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INTRODUCTION

The search for an effective weight loss agent has been the focus of the general and medical community for many years. Never before have we been inundated with so many advertisements for fad diets, nutritional supplements, low calorie foods, exercise regimes and pharmaceutical drugs. Despite all of these options, the numbers of overweight and obese individuals continues to rise around the globe. Currently there are only 3 medications that are approved in the United States for the treatment of obesity: sibutramine, orlistat, and phentermine. To date, these drugs have been disappointing for the majority of users, resulting in only 5%-10% loss of body weight (1). Clearly, there is a need for novel drug therapies. In this chapter, we focus on several novel drug targets that are currently being developed, including both peripherally produced proteins and centrally expressed neurotransmitters and receptors. While by no means comprehensive, we feel that these represent the forefront of obesity drug target research.

Peripherally produced peptides

Ghrelin

Ghrelin is a hormone that is released into the circulation primarily by endocrine cells of the stomach and gastrointestinal tract (2). It was first identified in 1999 as the endogenous ligand of the growth hormone secretagogue receptor (GHSR; ghrelin receptor) and was named for its potent growth hormone-secreting properties (3). In addition to its role as a growth hormone secretagogue, ghrelin stimulates gastrointestinal motility and gastric acid secretion, affects blood pressure and heart rate, and affects the release of many hormones, including insulin (3-11). However, it is ghrelin’s ability to act in the central nervous system (CNS) to reverse states of energy insufficiency that has received the most attention (12). For instance, ghrelin levels rise prior to meals, following food deprivation and in response to weight loss resulting from many different situations, including chronic exercise, anorexia nervosa and cancer anorexia (13-18). Importantly, ghrelin administration potently stimulates feeding and lowers energy expenditure (11, 19, 20). In addition, ghrelin shifts food preference towards diets rich in fat and at the same time shifts fuel preference away from metabolic utilization of fat as an energy source (11, 21). Ghrelin also increases the mRNA expression of many fat storage-promoting enzymes in white adipocytes (22). Still other studies suggest that one mechanism by which ghrelin affects feeding and body weight is to increase the rewarding value of food via direct engagement of midbrain dopaminergic pathways originating in the ventral tegmental area and projecting to various areas in the forebrain, including the nucleus accumbens. For instance, centrally- and peripherally-administered ghrelin induces dopamine overflow in the nucleus accumbens, and ghrelin increases action potential frequency in ventral tegmental area dopamine neurons (23-25). Furthermore, direct microinjection of ghrelin into the ventral tegmental

area increases food intake while direct injection of a GHSR antagonist decreases food intake in response to intraperitoneal ghrelin (23, 26). Also, although only reported as abstracts, ghrelin has been shown to significantly increase appetitive lever pressing for food rewards by rats and mice (27, 28). The end result of all these ghrelin-induced physiologic changes and behaviors is increased body weight, with a predominant effect of increasing adiposity (11, 20).

Interestingly, in most forms of obesity, ghrelin is not presumed to be causative since its levels are usually lower than those in lean individuals (29, 30). Rather, in individuals with the “common” form of obesity, ghrelin levels become elevated only after weight loss by dieting; this elevation in ghrelin is thought to contribute to the rebound weight gain commonly observed in dieters (31). The obesity and accompanying excessive eating of Prader-Willi Syndrome (PWS), however, seem to be a unique situation (32). The hyperphagia of PWS is extreme such that PWS individuals often display a significant obsession with food, pica behavior and nearly constant hunger, as well as other disadvantageous feeding behaviors such as food stealing, stealing money to buy food, hoarding, foraging and binge eating (32, 33). A potentially significant advance in PWS research was made just a few years ago with the report of marked elevations of circulating levels of ghrelin in obese adults with PWS (34). This initial finding was confirmed in a handful of other studies on adult PWS individuals as well as in obese children and teenagers with PWS (34-38). In fact, plasma ghrelin levels in obese PWS individuals have been found to exist at levels 3 to 4.5-fold higher than obese controls (34-38). Furthermore, ghrelin cell density is higher in the stomachs of PWS individuals as compared to obese control individuals (39). It has been postulated that these high ghrelin levels directly contribute to the voracious appetite, hyperphagia, obesity and extreme food-seeking behaviors that characterize this syndrome (34, 36).

