The Melanocortin-4 Receptor As Target for Obesity Treatment: a Systematic Review of Emerging Pharmacological Therapeutic Options

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REVIEW The melanocortin-4 receptor as target for obesity treatment: a systematic review of emerging pharmacological therapeutic options

L Fani1,3, S Bak1,3, P Delhanty2, EFC van Rossum2 and ELT van den Akker1

Obesity is one of the greatest public health challenges of the 21st century. Obesity is currently responsible for B0.7–2.8% of a country’s health costs worldwide. Treatment is often not effective because weight regulation is complex. Appetite and energy control are regulated in the brain. Melanocortin-4 receptor (MC4R) has a central role in this regulation. MC4R defects lead to a severe clinical phenotype with lack of satiety and early-onset severe obesity. Preclinical research has been carried out to understand the mechanism of MC4R regulation and possible effectors. The objective of this study is to systematically review the literature for emerging pharmacological obesity treatment options. A systematic literature search was performed in PubMed and Embase for articles published until June 2012. The search resulted in 664 papers matching the search terms, of which 15 papers remained after elimination, based on the speciﬁc inclusion and exclusion criteria. In these 15 papers, different MC4R agonists were studied in vivo in animal and human studies. Almost all studies are in the preclinical phase. There are currently no effective clinical treatments for MC4R-deﬁcient obese patients, although MC4R agonists are being developed and are entering phase I and II trials.

International Journal of Obesity (2014) 38, 163–169; doi:10.1038/ijo.2013.80; published online 18 June 2013 Keywords: MC4R; treatment; pharmacological; drug

INTRODUCTION appetite by expressing anorexigenic polypeptides such as Controlling the global epidemic of obesity is one of today’s pro-opiomelanocortin and cocaine- and amphetamine-regulated most important public health challenges. Obesity accounts for transcript. Pro-opiomelanocortin is a prohormone that is B0.7–2.8% of a country’s total healthcare expenditure.1 Its processed to produce g-melanocyte-stimulating hormone (MSH), prevalence has doubled or tripled in many countries in the adrenocorticotropic hormone (ACTH) and b-lipotropin. ACTH and European Union, United States and even in some developing b-lipotropin produce several substances including, respectively, countries.2 Treatment is often not successful because weight a-MSH and b-MSH. MSH is the ligand that binds and activates the regulation is very complex with many inﬂuencing factors such as MC4R. The MC4R has a central role in weight regulation. genetic makeup, environmental and behavioural factors,3 as well MC4R activation decreases food intake while elevating energy as social and psychological dimensions.4 Important for weight utilization.6 The importance of this receptor in appetite and regulation are appetite and energy control, which are energy regulation is illustrated by naturally occurring mutations predominantly regulated in the brain. that lead to partial or complete dysfunction of MC4R in patients. The brain control of appetite and energy balance is under the This MC4R defect leads to a clinical phenotype with lack of satiety, regulation of a complex interplay of several brain areas. The major extreme continuous hyperphagia, a decline in energy utilization, centres involved are multiple hypothalamic nuclei, for example, and consequently leads to severe early-onset obesity. In humans, the arcuate nucleus, paraventricular nucleus, lateral hypothalamic homozygous or compound heterozygous MC4R gene defects are area, dorsomedial hypothalamus and the ventromedial hypotha- rare. However, the prevalence of carriage of heterozygous MC4R lamus. Interrelationships between these centres provide tight mutations is estimated to be as high as 0.5–6% in obese homoeostatic regulation of body weight.5 individuals in the Netherlands.7 The role of MC4R gene mutation One of the key regulators of energy intake, expenditure, carriage in the development of obesity is unclear, although it is appetite and metabolism is the arcuate nucleus of the hypotha- conceivable that many of these mutations lead to a partial lamus (Figure 1). There are neurons in the arcuate nucleus that resistance of the receptor, which might be overcome by increased stimulate appetite. These express orexigenic polypeptides, includ- concentration of the MC4R ligand. The central role of MC4R in the ing neuropeptide Y and agouti-related peptide. Agouti-related regulation of appetite and energy expenditure makes MC4R peptide is a natural antagonist of the melanocortin-4 receptor agonist also an interesting target for studies on obesity drug (MC4R) activity. Other neurons in the arcuate nucleus decrease development in general.7 To date, B80 distinct mutations8 of

