Prevention and Treatment of Obesity and Diabetes and their Related Complications

Approximately 26 million patients in the United States are afflicted with Type 2 or Non-Insulin-Dependent Diabetes Mellitus (NIDDM) (National Diabetes Fact Sheet, Jan 26, 2011). Worldwide, 370-382 million patients are estimated to be diabetic [1,2]. The major defect in patients with type 2 diabetes (T2D) is the functional impairment of insulin action on peripheral tissues to stimulate glucose uptake, to decrease gluconeogenesis, and to inhibit hepatic glucose output. Although these defects are caused by both genetic and environmental factors, the ultimate outcome of diabetes is largely induced by environmental factors [3]. Insulin-mediated signal transduction mechanisms are extremely complex, involving more than 100 proteins and enzymes [4,5]. Parenteral insulin or oral hypoglycemic agents (to stimulate pancreatic insulin secretion), and metformin (to inhibit hepatic glucose output) are effective only for lowering blood glucose levels. Metformin can be toxic to humans [6]. Rosiglitazone and pioglitazone are thought to improve insulin sensitivity, but these agents pose possible cardiac enlargement, liver damage or bladder cancer [7,8]. Glucagon-like protein-1 (GLP-1) has been shown to reduce Hemoglobin (Hb) A1c levels by 0.98% during a 16 weeks trial, but was found to induce gastric discomfort [9]. It also has serious disadvantages to use for routine therapy due to parenteral treatment for low efficacy benefit [10]. Dipetidyl peptidase-4 (DPP-4) degrades GLP-1, and its inhibition increases circulating GLP-1. Thus, DPP4 inhibitor treatment resulted in decreased HbA1c by 0.39 to 0.56% (p<0.05) and fasting plasma glucose 11.0 to 17.2 mg/dL (p<0.05) in a 12 week randomized, double blind study with 100 mg daily dose [11]. DPP-4 inhibitor plus 2000 mg metformin treatment decreased HbA1c levels 2.07% whereas metformin treatment alone only reduced 1.3% [12]. Although the efficacy is not great, no serious side effect with the use of DPP-4 inhibitor was reported.


Introduction
Approximately 26 million patients in the United States are afflicted with Type 2 or Non-Insulin-Dependent Diabetes Mellitus (NIDDM) (National Diabetes Fact Sheet, Jan 26, 2011). Worldwide, 370-382 million patients are estimated to be diabetic [1,2]. The major defect in patients with type 2 diabetes (T2D) is the functional impairment of insulin action on peripheral tissues to stimulate glucose uptake, to decrease gluconeogenesis, and to inhibit hepatic glucose output. Although these defects are caused by both genetic and environmental factors, the ultimate outcome of diabetes is largely induced by environmental factors [3]. Insulin-mediated signal transduction mechanisms are extremely complex, involving more than 100 proteins and enzymes [4,5]. Parenteral insulin or oral hypoglycemic agents (to stimulate pancreatic insulin secretion), and metformin (to inhibit hepatic glucose output) are effective only for lowering blood glucose levels. Metformin can be toxic to humans [6]. Rosiglitazone and pioglitazone are thought to improve insulin sensitivity, but these agents pose possible cardiac enlargement, liver damage or bladder cancer [7,8]. Glucagon-like protein-1 (GLP-1) has been shown to reduce Hemoglobin (Hb) A1c levels by 0.98% during a 16 weeks trial, but was found to induce gastric discomfort [9]. It also has serious disadvantages to use for routine therapy due to parenteral treatment for low efficacy benefit [10]. Dipetidyl peptidase-4 (DPP-4) degrades GLP-1, and its inhibition increases circulating GLP-1. Thus, DPP-4 inhibitor treatment resulted in decreased HbA1c by 0.39 to 0.56% (p<0.05) and fasting plasma glucose 11.0 to 17.2 mg/dL (p<0.05) in a 12 week randomized, double blind study with 100 mg daily dose [11]. DPP-4 inhibitor plus 2000 mg metformin treatment decreased HbA1c levels 2.07% whereas metformin treatment alone only reduced 1.3% [12]. Although the efficacy is not great, no serious side effect with the use of DPP-4 inhibitor was reported.
More than 80% of the US population are either overweight or obese [13] with more than 80% of diabetic subjects overweight or obese [14,15]. Anti-obesity drugs are only minimally effective and pose moderate to severe side effects. It has been reported that a body weight gain of 11-16 pounds increases the incidence of T2D more than twice that of normal subjects while a gain of 17-24 pounds increases the risk thrice that of normal subjects [16]. Obesity and diabetes are the leading major causes of fatal health complications including hypertension, high cholesterol, stroke, heart disease, arthritis, and certain cancers [17]. Alzheimer's disease [18,19] and osteoporosis [20,21] are also diabetes-and obesity-related complications. Extensive counseling for diet and exercise improves glucose metabolism, lipid levels, and blood pressure [22]. The most effective method for preventing and treating these metabolic diseases is to educate patients to use beneficial dietary habits along with an exercise plan and to use medication to improve insulin resistance. Here we discuss the mechanism of a medication for diabetes and obesity including its availability, benefits and side effects. We also introduce our new drug development plan for the use of Cyclo (His-Pro) plus zinc (Cyclo-Z) for the prevention and treatment of diabetes and obesity.

