Current obesity treatments

Diet and exercise are still the cornerstones for obesity treatment. Clinicians have few pharmacological tools for obesity management, while available anti-obesity drugs achieve only relative short-term weight loss and are often followed by weight regain.^3,5,12

Pharmacotherapy for obesity includes drugs that induce weight loss by suppressing appetite or altering nutrient absorption. These drugs are usually able to induce 5 to 10% weight loss when compared with placebo, the minimum requirement for a drug to be approved by the regulatory authorities such as the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA). In addition to weight reduction, in order to be approved for obesity treatment, drugs should prove to confer additional health benefits to the patient such as improvement of cardiovascular risk factors and demonstrate to have a good safety profile.¹³ Currently available anti-obesity drugs approved by the FDA include, appetite suppressants and the lipase inhibitor orlistat, of which only the former is authorized by EMEA and marketed in Europe. Appetite suppressant drugs include central nervous system stimulants, such as phentermine, a noradrenergic sympathetic amine approved for short-term treatment of obesity, which is the most widely used drug owing to its low cost and the fact of being marketed for decades. Phentermine commonly associated side effects include insomnia, irritability and increase in blood pressure.⁵ The FDA has more recently approved two new drugs to treat obesity, lorcaserin, a selective 5-hydroxytryptamine receptor 2c agonist¹⁴ and a fixed dose combination of phentermine with topiramate.¹⁵ Lorcaserin is indicated as an adjunct therapy to diet and exercise for chronic weight management of obese or overweight individuals with BMI over 27 kg/m² associated with hypertension, dyslipidemia or diabetes, however it has been associated with several safety concerns that include cardiac valvulopathy and increased risk of psychiatric, cognitive, and serotonergic adverse effects.¹⁶ Topiramate is a sulfamate-substituted monosaccharide marketed since 1996 and formerly approved for seizure disorders and prevention of migraine headaches, which was shown to promote weight loss as a side effect of their therapeutic usage in clinical trials. Previously to FDA approval, topiramate was used off-label as adjuvant therapy in obesity, along with the anti-depressives, fluoxetine, sertraline and buproprion, and the anti-diabetic drug, metformin.¹² Orlistat is a lipase inhibitor that prevents hydrolysis of dietary triglycerides and consequently prevents the absorption of dietary fat that is excreted unaltered by the gastro-intestinal tract. Orlistat is the only drug approved for long-term use, however it produces only a modest weight loss and is associated with high rates of gastrointestinal side effects, such as steatorrea, faecal incontinence and flatulence.¹⁷

For patients with BMI over 40 kg/m² or over 35 kg/m² associated with high-risk co-morbid conditions, in which previous medical weight loss treatments have failed, obesity surgery is the only therapy that proved to provide significant and sustainable weight reduction¹⁸ and is recognized by the National Institutes of Health.^19,20 In severe obesity, bariatric surgical treatments, have shown to be cost-effective at producing and maintaining weight loss, since decrease the risk of development of new obesity associated co-morbidities and to improve the existing ones.²¹ Patients submitted to bariatric surgery display a significant reduction in health care use rates and total direct health care costs, as well as a significant decrease in overall 5-y mortality rate, with a reduction in the relative risk of death of 89%.⁹ Understandably, in response to the relative ineffectiveness of medical therapy and in spite of the potential risks associated with an invasive procedure, the demand for bariatric surgery has been increasing.²²

Somatostatin

The obese state is also characterized by a decreased growth hormone (GH) basal secretion and GH administration has been shown to reduce adiposity and increase lean mass.⁴⁸ Somatostatin is a peptide hormone produced in the hypothalamus as well as in other tissues such as the gastro-intestinal system, which inhibits GH and insulin-like growth factor 1 (IGF-1) secretion. Somatostatin based immunization procedures where formulated with the aim of removing the physiological inhibitory effects of somatostatin on the somatotropic axis in order to increase the endogenous levels of GH and IGF-1, without the need of direct usage of these anabolic hormones. In diet induced obese mice, anti-somatostatin vaccination has shown not to interfere with food intake nevertheless allowed to decrease body weight gain by 10% despite continuous feeding with high fat diet⁴⁹ (Table 1). This vaccination approach, although not aimed at a direct regulatory pathway of energy homeostasis, has the benefit of having a specific molecular target, somatostatin, which is capable to up regulate several hormones, in particular GH that has advantageous metabolic effects and decreases fat mass. However, there is still a need to assess the possible shortcomings on glucose metabolism secondary to the induced GH excess and to evaluate the possible risk of promoting neuroendocrine cell proliferation that are usually inhibited by somatostatin.