1Department of Pediatrics, Division of Endocrinology, Erasmus MC, Rotterdam, The Netherlands and 2Department of Internal Medicine, Division of Endocrinology, Erasmus MC, Rotterdam, The Netherlands. Correspondence: Dr ELT van den Akker, Department of Pediatrics, Division of Endocrinology, Erasmus MC- Sophia, Ofﬁce sp 1536, Dr Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands. E-mail: [email protected] 3These authors contributed equally to this work. Received 7 December 2012; revised 1 May 2013; accepted 12 May 2013; published online 18 June 2013 MC4R as target for obesity treatment L Fani et al 164 were: (1) articles that merely described the molecular or biochemical bases of the therapeutics without investigating the effect of using it in vivo and (2) articles studying outcome parameters other than weight and energy expenditure.

RESULTS Systematic review results The primary search resulted in 357 articles in Pubmed and 635 articles in Embase, with the elimination of redundant articles leading to 664 articles in total for analysis. After applying our inclusion and exclusion criteria to the search results, 15 articles remained. The MC4R agonists from these articles comprise the following: melanotan II (MTII), tetrapeptides, MK-0489, MK-0493, urea-based piperazine, Ro27-3225, cyclophanes, ACTH-derivates, compound 1, pyrrolidine diastereoisomer, BIMs (BIM-22493 and BIM-22511) and Figure 1. Schematic representation of the regulation of food intake b-MSH analogues. We will analyse the articles by showing the and energy expenditure by the MC4-receptor. results of the studies by compounds. An overview of the studies and the main results is presented in Table 2. An overview of the agents’ effectivity and specificity is presented in Table 3. Table 1. The search strategy used for (a) the Pubmed search (25 June 2012) and (b) the Embase search (25 June 2012) Melanotan II In the study by Hansen et al.,10 the effect of the MC3/4R agonist Search Query MTII was investigated. Six-week-old male Sprague–Dawley rats were randomly divided into two groups. The first group was (a) administered high-fat chow food supplemented with a cafeteria #1 melanocortin-4 receptor* OR MC4R OR MC4 Receptor* OR Receptor, Melanocortin, Type 4[mesh] OR diet (30% fat) and the control group was chow fed (5% fat), both (Melanocortin Receptor* AND ‘type 4’[tw]) for 12 weeks. The rats were maintained under a 12-h light/dark #2 Melanocortin Receptor* AND ‘type 4’[tw] cycle (lights on at 0600 hours). After intracerebroventricular (ICV) #3 #1 OR #2 MTII administration, an inhibitory effect on food intake was found #4 Drug therapy[mesh] OR Drug therapy[tw] OR drugs[tw] OR in both groups. The chronically overfed animals had a stronger drug[tw] OR pharmacotherap*[tw] OR pharmaceutic*[tw] inhibitory feeding response 15 and 24 h after MTII injection and #5 body weight[mesh] OR obesity[tw] OR obese[tw] OR lost more body weight (15±3 g) compared with control rats. In overweight[tw] OR weight[tw] OR BMI[tw] OR appetite[tw] conclusion, MTII inhibited food intake and decreased body weight OR food intake[tw] OR eating[tw] in overfed rats. One of the remaining issues of this compound is #6 #3 AND #4 AND #5 that it is non-specific for the MC4R, which might limit its use in (b) humans owing to potential side effects. One of the described side #1 ‘melanocortin-4’ NEXT/1 receptor*):de,ab,ti OR effects is conditioned taste aversion, which may be due to mc4r:de,ab,ti OR (mc4 NEXT/1 receptor*):de,ab,ti OR off-target effects. ((melanocortin NEAR/4 receptor*):de,ab,ti AND ‘type 4’:de,ab,ti #2 ‘drug therapy’/exp OR drugs:de,ab,ti OR drug:de,ab,ti OR Tetrapeptides: compound 10 and BL3020 pharmacotherap*:de,ab,ti OR pharmaceutic*:de,ab,ti Several studies analysed the effects of tetrapeptides derived from #3 obesity/exp OR obesity:de,ab,ti OR obese:de,ab,ti OR a-MSH generated by creating analogues of the His-Phe-Arg-Trp overweight:de,ab,ti OR weight:de,ab,ti OR BMI:de,ab,ti OR sequence. This sequence is commonly shared by all the appetite:de,ab,ti OR ‘food intake’:de,ab,ti OR eating:de,ab,ti melanocortins and is critical for the activation of melanocortin #4 #1 AND #2 AND #3 receptors (MCRs) by a-MSH.11 Modification of this sequence has been shown to affect selectivity of the peptide for MC3R versus MC4R have been reported in humans.9 This systematic review MC4R. focuses on MC4R agonists that have potential as future Ye et al.12 studied the pharmacological characteristics of several pharmacological obesity treatment. compounds based on the His-Phe-Arg-Trp analogue Tic-DPhe-Arg- Trp in vitro for their binding affinity, agonist activity and MC4R receptor selectivity. Tic and DPhe are synthetic amino MATERIALS AND METHODS acids designed to modulate the activity of the His-Phe-Arg-Trp The present systematic review aims at finding original articles published in sequence. These in vitro studies lead to development of the linear the international medical literature, particularly those describing pharma- tetrapeptide compound 10, which was studied in rats. ICV cological compounds with MC4R agonist effects in (pre)clinical studies. The administration of peptide 10 to Sprague–Dawley rats induced search was performed in the databases PubMed and Embase. Pubmed and inhibition of food intake. Feeding duration reduced significantly Embase were searched on 25 June 2012. Articles in PubMed were sought over 18 h, whereas time between feeding events increased. The using the search terms described in Table 1. All articles were then recruited cumulative overnight food intake was reduced by 24% compared into Endnote X3(1988–2009 Thomson Reuters, New York, NY, USA) in order with control rats. to sort the articles using inclusion and exclusion criteria, as listed below. Hess et al.6 studied tetrapeptide analogues by changing the This was primarily performed by analysing the article titles. When the titles were not clear enough, abstracts were also scanned. When still in doubt, molecular structure of the tetrapeptide sequence by backbone the whole article was read. The inclusion criteria were: (1) articles published peptidomimetic cyclization. In vitro analyses showed that one in the english language in peer-reviewed medical journals and (2) original of the analogues tested from the BL3020 library, BL3020-1, had studies aimed at studying the effect of MC4R agonist therapeutics in the best characteristics concerning high MC4R selectivity, obese animals or humans with MC4R mutations. The exclusion criteria intestinal metabolic stability, permeability and a relatively high

