PANCREATIC BETA-CELL MELATONIN RECEPTOR ACTIVATION AS A TREATMENT FOR PRESERVATION OF BETA-CELL MASS AND FUNCTION IN TYPE 2 DIABETES

GOVERNMENT RIGHTS

[0001] This invention was made with Government support of Grant No. DK063491 awarded by the National Institutes of Health. The Government has certain rights in this invention

BACKGROUND OF THE INVENTION

[0002] Type 2 diabetes mellitus is a multigenic disease; its already high prevalence in adults worldwide and its increasing prevalence in both adults and children indicate an urgent need to assess the susceptibility to this disease as early in life as possible and to identify therapeutic interventions to prevent the onset of type 2 diabetes mellitus and to treat the disease.

[0003] Poorly controlled diabetes can lead to a number of complications including heart disease, kidney disease and both macro and micro vascular damage often leading to amputation of limbs and blindness. In the United States, the total annual economic cost of diabetes in 2007 was estimated to be $174 billion with the cost of care expected to rise in the next 5 years at an exponential rate. The mechanisms and effective treatment of this devastating and costly disease remain, in part, elusive.

[0004] T2DM is a complex polygenic decease that affects multiple organ systems. Primary metabolic abnormalities in T2DM include the loss of beta-cell function and decline in beta- cell mass. However, T2DM is also associated with induction of hepatic and extrahepatic insulin resistance. Subsequently, diabetes therapy to date has been primarily focused on ameliorating impaired beta-cell insulin secretion (e.g. sulphonylureas, GLP-1 analogs and DPP-4 inhibitors) or on improvements in insulin sensitivity (Biguanides and Glitazones).

[0005] The sulfonylurea class of drugs was the first commercial diabetes treatment in the US, originally introduced more than 50 years ago. Sulphonylureas work by binding to the SUR1 subunit of the K _ATP channel which leads to closure of the channel and beta-cell depolarization. Closure of beta-cell K _ATP channels and subsequent induction of cell depolarization stimulates exocytosis of insulin granules and thus enhances insulin secretion. Although, this class of drugs is effective in stimulating insulin secretion in the short term (1 -5 years post diagnosis), prolonged use of these drugs leads to deterioration of beta-cell function leading to patients eventually being placed on insulin injection therapy. More recently studies have shown that sulphonylureas can promote beta-cell apoptosis in human islets thus raising an issue whether these drugs should be used for the treatment of T2DM. Additionally, use of sulphonylureas is associated with the risk for hypoglycemia, which can be a debilitating and sometimes deadly side effect for patients with T2DM. [0006] Biguanides such as metformin have been used for treatment of T2DM for over 50 years in Europe and over 20 years in the US. Primary actions of metformin are to enhance hepatic insulin sensitivity which leads to reduction of hepatic glucose production at fasting and following meal ingestion. Specifically, metformin decreases hepatic glucose production through its actions on AMP-activated kinase (AMPK) and consequent reduction in hepatic gluconeogenesis. Although, metformin is effective in lowering plasma glucose levels in T2DM patients, most patients require additional therapies as the disease progresses. Furthermore, use of metformin in elderly population with renal failure (common in T2DM) is associated with the risk of potentially deadly side effect of lactic acidosis.

[0007] Thiazolidinediones (TZD) are high affinity ligands for peroxisome proliferator- activated receptor- 1 (PPAR- 7 ) which is a member of the nuclear hormone receptor superfamily of ligand-activated transcriptional factors. This class of drugs improves skeletal muscle and adipose tissue insulin sensitivity through enhanced activation of the insulin signaling cascade, specifically by increasing insulin-induced P-13 kinase and Akt activation in those tissues. In human trials, TZD's have shown to be effective in treatment of T2DM, but recently have come under increased scrutiny due to its potential side effects such as heart failure.

