 vehicle -treated obese vs. vehicle -treated lean mice; *p<0.05 L-4F-treated obese vs. vehicle -treated obese mice.

[0055] FIGS. 5A-5B: (A) Body weight (g) of vehicle -treated lean mice, vehicle -treated ob mice, L-4F-treated ob mice, metformin-treated ob mice, and ob mice treated with both L-4F and metformin (n = 6 mice per group). Weights are presented in grams as mean ± SEM. (B) The data represented in the table are reflected graphically in (A). The table also provides the p values for each group compared to ob mice treated with vehicle.

DETAILED DESCRIPTION

[0056] The invention provides methods of preventing or treating (including management of, amelioration of symptoms of, and preventing the progression of) type 2 DM and the metabolic syndrome and its individual components, comprising the administering to a subject in need thereof of a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0057] The combination of a therapeutically effective amount of L-4F and a therapeutically effective amount of metformin is also contemplated to prevent or treat insulin resistance, impaired insulin secretion, increased glucose production, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, and excessive hepatic glucose production in a subject in need thereof. [0058] The combination of a therapeutically effective amount of L-4F and a therapeutically effective amount of metformin is also contemplated to prevent or treat cardiovascular disease, and diseases caused by inflammation.

[0059] In other embodiments, the combination of a therapeutically effective amount of L-4F and a therapeutically effective amount of metformin is contemplated to prevent or treat obesity, to reduce or maintain body weight, reduce or maintain food intake, and reduce or maintain appetite in a subject in need thereof.

[0060] In yet other embodiments, the combination of a therapeutically effective amount of L- 4F and a therapeutically effective amount of metformin is contemplated to prevent or treat Alzheimer's Disease, Attention-Deficit Hyperactivity Disorder (preferably in adults), and other disorders of the brain that are associated with metabolic disorders, a subject in need thereof.

[0061] In other embodiments, methods are provided for treating or preventing type 2 DM, insulin resistance, impaired insulin secretion, increased glucose production, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, excessive hepatic glucose production, Alzheimer's Disease, Attention-Deficit Hyperactivity Disorder, and obesity, and for reducing or maintaining body weight, food intake, and appetite, comprising administering a therapeutically effective amount of L-4F and a therapeutically effective amount of a compound that suppresses hepatic glucose production or a therapeutically effective amount of a compound that enhances peripheral glucose uptake. In other embodiments, the methods comprise administering a therapeutically effective amount of L-4F and a therapeutically effective amount of a biguanide compound.

[0062] In yet other embodiments, methods are provided for treating or preventing type 2 DM, insulin resistance, impaired insulin secretion, increased glucose production, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, excessive hepatic glucose production, Alzheimer's Disease, Attention-Deficit Hyperactivity Disorder, and obesity, and for reducing or maintaining body weight, food intake, and appetite, comprising administering a therapeutically effective amount of metformin and a therapeutically effective amount of a compound that enhances or mimics the activity of apolipoprotein Al . [0063] In yet other embodiments, methods are provided for reducing serum or plasma levels of inflammatory cytokines comprising administering the pharmaceutical compositions of the invention, and preferably, comprising administering a therapeutically effective amount of metformin and a therapeutically effective amount of L-4F.

[0064] In yet other embodiments, methods are provided for elevating serum or plasma levels of adiponectin comprising administering the pharmaceutical compositions of the invention, and preferably, comprising administering a therapeutically effective amount of metformin and a therapeutically effective amount of L-4F.

[0065] In another embodiment, methods are provided for sustained reduction in body weight, comprising administering the pharmaceutical compositions of the invention, and preferably, comprising administering a therapeutically effective amount of metformin and a therapeutically effective amount of L-4F. Sustained reduction in body weight comprises any amount of body weight loss from the time of administration of the pharmaceutical compositions of the invention until between about two weeks and about twenty years. In some embodiments, the subject reduces their body weight by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, or more.

[0066] In some embodiments, the reduction in body weight is maintained for more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, or more.

[0067] In another embodiment, methods are provided for preventing body weight gain, comprising administering the pharmaceutical compositions of the invention, and preferably, comprising administering a therapeutically effective amount of metformin and a therapeutically effective amount of L-4F.