International Journal of Obesity (2014) 163 – 169 & 2014 Macmillan Publishers Limited MC4R as target for obesity treatment L Fani et al 165 Table 2. Review of literature from systematic review on MC4R agonist (pre)clinical studies in animals or humans

Article MC4R agonist compound Type of study Subjects Outcome effects of compounds compared with vehicle

Hansen Melanotan II Single ICV injection Rats on high-fat and An inhibitory effect on food intake until 24 h and a reduction of body weight et al.10 Phoenix Pharmaceutical normal-fat diet was found in both high-fat and normal-fat diet rats

Ye et al.12 Tetrapeptide: compound 10 Single ICV injection Rats Feeding duration reduced signiﬁcantly over 18 h, whereas time between Not commercially available 1 h before dark cycle feeding events increased. The cumulative overnight food intake was reduced by 24% compared with the control rats

Hess S Tetrapeptide: BL3020 Daily oral dose for 12 Mice Acute effect: Up to 48% less food consumption. This effect lasted for 5 h. et al.6 Not commercially available days Chronic effect (12 days): reduction of body weight

He S MK-0489 DIO mice: single oral Diet-induced obesity Acute effect: reduced food intake up to 56% at the 4-h time point, followed by et al.13,14 Merck and Co. dose mice (acute effect) and 28 and 15% at 18 h Not commercially available DIO rats: twice daily rats (14 days) Chronic effect (14 days): lowered body weight oral dose for 14 days

Krishna R MK-0493 Once daily oral dose Obese humans (n ¼ 29) Signiﬁcant reduction in energy intake and insigniﬁcant weight loss et al.15 Merck and Co. for 12 weeks phase I and Not commercially available II trial

Hong Urea-based piperazine Twice daily oral dose Mice and rats Reduced food intake and weight reduction after 14 days et al.16 Not commercially available for 14 days

Benoit Ro27-3225 and Ro27-4680 Single ICV injection Rats db/db mice Reduced food intake at 4 h et al.17 Sigma-Aldrich Single IP injection

Conde- Cyclophane: Macrocycle 7 Single IP injection Rats trained to consume Reduced food intake ﬁrst 5 h after IP injection Frieboes Novo Nordisk a 24-h ration during 5 h et al.18 Not commercially available