[0008] Glucagon like peptide 1 (GLP-1 ) analogs and dipeptidyl peptidase 4 (DPP-4) inhibitors are the new class of antidiabetic drugs that function by elevating plasma levels of incretin hormones GLP-1 and GIP. Glucagon like peptide 1 is a hormone released from the distal ileum after meal ingestion which amplifies insulin secretion in response to the meal (termed an incretin effect). Thus intravenous administration of GLP-1 to humans with T2DM improves insulin secretion making incretin therapy an attractive antidiabetic strategy. Because GLP-1 is rapidly degraded by the enzyme dipeptidyl peptidase 4, available "incretin" therapies utilize either long acting GLP-1 analogs (Exenitide or Liraglutide) or DPP-4 inhibitors (Sitagliptin or Vidagliptin). Although, these therapies do provide moderate lowering of plasma glucose levels there is no evidence in humans that these drugs can preserve beta-cell mass in humans. Furthermore, recently studies have shown that incretin "mimetic therapy" may be associated with induction of pancreatitis and pancreatic cancer thus questioning the safety of these drugs.

[0009] In view of the current state of therapies for T2DM, there is clinical and research interest in novel therapies. The present invention addresses this issue.

SUMMARY OF THE INVENTION

[0010] Compositions and methods are provided for the treatment of Type 2 diabetes mellitus (T2DM). The present invention provides a novel treatment to prevent the decline in beta-cell mass and function in patients with T2DM. This treatment employs the use of oral melatonin supplementation or melatonin receptor agonist administration to increase nightly melatonin levels and activate pancreatic beta-cell receptors which result in preservation of beta-cell mass and function. In some embodiments of the invention, an individual is assessed for development of T2DM, including early stages of the disease, e.g. increased insulin resistance, loss of beta-cell function, hyperglycemia, and the like, and if diagnosed with T2DM, treating the individual with an oral melatonin supplementation or melatonin receptor agonist. In other embodiments of the invention, an individual suspected of a predisposition to T2DM is treated with oral melatonin supplementation or melatonin receptor agonist, and assessed for preservation of beta-cell mass and function following treatment. In other embodiments, compositions for use in such methods are provided. In some such embodiments, melatonin or a melatonin receptor agonist is the sole therapeutic agent for treatment of T2DM in the formulation or method of treatment.

[0011] The pathogenesis of T2DM was originally thought to be caused primarily by the induction of insulin resistance at the level of the peripheral tissues, such as skeletal muscle. However it is now better appreciated that T2DM develops from the failure of beta-cells to maintain adequate insulin secretion to meet the demands of prevailing insulin sensitivity. More specifically, the loss of beta-cell mass (number of beta-cells per pancreas) and beta- cell function (ability of beta-cell to secrete insulin in response to hyperglycemia) is now believed to be the central cause of Type 2 diabetes.

[0012] The above summary is not intended to include all features and aspects of the present invention nor does it imply that the invention must include all features and aspects discussed in this summary.

BRIEF DESCRIPTION OF TH E DRAWINGS

[0013] Figure 1 . Melatonin receptor expression in human, rodent and cultured beta cells.

Representative images (20X) of islets from a human (top) rat, and cultured INS-1 cells stained for insulin (green), melatonin receptor 2 (red) and nuclear stains Dapi (blue). Note conserved high expression of the melatonin receptors in beta-cells of all species.

[0014] Figure 2. Differential mechanisms of acute vs. prolonged melatonin receptor action.

Acute activation (A) leads to inhibition of the cAMP-PKA-pCREB pathway while prolonged activation of melatonin receptors (B) sensitizes the cAMP-PKA-pCREB.

[0015] Fig. 3. Mechanisms of beta-cell apoptosis in T2DM. The diagram emphasizes the conversions of pro-apoptotic stimuli (glucotoxicity, lipotoxicity, and IAPP) on activation of the intrinsic pathway of beta-cell apoptosis. The intrinsic pathway of apoptosis is activated through sequestration and decreased expression of the primary anti-apoptotic protein Bcl-2 resulting in induction of mitochondrial memebrane damage induced by pro-apoptotic proteins Bak and Bax, Cytochrome C leakage and activation of the caspace cascade. Specifically, glucolipotoxicity and human IAPP contribute to beta-cell apoptosis in Type 2 diabetes through inhibition of anti-apoptotic Bcl-2 and activation of pro-apoptotic Bax and Bak expression.

[0016] Figure 4. Pre-treatment with melatonin sensitizes cAMP-PKA pathway at low and high glucose concentrations. (A) Phosph-(Ser/Thr) PKA substrate protein expression at basal (2.8mM) and at glucose-stimulatory (16.7mM) glucose levels with or without pre- treatment with overnight melatonin (0.1 uM). (B) pCREB and pMAP Kinase protein expression at basal and at glucose-stimulatory glucose levels with or without pre-treatment with overnight melatonin (0.1 uM).