Glucose-dependent insulinotropic polypeptide

Glucose-dependent insulinotropic polypeptide formerly known as gastric inhibitory peptide (GIP) is a gastro-intestinal hormone secreted by the intestinal K-cells in response to the ingestion of carbohydrate and fat that stimulates glucose-dependent insulin release and secretion.⁵⁰ Besides the pancreas, the GIP receptor (GIP-R) is widely distributed in peripheral organs including the adipose tissue where it holds a key role in fat deposition and lipid metabolism.⁵¹ In wild type animals, GIP-R knockout is associated with mild impairment of glucose tolerance and insulin secretion, conversely in mice exposed to high fat diet, GIP-R knockout results in decreased weight gain, decreased tissue triacylglyceride storage and increased uncoupling protein-1 in brown adipose tissue suggesting the existence of increased energy expenditure.⁵² Functional GIP blockade using receptor antagonists have also shown to prevent or reverse obesity in rodent models, as well as increase fat oxidation in the liver and muscle and improve insulin resistance.⁵³

A vaccination approach for GIP inhibition has also been attempted. To induce the production of anti-GIP neutralizing antibodies, an immunoconjugate of GIP covalently attached to the Qβ bacteriophage has been developed. Anti-GIP vaccine inoculation has been shown to protect against diet induced weight gain due to decreased fat accumulation and increased energy expenditure secondary to a rise in basal metabolic rate and without changes in locomotor activity, results which were similar to the effects produced by chemical blockade of the GIP receptor⁵⁴ (Table 1).

Despite GIP not having a role in food intake regulation, GIP blockade was shown to be able to deplete adipose deposits and improve insulin resistance, therefore it has been considered a potential drug target for obesity related diabetes treatment.⁵⁵ By antagonizing GIP, there is also the possibility of inducing a negative insulinotropic effect, due to GIP role as incretin hormone that promotes glucose mediated insulin secretion. Since GIP does not has a role in food intake regulation and hyperglycemia has been shown to induce pancreatic resistance to the insulinotropic effects of the GIP hormone, this anti-obesity vaccine is particularly targeted for the obese patient with type 2 diabetes.

Ghrelin

Since ghrelin is the only identified orexigenic hormone, it has been considered a most promising target for the development of new obesity treatments,⁵⁶ which gave origin to several different research approaches in order to achieve neutralization of ghrelin biologic effects in energy homeostasis. As a proof of this concept, the effect of ghrelin blockade has been assessed in several experimental models which included genetic deletion of ghrelin or ghrelin receptor, antagonists of ghrelin receptor and GOAT inhibition.

Genetic deletion of ghrelin or GHS-R1a, the receptor that mediates ghrelins’ stimulatory effects on GH release and appetite, failed to show any significant defect in GH levels, linear growth and food intake or body weight in adult mice.^57-59 Nevertheless, ghrelin-deficient (Ghrl−/−) mice showed a decreased feed efficiency and were protected from the rapid weight gain induced by early exposure to high-fat diet, presenting decreased adiposity, improved serum profile of glucose and lipids, increased energy expenditure and locomotor activity.⁶⁰ In addition, ghrelin knockout mice compared with wild type mice also presented a decrease in respiratory quotient when fed with high fat diet, suggesting that endogenous ghrelin plays a more prominent role in determining the type of metabolic substrate that is used for maintenance of energy balance, decreasing fat utilization, than in the regulation of food intake.⁵⁷