Al- ACTH peptides Single ICV injection Previously fasted rats or Reduced food intake within 2 h following injection, lasting for 16 h. Effect more Barazanji Bachem Inc. ad libitum-fed rats pronounced in fasted rats et al.19

Cepoi Compound 1 Single ICV injection Mice Decreased food consumption after an overnight fast, with the greatest effect in et al.2 Not commercially available the ﬁrst hour after administration. Reduced nocturnal food intake when administered at night, 4 h after injection, lasting for 6 h

Chen Pyrrolidines: compounds Oral dosing once daily Deprivation-induced Acute: decreased food intake at until 24 h after oral administration et al.21 13b-1 for 1 week food intake in diet- Chronic (1 week): reduced food intake and weight on all days tested, reduced Not commercially available induced obese rats fat mass and decreased fasting glucose after 1 week

Kumar BIM-22493, BIM-22511 Continuous osmotic Diet-induced obese Acute reduction of food intake, improved obesity, hyperinsulinaemia et al.22 Biomeasure Inc., IPSEN mini-pump in leptin-deﬁcient mice (B À 50%) and fatty-liver disease Not commercially available subscapular region

Hsiung Human beta-MSH analogues Single ICV injection Ad libitum chow-fed rats Decreased food intake and negative energy balance, increased fat utilization, et al.23 Not commercially available 24 h after injection

Hsiung Cleaved human beta- Single ICV injection Diet-induced obese rats Induced an acute negative energy balance in indirect calorymetry 24 h after et al.24 MSH(5–22) analogue injection Bachem Inc. Abbreviations: ACTH, adrenocorticotropic hormone; MC4R, melanocortin-4 receptor; MTII, melotan II; ICV, intracerebroventricular; IP, intraperitoneal injection.

Table 3. Compounds’ half maximal effective concentration (EC50) to the MC4R in vitro and the relative potency to other MCRs

Compound hMC4R EC50 Effective Action Relative potency to other Anti-obesity function Reference in vitro (nM) treatment method MCRs (based on EC50) in Mc4r KO mice

Melanotan II 3 ICV Agonist Not speciﬁc NT 10 Compound 10 4 ICV Agonist 450 (MC1,3,5 R) NT 12 BL3020-1 4 Oral (Ra, Mu) Agonist 42 (MC5R); 415 (MC1R); 4190(MC3R) NT 6 MK-0489 5 Oral (Mu) Agonist 4200 (MC1R) Not active 13,14 MK-0493 — Oral (Hu, Mu) Agonist — - 15 Piperazine compound 1 22 Oral Partial agonist 4100 (MC1,3,5R) Not active 16 Ro27-3225 1 ICV (Ra), IP (Mu) Agonist 48 (MC1R), 4600 (MC3,5R) NT 17 Macrocycle 7 470 IP (Ra) Agonist oMC1R; 410 000 (MC3,5R) NT 18 a-MSH 6.5 ICV (Ra) Agonist Not speciﬁc NT 19 Compound 1 11.3 ICV (Mu) Agonist *(MC1,3,5R) Not active 20 Pyrrolidine 13b-1 10 Oral (Ra) Agonist 4260 (MC1,3,5R) NT 21 BIM-22493 0.27 IP, SC-pump (Mu) Agonist 420 (MC1,3R); 45000(MC5R) Not active 22 BIM-22511 0.1 IP, SC-pump (Mu) Agonist 480 (MC1,3R); 440 000(MC5R) Active 22 Ac-YR(CEHdFRWC)amide 0.6 ICV (Ra), SC (Ra, Mu) Agonist 430 (MC1R); 450 (MC3,4 R) (Ki) Not active 23 Beta-MSH(9–22) 1.1 ICV (Ra) Agonist E0.5 (MC3R) NT 24 Abbreviations: Hu, human; ICV, intracerebroventricular injection; IP, intraperitoneal injection; MCR, melanocortin receptor; MC4R, melanocortin-4 receptor; Mu, mouse; NT, not tested; Ra, rat; SC, subcutaneous.