Materials and Methods
Literature searches were conducted using the PubMed (http:// www.nih.gov/pubmed ) and Google websites (www.google.com ) with the following terms, key words, or phrases: Prevalence of diabetes and obesity, fact sheets of diabetes and obesity, diabetes drugs, obesity drugs, diabetes and obesity treatments, diabetes and obesity complications, diabetes and obesity co-existing diseases, relationship between diabetes and obesity, relationship between diabetes, obesity, Alzheimer's disease and osteoporosis. Clinical recommendations were synthesized based on good scientific bases from the published literature.

Diabetes
It was estimated that 2.8% of the world population was diabetic in 2000 and predicted to reach 4.4% by 2030 [23]. The risk for developing diabetes for individuals born in the year 2000 is 32.8% for males and 38.5% for females in their lifetime [24]. About 800,000 people are newly diagnosed with diabetes each year, and 200,000 people die annually from diabetes-related complications [25]. Mortality from diabetes is approximately 71,300 people each year in the US and more than 3.4 million people worldwide [26]. Global diabetes-caused deaths are also likely to increase with more than 80% of diabetes mortality occurring in low and middle income countries [27]. Total national health care expenditure in the US was $865 billion in the year 2002, of which $160 billion was incurred for the treatment of diabetes [28]. Diabetes treatment costs increased to $176 billion and loss of productivity due to diabetes was estimated to be $69 billion in the US in 2012, resulting in a total cost $245 billion. Diabetes related health expenditure was 2.3 times greater compared to the amount spent on other health related expenses of the population without diabetes (ADA report on March 6, 2013). The global healthcare expenditure on diabetes in 2010 was estimated to be $376 billion and expected to increase to $490 billion in 2030 [29]. The drug market for diabetes treatment is estimated to be over $20 billion per year in the US alone [30].

Regulation of glucose homeostasis
Blood glucose control mechanisms are outlined in Figure 1. When glucose is absorbed through the small intestine, it is supplied to all cells through the vascular system. Pancreatic beta cells are stimulated to secret insulin, and glucose is absorbed by all cells even those without insulin-mediated uptake systems. Only muscle and fat cells require insulin for glucose uptake. Insulin also inhibits hepatic glucose output. When blood glucose is low, pancreatic alpha cells secret glucagon which stimulates hepatic glucose output. Thus, the body is able to maintain normal blood glucose levels even in the starved state. Under certain conditions such as aging, muscle cells begin to require more insulin to reach the same level of normal glucose uptake. This condition is called pre-diabetes which occurs 10-15 years prior to development of full blown diabetes and is characterized by blood glucose levels that are higher than normal even though pancreatic production and secretion of insulin is at 100% capacity.

Mechanisms of insulin activity
When insulin binds to the α-subunit of the insulin receptor (IRα), the β-subunit (IR-β) is auto-phosphorylated to activate its kinase activity ( Figure 2). This results in a signaling cascade that leads to glucose uptake. Insulin receptor substrates (IRS) associated with src homology 2 (SH2) activate the enzyme phosphatidylinositol 3-kinase (PI-3-kinase) which then activates Akt/protein kinase B (PKB), protein kinase C (PKC) and several unknown factors to synthesize or translocate the glucose transporter-4 (GLUT-4) to the cell membrane Figure 1: Blood glucose control mechanisms. When blood glucose is absorbed through the small intestine, glucose uptake activates pancreatic β-cells to synthesize and secrete insulin. Insulin signals muscle and fat cells to uptake glucose by inducing expression and translocation of the GLUT-4 transporter to the cell membrane. Insulin also blocks hepatic glucose output when blood glucose levels are higher than the normal range. When blood glucose levels are lower than the normal range, pancreatic α-cells produce glucagon and stimulate the liver to produce glucose. When insulin resistance and diabetes develop, insulin sensitivity in the insulin requiring cells are decreased, and insulin production in the pancreas will also stop in the severe diabetic condition.
2. Short acting insulin: To use 30 minutes before a meal to prevent blood glucose elevation after food consumption. This type of insulin is may also be used with longer-acting insulin Regular (R).
3. Intermediate acting insulin: This type of insulin is used with rapid-or short-acting insulin. It is normally used twice a day. NPH (N) is a prototype of intermediate acting insulin.
4. Long acting insulin: This type of insulin is also used with rapid or short acting insulin.