[0017] Figure 5. Pre-treatment with melatonin sensitizes cAMP-PKA pathway in response to known CAMP-PKA agonist such as Gastric Inhibitory peptide (GIP) and Glucagon-like peptide 1 (GLP-1 ). (Top panel) Representative examples of pCREB protein expression at basal (2.8mM) and following exposure to either GIP or GLP-1 with or without pre-treatment with overnight melatonin (0.01 uM). (Bottom panel) pCREB protein expression at basal (2.8mM) and following exposure to either GIP or GLP-1 with or without pre-treatment with overnight melatonin (0.01 uM).

[0018] Figure 6. Melatonin reduces beta-cell apoptosis in INS-1 cells transduced with h- IAPP. (A) Caspase-3 protein expression in INS-1 cells transduced with either cytotoxic human IAPP (h-IAPP) or non-cytotoxic rodent IAPP (r-IAPP) with or without pre-treatment with melatonin (n=3-4) independent experiments. (B) Melatonin attenuates beta-cell apoptosis in INS-1 cells transduced with h-IAPP through increase in Bcl-2 expression.

[0019] Figure 7. Melatonin reduces beta-cell apoptosis in isolated human islets from patients with T2DM. (A-top) Examples of T2DM islets untreated (left) or overnight treated (right) with 0.1 uM melatonin stained for propidium iodide (a marker of cell death). (A- bottom) T2DM islets untreated (left) or overnight treated (right) with 0.1 uM melatonin stained for insulin (green) and nuclei (blue). (B) Quantification of islet cell apoptosis in T2DM human islets untreated (white) or overnight treated with either 0.1 uM (black) or 1 uM (grey) melatonin.

[0020] Figure 8. Melatonin reverses deleterious effects of glucotoxicity on insulin secretion.

(TOP) representative profiles of insulin concentration profiles from perifused human islets incubated in either normal 5mM glucose (A), glucotoxic 16mM glucose (B) or glucotoxic 16mM glucose plus 1 uM melatonin (C). (Bottom) Mean insulin secretory pulse mass (D), measure of regularity of insulin secretion (E) and mean pulse interval in n=3-5 perifusion runs (F). [0021] Figure 9. Daily melatonin treatment in-vivo improves prevailing glycemia and reduces beta-cell apoptosis in diabetic h-IAPP transgenic rats. (A) Fasting and (B) Fed glucose concentrations prior (0 weeks) and after (10 weeks) of either saline or daily melatonin treatment in h-IAPP transgenic rats. (C) beta-cell apoptosis and (D) beta-cell mass prior after (10 weeks) of either saline or daily melatonin treatment in h-IAPP transgenic rats.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] Methods for the treatment and diagnosis of diseases related to hyperglycemic conditions, including diabetes, and the like are provided. Methods of the invention include administering to an individual having type 2 diabetes, or diagnosed as susceptible to development of type 2 diabetes, a melatonin agent, which increases beta cell mass and/or function. An individual may be diagnosed prior to initiation of treatment. An individual may be evaluated for beta-cell mass or function following a course of treatment.

[0023] Unless otherwise apparent from the context, all elements, steps or features of the invention can be used in any combination with other elements, steps or features.

[0024] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0025] The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence without affecting the protein sequence. Moreover, due to biological functional equivalency considerations, changes can be made in protein structure without affecting the biological action in kind or amount. All such modifications are intended to be included within the scope of the appended claims. [0026] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0027] Diabetes mellitus is impaired insulin secretion and variable degrees of peripheral insulin resistance leading to hyperglycemia. Early symptoms are related to hyperglycemia and include polydipsia, polyphagia, and polyuria. Later complications include vascular disease, peripheral neuropathy, and predisposition to infection. Diagnosis is by measuring plasma glucose. Conventional treatment is diet, exercise, and drugs that reduce glucose levels, including insulin and oral antihyperglycemic drugs. Prognosis varies with degree of glucose control.

Diagnostic Criteria for Diabetes Mellitus and Impaired Glucose Regulation

FPG = fasting plasma glucose; OGTT = oral glucose tolerance test, 2 h glucose level.

Note: All values refer to glucose levels in mg/dL [mmol/L].