& 2014 Macmillan Publishers Limited International Journal of Obesity (2014) 163 – 169 MC4R as target for obesity treatment L Fani et al 166 bioavailability of 8.5%. In rats, BL3020-1 was detected in the brain on a phase I/II trial in humans treated with an MC4R agonist. following oral administration. In mice, a single oral dose led to In contrast to the effect in rodents, the effect on weight loss in reduced food consumption. Up to 48% less food consumption was humans was insignificant. found in the BL3020-1 group compared with the control group. This effect lasted for 5 h. When administered for 12 days in a Urea-based piperazine partial MC4R agonist once daily oral dose, these mice showed reduced weight gain Hong et al.16 tested a urea-based piperazine, highly selective compared with controls. BL3020 was among the first publications (4100-fold relative to other MCRs) MC4R partial agonist, reporting on a MC4R compound that showed effect of reducing compound 1. With its good oral bioavailability in rats, it also weight gain when administered orally and chronically. No further showed a positive pharmacokinetic profile, low clearance and studies on BL3020 analogues were published. high plasma drug exposure. In wild-type mouse models, 4 h after administration of compound 1, food intake was reduced by Spiroindane-based acetyl amide compounds: MK-0489 and MK-0493 37% compared with no effect in MC3R/MC4R KO mice. A study by He et al.13 investigated the function of the Merck Importantly, at high doses compound 1 exhibited no penile compound MK-0489 (Rahway, NJ, USA), a spiroindane-based erectile activity in mice. The efficacy of compound 1 was also acetyl amide small molecule MC4R agonist, and several investigated in a rat model of obesity. Compound 1 was analogues. Initially, it was found that an N-methyl derivative of administered orally for 14 days and compared with vehicle and MK-0489 had good pharmacokinetic profiles in rats with a half-life MC4R-full agonist compound A23 as controls. Compound 1 of 15.1 h and good oral bioavailablity. This encouraged the authors reduced body weight more compared with the vehicle. It was to develop further amide analogues of this compound to improve comparable to MC4R-full agonist A23 in reducing the body MC4R specificity and bioavailability. In this study, MK-0489 itself weight. Piperazine compound 1 is a partial agonist with a was evaluated in rodent obesity models. relatively high half maximal effective concentration compared After oral dosage of MK-0489, reduction of food intake and with the other compounds described in this review. However, its body weight was observed in wild-type mice. Specificity was oral bioavailability is good and it is highly specific for the MC4R, tested by treatment of MC4R/3R knockout (KO) mice in which this which could be advantageous for its side effect profile. compound had no effect on food intake or body weight. The same compound was also tested in a 14-day diet-induced obese (DIO) Ro27-3225 rat model. In this study, following 13 days of twice daily oral Benoit et al.17 studied a compound, Ro27-3225, that is highly dosing, the body weight of the rats was significantly decreased in selective for MC1R and MC4R. When administered ICV to rats, it the highest dosing group (20 mg kg À 1), to an extent that increased Fos-like immunoreactivity in many parts of the nervous was comparable to dexfenfluramine treatment at 3 mg kg À 1 system, such as the nucleus of the solitary tract, central nucleus twice daily. of the amygdala, paraventricular nucleus and the area postrema. Following this, He et al.14 published another study that tested This increase is a pattern shown similarly by non-selective MC3/4R two other analogues of MK-0489 in vivo. Two models were used: a agonists. Ro27-3225 dose dependently reduced 4 h food intake DIO mouse model and an MC4R/3R double KO mouse model. when it was centrally given to rats or peripherally to db/db mice It turned out that two of the analogues tested, analogues 26 and that lack functional leptin receptors. Although this compound is 30, lowered food intake and body weight in the DIO mice. not specific for MC4R, no adverse effects were found in contrast to However, the same effect was present in the MC4/3R KO mouse MTII (a non-selective MC3/4R agonist) that caused taste aversion. models, suggesting non-MC4R-mediated effects. Analogue 32 was efficacious in the DIO mouse obesity model without an effect in A novel class of cyclophanes the KO mice, suggesting that the efficacy was mediated through Conde-Frieboes et al.18 studied a new class of cyclophanes as MC4R. When dosed orally in the DIO mouse study, analogue MC4R agonists that showed competition with the antagonist 32 reduced food intake up to 56% at the 4-h time point, followed SHU9119 over the MC4R. Compound macrocycle 7, which showed by 28 and 15% at 18-h time point. It also lowered body weight threefold selectivity for MC4R compared with MC3R in in vitro- significantly. binding assays, was studied in scheduled fed male Sprague– Krishna et al.15 described for the first time the effect of the Dawley rats, which had been trained to consume a 24-h food MC4R agonist MK-0493 in obese humans (see Table 2). In earlier ration during 5 h. Macrocycle 7 was administered intraperitoneally studies, oral administration of this drug to rodent models of to eight rats just before food was presented. Sibutramine was diet-induced obesity caused a reduction in weight and energy used as a positive control. Food intake was followed for 3 h and intake. The effect of this drug in obese humans was compared compared with a vehicle-treated group. Compared with controls, with sibutramine, which is a serotonin and a norepinephrine macrocycle 7 caused a 50% decrease in food intake within 3 h reuptake inhibitor developed for the treatment of obesity. Phase I after administration. The authors suspect that this effect suggests and II clinical trials were performed to qualify an ad libitum energy high penetration rate of the blood–brain barrier, but studies on intake model and to determine the acute effects of two doses of oral bioavailability, side effects and effect on body weight change MK-0493 as compared with placebo and sibutramine. In addition, after chronic administration have to be awaited. the evaluation of the effects of chronically administered MK-0493 on weight loss was performed. It turned out that the administration of 30 mg of sibutramine caused a statistically significant ACTH and its derivates greater reduction in energy intake when compared with a lower Al-Barazanji et al.19 studied the central effect of ACTH and its dose of 10 mg sibutramine and a placebo. However, neither derivates. The effect of the administration of ACTH 1–39 and dose of MK-0493 caused significant reductions in the energy peptides derived from the N-terminus (ACTH 1–10, ACTH 1–13 intake or weight after 12 weeks of treatment when compared amide ¼ a-MSH) and C-terminus (ACTH 18–29 and ACTH 22–39) of with placebo. ACTH on food consumption was studied in fasted rats and ad In conclusion, MK-0489 and MK-0493 demonstrated libitum-fed rats. ACTH reduced food intake in fed and fasted rats, MC4R-mediated efficacy on food intake and weight reduction in the effect being more pronounced in the fasted rats. The rodent models of diet-induced obesity, but in obese humans N-terminal-derived a-MSH peptide (ACTH 1–13), but not ACTH MK-0493 showed no significant reduction on food intake or 1–10, reduced food consumption over 2 h after being injected ICV weight compared with placebo. MK-0493 was the first publication in rats that had been fasting for 16 h, but did not reduce food