[0068] Metformin (Glucophage®) is an insulin-sensitizing drug that is often used in the treatment of type 2 diabetes and related disorders, and it acts at least in part by suppressing hepatic glucose production. Metformin and its pharmaceutically acceptable salt forms are contemplated for use in the combinations used in the methods and pharmaceutical compositions of the invention.

[0069] In one embodiment, the dose of metformin is from about 1500 mg/day to about 2500 mg/day. In other embodiments, the dose of metformin is from about 1500 mg/day to about 2000 mg/day. In a preferred embodiment, the dose of metformin is 2000 mg/day.

[0070] Apolipoprotein Al (Apo Al) is the major protein present on the surface of high density lipoprotein (HDL) particles. The protein promotes cholesterol efflux from tissues to the liver for excretion. It is also a cofactor for lecithin cholesterolacyltransferase (LCAT) which is responsible for the formation of most plasma cholesteryl esters. HDL elevators and mimetics are emerging therapies for atherosclerosis. Mimetics of Apo Al such as L-4F and D-4F are contemplated for use in the combinations used in the methods and pharmaceutical compositions of the invention. In a preferred embodiment, the Apo Al mimetic used in the invention is L-4F.

[0071] In one embodiment, the dose of L-4F is from about 0.2 mg/kg/day to about 1.2 mg/kg/day. In a preferred embodiment, the dose of metformin is 2000 mg/day. In a preferred embodiment, the dose of L-4F is about 0.5 mg/kg/day.

[0072] In yet another embodiment, methods comprise administering L-4F at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day in combination with metformin at a dose from about 1500 mg/day to about 2000 mg/day for prevention or treatment of the disorders described herein for a subject in need thereof.

[0073] In some embodiments, metformin is administered once a day, in other embodiments it is administered twice a day, in other embodiments it is administered three times a day, and in yet other embodiments it is administered four times a day. In some embodiments, when a higher total dose of metformin is administered to a subject within one day, the total amount is not administered all at once, but rather over different time periods during the day.

[0074] In some embodiments, L-4F is administered once a day, in other embodiments it is administered twice a day, in other embodiments it is administered three times a day, and in yet other embodiments it is administered four times a day. In some embodiments, when a higher total dose of L-4F is administered to a subject within one day, the total amount is not administered all at once, but rather over different time periods during the day.

[0075] It is contemplated that any of the compounds used in the invention can be administered either in one bolus or at numerous times throughout a day, depending on side effects and what causes the compound to work most efficaciously either alone, or in combination with other compounds.

[0076] In yet another embodiment, methods comprise administering L-4F at a dose of about 0.5 mg/kg/day in combination with metformin at a dose from of about 2000 mg/day for prevention or treatment of the disorders described herein for a subject in need thereof.

[0077] In one embodiment, L-4F and metformin are administered at a ratio of L-4F to metformin of about 1: about 1000.

[0078] In other embodiments of the invention, the methods of the invention further comprise administering one or more of a thiazolidenodione compound, a DPP-IV inhibitor, an incretin mimetic, an insulin secretagogue, an additional biguanide, and an additional apolipoprotein Al mimetic, a compound that suppresses hepatic glucose uptake, and a compound that enhances peripheral glucose uptake, among other therapies.

[0079] In another embodiment, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

[0080] In one embodiment, the pharmaceutical composition comprises metformin administered at a dose from about 1500 mg/day to about 2500 mg/day.

[0081] In another embodiment, the pharmaceutical composition comprises metformin administered at a dose of about 2000 mg/day.

[0082] In another embodiment, the pharmaceutical composition comprises L-4F administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day. [0083] In another embodiment, the pharmaceutical composition comprises L-4F and metformin at a ratio of L-4F to metformin of about 1 : about 1000.

[0084] In other embodiments of the invention, the pharmaceutical compositions of the invention further comprise one or more of a thiazolidenodione compound, a DPP-IV inhibitor, an incretin mimetic, an insulin secretagogue, an additional biguanide, and an additional apolipoprotein Al mimetic, a compound that suppresses hepatic glucose uptake, and a compound that enhances peripheral glucose uptake, among other therapies.