Ghrelin’s mechanism of action on body weight likely involves interactions with its receptor in many areas of the CNS. Many of the initial studies have focused on the role of the orexigenic NPY/AgRP neurons present in the arcuate nucleus (Arc) of the hypothalamus as being a key target for ghrelin action. Consistent with this notion, ablation of the Arc with monosodium glutamate significantly blunts the ingestive behaviors normally stimulated by central delivery of ghrelin, while microinjection of ghrelin directly into the Arc in non-monosodium glutamate-treated brains stimulates food intake (20, 40, 41). Ghrelin and/or GHSR agonists induce c-fos and augment NPY and AgRP transcription in NPY/AgRP neurons, stimulate [Ca2+]i increases in isolated arcuate NPY/AgRP neurons, and directly depolarize these NPY/AgRP neurons, all of which indicate that ghrelin activates NPY/AgRP neurons (42-49). Also, a few studies suggest that extra-arcuate nucleus neurons are direct targets of ghrelin. These neurons include ones that comprise the dorsal vagal complex of the caudal brainstem. Ghrelin receptor mRNA is expressed in and ghrelin has been shown to induce c-fos in all three branches of the dorsal vagal complex (47, 50) (51). Additionally, delivery of ghrelin to the caudal brainstem, via injection into the 4th ventricle or direct

microinjection into the dorsal vagal complex, increases cumulative food intake, number of meals and size of meals during the first few hours after treatment and decreases the time till first meal onset (52).

Several studies now support a model that predicts a physiologically relevant role for naturally-occurring ghrelin in the complex circuitry responsible for coordinated body weight control. Such a notion had been challenged in the first published studies using ghrelin- and GHSR-knockout mice, in which no or only modest differences in body weights were noted between mice lacking ghrelin or the ghrelin receptor and wild-type animals (53-55). However, several recent papers support the idea for a requirement of intact ghrelin signaling for normal body weight homeostasis and the development of diet-induced obesity. For example, in our own study using GHSR-null mice, we found that ghrelin receptor deficiency was associated with reduced body weight in animals exposed to either high-fat diet (both males and females) or standard chow (females only); this reduced body weight was due to selective decreases in adiposity, and was associated with both reduced feed efficiency (defined as weight gain per energy consumed) and reduced food intake (56). Wortley et al. demonstrated that mice genetically deficient in ghrelin were leaner than wild-type mice after early exposure to high-fat diet; this was due to an effect on adiposity alone (and not lean mass) and was the result of increased energy expenditure, without any changes in food intake when studied over the short-term (57). Selective knockdown of GHSR expression in transgenic rats expressing an antisense GHSR transcript (under the control of a tyrosine hydroxylase promoter) also resulted in decreased adiposity and reduced food intake (58).

Perhaps even more exciting – especially in the context of practical, non-surgical treatment strategies for obesity – are two recent studies which have examined the effect of reducing the bioavailability of naturally occurring ghrelin (59, 60). In the first of these studies, a polyethylene glycol-modified L-RNA oligonucleotide (NOX-B11-2) that was designed to specifically bind to acylated ghrelin with high affinity was introduced to diet-induced obese mice. This compound resulted in weight loss and reductions in adiposity, food intake and feed efficiency (59). In the second of these studies, a vaccination approach was taken in order to reduce ghrelin bioavailability (60). The vaccines consisted of molecules mimicking the structure of the acylated form of ghrelin and resulted in the production of antibodies that specifically recognized n-octanoyl-modified ghrelin. Animals that developed high anti-acylated ghrelin antibody titers had a decreased ratio of brain/plasma total ghrelin levels. Importantly, high anti-acylated ghrelin antibody titers were associated with decreased body weight gain, decreased adiposity and decreased feed efficiency. There did not appear to be an effect on reducing food intake within the short observation period of the study.

The effect of pharmacologic blockade of GHSR using small molecule antagonists has also been examined. For instance, one such compound currently in development, when delivered orally to high-fed diet-fed mice, was reported to

cause a sustained decrease in body weight without a change in food intake (61). These animals also had a 40% reduction in liver fat content and improved insulin sensitivity. In a separate study, daily oral administration of another GHS-R1 antagonist to diet-induced obese mice led to reduced food intake and weight loss (up to 15%) due to selective loss of fat mass (62). In this study, pair-feeding experiments indicated that the observed weight loss was largely a consequence of reduced food intake.

Although these studies used different methods of inactivating normal ghrelin signaling pathways, they all had in common decreased body weight (with a specific effect on fat mass and no effect on lean mass); the effects on energy expenditure and food intake were variable, although the methodology by which these were measured were not standardized among the studies. As such, inhibition of ghrelin action has been touted and is currently being actively developed as a feasible strategy to reduce body weight and food intake (63, 64).

Preproglucagon derivatives

Glucagon-like peptide-1 (GLP-1) is an incretin hormone derived from the larger preproglucagon prohormone (65). It is produced by the endocrine L cells of the distal small intestine and colon and also by neurons found within the nucleus of the solitary tract, the dorsal and ventral medulla and the olfactory bulb (65, 66). A long-lasting analog of GLP-1, exenatide, is currently marketed in the United States to improve glycemic control in individuals with Type 2 diabetes mellitus. GLP-1’s and exenatide’s glucoregulatory actions include augmentation of both first-phase and second-phase insulin secretion (67). Sitagliptin, an inhibitor of the dipeptidyl peptidase IV which normally contributes to a rapid degradation of endogenous GLP-1, is also now available in the United States for blood glucose control in Type 2 diabetes mellitus (68-70).