International Journal of Obesity (2014) 163 – 169 & 2014 Macmillan Publishers Limited MC4R as target for obesity treatment L Fani et al 167 consumption in fed rats. The anorectic effects of the N-terminal cAMP assays indicated B10-fold selectivity of the compounds for fragments of ACTH are mediated through the MC4R, whereas the MC4R compared with MC3R. Specificity of function was further C-terminal fragments of ACTH stimulated feeding in fasted, but determined using MC3R KO and MC4R KO female mice. When not fed, rats via an MC4R-independent mechanism. This study administered peripherally, the compounds had been shown to underlines previous findings that the natural ligand for MC4R cross the blood–brain barrier and carry out their effects. leading to satiety is a-MSH (ACTH 1–13) and not the other splice Intraperitoneal injection of BIM-22493 led to acute reduction of variants of its prohormone pro-opiomelanocortin. One of the food intake in wild-type and MC3R KO, but not MC4R KO mice. issues for the use of natural a-MSH in obesity treatment is that it is Fourteen days of BIM-22493 and BIM-22511 administration using non-specific for the MC4R. subcutaneous mini-osmotic pumps led to weight loss in wild-type and MC3R KO, but not MC4R KO mice. However, only BIM-22493 suppressed food intake in wild-type mice. BIM-22493, but not Compound 1 20 BIM-22511, also ameliorated lipid profile in DIO wild-type mice. In a study by Cepoi et al. a small peptide mimetic MC4R-specific Interestingly, BIM-22551-treated MC4R KO mice showed a agonist was tested to determine its effects on inhibiting substantial reduction (B50%) in fasting insulin levels. feeding in mice. The original pharmacological name was Overall, these results suggest that the two MC4R agonists 1,2,3R,4-tetrahydroisoquinoline-3-carboxylic acid and is renamed 20 regulate body weight, liver metabolism and glucose homoeostasis for simplification by Cepoi et al. as compound 1. In vitro assays through independent pathways. MC4R is necessary for melano- with HEK293 cells expressing human and mouse MCRs, using cortin agonist-induced weight loss and improvements in the liver cyclic AMP levels and binding assays as end points, showed that metabolism. However, MC4R is not required for the amelioration compound 1 has high potency and selectivity for human MC4R. of hyperinsulinaemia by BIM-22511 in obese MC4R KO mice. When given ICV to mice, compound 1 decreased food The BIM-22493 and BIM-22551 have the highest in vitro consumption after an overnight fast, with the greatest effect in potency, while remaining highly specific for the MC4R, compared the first hour after administration. When administered at night, with the other compounds described in this review. wild-type mice had significantly reduced food intake 4 h after the ICV injection of compound 1. This inhibition, under normal nocturnal feeding, persisted for over 6 h after administration, but Beta-MSH analogue was negligible after 18 h. Compound 1 did not reduce food intake This structure–activity relationship studied by Hsiung et al.23 used in MC4R KO mice, indicating an MC4R-specific mechanism of human b-MSH as a lead sequence to develop potent and selective action. Since their first publication in 2004, no further studies were agonists of MC4R and MC3R. A potent and selective agonist was published on this compound to our knowledge. discovered, namely, Ac-YRcyclo(CEHdFRWC)amide that reduced food intake and body weight in DIO rats and normal chow-fed Pyrrolidine diastereoisomer rats. This was mediated through its MC4R activity, as shown by 21 its lack of activity in MC4R-deficient mice. The study of Chen et al. investigated the activity of several Further study24 showed in vitro enzymatic cleavage of native pyrrolidine diastereoisomers. These