[0085] In some embodiments of the invention, it is contemplated that the combination of metformin and L-4F act synergistically in preventing or treating the disorders contemplated by the invention for prevention and treatment thereof in a subject in need thereof.

[0086] It is also contemplated that the combinations of the invention lead to fewer or reduced side effects than the compounds when given alone. In some embodiments of the invention, when compounds of the invention are administered in combination, lower doses of each of the individual compounds may be used than if they were administered separately.

[0087] In some embodiments, administration of the compounds contemplated in the methods and pharmaceutical compositions of the present invention results in the control and maintenance of blood glucose levels. In one embodiment, the methods of the invention achieve plasma glucose of less than 200 mg/dL. In a preferred embodiment, the methods of the invention achieve plasma glucose of less than 100 mg/dL. The target goal for plasma glucose levels, however, can vary greatly depending on the individual subject and their body weight, and whether the plasma is obtained from a fasting subject, or in a subject postprandially.

[0088] Glycemic control, as reflected by glycated hemoglobin (HbAIc) predicts the development of diabetes-related complications. HbAIc values are expressed as the percentage of an individual's total hemoglobin content that is glycated. In one embodiment, the methods of the invention achieve HbAIc of less than about 7%.

[0089] In some embodiments, the methods of the invention reduce a subject's HbAIc by 1%, 2%, 3%, 4%, or 5%. [0090] In other embodiments, the methods of the invention reduce the amount of insulin necessary to be administered to patients with type 2 DM. The amount of insulin administered to a diabetic patient depends on the severity of the patient's hyperglycemia. For example, the methods of the invention are contemplated to reduce the dose of insulin therapy to below between about 0.3 and about 0.4 U/kg/day.

[0091] In some embodiments, administration of the compounds contemplated in the methods and pharmaceutical compositions of the present invention results in reducing the body mass index (BMI) of a subject to between about 18.5 and about 24.9, or results in maintaining the BMI of a subject to between about 18.5 and about 24.9. In some embodiments, the methods of the invention reduce a subject's BMI to less than 40 or results in maintaining the BMI at less than 40. In other embodiments, the methods of the invention reduce a subject's BMI to less than 35 or results in maintaining the BMI at less than 35. In yet other embodiments, the methods of the invention reduce a subject's BMI to less than 30 or results in maintaining the BMI at less than 30. In yet other embodiments, the methods of the invention reduce a subject's BMI to less than 25 or results in maintaining the BMI at less than 25. In some embodiments, the reduction in BMI is maintained for more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, or more.

[0092] In some embodiments, administration of the compounds contemplated in the methods and pharmaceutical compositions of the present invention results in reducing a subject's waist size. In some embodiments, the waist size is reduced to less than 80 inches, in some embodiments it is reduced to less than 70 inches, in some embodiments it is reduced to less than 60 inches, in some embodiments it is reduced to less than 50 inches, in some embodiments it is reduced to less than 40 inches, in some embodiments it is reduced to less than 35 inches, in some embodiments it is reduced to less than 30 inches, in some embodiments it is reduced to less than 25 inches, and in some embodiments it is reduced to less than 20 inches.

[0093] In a preferred embodiment, the subject's waist size is reduced to less than 40 inches if the subject is male, and reduced to less than 35 inches if the subject is female. [0094] In some embodiments, the reduction in waist size is maintained for more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, or more.

[0095] In some embodiments, administration of the compounds contemplated in the methods and pharmaceutical compositions of the present invention results in reducing a subject's hip-to- waist ratio. In some embodiments, the hip-to-waist ratio is reduced to less than 1.0, in some embodiments it is reduced to less than 0.95, in some embodiments it is reduced to less than 0.85, in some embodiments it is reduced to less than 0.82, and in some embodiments it is reduced to less than 0.81. In some embodiments, the reduction in hip-to-waist ratio is maintained for more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, 50 years, or more.