An attractive feature of GLP-1 mimetics for diabetics using these agents for their glucoregulatory properties is their ability to affect food intake and body weight. The effects of GLP-1 on food intake have been reported in several animal studies. For instance, six days of daily intracerebroventricular delivery of GLP-1 was shown to significantly reduce food intake and body weight whereas similar infusion of a GLP-1 receptor antagonist was shown to have the opposite effects (71). GLP-1 also reduces gastric acid secretion and delays gastric emptying both when given centrally or peripherally (65, 72). More chronic treatment with exenatide or other long-acting GLP-1 mimetics also has been shown to reduce food intake, body weight and fat deposition in several different rodent models of obesity (73-76). Furthermore, one of these GLP-1 mimetics was shown to shift the food preference of rats exposed to both candy and chow away from candy and towards chow (76).

GLP-1’s effectiveness in reducing food intake has also just recently been reported in another animal model -- obese Göttingen minipigs (77). In this latter

study, a new, once-daily human analog of GLP-1, named Liraglutide, was administered subcutaneously (s.c) once daily to the minipigs. It was shown to strongly suppress (by > 60%) food intake throughout the 7-week treatment period, without any indication of desensitization. There was a trend towards a reduction in meal number and a significant decrease in average meal size and meal duration. In addition, body weight decreased significantly (4% – 5%). Upon discontinuation of the treatment, food intake returned to its pre-treatment levels. The researchers noted that minor signs of nausea and discomfort were observed only during the first three days of treatment, but that afterwards, there were no visible or measurable side effects (77).

Clues as to GLP-1’s mechanism of action come from the distribution of GLP-1 receptors, which involves multiple sites within the CNS known or postulated to be involved in body weight homeostasis, including several hypothalamic nuclei (such as the arcuate nucleus, the paraventricular nucleus, and the dorsomedial nucleus), all three branches of the dorsal vagal complex, and the ventral tegmental area (66, 78). Central administration of GLP-1 to rats induces c-fos protein in several areas important in body weight homeostasis, including the paraventricular hypothalamic nucleus, the central nucleus of the amygdala, the supraoptic nucleus, the arcuate nucleus, the area postrema and the nucleus of the solitary tract (79, 80). Direct injections of GLP-1 into the paraventricular hypothalamic nucleus has been shown to cause anorexia without concomitant taste aversion (79). Interestingly, exentaide’s anorexic actions may also involve interactions with ghrelin. As such, exentatide was recently described to significantly reduce ghrelin levels in fasted rats by up to 74% (81).

Several studies have now shown that exenatide has similar food intake- and body weight-reducing actions in humans as it does in animal models. For instance, in several of the initial long-term trials set-up primarily to examine the effects of exenatide on glycemic control, body weight reduction also was observed (82, 83). Interim analyses of studies in which individuals received exenatide during a 30-week placebo-controlled trial and then subsequently for 52 weeks in an open-label extension study demonstrated progressive reduction in weight over the 82 weeks and statistically significant improvement in some cardiovascular risk factors (84, 85).

It is interesting that another peptide derived from the preproglucagon prohormone, oxyntomodulin, which also works at least in part through interaction with GLP-1 receptors, also has weight-reducing properties (86). For instance, peripheral administration of oxyntomodulin can dose-dependently inhibit both fast-induced and dark-phase food intake without delaying gastric emptying (87). Furthermore, prolonged central administration of oxyntomodulin to rats also reduced body weight to a greater degree than that observed in pair-fed control mice, suggesting effects on behaviors and processes other than simply food intake (88). Similar effects have been observed in both lean and obese humans (89, 90). For instance, a 4-week-long, double-blind, randomized, controlled trial

in overweight and obese subjects demonstrated an oxyntomodulin-induced significant decrease in body weight, which on average amounted to an additional 0.45 kg weight loss per week as compared to the placebo control group (90). Energy intake was significantly reduced by oxyntomodulin, and was maintained over the 4-week duration of the trial, without any change in subjective food palatability (90).

Thus, research trials are ongoing in order to obtain approval for the use of these two proglucagon-derived hormones specifically in the treatment of obesity.

Amylin

Amylin is a 37-amino acid peptide that is co-secreted with insulin from pancreatic beta cells in response to eating and hyperglycemia (91). Recently, a synthetic analog of amylin, pramlintide (which differs from the human isoform by 3 amino acids), was approved by the United States Food and Drug Administration (FDA) for use by type 1 and type 2 diabetics as an anti-hyperglycemic agent as adjunctive therapy to insulin. This approval was based on several studies demonstrating beneficial glucoregulatory effects, including reductions in total daily insulin use, postprandial glucose excursions and hemoglobin A1c. Its positive effects on blood glucose homeostasis are thought to involve actions to slow gastric emptying, and thus a protracted delivery of ingested nutrients to the bloodstream, as well as a suppression of glucagon secretion (72, 92-94).