compounds were tested in human b-MSH(5–22) with two ubiquitous dipeptidyl peptidases binding assays using membranes from HEK293 cells expressing (DPP), DPP-I and DPP-IV, which generated two potent MC3/4R MC4R and a radiolabeled ligand. The best compounds were then peptide analogues: b-MSH(7–22) and b-MSH(9–22). On the test tested in a whole cell functional agonist assays using cAMP day, animals were injected ICV with compound or vehicle and accumulation and competition-binding assays as end points. In indirect calorimetry was performed for 22–24 h after injection. the competitive-binding assays using MCR-expressing HEK293 The DPP-I/DPP-IV cleaved peptide, b-MSH(9–22), induced an acute cells, NDP-MSH was used as the radiolabeled ligand. Functional negative energy balance in DIO rats, whereas its parent molecule, cAMP assays were used to further characterize compounds with b-MSH(5–22), administered at the same dose, did not have any highest affinity for MC4R. In competition-binding assays, both effect. For b-MSH, further studies on oral bioavailability, 13b-1 and 13b-2 were highly selective for hMC4R over other blood–brain barrier permeability and effect after chronic admin- melanocortin receptor subtypes. The effect of pyrrolidine 13b-1 istration have to be awaited. was subsequently examined in a model of deprivation-induced food intake in DIO rats. Both the 30-mg kg À 1 dose and the positive control (fenfluramine) decreased food intake within 24 h DISCUSSION AND CONCLUSION after oral administration. Authors state that despite its high tissue distribution, which might be related to its disbasic structure, In this study, we aimed to explore future therapeutic possibilities 13b-1 had delayed brain penetration. When administered daily, for obese patients. This systematic review shows that most both the fenfluramine and 13b-1 significantly reduced food intake compounds are in the preclinical phase. However, several and weight for up to 1 week. After a week of administering 13b-1, promising pharmacological compounds influencing MC4R path- fasting glucose and body composition were measured. ways have been found. Compound 13b-1 was proven to dose dependently increase fat These animal studies show that a variety of pharmacologically loss compared with baseline measurements. Unlike fenfluramine, modified MC4R agonists are able to reduce food intake, reduce compound 13b-1 did not significantly affect lean mass. body weight and some have been shown to increase energy Compound 13b-1 also significantly decreased fasting glucose, expenditure. Most studies analysed acute effects (hours–24 h) and whereas this was not the case with administration of fenfluramine. some analysed effects after chronic administration (days–2 weeks). This compound, 13b-1, is one of the few compounds, together An overall theme of these studies was the goal of generating with the compound described in the next paragraph, that show an compounds with improved specificity for MC4R in causing these effect on glucose metabolism besides weight reduction. metabolic effects. Crossing the blood–brain barrier is also necessary to reach the MC4R. Of the 15 reviewed studies, 5 studies administered the compounds ICV only, cross-passing BIM-22493 and BIM-22511 the blood–brain barrier. The other 10 studies administered Kumar et al.22 investigated the MCR agonists BIM-22493 and the compounds peripherally, either orally or subcutaneously. BIM-22511 both in vitro and in vivo. Radioligand-binding assays In 4/15 studies, blood–brain barrier penetration was mentioned in showed relative non-selectivity of these compounds for MC3R and the article, of which 2 (compound 13b-1 and BIM-22493/ BIM- MC4R in CHO-K1 cells transfected with MCRs. However, functional 22511) studied it.