[0028] In type 2 DM (previously called adult-onset or non-insulin-dependent), insulin secretion is inadequate. Often insulin levels are very high, especially early in the disease, but peripheral insulin resistance and increased hepatic production of glucose make insulin levels inadequate to normalize plasma glucose levels. Insulin production then falls, further exacerbating hyperglycemia. The disease generally develops in adults and becomes more common with age. Plasma glucose levels reach higher levels after eating in older than in younger adults, especially after high carbohydrate loads, and take longer to return to normal, in part because of increased accumulation of visceral and abdominal fat and decreased muscle mass.

[0029] Type 2 DM is becoming increasingly common in children as childhood obesity has become epidemic: 40 to 50% of new-onset DM in children is now type 2. Over 90% of adults with DM have type 2 disease. There are clear genetic determinants, as evidenced by the high prevalence of the disease within ethnic groups (especially American Indians, Hispanics, and Asians) and in relatives of people with the disease.

[0030] Pathogenesis is complex and incompletely understood. Accumulating data suggests that T2DM develops as a result of failure of beta-cells to adequately compensate for insulin resistance. Consistent with this, genes linked to T2DM by genome wide association scans, influence beta-cell mass and/or function. The primacy of beta-cell failure in the pathophysiology of T2DM is also supported by islet pathology that reveals a beta-cell deficit of ~ 50 and 65% in individuals with IFG and T2DM, respectively. The cause of this deficit is unknown but is likely mediated in part by the increase in beta-cell apoptosis. Furthermore, people with type 2 DM and those at risk for it, display impaired insulin secretion, including impaired first-phase insulin secretion in response to IV glucose infusion, a loss of normally pulsatile insulin secretion, an increase in proinsulin secretion signaling impaired insulin processing, and an accumulation of islet amyloid polypeptide (a protein normally secreted with insulin). Hyperglycemia itself may impair insulin secretion and further promote beta-cell apoptosis, because high glucose levels desensitize β cells ambient glucose levels, cause β- cell dysfunction and beta-cell apoptosis (glucose toxicity).

[0031] Patients with type 2 DM may present with symptomatic hyperglycemia but are often asymptomatic, and their condition is detected only on routine testing. In some patients, initial symptoms are those of diabetic complications, suggesting that the disease has been present for some time. In some patients, hyperosmotic coma occurs initially, especially during a period of stress or when glucose metabolism is further impaired by drugs, such as corticosteroids.

[0032] DM is indicated by typical symptoms and signs and confirmed by measurement of plasma glucose. Measurement after an 8- to 12-h fast (fasting plasma glucose [FPG]) or 2 h after ingestion of a concentrated glucose solution (oral glucose tolerance testing [OGTT]) is best. OGTT is more sensitive for diagnosing DM and impaired glucose tolerance but is less convenient and reproducible than FPG.

[0033] In practice, DM or impaired fasting glucose regulation is often diagnosed using random measures of plasma glucose or of glycosylated hemoglobin (HbA1 c). A random glucose value > 200 mg/dL (> 1 1 .1 mmol/L) may be diagnostic, but values can be affected by recent meals and must be confirmed by repeat testing; testing twice may not be necessary in the presence of diabetic symptoms. HbA1 c measurements reflect glucose levels over the preceding 2 to 3 mo. Values > 6.5 mg/dL indicate abnormally high plasma glucose levels. However, assays and reference ranges are not yet standardized, and values may be falsely high or low. For these reasons, HbA1 c is not yet considered as reliable as FPG testing or OGTT for diagnosing DM and should be used mainly for monitoring DM control.

[0034] Risk factors for type 2 DM include age > 45; obesity; sedentary lifestyle; family history of DM; history of impaired glucose regulation; gestational DM or delivery of a baby > 4.1 kg; history of hypertension or dyslipidemia; polycystic ovary syndrome; and black, Hispanic, or American Indian ethnicity. Risk of insulin resistance among overweight patients (body mass index≥ 25 kg/m2) is increased with serum triglycerides≥ 130 mg/dL (≥ 1 .47 mmol/L); triglyceride/high density lipoprotein (HDL) ratio≥ 3.0 (≥ 1 .8); and insulin≥ 108 pmol/L. People with these characteristics are at particularly high risk and should be screened for DM with a fasting plasma glucose level at least once q 3 yr as long as plasma glucose measurements are normal and at least annually if results reveal impaired fasting glucose levels.

[0035] "Inhibiting" the onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.