Inhalation insulin powder: Human insulin powder (Exubera®) is inhaled instead of subcutaneous injection and it is a rapid acting insulin.
Sulfonylurea derivatives: Sulfonylurea compounds have been the backbone of oral hypoglycemic therapy in T2D treatment for more than 50 years. These drugs work by discharging more insulin from the pancreas. Sulfonylurea binds to an ATP-dependent potassium (K + ) channel on the cell membrane of pancreatic beta cells. This binding action induces the depolarization of the cell membrane resulting in the opening of voltage-gated calcium channels. The rise in intracellular calcium increases fusion of insulin granules with the cell membrane resulting in increased insulin secretion. A potential side effect is hypoglycemia. Sulfonylurea derivatives are also teratogenic and therefore cannot be used in pregnant women. However, the side effects are relatively minor compared to other anti-diabetes drugs. There are five first generation drugs and eight second generation sulfonylurea drugs. The major sulfonylurea drugs are Glyburide®, Glimepiride®, Glipizide®, and Gliclazide®.

Meglitinides:
Meglitinides are a new class of drugs (phenylalanine derivative) which act similar to sulfonylurea compounds on insulin secretion. There are two commonly prescribed formulations of these drugs: Nateglinide (Starlix®) and repaglinide (Prandin®) which are rapidly absorbed within one hour and effective in lowering postprandial blood glucose. Therefore, it is recommended to administer the drugs 1-30 min before each meal. The normal dosage of nateglinide is 60-120 mg, 3 times daily before each meal. Repaglinide dosage is 0.5-2 mg before each meal with a maximum dosage of 16 mg/day. Nateglinide is the only D-phenylalanine derivative currently approved by the FDA.
Biguanides: Metformin is a biguanide available under several brand names: Glucophage®, Glucophage XR®, Fortamet®, Glumetza®, and Riomet® in 500, 850, and 1000 mg dosage units. It is recommended to take 500 mg twice a day or 850 mg once daily with meals. The maximum dosage for an adult is 2550 mg daily. Glucophage XR®, Fortamet®, and Glumetza® are extended release formulas. Riomet® is a liquid form. Five mL of Riomet® is equal to a 500 mg tablet form of metformin. The main effects of this agent are to acutely decrease hepatic glucose output by inhibiting mitochondrial respirator chain reactions. This causes hepatic energy production resulting in activation of AMP-activate protein kinase (AMPK). The exact mechanism by which metformin increases AMPK remains uncertain although it has been suggested that it does this by inhibiting glucagon-induced elevation of cyclic-AMP resulting in activation of protein kinase A [32,33]. Activation of AMPK requires an increase of small heterodimer partner (SHP), which in turn inhibits the expression of the hepatic gluconeogenic genes, phosphoenolpyruvate carboxy kinase (PEPCK), and Glucose-6 phosphatase (Glc-6-Pase) [34]. Metformin exhibits When insulin binds to the α-subunit of the insulin receptor located outside of cell membrane, it induces auto-phosphorylation of the β-subunit generating protein, lipid, and glycogen synthesis, growth, and gene expression. At the same time the signal proceed through the phosphoinositol 3' kinase, atk/protein kinase B, and protein kinase C pathways eventually inducing expression and translocation of the glucose transporter-4 (GLUT-4) to the cell membrane. GLUT-4 can then transport glucose across the cell membrane. If these signaling processes are interrupted, insulin resistance and diabetes will be induced. some toxicity to humans [6] with the most common adverse side effects being gastrointestinal symptoms, including diarrhea, cramps, nausea, vomiting, and increased flatulence [35]. Lactic acidosis can be fatal with metformin treatment at a frequency of 0.06 per 1000 patients per year [36].
Thiazolidinediones: Thiazolidinediones (TZDs) are novel treatment modalities for T2D. Fasting blood glucose and HbA1c can be improved on average about 40 mg/dL and almost 1%, respectively. Three compounds of this class troglitazone (Rezulin®), rosiglitazone (Avandia®), and pioglitazone (Actos®) have been approved by the FDA. These drugs are potent agonists for the peroxisome proliferatorsactivated receptor-gamma (PPARγ) receptor. Activation of the PPARγ nuclear receptor stimulates gene expression and synthesis of the glucose transporter -4 (GLUT-4). Translocation of GLUT-4 to the cell membrane stimulates glucose uptake by muscle and fat cells. This class of drug has little effect on hepatic glucose output but lowers plasma insulin levels and therefore improves insulin sensitivity in the muscle cells [37]. GLUT-4 gene expression can also stimulate gene expression of other unwanted genes. While TZDs are excellent anti-diabetic agents, they can pose moderate to severe untoward reactions. The first generation TZD, Rezulin®, was marketed in 1999 by Parks and Davis Pharmaceutical Co. One million patients used this drug and it was fatal in about 80 subjects. Avandia® entered the market in 1999 and almost 4 million people in the US used it. Although it did not cause any fatal incidences, moderate to severe side effects associated with congestive heart failure, pulmonary edema, cardiac hypertrophy, and liver damage were documented [7,8,37,38]. It was estimated that the risk of hip and non-vertebral osteoporotic fracture increased 2-3 fold compared to those who did not take Avandia® [39], and drug intake was correlated with liver failure and severe hepatotoxicity [40]. Peripheral edema was shown to increase 3-fold with Avandia® treatment in comparison to control [41,42]. Macular edema incidence was also high with the consumption of Avandia® [43,44]. In addition, upper respiratory tract infection, headache, and cough symptoms were also noted. Similar adverse reactions and a link with bladder cancers were also found to be associated with Actos® [45,46].