[0096] In other embodiments, methods are provided for treating or preventing type 2 DM, insulin resistance, impaired insulin secretion, increased glucose production, hyperglycemia, hyperinsulinemia, impaired glucose tolerance, excessive hepatic glucose production, Alzheimer's Disease, Attention-Deficit Hyperactivity Disorder and obesity, and for reducing or maintaining body weight and food intake, comprising administering a therapeutically effective amount of L-4F.

[0097] In some embodiments, administration of the compounds contemplated in the methods and pharmaceutical compositions contemplated by the invention are directed to treating or preventing the diseases and conditions described herein, in patients who have undergone bariatric surgery.

[0098] In other embodiments, administration of the compounds contemplated in the methods and pharmaceutical compositions contemplated by the invention are directed to treating or preventing the diseases and conditions described herein, in patients who are obese and do not have diabetes. [0099] As used herein, the term "in combination" refers to the use of more than one prophylactic and/or therapeutic agents. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject in need thereof. A first prophylactic or therapeutic agent can be administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject. The prophylactic or therapeutic agents are administered to a subject in a sequence and within a time interval such that the agent of the invention can act together with the other agent to provide an increased benefit than if they were administered otherwise. Any additional prophylactic or therapeutic agent can be administered in any order with the other additional prophylactic or therapeutic agents.

[00100] As used herein, the term "synergistic" refers to a combination of therapies (e.g., a combination of prophylactic or therapeutic agents) which is more effective than the additive effects of any two or more single agents. A synergistic effect of a combination of therapies permits the use of lower dosages of one or more of the therapies and/or less frequent administration of said therapies (e.g., agents) to a subject with a disease or disorder. The ability to utilize lower dosages of therapies and/or to administer said therapies less frequently reduces the toxicity associated with the administration of said therapies to a subject without reducing the efficacy of said therapies in the prevention, management, or treatment of a disease or disorder. In addition, a synergistic effect can result in improved efficacy of therapies in the prevention, management, or treatment of a disease or disorder, or a condition or symptom associated therewith. Finally, synergistic effect of a combination of therapies may avoid or reduce adverse or unwanted side effects associated with the use of any single therapy.

[00101] As used herein, the phrase "side effects" encompasses unwanted and adverse effects of a prophylactic or therapeutic agent. Adverse effects are always unwanted, but unwanted effects are not necessarily adverse. An adverse effect from a prophylactic or therapeutic agent might be harmful or uncomfortable or risky.

[00102] By "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" herein is meant a dose that produces therapeutic effects for which it is administered, in the context of the combination therapy in which it is administered. Often, the therapeutically effective or sufficient amount or dose of the compounds comprising the pharmaceutical compositions of the invention will be lower when administered in the specific combinations, than the doses that would be therapeutically effective or sufficient when the compounds are administered separately. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington. The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[00103] The dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired. Doses can be given daily, or on alternate days, as determined by the treating physician.

[00104] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 20th ed., 2003).

[00105] The compounds of the invention may be formulated into pharmaceutical compositions as natural or salt forms. Pharmaceutically acceptable non-toxic salts include the base addition salts (formed with free carboxyl or other anionic groups) which may be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as, for example, hydrochloric, sulfuric, or phosphoric acids, or organic acids such as acetic, p-toluenesulfonic, methanesulfonic acid, oxalic, tartaric, mandelic, and the like. Salts of the invention include amine salts formed by the protonation of an amino group with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like. Salts of the invention also include amine salts formed by the protonation of an amino group with suitable organic acids, such as p-toluenesulfonic acid, acetic acid, and the like. Additional excipients which are contemplated for use in the practice of the present invention are those available to those of ordinary skill in the art, for example, those found in the United States Pharmacopeia Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention, Inc., Rockville, Md. (1989), the relevant contents of which is incorporated herein by reference.

[00106] In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like.

[00107] Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In a preferred embodiment, the pharmaceutical compositions are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.

[00108] In yet another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used to achieve controlled or sustained release (see e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1 :105); U.S. Pat. No. 5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; International Publication No. WO 99/15154; and International Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In yet another embodiment, a controlled or sustained release system can be placed in proximity of the therapeutic target, i.e., the lungs, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

[00109] Controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more compounds of the invention. See, e.g., U.S. Pat. No. 4,526,938, International publication No. WO 91/05548, International publication No. WO 96/20698, Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel," Radiotherapy & Oncology 39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application," Pro. Int'l. Symp. Control. ReI. Bioact. Mater. 24:853-854, and Lam et al., 1997, "Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery," Proc. Int'l. Symp. Control ReI. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in their entirety.