[0036] "Treating" a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself. As used herein, ameliorating a disorder and treating a disorder are equivalent.

[0037] The administration of the agents can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermal^, intraperitoneal^, intramuscularly.

[0038] Melatonin has the structure as shown:

melatonin

[0039] Methods of the invention employ the use of oral melatonin supplementation or melatonin receptor agonist administration to increase nightly melatonin levels and activate pancreatic beta-cell receptors which result in preservation of beta-cell mass and function. In some embodiments of the invention, an individual is assessed for development of T2DM, including early stages of the disease, e.g. increased insulin resistance, loss of beta-cell function, hyperglycemia, and the like. In some cases a patient is diagnosed as having impaired glucose regulation and has fasting plasma glucose level of 5.6-6.9 mmol/L and/or oral glucose tolerance test glucose levels of 7.7-1 1 .0 mmol/L. A patient may be diagnosed as susceptible to the development of T2DM based on body mass index≥ 25 kg/m ² ; serum triglycerides≥ 130 mg/dL (≥ 1 .47 mmol/L); triglyceride/high density lipoprotein (HDL) ratio≥ 3.0 (≥ 1 .8); and/or insulin≥ 108 pmol/L. A patient may be diagnosed as diabetic based on blood glucose levels of > 1 1 .1 mmol/L; HbA1 c measurements > 6.5 mg/dL. [0040] An individual having one or more of these indicia may be treated with an oral melatonin supplementation or melatonin receptor agonist, e.g. by daily oral administration. Preferably the therapeutic agent consists of oral melatonin supplementation or melatonin receptor agonist in a suitable excipient, at a dosage range of from 0.1 to 100; 0.1 to 1000 mg per patient per day. The individual thus treated may be monitored for clinical factors including, without limitation, blood glucose, HbAl c levels, insulin levels, and the like to determine the efficacy of treatment, where maintenance or reduced rate of loss of function is indicative that the treatment is effective.

[0041] Treatment may be maintained for periods of time, e.g. daily administration for at least one week, at least two weeks, at least one month, at least two months, at least three months, or longer, e.g. at least six months, at least one year, and the like. Following a course of treatment the individuals' beta cell activity may be monitored at time points of interest, e.g. following a time course as set forth above. Indicia of beta cell function may conveniently include determination of serum insulin levels following an oral glucose challange, determination of blood glucose levels, determination of HbAl c levels, and the like, where the levels are within about 50% of the levels prior to treatment, within about 75% of the levels prior to treatment, within about 85% of the levels prior to treatment, within about 95% of the levels prior to treatment, and may be equivalent to the levels prior to treatment, indicating a maintenance of beta cell function.

[0042] In one embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly useful are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. [0043] The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.

[0044] The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges. It is recognized that compositions of the invention when administered orally, should be protected from digestion.

[0045] The compositions for administration will commonly comprise an agent in a powdered formulation, e.g. a tablet, capsule, etc. A variety of carriers can be used. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on volumes, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g., Remington's Pharmaceutical Science (15th ed., 1980) and Goodman & Gillman, The Pharmacological Basis of Therapeutics (Hardman et al., eds., 1996)).

[0046] Thus, a typical pharmaceutical composition for oral administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art, e.g., Remington's Pharmaceutical Science and Goodman and Gillman, The Pharmacological Basis of Therapeutics, supra.

[0047] The compositions containing melatonin agents can be administered for therapeutic treatment. Compositions are administered to a patient in an amount sufficient to substantially enhance beta cell function, as described herein. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. The particular dose required for a treatment will depend upon the medical condition and history of the mammal, as well as other factors such as age, weight, gender, administration route, efficiency, etc.

[0048] An effective amount for a particular patient may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects (see, e.g., Maynard, et al. (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001 ) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

[0049] A variety of individuals are treatable according to the subject methods. Generally such individuals are mammals or mammalian, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the individuals will be humans.

[0050] Subject treatment methods are typically performed on individuals with such disorders or on individuals with a desire to avoid contracting such disorders. The invention also includes preventing or reducing the risk of a Type II diabetes by administering a pharmaceutical composition comprising a melatonin receptor agonist, including melatonin.

[0051] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to ensure accuracy with respect to the numbers used (e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees centigrade; and pressure is at or near atmospheric.