Alpha-glucosidase inhibitors:
Alpha-glucosidase inhibitors (AGIs) are saccharides that competitively bind to the membrane bound alpha-glucosidase. This binding inhibits carbohydrate digestion and conversion to glucose that can be easily absorbed by the small intestine. Hence, AGIs reduce glucose absorption and blood glucose levels. In healthy subjects, the AGIs are not only effective in delaying carbohydrate digestion, but are also effective in slowing down metabolic fermentation of carbohydrates in the colon [47] acarbose (Precose®) is the most widely prescribed drug. Other AGIs include miglitol (Glyset®) and voglibose (Volix®, Basen®). Compared to sulfonylurea, AGIs are inferior in glycemic control. However, these drugs can reduced fasting and postprandial plasma insulin and glucose levels, and are effective in decreasing HbA1c levels 0.77% with acarbose and 0.68% with miglitol treatment [48]; and may therefore delay or prevent diabetic complications [49]. It is generally recommended to start at a lower dose of acarbose and titrate up slowly to an effective dosage to avert gastrointestinal side effects found at the highest dosing.

GLP-1 and DPP-4 inhibitor: GLP-1 was effective in reducing
HbA1c levels by 0.98% during a 16 week trial, but patients did experience gastric discomfort [9]. Another disadvantage of using this agent is that it must be injected subcutaneously [10]. Both GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) are in a group of compounds called incretins, which are gastrointestinal hormones that increase insulin release from the pancreatic beta cells after eating. The mechanism of these hormones is to inhibit glucagon release from pancreatic alpha cells. The most commonly available GLP-1 product is Byetta®. There is a risk of acute pancreatitis with the use of GLP-1 [50]. GLP-1 is rapidly deactivated by DPP-4. DPP-4 inhibitors are commercialized as sitagliptin or Januvia®. The combination of 100 mg DDP-4 inhibitor plus 2000 mg metformin (Janumet®) treatment has been shown to decreased HbA1c levels 2.07% whereas metformin treatment alone only reduced HbA1c levels 1.3% (11). Since there is no serious side effect associated with the ingestion of DPP-4 inhibitor, this agent is considered a novel anti-diabetes agent for blood glucose control.
Bile acid sequestrants (BAS): BAS have been approved by the FDA for both glycemic and lipid control [51]. BAS are associated with the liver X receptors (LXR) and can lower blood glucose levels [52]. LXR activation improves insulin sensitivity by inhibiting the activity of PEPCK and G6Pase and therefore prevents gluconeogenesis [53]. LXR also improves glucose metabolism by promoting glucokinase and GLUT-4 gene expression in adipocytes [54] and by promoting insulin secretion from the islet beta cells [55]. Bile acids are natural ligands for the Farnesoid X receptor (FXR) and its activation in the liver increases the production of small heterodimer partners (SHP), which increases glucose metabolism by inhibiting production of PEPCK [56]. FXR activation also decreases blood glucose in rats [57]. Thus, FXR deactivation induces LXR activation and appears to have competitive effects on PEPCK. Also, LXR activity may overcome the effects of FXR deactivation [58]. BAS may also bind fat-soluble vitamins (Vitamins A, D, E and K) preventing uptake by the body [59].