Importantly, amylin also has been shown to reduce food intake. It likely does this at least in part through direct action on neurons whose cell bodies are located in the area postrema. The area postrema is well-suited to respond to hormones made in the periphery, including not only amylin, but others such as leptin and ghrelin (52), because it lacks a blood-brain barrier. In fact, amylin is known to bind with high affinity to receptors located within the area postrema (95) and also has been shown by electrophysiology to activate about half of all area postrema neurons tested. Interesting, nearly all the amylin-engaged area postrema neurons also seem to be glucose-responsive (96). Supporting the assertion that amylin acts at the area postrema to effect its food-intake reducing actions, immunohistochemical analysis has revealed that peripheral administration of amylin strongly induces area postrema c-fos expression, which, as discussed above, is a marker of neuronal activation (97). C-fos induction was also observed within the central nervous system in the nucleus of the solitary tract, the external part of the lateral parabrachial nucleus and the central nucleus of the amygdala (97). Importantly, lesioning of the area postrema not only blunts c-fos induction in those sites but also blocks the anorexigenic actions of peripherally-administered amylin (97, 98); amylin-associated food-reduction is also blunted following lesioning of the lateral parabrachial nucleus (99). Downstream effectors of amylin action likely include the anabolic, arcuate POMC neurons, since amylin administration has been shown to significantly increase hypothalamic POMC mRNA levels (91).

Amylin’s ability to acutely decrease food intake has been observed in several animal models. Initial studies used lean rodent models. For instance, in lean rats exposed to standard chow diets, amylin was shown to decrease meal size without a compensatory increase in meal frequency, thus suggesting a primary satiating effect of amylin (100). Amylin is equally effective at reducing food intake in genetically obese (ob/ob) mice (including ob/ob and db/db) mice and lean mice (101). Amylin also has been shown to selectively decrease the intake of highly palatable foods (102). Other animal studies have demonstrated that amylin specifically does not produce taste aversion, nor was it shown to be associated with behaviors that serve as markers for nausea in animals (100).

These actions on food intake translate to a reduction in body weight. For instance, in a recent study, continuous infusion of amylin over a 3 week period significantly slowed body weight gain and significantly decreased food intake, (with the effect being most pronounced during the first week of treatment) in both lean rats and diet-induced obese-prone rats (91). Interestingly, diet-induced obese-prone rats maintained on a moderately high-fat diet (32% kcal from fat) experienced 3-fold greater loss of fat with amylin treatment than did pair-fed control rats; there was relative preservation of lean body mass. Thus, amylin treatment produced greater fat loss than caloric restriction alone, which suggests the possibility that amylin may also affect energy expenditure. In fact, a relative preservation of energy expenditure was observed with amylin treatment.

Many studies have confirmed that amylin also is effective at reducing food intake and body weight in humans. For instance, a single dose of pramlintide has been shown to cause a statistically significant (16%) reduction in total caloric intake by obese individuals challenged with a buffet meal (103). In addition, these individuals reported experiencing enhanced satiety both during and after the buffet meal in response to amylin (103).

One of the first major long-term clinical trials examining the effectiveness of amylin in reducing weight was published in 2003 (104). This was a 52-week, double-blind, placebo-controlled, parallel-group, multicenter study, which included 656 patients with insulin-treated type 2 diabetes, most of whom were obese. These individuals were randomized to receive either placebo or pramlintide. For this study, change from baseline in HgbA1c at week 26 was the primary endpoint, whereas body weight change was a secondary end point. Those individuals in the pramlinitide group were found to have a sustained reduction from baseline in HgbA1c as well as a significant reduction in mean body weight after one year (-1.4 kg). Mild-to-moderate nausea was the most common adverse event associated with pramlintide (104)

A just-published Phase 2, randomized, placebo-controlled, dose-escalation, multi-center study set out to specifically assess the efficacy of pramlintide in reducing body weight, as well as its safety and tolerability, in non-insulin-treated

obese subjects both with and without type 2 diabetes (105). Either pramlintide or vehicle was self-administered s.c. three times a day prior to meals. Those individuals receiving pramlintide had placebo-corrected body weight reductions of 3.7%, and nearly a third of the pramlintide group had even higher reductions (≥5%). Furthermore, a larger percentage of individuals in the pramlintide group as compared to the control group reported improvements in appetite control and overall well-being. In this study, the most common adverse event was mild, transient nausea, although those not reporting nausea lost a similar amount of weight as those who did not experience nausea (105).

In another recently-published, randomized, blinded, placebo-controlled, multicenter study examining the effects of pramlintide on body weight in obese, non-diabetic individuals, individuals self-administered compound s.c. 15 min prior to meals, for 6 weeks (102). The pramlintide group was shown to have a progressive and significant decrease (about 2%) in body weight, and significant reductions in 24-hour caloric intake, portion sizes, and binge-eating tendencies. The reduction in food intake was seen both in the beginning of the study (before there had been significant weight loss) and at the end of the study (after significant weight loss). Furthermore, pramlintide significantly reduced caloric intake during a “fast-food challenge” during which subjects were provided with a lunch consisting of deep-dish pizzas, ice cream, and high fructose corn syrup-sweetened soft drinks. In this study, there were no major differences in nausea ratings between pramlintide and placebo-treated subjects (102).