These data may suggest that ghrelin is not an essential orexigenic signal. However, genetic deletions have the limitation of being prone to the development of adaptive mechanisms in neuroendocrine pathways that may have compensated the congenital lack of ghrelin signaling. Furthermore, unintentional ablation of the obestatin gene, also encoded by the ghrelin gene, which was shown to supress food intake, might further mask the potential of ghrelin in energy homeostasis.⁶¹ Moreover, the ghrelin receptor has been demonstrated to maintain a high level of constitutive activity even in the absence of the endogenous ligand,⁶² and its physiological relevance has been revealed by patients harboring missense mutations of the GHS-R with loss of its constitutive activity that are characterized by short stature and obesity.⁶³

Ghrelin receptor antagonists

Ghrelin receptor antagonists have demonstrated to decrease food intake, body weight and improve glucose tolerance due to increased glucose-dependent insulin secretion,⁴³ thus confirming the potential of ghrelin blocking as a potential treatment target for obesity and type 2 diabetes. Two ghrelin receptor antagonists (YIL-870 and YIL 871), despite the low circulating ghrelin levels, were found to promote weight loss and improve glucose tolerance in diet induced obesity (DIO) mice, suggesting that there is still sufficient ghrelin tone to allow the GHS-R1a antagonists to have a therapeutic effect not only in obesity but also in diabetes.⁴³ Ghrelin antagonism has been found to be specifically beneficial in the context of obesity combined with estrogen deficiency, being able to decrease food intake and body weight and increase uncoupling protein 1 (UCP-1) in brown adipose tissue, suggesting enhanced energy dissipation, in a mouse model of postmenopausal obesity.⁶⁴ The fact that ghrelin receptor is characterized by a high degree of constitutive signaling activity, raises questions about the physiologic importance of ghrelin in receptor activity regulation and whether an unknown endogenous inverse agonist exists, suggesting that the development of inverse agonist compounds could represent alternative valuable pharmacological tools potentially more efficient than neutral antagonists.⁶²

A different approach of neutralizing ghrelin consist in using ribonucleic acid Spiegelmer (SPM), a non-natural nucleic acid with specific binding activity toward a given molecule, synthetized as a mirror image of the biological target, which was shown to decrease food intake, promote weight loss and decrease food efficiency (weight gain per kilocalorie ingested), an indirect evidence of increased energy expenditure.⁶⁵ Anti-ghrelin SPM does not cross the blood-brain barrier due to its charge and size, thus ghrelin neutralization is thought to occur mainly in the periphery, though direct action in the ARC cannot be excluded, since the circunventricular area of the median eminence is characterized by an incomplete blood-brain barrier.⁶⁶

Ghrelin O-acyltransferase inactivation

The inactivation of GOAT, the enzyme that activates ghrelin, has been proposed as an alternative tactic to block ghrelin activity,⁶⁷ since antibodies targeted to hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin, which has no biological activity, resulted in increased metabolic rate and suppressed re-feeding after food deprivation.⁶⁸ A peptide-based GOAT specific antagonist inhibitor, the bisubstrate analog GO-CoA-Tat, composed of three elements: ghrelin, octanoyl Co-A and a Tat sequence that allows the analog to penetrate within the cell cytoplasm where ghrelin acylation occurs, has also been recently developed. The GO-CoA-Tat was shown to decrease serum levels of acyl ghrelin and prevent body weight gain; additionally, also demonstrated to increase insulin secretion and improve glucose tolerance.⁶⁹

Passive immunization

After a first attempt to produce a polyclonal anti-ghrelin antibody that dose-dependently inhibited fast-induced feeding and supressed dark phase food intake after intracerebroventricular administration,⁷⁰ Lu et al., produced the anti-ghrelin antibody, so called 33A, which binds with high affinity to the active form of the molecule, acylated ghrelin but not to des-acyl ghrelin.⁷¹ The inoculation of this monoclonal anti-ghrelin antibody inhibited acute ghrelin-mediated orexigenic effects, but was unable to change long-term food intake in mice. The antibody demonstrated to prevent the increase in food intake induced by exogenous administration of acyl-ghrelin, showing that 33A specifically blocked the activity of acylated ghrelin, however was unable to prevent the increase in food intake after a fast-refeeding challenge that is mediated by endogenous ghrelin. Since this anti-ghrelin antibody was shown to increase the ratio of acylated to des-acylated ghrelin, compensatory mechanisms might have been activated to counteract the blockage of peripheral ghrelin which suggests that blocking the peripheral ghrelin pathway alone may not be sufficient in the long-term.⁷¹