Obesity
More than 80% of diabetic subjects are overweight or obese [14,15]. It has been reported that a body weight gain of 11-16 pounds increases the incidence of T2D more than twice that of normal subjects while a gain of 17-24 pounds increases the risk three times that of normal subjects [16]. It was also found that extensive counseling for diet and exercise can improve weight loss in obese adults, and improved glucose metabolism, lipid levels, and blood pressure. The prevalence of obesity doubled among adults between 1980 and 2004, and doubled again in between 2007 and 2008. Currently, the prevalence of obesity is 32.2% among adult men and 35.5% among adult women [60]. Obesity is associated with an increased risk of diabetes, cardiovascular disease [61], and is a major cause of morbidity and mortality in the US [14]. Each year, approximately 300,000 US adults die from obesity-related causes [14].
The estimated medical cost of obesity in the US was $78.5 billion in 1998 and increased to $147 billion in 2008 [62], and consumers spend about one-third of this amount for weight loss products and services [63]. The medical costs associated with obesity represents 9.1% of annual medical healthcare spending [63]. Healthcare costs for obese adults are 56% higher than for normal weight adults. In a study conducted at Cornell University in 2012 and published recently in Medical Daily, it was estimated that obesity now costs $190 billion and accounts for almost 31% of the US healthcare cost. In another publication of Fair Food Network published in 2013, Tang et al. estimated that obesityrelated diseases and health problems accounts for 61% of healthcare cost in the US. Obesity is a preventable disease which is the leading cause of death worldwide [64]. It is estimated to account for 111,909 to 365,000 deaths/year in the US. Large scale studies have found that mortality risk is lowest in a population of people with a BMI in the normal range (20-25 kg/m 2 ) [65,66]. On average, obesity reduces life expectancy by six to seven years. Obese subjects with a BMI of 30-35 kg/m 2 have a shortened life expectancy by two to four years and with a BMI >40 kg/m 2 , it is shortened by ten years [7,67]. The main cause of obesity is a combination of excessive food intake and a lack of physical activity due to easy access to high fat content foods and increased sedentary lifestyle which includes reliance on automobiles for even short trips and the increasing use of computers [68].
Pathophysiological mechanisms in developing obesity are attributed to hormonal effects [69]. Leptin is produced by adipose tissue and regulates fat storage in the body. It acts on a specific region of the brain, the satiety center, to regulate food intake, energy expenditure, and neuroendocrine function [70]. The leptin pathways are hormonal signaling pathways which signal from the adipose tissue to the brain to regulate energy balance [71. Human obesity is typically not due to a deficiency of leptin but due to central or peripheral leptin resistance or decreased leptin sensitivity. Leptin resistance is very similar to insulin resistance in muscle and fat cells. Thus, therapeutic augmentation of circulating leptin levels results in reduction of food intake, body fat mass, and body weight in many obese patients [72,73]. Circulating leptin levels are proportional to adiposity and increase with increasing body fat [74]. In addition to leptin, adiponectin, insulin, orexin, and ghrelin are also considered to influence appetite. Insulin also enters the brain, binds to its receptors in the hypothalamic nuclei and regulates central neural networks and energy balances. A schematic presentation of the key roles of brain insulin and leptin actions in the brains are shown in Figure 3.

Currently recognized major anti-obesity agents
Currently available drugs for the treatment of obesity either have an anorexic effect or inhibit lipase activity. Phentermine (ADIPEX-P®), diethylpropion (Tenuate®) and sibutramine (Meridia®) are anorectic agents and orlistat (Xenical®) is a lipase inhibitor. Sibutramine was withdrawn from the US market in October 2010 due to serious side effects [75]. A meta-analysis concluded that none of these drugs give more than a 4 kg (4% or 4 kg) weight loss greater than placebo [76].
Phentermine hydrochloride: It acts on the hypothalamus to stimulate adrenal glands to release epinephrine, which in turn signals reduced hunger and also induces fat cells to break down stored fat. However, the major effect is hunger reduction. Phentermine is generally considered well tolerated, although, since it acts through the sympathomimetic pathways, it can induce tachycardia, elevated blood pressure, palpitations, restlessness, and insomnia. Patients also reported dryness of mouth, unpleasant taste, diarrhea, and constipation. Long term use data showed no net weight loss. Thus, one must carefully weigh the benefits of phentermine against its side effects. It is also distributed under the tradename Adipex-P® in 37.5 mg tablets and in capsules of 15 mg, 30 mg and 37.5 mg. The time for maximum plasma concentrations after administration (T max ) is 3 to 4.4 hrs.

Diethylpropion (Tenuate®):
It is used for short-term weight reduction. It acts on the satiety center in the brain to cause a temporally reduction in appetite or hunger. This compound may indirectly elevate levels of leptin which is also known to suppress hunger. However, patients treated with diethylpropion can develop leptin resistance, in which high levels of leptin are not as active as in lean subjects. Diethylpropion stimulates the release of catecholamines, which suppress hunger signals and appetite. Catecholamine is known to halt neuropeptide Y, whose function is to initiate eating and decrease energy expenditure. Catecholamine doses for body weight control are variable. Its half-life biologically is 4 to 6 hours. Its adverse effects are tachycardia, arrhythmias, hypertension, and hypotension. It may induce dizziness, insomnia, euphoria tremor, and headache as-well-as dermatological, gastrointestinal, hematologic, and other miscellaneous minor side effects.

Orlistat (Xenical®):
It is the saturated derivative of lipstatin,  from the bacterium Streptomyces toxytricini which is an inhibitor of pancreatic lipases. It is also known as tetrahydrolipstatin [77]. It functions by preventing absorption of fats by acting as a lipase inhibitor. Patients using orlistat lose about 2-3 kg more compared to controls over the course of a year [78]. Orlistat has been shown to moderately reduces blood pressure and prevent the onset of T2D by nearly 40% in obese people [79]. It has been shown in rare cases to induce acute kidney injury, possibly due to the increased fat malabsorption, and the formation of calcium soaps resulting in increased free oxalate absorption and enteric oxalosis, thus precipitating hyperoxaluric renal disease [80].
Methamphetamines: These are central nervous system stimulants used to treat attention deficit hyperactivity disorder, narcolepsy, and treatment-resistant depression under the trade name Desoxyn® [81], and in rare cases, for the treatment of obesity; although, its effect on obesity is mild. Obesity drug safety has been a major concern with the use of methamphetamines. Lorcaserin is classified as a controlled substance Schedule IV drug, fenfluramine was withdrawn from the market due to cardiac valvulopathy, phenylpropanolamine for hemorrhagic stroke, and ephedrine for cardiovascular and neurologic side effects [76].