EXPERIMENTAL

[0052] Melatonin and control of circadian rhythms. During evolution, mammals developed an intricate, yet robust system for adapting to changes in light and dark. This system, termed "the circadian rhythm" allows the organism to adapt its internal metabolism to changes in the external environment. It creates daily circadian oscillations in many known physiological functions such as cardiovascular function, thermoregulation, glucose and lipid metabolism and cell turnover. The so-called "master clock regulator" of the circadian system is located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus and has long been believed to serve as a pacemaker to regulate circadian oscillations throught the body. One of the primary ways that SCN regulates daily circadian function is through the circadian release of hormone melatonin, which is synthesized and secreted from the endocrine cells located in the pineal gland. The neurons of the SCN are entrained to the light-dark cycle by light cues carried from a subgroup of retinal ganglion cells, which serve as sensory inputs to the SCN by the retinohypothalamic tract. Consequently, the SCN regulates melatonin synthesis/secretion through the "darkness-induced" increase in the noradrenergic input to the pineal gland leading to the increase in the synthesis and secretion of melatonin. Thus, melatonin secretion in mammals peaks in the night where it's concentrations are -10-15 fold higher than during the day. Melatonin also entrains daily circadian rhythms that synchronize with the light-dark cycle and has, therefore, been successfully used to treat circadian rhythm disorders such as shift-work and variety of sleep dysfunctions. In addition to its known circadian functions, recent evidence suggests that melatonin is also involved in the regulation of diverse physiological functions such as the modulation of cellular and humoral immunity, regulation of blood pressure, glucose and lipid metabolism and regulation of cell turnover.

[0053] Melatonin secretion and action in health. Melatonin's diverse physiological functions are mediated through the activation of two distinct high-affinity G-coupled receptors, the MT1 receptor (encoded by MTNR1 A gene) and MT2 receptor (encoded by MTNR1 B gene). These receptors are 60% homologous, have similar pharmacological characteristics, and are ubiquitously expressed in mammals. More recently, melatonin receptors have been reported to be expressed by beta-cells in humans and rodents, an observation we have confirmed in pancreas of humans, rodents and cultured beta-cells (Fig. 1).

[0054] The signal transduction pathways of melatonin receptor 1 and 2 vary between different tissue types and with the length of the activation, i.e. acute (<2 hours) vs. prolonged (6-16 hours) stimulation. With acute exposure to melatonin, both MT1 and MT2 receptors are coupled to pertussis sensitive inhibitory G-proteins leading to inhibition of adenylyl cyclase with consequent decreased activation of protein Kinase A (PKA) and cAMP responsive element binding protein (CREB) phosphorylation (Fig. 2). In contrast more chronic (6-16 hours) melatonin exposure has different actions. This has been best characterized in the pars tuberalis (PT), a region on the pituitary gland that like pancreatic beta-cells, has high expression of melatonin receptors. Prolonged (6-16 hours) melatonin receptor activation in the PT leads to sensitization of the cAMP-PKA-pCREB pathway once melatonin is withdrawn (Fig. 2). For example, in ovine PT cells, melatonin pre-treatment (up to 16 hr) leads to an ~a 10 fold increase in the cAMP content upon subsequent activation with cAMP-agonist forkskolin. Furthermore, increased activation of the cAMP- PKA-pCREB pathway following pre-treatment with melatonin persists up to 16hr following the removal of melatonin. Interestingly, the duration of melatonin pretreatment required to sensitize the cAMP-PKA-pCREB pathway is comparable to the period of peak melatonin secretion at night.

[0055] This melatonin-induced sensitization of the cAMP-PKA pathway has been proposed to be the key mechanism mediating melatonin receptor action in vivo. [0056] Melatonin and treatment of T2DM. Plasma melatonin levels are reduced in both humans with T2DM and in the GK rat, suggesting that hyperglycemia per se, may also lead to impaired melatonin secretion. Furthermore, ageing-associated decline in glucose tolerance and insulin secretion coincides with an exponential decline in diurnal melatonin rhythms.

[0057] Beta cell apoptosis in T2DM and the role for melatonin signaling in beta-cell survival.

The deficit in beta-cell mass and increased beta cell apoptosis is central to development of T2DM. Potential mechanisms that have been proposed for the increased beta cell apoptosis in T2DM include toxicity due to prolonged exposure to high glucose levels (or glucotoxicity), toxicity due to exposure to high levels of free fatty acids (or lipoptoxicity), and toxicity due to formation of human islet amyloid polypeptide (h-IAPP) oligomers (proteotoxicicty).