More recently, a mixture of monoclonal antibodies targeting different ghrelin haptens was shown to be required to maintain increased energy expenditure during fasting and deprivation-induced food intake, as well as to reduce overall food intake upon re-feeding. Passive immunization with a single high-affinity monoclonal antibody that targeted specifically the ghrelin N-terminus (JG4) did not in alter energy expenditure, fuel substrate utilization, or food intake in fasted mice. In contrast, combining JG4 with a C-terminally directed anti-ghrelin antibody (JG3), was able to increase energy expenditure and maintain the utilization of carbohydrate as a fuel substrate in fasted mice. Moreover, a “triplet” mAb cocktail combination that included the additional C-terminally directed antibody (JG2) that had been comparably ineffective in conjunction with JG3 or JG4 alone, not only promoted increased energy expenditure, but also reduced deprivation-induced food intake. Thus, suggesting that an oligoclonal response is necessary to maintain an increased whole body energy expenditure during fasting and re-feeding over a 24-h period, as well as to reduce overall food intake after fasting.⁷²

The main limitations of passive immunization approaches include development of acquired tolerance and lack of long-term effectiveness, due to the reduced half-lives of the antibodies and need of periodic administration, as well as the possibility of activation of compensatory pathways of ghrelin production in common with the other methods used for ghrelin inactivation.

Active immunization

Anti-ghrelin vaccines were developed with the rationale of inducing an immune response to prevent the interaction of ghrelin with its receptor and suppress endogenous ghrelin bioactivity. The first ghrelin immunization attempts included vaccines which used bovine serum albumin (BSA)⁷³ and keyhole limpet hemocyanin (KLH)⁷⁴ as carrier proteins and immunogenic substances (Table).

Ghrelin conjugated to BSA was used to immunize 19 wk old pigs in the presence of Freund’s incomplete adjuvant and diethylaminoethyldextran (DEAE). Two boost immunizations occurred 20 and 40 d after the first immunization and 14 d after primary immunization anti-ghrelin antibodies could be found in plasma of vaccinated animals. This immunoconjugate was able to decrease voluntary food intake by 15% and by the end of the experiment vaccinated animals weighed 10% less than control animals. As ghrelin is a known secretagogue of growth hormone, there were concerns associated with the effects of vaccination on the somatotropic axis, nonetheless such evidence was not found as growth hormone levels actually increased in vaccinated animals that was attributed to the normal feedback mechanism in response to dietary restriction and decreased fat mass.⁷³ Zorrilla, et al., produced 3 immunoconjugates using different ghrelin haptens: Ghr1 with N-terminal amino acids 1–10 of ghrelin, Ghr2 with C-terminal amino acids 13–28, and Ghr3 with the full-length rat ghrelin sequence (1–28) that were conjugated to KLH. Age- and weight-matched rats received immunizations on experimental days 0, 21, 35, 56, and 84. The first three immunizations consisted of Ribi emulsion adjuvant containing Ghr1-KLH, Ghr2-KLH, Ghr3-KLH, or KLH, while the last two inoculations used alum as adjuvant. Immunized rats ate normally but, once anti-ghrelin antibodies titers increased, body weight gain decreased with a decreased circulating leptin levels and preferential reduction of body fat due to decreased feed efficiency, indicating a reduced energy thrift. The ratio of brain to serum ghrelin levels was also lower in rats with stronger anti-ghrelin immune responses.⁷⁴

In contrast, ghrelin vaccination using ghrelin-PADRE peptide, a synthetic Pan-DR helper T cell epitope which binds with high affinity to HLA-DR molecules being able to induce a high IgG immune response to various antigens coupled to PADRE, was able to induce the development of high anti-ghrelin antibody titers although it failed to show any effect on body weight or food intake.⁷⁵

The main limitation of these anti-ghrelin vaccination strategies are the need to use adjuvants in order to achieve an appropriate antibody response which may be associated with inflammatory responses, risk of exacerbated immune response or have limited use in humans.