Rimonabant:
It is marketed in Europe and other countries. However, it was not approved in the US and has been withdrawn from the market due to the safety issue because of concerns over sucidailty, depression, and other related sides effects associated with th euse of this drug (http:// www.accompliarreport.com/News/news-061807.htm ) As Greenway and Caruso reported, excellent safe and effective anti-obesity drugs do not exist [82]. However, Bray [83] suggested that we should not abandon the currently available drugs and hope for future more effective drugs. Thus, a safe, effective, and affordable obesity medication is urgently needed.

Study Results from the New Drug Development Processing for the Prevention and Treatment of Diabetes and Obesity. 6=
Animal studies Blood glucose decreasing rates: Zinc treatment decreased fed blood glucose levels both in genetically T2D G-K rats without obesity ( Figure 4) and in genetically obese T2D mice ( Figure 5). Zinc or CHP alone was minimally effective in decreasing blood glucose levels. The optimal effective dose of CHP was 0.5 mg/L with 10 mg/L zinc, which is 0.1 mg CHP/kg BW/day based on the BW and daily water intake. This optimal dose of CHP is similar to that for the Three Hour Average above Fasting Glucose (TAFGC) reduction ( Figure 6). However, the acute optimal dose of CHP is 0.5 mg/Kg BW [84]. Blood glucose levels neither drop immediately nor do they decrease in a linear manner ( Figure 5). It was also noticed that genetically heterozygous lean mice had slightly increased blood glucose during the 3-week treatment period. This means that Cyclo-Z does not appreciably alter blood glucose levels in non-diabetic animals.

Plasma insulin level changes by ZC treatment:
Since ZC-treated diabetic rats and mice have decreased TAFGC and blood glucose levels, plasma insulin levels must increase if ZC effect is merely insulinemic. However, plasma insulin levels significantly decreased (Table 1), along with TAFGC and blood glucose levels in diabetic G-K rats (Figures 4 and 6). Zinc or CHP treatment alone significantly increased plasma insulin levels in G-K rats showing zinc and CHP are insulin secretagogues. However, CHP plus zinc treatments invariably decreased plasma insulin levels in a CHP concentration dependent   manner with a fixed amount of zinc (Table 1). This phenomenon suggests that CHP plus zinc treatment improves insulin sensitivity and plasma insulin levels are low in untreated hyperglycemic rats because prolonged hyperglycemia blocks insulin secretion from pancreatic beta cells. The similar decrease of plasma insulin levels were also observed in ob/ob mice with zinc treatment [85].
Body weight control by ZC treatment: When 10 month old G-K rats were treated with zinc only and with ZC for 4 weeks, body weight gain rates significantly decreased compared to the control (Figure 7) [86]. However, when these rats were given normal tap water for another 4 weeks; reduced body weight gain rate was still maintained only in ZC treated rats (Figure 8). These findings are similar to previous findings where improvement of insulin resistance in aged overweight S-D rats was maintained until one week after the cessation of ZC treatment [84].

Determination of optimal dose of CHP for reduction of BW:
We established the optimal dose of CHP on BW reduction with different doses of CHP (1, 3, or 6 mg/L CHP plus 10 mg/L zinc) in drinking water for 15 days in overweight (650-850 g) S-D rats. The optimal dose for the reduction of BW in these rats was 3 mg/L CHP with 10 mg zinc/L in drinking water (Figure 9), which was calculated as 0.1 mg CHP/kg BW/day, similar for glucose control (Figures 4-6).
Baseline BW-dependent BW reduction by CHP plus zinc treatment: As shown in Figure 10, BW decreased most in the rats weighing over 1000 g (2.61 ± 0.35 g/day; p<0.001), and less in the rats weighing 650-850 g (0.82 ± 0.02 g/day; p<0.05). In contrast to the weight loss seen in overweight and obese rats, lean S-D rats weighing 350-450 g slightly gained BW at a low rate although the differences were still significant (0.52 ± 0.02 g/day; p<0.001) ( Figure 10). These data suggest that severely obese subjects could have more benefit than mildly obese subjects from ZC treatment, with little or no effect on lean non-obese subjects.