[0058] These three pro-apoptotic mechanisms induce beta-cell apoptosis through activation of the caspase family of cystine proteases, mediated by activation of pro-apoptotic and downregulation of pro-survival members of Bcl-2 family of mitochondrial proteins (Fig. 3). The Bcl-2 family consists of pro-survival proteins Bcl-2 and Bcl-xL, and pro-apoptotic members such as Bid, Bax and Bak. The interaction between pro- and anti-apoptotic proteins determines whether a cell remains viable or proceeds to apoptosis. Initiation of apoptosis requires activation of the pro-apoptotic Bid, Bax and Bak proteins, which upon translocation to the mitochondrial membrane have the potential to oligomerize and induce mitochondrial membrane pores. This leads to leakage of cytochrome c from the mitochondria to cytosol with consequent activation of the caspase protease cascade. It has been proposed that the primary function of Bcl-2 is to protect the mitochondrial membrane integrity by antagonizing Bax and Bak oligomerization. Therefore, maintaining adequate Bcl-2 expression is critical for beta-cell survival.

[0059] cAMP-responsive element-binding protein (CREB) is a transcription factor that promotes beta-cell survival via upregulation of Bcl-2 gene expression. Activation of CREB is mediated by phosphorylation of the 133-serine residue and is dependent on activation of PKA. Once activated, pCREB positively regulates the CRE site in the 5'-flanking region of the Bcl-2 gene promoter, which promotes beta-cell survival through restraint of beta-cell apoptosis. Thus, mice deficient in CREB expression develop diabetes characterized by reduced beta-cell mass and increased beta-cell apoptosis. Furthermore, expression of CREB dominant negative mutant in isolated human islets leads to increased beta-cell apoptosis, where overexpression of CREB reverses beta-cell apoptosis by raising Bcl-2 levels. In conclusion, PKA-dependent activation of CREB is critical for beta-cell survival through its actions on modulating beta-cell Bcl-2 levels. [0060] Beta-cell toxicity in response to high glucose, free fatty acids and accumulation of toxic IAPP oligomers likely each contribute to beta-cell loss in T2DM. Each of these pro- apoptotic factors ultimately induce beta-cell death via modulation of Bcl-2 family gene expression (Fig. 3).

[0061] Because prolonged exposure to melatonin has been shown to sensitize the activation of the cAMP-PKA pathway and subsequently lead to increase in pCREB expression, melatonin treatment may reduce beta-cell loss through increased expression of CREB and Bcl-2 leading to reduction in Caspase 3 activation and consequent decrease in beta-cell loss. To address this we first examined the potential of melatonin to activate PKA activity at basal and glucose-stimulated conditions. We used INS-1 beta-cell line expressing the human insulin gene to examine effects of melatonin on PKA activation by employing Phosph- (Ser/Thr) PKA substrate antibody, which has been shown to accurately detect PKA cellular substrates and thus has been used as an accurate marker of PKA activation. Overnight exposure to melatonin led to increased expression of the PKA substrate protein at both low (2.8mM) and high glucose (16.7mM) concentrations (Fig. 4A). Furthermore, overnight exposure to melatonin led to a increased expression of pCREB and p44/p42 Mitogen-Activated Protein Kinase (MAPK), both of which are PKA substrates involved in regulation of beta-cell function (Fig. 4B). Additionally, overnight exposure to melatonin led to an increased expression of pCREB in response to known activators of the cAMP-PKA pathway such as GIP and GLP-1 thus further emphasizing that exposure to melatonin increases the activation of the PKA-pCREB pathway in beta-cells (Fig. 5)

[0062] To test whether melatonin has the capacity to attenuate beta-cell apoptosis induced by h-IAPP (a toxin known to contribute to beta-cell loss in humans with T2DM), INS-1 cells were transduced with adenovirus expressing either cytotoxic human IAPP (h-IAPP) vs. non- cytotoxic rodent IAPP (r-IAPP) for 48 hours and then exposed to overnight (12-16hr) incubation with melatonin (0.1 μΜ and 1 μΜ). Overexpression of h-IAPP led to induction of beta-cell apoptosis mediated by increased expression of cleaved caspase-3 (Fig. 6A). Overnight incubation with melatonin (at both 0.1 μΜ and 1 μΜ) resulted in 40-60% suppression of beta cell apoptosis characterized by reduced caspase-3 expression in INS-1 cells transduced with h-IAPP (Fig. 6A, P<0.05 for both 0.1 and 1 μΜ melatonin). Importantly, prolonged treatment with melatonin resulted in increased expression of a key cellular pro-survival protein Bcl-2 (Fig. 6B), thus demonstrating that melatonin may antagonize the activation of the intrinsic (mitochondrial) pathway of apoptosis.