Neurotransmitters, neuropeptides and their receptors

Serotonin receptors 5-HT2CR and 5-HT1BR

The central serotonin (5-hydroxytryptamine; 5-HT) system has been known for a long time to be associated with food intake and body weight homeostasis (106). For instance, several studies in the 1970s and 1980s reported observations of a marked inverse relationship between serotonin levels and food consumption (107). However, for a number of reasons, including the widespread projection pattern of serotonergic neurons, the existence of multiple serotonin receptors and the relative unavailability of drugs selective for each of these receptors, progress towards determining the specific neuroanatomical circuitry through which serotonin works to affect body weight has until recently been hindered (106). The complex nature of this circuitry also had slowed the rationale design of pharmaceuticals that might effectively and selectively target the serotonergic system without causing the severe side effects observed with previously-used medications such as d-fenfluramine.

D-fenfluramine (d-FEN) blocks the reuptake of serotonin and also directly stimulates its release. It is a potent and very effective medication to reduce food intake and body weight. In the mid-1990s, it was widely prescribed in combination with phentermine (“fen/phen”) for the treatment of obesity, however

it was taken off the market by the FDA in 1997 due to reports of valvular heart disease (108). The mechanism by which d-FEN caused these valvular problems is still not completely known, however there is convincing evidence now to suggest that stimulation of the 5-HT2BR is at least partially to blame (109).

Sibutramine is another medication that affects serotonin. It not only blocks the reuptake of serotonin, but also blocks the reuptake of noradrenaline. As mentioned earlier, it is currently one of two medications currently available in the US with an indication for the long-term management of obesity. Although it is effective at inducing moderate weight loss, adverse side effects including small increases in blood pressure limit the ability to use the doses previously shown to be maximally effective.

Recently, there has been renewed interest in the serotonergic system. This at least in part is due to new research that has been able to dissect out some of the key molecular players in the central serotonergic system. It is now known that d-fenfluramine dose-dependently induces c-fos expression in the anorexigenic, anabolic POMC neurons located in the lateral regions of the hypothalamic arcuate nucleus of the rat (110); this included threshold anorexic doses of d-FEN. Neuronal activation of these POMC neurons by d-FEN was also confirmed electrophysiologically (110). It turns out that up to 80% of the arcuate POMC neurons co-express the 5-HT2CR, thus suggesting a role for this specific serotonin receptor in d-FEN’s anorexic actions (110). Interestingly, mice lacking 5-HT2CRs previously had been shown to be hyperphagic, obese and resistant to threshold anorexic doses of d-FEN (111-113). Confirming the key role of 5-HT2CR in serotonin-related anorexia, 1-(m-chlorophenyl)piperazine (mCPP), which is a selective 5-HT2C/1BR agonist, also was able to depolarize POMC neurons as well as decrease acute food intake when administered to rats (110). Not only does serotonin directly activate the anorexigenic POMC neurons by binding to 5-HT2CRs on the cell membrane of POMC neurons, but it also indirectly activates them by binding to 5-HT1BRs present on adjacent AgRP/NPY neurons (107). Activation of these 5-HT1BRs has the dual effect of both reducing inhibitory input from adjacent AgRP/NPY neurons onto POMC neurons and limiting the release of AgRP in the vicinity of MC4-Rs (107). A key role for the central melanocortin system in mediating the anorexic actions of d-FEN was further confirmed by experiments in which genetic and pharmacologic blockade of MC4-Rs significantly blunted the usual anorexic response to d-FEN and by other experiments in which an MC4-R/MC3-R agonist was shown to be active in reducing food intake in 5-HT2CR-knockout mice (110). Thus, it appears as if a major mechanism thru which serotonin acts centrally to reduce food intake and body weight is via direct interaction with 5-HT2cRs and 5-HT1BRs on arcuate POMC and AgRP/NPY neurons, respectively, with a resulting activation of the downstream melanocortin system.

This new research likely will permit the return of pharmaceuticals to the market that target specific parts of the serotonergic neurocircuitry to fight obesity. These

likely will include selective agonists of 5-HT1BRs and 5-HT2CRs. For instance, Lorcaserin is one such selective 5-HT2CR agonist currently in development the treatment of obesity (114).This agent has approximately 15-fold and 100-fold selectivities in vitro for the 5-HT2CR as compared to the 5-HT2AR and 5-HT2BR, respectively. A 12-week, double blind, randomized, placebo-controlled trial examining the efficacy and safety of lorcaserin in obese individuals was completed in 2005 and demonstrated dose-dependent weight loss of up to 3.5 kg, reductions in BMI, waist circumference, hip circumference, and total cholesterol. Furthermore, no adverse effects on heart valves were observed during the short study duration. In September 2006, Arena Pharmaceuticals began their Phase III trial with Lorcaserin.