CHP plus zinc treatment decreased plasma leptin levels and increased plasma adiponectin levels in obese S-D rats:
When non-diabetic obese aged S-D rats were treated with drinking water containing 3 mg/L CHP plus 20 mg/L zinc (ZC) for 4 weeks, the plasma leptin levels significantly decreased compared to controls (P<0.01) ( Figure 11A). On the other hand, plasma adiponectin levels in these rats significantly increased compared to controls ( Figure 11B). When non-obese lean diabetic mature G-K rats were treated with drinking water containing the same amount of ZC as used for the obese S-D rats, plasma leptin levels slightly decreased compared to controls (p<0.05) ( Figure 11A). However, plasma adiponectin levels did not increase in mature non-obese diabetic G-K rats ( Figure 11B). The baseline values of leptin are higher in overweight aged S-D rats than in lean non-obese G-K rats, but adiponectin levels are lower in overweight aged S-D rats than in lean non-obese G-K rats. ZC treatment reversed these trends in       overweight S-D rats. These data suggest that CHP plus zinc treatment is very effective in the treatment of obese animals. BW reduction with decreasing plasma leptin levels and increasing adiponectin levels are an indication of improvement of leptin sensitivity similar to that seen with insulin sensitivity in diabetic G-K rats treated with ZC noted above.

Stimulation of insulin degradation in the cytosol of brain tissue in human amyloid precursor protein (APP) transgenic mice model:
Insulin and amyloid proteins are degraded by Insulin degrading enzyme (IDE). Zinc is an integral part of IDE and is absolutely required for IDE enzymatic activity. IDE in the cytosol of brain samples from control animals require more than 20 minutes to degrade radiolabeled insulin whereas the cytosol from ZC treated animals degraded the majority of insulin within 15 minutes (left) (Figure 12). This is an approximately a 30% enhancement of IDE activity by ZC therapy (right) (n=6). Hence, Figure 12 supports the hypothesis that internalized insulin clearance may ameliorate diabetes.

Human Studies
Clinical trial with animal prostate substance plus zinc: Previous animal studies demonstrated that rabbit prostate extract (PE) stimulated intestinal zinc absorption and reduced blood glucose levels *P < 0.05, ***P < 0.001 from regression analyses. ** * *P < 0.05; **P < 0.01 versus controls Figure 11A: Comparison of plasma leptin levels between non-diabetic obese S-D rats and non-obese diabetic G-K rats. Fourteen 15-months-old male S-D rats weighing 650-850 g were given drinking water containing no additive or 3.0 mg CHP plus 10 mg zinc/liter and 3-month-old G-K rats were given drinking water containing either no additive or 1.0 mg CHP plus 10 mg zinc/liter for 4 weeks. Rats with the ZC treatments showed reduced leptin level in both aged overweight S-D and lean diabetic G-K rats.

Obese S-D Rats
Lean  in streptozocin-induced diabetic rats [87]. In an attempt to translate these results to humans, we performed a clinical trial with 200 mg prostate powder plus 20 mg zinc containing gel capsule (Pro-Z) with FDA IND approval (IND # 48,348) for diabetes treatment [88]. In this study, treatment of diabetic patients with 2-4 capsules of Pro-Z/day for three months significantly decreased TAFGC, HbA1c, and urine glucose levels as shown in Table 2. The patients reported no adverse side effects with Pro-Z treatment. These data demonstrate that Pro-Z is a potentially superior diabetes treatment agent compared to any of the other commercially available diabetes medications. The double-blinded study showed no adverse clinical side effects and no changes in blood chemistry data of subjects administered a onetime oral dose of 0, 2, 4, or 8 capsules of Cyclo-Z in chemistry data and blood cell counts. Although subjects (n = 12) who took 8 capsules of Cyclo-Z before breakfast showed significantly reduced blood glucose levels at 8 hours, their plasma glucose levels were within normal range [89]. Blood glucose levels at twenty-four hours had returned to baseline levels prior to Cyclo-Z intake.
Phase2a clinical trials with Cyclo-Z: A Phase 2a clinical trial with Cyclo-Z was initiated and prematurely terminated as the study subjects were mainly recruited from obese subjects with very mild diabetes (HbA1c levels between 6.1 and 8.0). In this first Phase2a trial,   improvement of diabetic parameters was significant only in those patients with basal HbA1c levels above 6.9. However, the Body Mass Index (BMI) was significantly improved in these severely obese, mildly diabetic patients during the three months trial period with Cyclo-Z.
We have recently initiating a second Cyclo-Z clinical trial to be focused on severely obese, diabetic subjects with HbA1c levels above 8.0.
These studies strongly suggest that these two drugs are potentially very desirable anti-diabetes and anti-obesity agents. Most importantly, no side effects have ever been reported in patients enrolled in the clinical trials or by consumer's correspondence after more than 10 years of use by many diabetic subjects.

Mechanisms of improving insulin sensitivity by Cyclo-Z treatment
IDE is a zinc containing enzyme and plays a major role in the regulation of internalized insulin degradation [90][91][92]. However, diabetic animals and humans are zinc deficient [93,95] due to impaired intestinal zinc absorption and hyperzincuria [95][96][97][98][99]. Insulin-bound insulin receptor is transported to the endosome where it is separated from the receptor and the inactive insulin is degraded into peptides by IDEs and the peptides will be completely degraded into amino acids by lososomal enzymes. Insulin-receptors are then recycled or degraded into amino acids resetting the signal transduction pathway ( Figure  13). If endosomal IDE levels are inadequate, undigested used insulin will remain in the cytosol preventing insulin signal transduction and synthesis or translocation of the glucose transporter-4 (GLUT-4) to the cell membrane for glucose uptake. IDE is therefore essential in maintaining insulin sensitivity [90][91][92]. Cyclo-Z enhances IDE synthesis and stimulates insulin degradation ( Figure 13). In addition, CHP enhances gene expression of factors that ameliorate diabetes and antagonizes factors that induce diabetes [100]. Although CHP or zinc alone is somewhat effective in the control of blood glucose metabolism, Cyclo-Z may be the most effective agent in ameliorating insulin resistance in diabetes and obesity since it stimulates IDE synthesis and enhances beneficial gene expression for diabetes improvement.