[00110] Pharmaceutical compositions of the invention are formulated to be compatible with its intended route of administration. Examples of suitable routes of administration include, but are not limited to, oral, parenteral (e.g., intravenous, intramuscular, intradermal, intra-tumoral, intra-synovial, and subcutaneous) intranasal, transdermal (topical), transmucosal, intra-tumoral, intra-synovial, vaginal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intra-tummoral, intra synnovial, intranasal or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocamne to ease pain at the site of the injection.

[00111] In a preferred embodiment, the pharmaceutical compositions of the invention are administered orally.

[00112] If the compositions of the invention are to be administered orally, the compositions can be formulated orally in the form of, e.g., gum, tablets, capsules, cachets, gelcaps, solutions, suspensions and the like. Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, micro crystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well-known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release or sustained release of a prophylactic or therapeutic agent(s).

[00113] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

11 [00114] Kits

[00115] The invention provides a pharmaceutical pack or kit comprising one or more containers filled with metformin and L-4F or D-4F. The pharmaceutical pack or kit may also further comprise one or more additional therapeutic or prophylactic agents useful for the treatment of a disease or disorder. The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. The pharmaceutical pack or kit may also comprise one or more containers filled with a compound that suppresses hepatic glucose production or that enhances peripheral glucose uptake, in addition to L-4F or D-4F. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00116] The present invention provides pharmaceutical packs or kits that can be used in the above methods of the invention. In one embodiment, a kit comprises at least L-4F or D-4F and at least compound that suppresses hepatic glucose production or that enhances peripheral glucose uptake, in one or more containers. The kit may further comprise one or more other prophylactic or therapeutic agents, or active ingredients useful for the treatment of the diseases and disorders described herein. Examples of such agents and compounds are disclosed above.

EXAMPLES

[00117] It is understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggestive to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0100] The following methods apply to all of the Examples utilizing animal subjects.

[0101] L-4F and all reagents were from sources previously reported (See, e.g., Peterson, S. et al, 2007, J Pharmacol. Exp. Thor. 322: 514-520; and Kruger, A. et al. 2005, Circulation 111: 3126-3134) and all other reagents were of the highest available purity. [0102] Mice and protocols

[0103] Male obese (ob) mice (B6v-Lep ob/J) were purchased from Harlan (Chicago, IL) at the age of 7 weeks, allowed to acclimatize for one week and used at the age of 8 weeks. Age- and sex-matched lean mice (B6.V, lean, Harlan, Chicago, IL) were used as controls. Mice were fed a normal chow diet and had free access to water and food. Body weight of ob and lean mice at the beginning of the treatment was 34±5 g and 26± 3 g, respectively.

[0104] Beginning at 9 weeks of age when all ob mice had established diabetes, L-4F (i.e.,

Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂ synthesized from L-amino acids only as previously described (See, e.g., Navab, M., et al. 2,002, Circulation 105: 290- 292) was injected at a dose of 200 μg/100 gm daily in 200 μl vehicle), or vehicle (ABCT: ammonium bicarbonate buffer at pH 7.4 containing 0.01% Tween20) were administered intraperitoneally for 6 weeks (blood was collected (50-100 μl) from tail veins of anesthetized ob mice following L-4F or vehicle solution). Glucose monitoring was performed using an automated analyzer (Lifescan Inc., Milpitas, CA). Levels were 229±21 and 154±9 mg/dl for ob and lean mice, respectively. There were four groups of animals in the studies described in Examples 1-4: (A) lean, (B) lean+L-4F, (C) ob and (D) ob+L-4F. In a separate experiment vehicle-treated and L-4F-treated animals were housed in metabolic cages. Food intake was monitored daily and was not different in mice receiving vehicle or L-4F. Food intake was monitored less frequently thereafter by measuring remaining chow and body weight. The Animal Care and Use Committee of New York Medical College approved all experiments.