CB1R

Cannabis, or marijuana, while most readily known for its psychoactive properties, is also a powerful analgesic and appetite stimulant. Cannabinoid receptors (CB1R and CB2R), are G-protein coupled receptors expressed throughout the body, and bind to Δ9-tetrahydrocannabinol, which is the active component of marijuana, as well as to the endogenously produced ligands, anandamide and 2-arachidonoyl glycerol (115). CB1R has been postulated to play an important role in appetite regulation because of its expression in tissues known to be important in energy homeostasis. CB1R is predominantly expressed in the brain, including the hypothalamus, forebrain, brainstem and the nucleus accumbuns (116), with some expression peripherally in the gastrointestinal tract, adipose tissue, liver, muscle, thyroid and pancreas (117). This widespread distribution makes CB1R an ideal drug target, as pharmaceutical manipulation could potentially affect multiple levels of the energy homeostasis network simultaneously, reducing compensatory adaptations (118).

Numerous studies have detailed the effects of CB1R and the endocannabinoids on energy homeostasis (119).These include the regulation of orexigenic and anorexigenic neuropeptides in the hypothalamus, nausea and hunger/satiation signals in the hindbrain, and reward aspects of food in the mesolimbic system. Peripherally, the endocannabinoid system also influences nutrient absorption, lipogenesis and decreases glucose uptake in muscles.

CB1R knock out mice show reduced food intake, decreased body weight, leanness, and enhanced leptin sensitivity through both hypothalamic and adipocyte action (120, 121). The cannabinoid antagonist SR 141716, decreased appetite and caused weight loss (122), and these effects were absent in CB1R knock out mice (123). Furthermore, this antagonist reduced food intake, body weight and adiposity, while improving serum lipid profiles and lipolysis in diet-induced obese mice (124-126). Administration of SR 141716 to obese, leptin deficient (ob/ob) mice increased oxygen consumption and thermogenesis, suggesting a direct effect on energy expenditure (127). Together, these

experiments suggest that the endocannabinoid system is amendable to drug manipulation.

SR 141716, is marketed as Rimonabant, by Sanofi-Aventis, and its efficacy in humans was tested in four Phase III clinical studies. These trials, known as Rimonabant in Obesity (RIO), involved over 66000 overweight and obese participants for up to 2 years (118). The RIO studies were randomized, double-blind, placebo-controlled trials studying the effects of a low dose (5 mg) or high dose (20 mg) of rimonabant on different patient groups. In addition to treatment with rimonabant, participants reduced their food intake by 600 kcal/day and were instructed to increase their physical activity levels. RIO-Europe studied the effects of rimonabant on weight loss and cardiovascular risk factor on non-diabetic, obese subjects for a 1 year period (128). RIO-North America extended the European study to 2 years, with some participants being switched to the placebo in their second year (129). RIO-Lipids measured body weight, cardiovascular risk factors, and additional metabolic markers such as LDL, C-reactive protein and adiponectin in patients with a combination of obesity and dyslipidemia (130). Finally, RIO-Diabetes studied the effect of rimonabant on weight lose in obese diabetics (131). These trials showed that rimonabant is effective at promoting and maintaining weight loss, and compared to the placebo group, those given 20 mg of rimonabant lost an average of 10.8 lbs after 1 year (132). Rimonabant also decreased waist circumference, increased insulin sensitivity, improved glucose tolerance, increased HDL cholesterol, decreased triglycerides, decreased plasma leptin and decreased C-reactive protein relative to the placebo group (118, 133). Importantly, the metabolic effects were greater than those predicted by the amount of weight lost. While rimonabant’s weight loss effects plateaued at about 5%-10% of total body weight, it decreased the incidence of metabolic syndrome by 50% in these trials. Furthermore, in the RIO-North America trial, patients who were switched from rimonabant to placebo in the second year regained their lost weight, whereas those who continued with the rimonabant maintained their weight loss, suggesting that treatment must be continuous for long term benefits (129).

Recently, concerns have been raised over the safety of rimonabant, highlighted by the FDA’s Endocrine and Metabolic Drugs Advisory Committee advising against rimonabant as an anti-obesity treatment (134).While no effects on plasma lipids or blood pressure were detected, some dose related side effects were reported, the most common being upper respiratory tract infection, nausea, and diarrhea (128-130, 132). These side effects generally occurred during the early stages of the trial, and declined during the second year (129). Significantly, depression or depressed mood disorder were reported in the RIO-Lipids and Diabetes trials, and were responsible for 2.9% of the withdrawals from the RIO-Lipids study (130, 131). It has been suggested that obese patients seeking medical treatment may have increased tendencies towards depression (135), and whether this is exacerbated by rimonabant remains to be seen. Despite these published findings, the FDA advisory committee cited concerns about the

increased incidence of depression and suicidal thoughts during rimonabant treatment, and advised against the approval of rimonabant (134, 136). As of June 29, 2007, Sanofi-Aventis has withdrawn their new drug application in the United States until they can further address the concerns of the FDA advisory committee (137).