Mechanism of improving body weight control
Zinc deficiency results in hypoleptinemia in animals [101] and in humans [102], and supplementation with zinc increases leptin synthesis [103]. Secretion of leptin is regulated by zinc in cultured rat adipocytes [104]. Leptin deficiency is associated with hyperphagia, hyperinsulinemia, and insulin resistance [105]. Administration of leptin reverses all of these findings in rodents [106]. Although leptin deficiency from a single homozygous gene mutation is associated with being overweight in humans, mutation of the leptin gene or its receptor is clinically rare [107]. However, in most obese animal models and humans, levels of plasma leptin are elevated [108,109], and age related diet-induced obese animals and humans generally exhibit leptin resistance [110]. CHP is found at concentrations as high as 0.3% (w/w) in parts of the hypothalamus (the satiety control center of the brain), and it has a cellular and subcellular localization distinct from thyrotropine releasing hormone (TRH) [111]. More importantly, CHP stimulates intestinal zinc absorption and muscle cell zinc uptake [112]. Thus, CHP may be an effective agent that stimulates zinc metabolism and regulates metabolic activities in the control of body weight. The effects of CHP plus zinc combinations on body weight was CHP dose dependent and related to plasma leptin levels and to adiponectin metabolism ( Figure 11). Our data demonstrated that the reduction of BW induced by ZC treatment was accompanied by decreased plasma leptin levels. These observations suggest that ZC treatment may improve leptin sensitivity.
Treatment with CHP decreased food intake in rats, specifically fat and to some degree carbohydrate intake [113]. CHP reduces food intake by interacting with serotonin and fenfluramine in the brain, which are known anti-obesity agents [114]. Additionally, intraventricular administration of CHP has been shown to significantly reduced water intake [115]. We also found decreased water intake in older G-K and S-D rats after ZC treatment [85,86]. Young rats showed no difference in water consumption regardless of ZC addition to drinking water. Human obesity is polyfactorial not caused by a single gene mutation [116]. Environmental factors are likely to represent prime causative factors in modern obesity. Eating too many calories for one's energy needs combined with reduced energy expenditure contribute to obesity. Currently, no available drug induces loss of BW more than 4 kg in 2 years of treatment, and all current drug treatments have side effects. Our experimental data collectively suggest that Cyclo-Z treatment may help to reduce BW or BW gain without any adverse side effects. The mechanisms by which Cyclo-Z treatment improves BW control may be via reduction of caloric intake and improvement of leptin sensitivity. The potential side effects of Cyclo-Z are not well understood. Side effects of zinc are well established during the last 50 years of study. In general its side effects are irritation of stomach other than megadose of more than 1 g of zinc element which could induce serious pathological condition and fatal if taken more than 10 g at a time. CHP side effects are not well established without any report of side effects but some studies of anti-toxicity of CHP have been published [100,117,118]. The long term side effect in humans will be established in our long term clinical trial and in the toxicological studies in dogs with high dose of Cyclo-Z for one year.

Conclusions
The most important anti-diabetes agents are those treating insulin resistances in diabetic patients. Both pioglitazone and rosiglitazone are effective in improving insulin sensitivity but pose several serious side effects [119]. Recent FDA rulings require warning labels for users of Avandia® cautioning for potential hypercardia and liver damage, and for users of Actos® for bladder cancer. Most anti-diabetic drugs merely lower blood glucose temporarily, but pose moderate to serious side effects. Thus, it is highly desirable to have safe nontoxic medications for the treatment of diabetes. GLP-1 and DPP-4 inhibitor are relatively non-toxic but have drawbacks in that they must be injected subcutaneously and yet has a low effect on blood glucose reduction. Cyclo-Z is a viable diabetes treatment that is similar to thiazolidinedione derivatives which improve insulin resistance but, most importantly, Cyclo-Z poses no known short-term side effects. Safety of anti-obesity drugs has also been a major concern. Essentially all anti-obesity drugs pose moderate to serious side effects and yet the most effective agent reduces body weight by only a maximum of 4 kg in the first year. Therefore, a safe, effective, and affordable anti-obesity medication would fulfill a presently unmet medical need. If we prove that Cyclo-Z is safe and effective in the treatment of obese diabetics, Cyclo-Z may become an integral agent for treating diabetic and/or obese subjects. However, this optimism must be proven in Phases 3 and 4 clinical trials.