[0063] Additionally we were able to test whether activation of melatonin signaling attenuates beta-cell apoptosis in islets obtained from organ donors with T2DM. Islet cell apoptosis was determined by measuring the fractional islet area positive for propidium iodide, a known marker of cell death. Melatonin treatment led to -70% suppression of propidium-iodide estimated islet cell apoptosis (P<0.05 at both 0.1 uM and 1 uM of melatonin vs. no treatment, (Fig. 7).

[0064] Deficit in insulin secretion is an early and progressive abnormality in T2DM. In T2DM, insulin secretion is decreased due to attenuation in the magnitude of insulin secretory burst size (pulse mass) and the orderliness of insulin secretion. In beta-cells, elevation in cAMP levels leads to activation of protein kinase A and a recently identified cAMP-dependent guanine nucleotide-exchange factor EPAC1 . PKA and EPAC both enhance insulin secretion by promoting translocation of insulin granules to the surface membrane and by enhancing the orderliness of insulin release. Thus, potentiation of insulin secretion through activation of the cAMP-PKA-EPAC1 pathway is an attractive therapeutic strategy for T2DM.

[0065] In a variety of cell types, prolonged exposure to melatonin (6-16 hr, mimicking the nightly exposure of cells) increases both basal and receptor-stimulated activation of the cAMP-PKA pathway by enhancing both intracellular cAMP levels and PKA activation. In health, overnight exposure to melatonin may potentiate and subsequently primes the beta- cell for subsequent activation of the cAMP-PKA pathway, which would enhance insulin secretion through increase in insulin secretory burst size and orderliness of insulin release. Thus in health melatonin, among other insulin secretion potentiating hormones (e.g. GLP-1 and GIP), appears to play an important role in potentiating insulin release. On the other hand, in T2DM, deficiency in diurnal melatonin release as a consequence of hyperglycemia, circadian rhythm disruptions, and/or aging contributes to the deficit in beta-cell function. Therefore melatonin would enhance insulin secretion by sensitizing the activation of the cAMP-PKA-EPACI pathway in islets and that these molecular events will result in amplification of insulin secretory pulses as well as promoting the orderliness of insulin release.

[0066] To address this we examined pulsatile insulin secretion in isolated human islets utilizing previously validated islet perifusion method. Insulin secretory pulse mass and the orderliness (analyzed with approximate entropy (ApEn)) were examined by deconvolution analysis of min-by-min sampled insulin concentration profiles from human islets pre- incubated at either normal 5mM glucose, glucotoxic 16mM glucose, and glucotoxic 16mM glucose+Ι μΜ melatonin. As expected, prolonged incubation (96hr) of human islets at 16mM glucose (glucotoxicity) led to significantly diminished (-80%) insulin secretory pulse mass and disturbed regularity of pulsatile insulin release with no change in the frequency of insulin pulses as illustrated in (Fig. 8). In contrast, pre-incubation with melatonin was able to reverse deleterious effects of high-glucose on both insulin pulse mass and the orderliness of insulin secretion (Fig. 8). 7] To test whether melatonin treatment can be beneficial in preserving beta-cell function and mass in vivo in a rodent model of Type 2 diabetes, we used h-IAPP transgenic rats at 9 months of age. This is the age at which HIP rats display impaired fasting (125 mg/dl) and postprandial (170mg/dl) glucose levels thus recapitulating early development of Type 2 diabetes in humans. We intervened at this stage and treated one group of rats with daily melatonin administration (3 mg/kg/day) and one group with saline as control for 10 weeks. While saline treated rats became progressively more hyperglycemic, daily melatonin treatment prevented further deterioration in both fasting and postprandial glucose levels (Fig. 9A and B). The beneficial effect of melatonin on prevailing glucose concentrations was associated with preservation of beta-cell mass due to decrease in beta- cell apoptosis (Fig. (C and D).