Melanocortin (α-MSH)

Energy homeostasis involves complex interactions between peripheral metabolic signals and the CNS (63, 138). Such interactions between the periphery and the CNS are relevant, for instance, in the discussion of the mainly gastrointestinal tract-derived hormone, ghrelin, mentioned above. This interaction is also of utmost importance in the anorexigenic, body weight loss-promoting actions of the white adipose tissue-derived hormone, leptin. In fact, many of the identified forms of human monogenic obesity and a majority of the spontaneous rodent obesity strains involve perturbances either directly to leptin and its receptor or to the melanocortin signaling pathway which is downstream of leptin (139). This leptin-melanocortin signaling pathway is crucial to the successful transmittal of metabolic cues carried by the peripheral hormonal signal, leptin, into coordinated behavioral, autonomic and endocrine responses in the CNS.

It has been suggested that the extreme obesity that results from leptin deficiency demonstrates the over-riding importance of the leptin signaling pathway in energy homeostasis. The cloning of the leptin gene in 1994 (140) was hailed as a breakthrough in the obesity field, and it was widely hoped that leptin therapies for obesity would soon follow. While there were rare cases of obesity due to leptin mutations in which leptin replacement restored normal body weight (141), it is now apparent that leptin mutations are the exception, rather than the norm. Most individuals with obesity have high levels of leptin, suggestive of a state of leptin resistance (142-144). Because of this resistance, even high doses of leptin have produced only modest success at reducing body weight in general obesity (145, 146).

Despite the ineffectiveness of leptin-replacement therapy, preliminary studies suggest that leptin may be effective in enhancing the maintenance of weight loss achieved by either lifestyle modifications or other drug therapies. Weight loss is often accompanied by drops in leptin levels, sympathetic nervous tone and thyroid hormone levels, which favor a rebound weight gain (147). These physiological responses to weight loss can be reversed by low doses of leptin (147, 148). Furthermore, rats that are treated with sibutramine show enhanced reductions in food intake, body weight, and fat mass when leptin is also given (149), thus suggesting a role for leptin in combination with other agents to fine-tune weight loss therapy.

Leptin resistance is thought to be due mainly to impairments in leptin action, through decreased blood-brain transit, reduced neuronal response, and reduced

intracellular signaling in response to leptin (118). While much research has been focused on overcoming these blockades in leptin action, another approach has been to investigate methods to enhance the second and higher order targets of leptin signaling. The melanocortin pathway is one of the most attractive of the downstream systems, particularly since mutations in this system result in profound obesity.

The MC4-R signally system is unique in that that is has an endogenous agonist, α-melanocyte stimulating hormone (α-MSH), a product of pro-opiomelanocortin (POMC), as well as an endogenous antagonist/inverse agonist, agouti-related protein (AgRP) (63, 106, 150). Both peptides are produced in the arcuate nucleus of the hypothalamus, and are regulated by leptin. Melanocortin neurons are targeted by these leptin-responsive neurons and in turn innervate CNS sites that control feeding behavior, endocrine and autonomic function.

Notably, mutations of melanocortin-4 receptor (MC4-R) are the most common causes of human monogenic obesity and are thought to account for 5%-10% of common morbid obesity (151). Furthermore, transgenic mouse experiments have also established the importance of MC4-R energy homeostasis. MC4-R deficient mice develop adult onset obesity accompanied by hyperinsulinemia and hyperglycemia that sometimes develops into diabetes (152). A decreased metabolic rate is suggestive of a role for the melanocortin system in regulating the autonomic nervous system. Heterozygous mice for the MC4-R show an intermediate level of weight gain. Unlike wildtype mice, MC4-R deficient mice are not able to respond to high fat diets by increasing thermogenesis and physical activity, suggesting that the melanocortin system is essential for these adaptive behaviors (153).

Antagonism of MC4-R from ectopic expression of Agouti protein, or endogenous expression of agouti-related protein (AgRP) also demonstrates the importance of melanocortin signaling in maintaining energy homeostasis (151, 154, 155). Hypothalamic injections of the agonist melanotan II (MTII) decrease feeding, whereas the antagonist SHU9119 blocks the effects of MTII (156). Numerous MC4-R agonists are being developed as potential targets for obesity therapy (157-159) One significant set back has been the incidence of increased penile erections that result from some of these agonists, especially the ones that are derived from MTII (160). While continuing to develop novel melanocortin receptor agonists, Palatin Technologies has begun clinical trials for one particular agonist, Bremelanotide (PT-141), a metabolite of MTII, for the treatment of sexual dysfunction (161).