[0105] Cytokine measurements

[0106] Adiponectin (high molecular weight, HMW), IL-6 and IL- lβ were determined in mouse serum using an ELISA assay (Pierce Biotechnology, Inc., Woburn, MA) and insulin was measured using an ELISA Kit (Millipore, Billerica, MA).

[0107] Glucose and insulin tolerance tests

[0108] After a 12 hour fast, mice were injected intraperitoneally with glucose (2.0g/kg body weight). Blood samples were taken at various time points (0-120 min), and blood glucose levels and serum insulin levels were measured. For determination of insulin tolerance, mice were injected intraperitoneally with insulin (2.0 U/kg). Blood samples were taken at various time points (0-90 min) and blood glucose levels were measured.

[0109] Statistical analyses

[0110] Statistical significance between experimental groups was determined by the Fisher method of analysis of multiple comparisons (p<0.05). For comparison between treatment groups, the null hypothesis was tested by a single-factor ANOVA for multiple groups or unpaired t-test for two groups and Data are presented as mean ± SEM.

EXAMPLE 1

Body weight response of obese mice following L-4F treatment

[0111] The effect of long-term L-4F treatment on body weight responses was examined (FIG. IA). Daily treatment with L-4F was well tolerated by obese mice. Food intake was not significantly decreased by L-4F treatment compared to vehicle -treated animals (FIG. IB). Activity and grooming were maintained during L-4F treatment and obesity associated hyperglycemia was decreased (FIG. IA). L-4F treatment reduced, but did not entirely prevent weight gain, in ob mice when compared to age matched vehicle treated obese mice. This suggests that L-4F may modulate both adipose and decreased lipid synthesis. The final weight after 6 weeks of vehicle and L-4F treated animals was 51±2.7 g and 44.2+1.2 g, respectively. L- 4F mediated prevention of weight gain was reversible when L-4F was withdrawn. As seen in FIG. IA, when L-4F was discontinued at week 10, obese mice gained weight at a faster rate than ob-vehicle animals. Recommencement of L-4F administration at 12 weeks decreased body weight to levels similar to that seen in ob animals continuously treated with L-4F. L-4F-treated lean mice also gained less weight than vehicle treated lean mice (final weight: 30±0.5 and 27.4±0.7 g for vehicle and L-4F treated lean mice, respectively). This minimum effect on the lean mice is due to a lower fat content compared to ob mice and increased L-4F efficacy in ob when compared to lean animals. [0112] As seen in FIGS. 1C and ID, fat and body appearance of obese mice confirmed the reduction in fat content and body weight loss. Visceral fat in obese mice was decreased by L-4F treatment from 7.07±0.32 gm to 5.70±0.64 gm (p<0.02). Subcutaneous fat in obese mice was decreased from 4.30±0.46 gm to 3.53±0.30 gm (p<0.04).

EXAMPLE 2

L-4F treatment improved glucose tolerance and increased insulin sensitivity

[0113] Currently one of the main treatments for type 2 diabetes consists of insulin- sensitizing therapy resulting in a lowering of blood glucose and insulin levels. Plasma glucose levels in ob mice treated with vehicle were significantly higher than those in ob mice treated with L-4F at all time points during the glucose tolerance test. Blood glucose levels in vehicle treated ob mice were significantly (p<0.05) elevated 30 minutes after glucose administration and remained elevated (p<0.05) at 120 minutes. In L-4F-treated ob mice, blood glucose levels were significantly (p<0.001) elevated 30 minutes after glucose administration but had returned to initial levels at 120 minutes, (FIG. 2A). Insulin administration to the L-4F treated ob mice resulted in a rapid decrease in glucose but not in the ob mice receiving vehicle alone, suggesting improved insulin sensitivity in the L-4F treated ob mice. Glucose levels were significantly (p<0.001) lower in L-4F treated ob mice compared to vehicle treated ob mice at all time points examined (FIG 2B).

EMAMPLE 3

Effect of L-4F treatment on glucose and insulin levels in ob mice

[0114] Plasma glucose levels in ob mice treated with vehicle for six weeks were 418±21 mg/dL compared to 250±27 mg/dL (p<0.002) in ob mice treated with L-4F for six weeks (FIG 3A). Consistent with the IPITT results, plasma insulin levels in ob mice which received L-4F for six weeks were lower than in the vehicle treated ob mice. FIG. 3B shows that ob mice treated with vehicle alone had insulin levels of 2840±339 pM compared to 1175±52 pM in ob mice treated with L-4F. Insulin levels in obese mice treated with L-4F were not significantly different from those found in vehicle treated lean mice (data not shown). EXAMPLE 4

L-4F treatment increases serum adiponectin levels and decreases IL-I β and IL-6 levels

[0115] Because an increase in serum adiponectin has been reported to follow a decrease in oxidative stress, we examined the effect of L-4F administration on serum adiponectin. As expected serum adiponectin levels in vehicle treated ob mice were significantly lower compared to age-matched vehicle treated lean mice (FIG. 4A); adiponectin levels in vehicle treated ob mice were 2.3 ± 0.32 μg/ml compared to 3.9 ± 0.45 μg/ml(p<0.027) in vehicle -treated lean animals. L-4F administration resulted in a significant increase in adiponectin in the ob mice to 3.70 ± 0.45 μg/ml compared to ob mice treated with vehicle alone (p<0.04). L-4F administration resulted in a significant increase in adiponectin in lean mice (results not shown).

[0116] Vehicle -treated obese mice had significantly (p<0.02) higher serum IL-lβ levels (121 ± 22 pg/ml) when compared to age-matched vehicle treated lean mice (54 ± 9 pg/ml) (FIG. 4B). Administration of L-4F resulted in a significant decrease in serum IL-I β levels to 48 ± 25 pg/ml (p<0.05) in the ob mice. The changes in serum IL-lβ were the reciprocal of those seen with serum adiponectin levels (FIG. 4A). As shown in FIG. 4C, vehicle -treated ob mice had significantly (p<0.05) higher serum IL-6 levels (402 ± 7 pg/ml) when compared to age-matched vehicle treated lean mice (138±27 pg/ml). Administration of L-4F resulted in a significant (p<0.05) decrease in serum IL-6 levels to 214 ± 41 pg/ml in the obese mice. The changes in serum IL-6 levels were, again, the reciprocal of those seen with serum adiponectin levels (FIG. 4A).

EXAMPLE 5

Combination of L-4F and metformin in human subjects

[0117] In a double-blind, placebo-controlled study, human study subjects are divided into four equal groups as follows: (1) Control group receiving placebo; (2) Metformin group receiving between 1500 mg/day and 2500 mg/day of metformin; (3) L4-F group receiving between 0.2 mg/kg/day and 1.2 mg/kg/day of L4-F; (4) Combination group receiving 2000 mg/day of metformin and 0.5 mg/kg/day of L4-F. [0118] Human subjects include obese individuals (men and women) between 21 and 65 years of age. Excluded from the study are individuals with known diabetes (or fasting blood glucose > 100 mg/dl or HbAIc of > 6.0%); known renal disease (or creatinine > 1.4); known severe pulmonary disease; congestive heart failure; and chronic acidosis.

[0119] At various time intervals throughout the course of the study, the following measures can be assessed: body weight; food intake; blood levels of glucose, insulin, adiponectin, lipids, HbAIc, and C-reactive protein; insulin sensitivity; glucose tolerance; body composition; inflammatory cytokines, including but not limited to IL- lβ, IL-6, and TNF-α; and levels of CB- 1.

[0120] In one phase of the experiment, subjects receive the treatments to which they are assigned daily for six months, and the measures described above are assessed at many intervals throughout the course of the six months, including at the end of six months. In another phase of the experiment, the subjects continue to receive their assigned treatments for another six months on a daily basis, over which time the measures described above are assessed at many intervals throughout the course of the additional six months. Based on the data obtained at the one year interval, a crossover trial is carried out, and a portion of the subjects in the Combination group are then assigned a placebo for six months, and a portion of the placebo group is then assigned the combination of metformin and L4-F for six months. If, for example, the Combination group loses weight after the one year of treatment, and then those subjects switched to the placebo group in the crossover trial gain weight back after six months, a portion of those subjects will be assigned back to receiving the combination of metformin and L4-F to assess if body weight was again reduced. This applies not only to body weight, but to any of the measures assessed in the initial one -year.