Miscellaneous

SIRT1

It has long been noted that despite a diet high in saturated fats, the French population has a very low incidence of coronary heart disease (162). This phenomenon has been dubbed the French paradox, and has been attributed to the high levels of wine consumption. One of the potential active ingredients in wine that may convey protective effects is resveratrol. A polyphenolic flavenoid found in numerous plants, most notably, in grapes and red wine, it first attracted attention in longevity studies. Resveratrol was shown to increase the lifespan of lower organisms, in a manner similar to that seen in extreme calorie restriction, by promoting the activity of the sirtuin/Sir2 family of NAD+-dependent histone deacetylases (163). Because of the decreased levels of NAD+ levels seen in obesity, resveratrol has become an attractive means for improving metabolic function.

Seven sirtuin genes have been identified in mammals. In particular, SIRT1, 3 and 4 have been shown to regulate metabolic responses to changing dietary conditions (164). While initially described as a transcriptional silencer in yeast, SIRT1 is now known to have an important role in modulating mammalian transcriptional regulators. Recent work has shown the importance of Sirt1 in regulating gluconeogenic genes, suppressing adipogenesis genes in white adipose tissue, and cell survival. (164-168). SIRT1 also upregulates adiponectin, an adipoctye hormone that is expressed at levels inversely proportional to adiposity levels, and which is important for the regulation of energy balance, and the metabolism of lipids and glucose (169). Importantly, SIRT1 deacetylates PGC-1α, a nuclear receptor coactivator critical for the biogenesis of mitochondria in brown adipose tissue and skeletal muscle, two tissues that can expend energy through adaptive thermogenesis (170). By acting on PGC-1α, SIRT1 can also suppresses glycolysis and increase hepatic glucose production (171).

Because resveratrol is a potent enhancer of Sirt1 activity in vitro (172), it was postulated that it could also promote metabolism through Sirt1 pathways. Recent work has shown that, at least in rodents, resveratrol can protect against high-calorie diets (173, 174). Mice on high fat diets typically develop obesity, numerous organ pathologies due to lipid accumulation, and insulin resistance. While the dosage and duration of resveratrol treatment differed between studies, in general, the investigators found an improvement in insulin sensitivity, circulating glucose levels, motor function and mitochondria numbers.

Interestingly, mice given short-term, high doses of resveratrol are resistant to diet induced obesity and have decreased amounts of white adipose tissue (174). These mice exhibit signs of increased adaptive thermogenesis in both brown adipose tissue and skeletal muscle. In muscles, increased ratios of slow-twitch/oxidative muscle fibers to fast-twitch/glycolytic fibers are accompanied by decreased exercise fatigue, and increased oxidative phosphorylation gene expression.

Long-term resveratrol treatment also appears to have positive effects, the most noticeable being an increase in the lifespan of mice fed high-calorie diets, so that their longevity is similar to that of mice fed on standard chow (173). Metabolic effects, such as improvements in hepatic glucose and insulin metabolism, and the prevention of hepatic steatosis were observed.

In these mouse studies, resveratrol acts to increase PGC-1α activity, although the mechanism of this is unclear. High doses of resveratrol show indirect activation by promoting the SIRT1 mediated deacetylation of PGC-1α (174). In contrast, chronic low doses show a direct effect on PGC-1α mRNA expression levels (173). Low doses were also associated with the activation of the Ser/Thr kinase AMPK pathway, which strongly inhibits gluconeogenesis and increases fatty-acid oxidation, and may explain the hepatic effects of resveratrol.

Despite these exciting and intriguing results in mouse models, the role of the SIRT1 and the effectiveness of resveratrol in humans are not clear. While there are no known mutations of SIRT1, 3 single nucleotide polymorphisms (SNPs) were found to be significantly associated with energy expenditure among healthy offspring of diabetics (174). Resveratrol is currently available as a nutritional supplement, but it has not been fully developed as a drug agent. Sirtris Pharmaceuticals has recently begun clinical trials with SRT501, a proprietary form of resveratrol (175). While currently in Phase Ib trials, early preclinical models of type II diabetes have found SRT501 to be effective at decreasing glucose and increasing insulin sensitivity. Sirtris is also developing additional SIRT1 activators that are unrelated to resveratrol. In addition to the beneficial effects seen with SRT501, these new activators also increase mitochondria numbers, and improve insulin sensitivity in muscle, liver and fat cells.

CONCLUSION

Because of its prevalence, health concerns and social stigma, obesity is one of the most active research fields today. According to the National Institutes of Health, there are currently 463 studies underway for the prevention, treatment or management of obesity (176). While numerous drugs are currently under development, it likely will turn out that certain of these newly-developed drugs will prove to be more effective at treatment of certain forms of obesity or the obesity occurring in certain populations than others. Furthermore, it is likely that no single medication will be completely effective in treating obesity because of the redundant pathways and controls that the body has to maintain energy homeostasis. Combination therapies of drugs that target different systems may prove to be the most effective method of reducing and maintaining body weight.