[0121] In a separate trial, the above procedures are followed for assessing the effects of the combination of metformin and L-4F, compared to metformin alone, L-4F alone, and placebo, at the doses described above, in patients who have undergone bariatric surgery. EXAMPLE 6

Combination of L-4F and metformin on body weight in mice

[0122] The effect of long-term L-4F treatment in combination with metformin treatment on body weight responses was examined in lean and ob mice (FIGS. 5A and 5B). Five groups of mice were used (n = 6 mice per group). Metformin was administered at a dose of 250 mg/kg, and L-4F was administered at a dose of 2 mg/kg. Mice were (1) lean and administered vehicle; (2) ob and administered vehicle; (3) ob and administered L-4F; (4) ob and administered metformin; (5) ob and administered L-4F and metformin. Treatment of ob mice with either L-4F or metformin, alone, significantly reduced body weight relative to ob mice treated with vehicle. Treatment of ob mice with the combination of metformin and L-4F significantly reduced body weight relative to ob mice treated with vehicle, and relative to ob mice treated with L-4F, and ob mice treated with metformin.

Claims

Claims:

1. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with therapeutically effective amount of metformin.

2. The method of claim 1, wherein L-4F is administered at a dose from about 0.2 mg/kg/day mg/day to about 10.2 mg/kg/day.

3. The method of claim 1, wherein metformin is administered at a dose from about 1500 mg/day to about 2500 mg/day.

4. The method of claim 1, wherein L-4F is administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day and metformin is administered at a dose from about 1500 mg/day to about 2000 mg/day.

5. The method of claim 1, wherein L-4F and metformin are administered at a ratio of L-4F to metformin of about 1: about 1000.

6. The method of claim 1, wherein L-4F and metformin are administered orally.

7. A method of treating or preventing insulin resistance, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

8. A method of improving glucose tolerance, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

9. A method of improving insulin sensitivity, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

10. A method of treating or preventing the metabolic syndrome, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

11. A method of treating or preventing hypertension, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

12. A method of treating or preventing obesity, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

13. A method of reducing body weight, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

14. A method of reducing food intake, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

15. A method of treating or preventing hyperlipidemia, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin

16. A method of treating or preventing coronary artery disease, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

17. A method of treating or preventing atherosclerosis, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin

18. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that suppresses hepatic glucose production.

19. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that enhances peripheral glucose uptake.

20. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of metformin in combination with a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al.

21. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al in combination with a therapeutically effective amount of a compound that suppresses hepatic glucose production.

22. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al in combination with a therapeutically effective amount of a compound that enhances peripheral glucose uptake.

23. A method of treating or preventing type 2 diabetes, comprising administering to a subject in need thereof a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a biguanide compound.

24. A pharmaceutical composition comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of metformin.

25. The composition of claim 24, wherein the metformin is administered at a dose from about 1500 mg/day to about 2500 mg/day.

26. The composition of claim 24, wherein metformin is administered at a dose of about 2000 mg/day.

27. The composition of claim 24, wherein L-4F is administered at a dose from about 0.2 mg/kg/day to about 1.2 mg/kg/day.

28. The composition of claim 24, wherein L-4F and metformin are administered at a ratio of L-4F to metformin of about 1: about 1000.

29. The composition of claim 24, further comprising a thiazolidenodione compound.

30. The composition of claim 24, further comprising a DPP-IV inhibitor.

31. The composition of claim 24, further comprising an incretin mimetic.

32. The composition of claim 24, further comprising an insulin secretagogue.

33. A pharmaceutical composition comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that suppresses hepatic glucose production.

34. A pharmaceutical composition comprising a therapeutically effective amount of L-4F in combination with a therapeutically effective amount of a compound that enhances peripheral glucose uptake.

35. A pharmaceutical composition comprising a therapeutically effective amount of metformin in combination with a therapeutically effective amount of a compound that enhances activity of or mimics activity of apolipoprotein Al .