& 2014 Macmillan Publishers Limited International Journal of Obesity (2014) 163 – 169 MC4R as target for obesity treatment L Fani et al 168 Many previously characterized MC4R agonists have side effects by Raun et al.31,32 from the Novo Nordisk Research Unit (Måløv, that are likely undesirable. For example, although both MTII10 and Denmark) describes the effects of a long-acting MC4R agonist. the urea-based piperazine compound16 cause reduced food intake They studied the in vivo effects in rats and minipigs after and weight reduction in rodent animal models, MTII also causes sub chronic treatment with a MC4R selective and long-acting penile erectile activity25 and conditioned taste aversion as an a-MSH analogue (MC4-NN1) and compared with vehicle. DIO off-target effect,18 which limits its use as a therapeutic for rats were injected daily for 3 weeks and minipigs were injected metabolic dysfunction. Other important undesirable effects of subcutaneously on every other day for 8 weeks. A dose- acute treatment with MC4R agonists are increased blood pressure dependent reduction in food intake, body weight reduction and and heart rate.26 increase in energy expenditure was found in both rats and Oral administration is the most convenient mode of drug intake minipigs. For a more detailed data description, we will have to for patients, and is therefore preferred for chronic diseases that wait for the publication as original article. require long-term treatment. A major drawback of current MC4R The fourth study did not meet the inclusion criteria because it agonists is that these have poor bioavailability, hence they cannot was published after we completed our systematic search. This be taken orally. The study by Hess et al.6 and Linde et al.27 indicate study of Kievit et al.26 reports the longest treatment with that by utilizing backbone cyclization it is possible to make promising results of the MC4R agonist BIM-22493, in non-human bioactive peptides that can cross the intestinal wall and are stable primates. This compound, BIM-22493, was previously mentioned in the intestinal environment, thus providing an improvement in in the study by Kumar et al.22 Treatment with BIM-22493 results in oral bioavailability. Similarly, the study by He et al.13 showed an temporary decreases in food intake (35%), with persistent weight enhanced oral bioavailability of 3 out of 16 different spiroindane loss over 8 weeks of treatment (13.5%) in DIO rhesus macaques. amides. However, it should be noted that these are molecular These animals significantly decreased adiposity and improved studies that are still far away from being clinically integrated. glucose tolerance. The investigators did not observe any increase The study of spiroindane was performed in both mice and rats in the blood pressure or heart rate. and showed good efficacy of the MK-0489 compound. None of the MC4R agonists, described in literature, reached The compounds described by Krishna et al.15 appear to be the beyond phase I or II trials. Currently, only three drugs are closest in terms of development for clinical use. Their phase I and approved by the Food and Drug Administration for the treatment II clinical trial is the only study that has tested MC4R agonists of obesity, and two of them influence appetite through the in obese humans. Unfortunately, the observed effect of MK-0493 brain.33 Phentermine plus extended-release topiramate is a fixed- on energy intake and weight loss in humans was not as positive as dose combination of the sympathomimetic amine phentermine, expected from preclinical studies. This is probably due to which is an anorectic agent, and the antiepileptic drug topiramate. differences in the potency of effect in rodent models of obesity Phentermine works through the hypothalamus by stimulating the and obese humans, and demonstrates the difficulty in translating sympathetic system to produce catecholamines by the preclinical studies to a clinical situation. adrenal glands. In the brain it also releases serotonine and There are four studies that did not meet the inclusion criteria for dopamine. Topiramate affects neurotransmitters in the brain. this review but are worth mentioning because of their high Both medications reduce appetite and, in some people, induce potential for the future. a negative energy balance. Lorcaserin is a selective The first study did not meet the inclusion criteria because it is 5-hydroxytryptamine-2 (serotonine)2C receptor agonist.34 The an in vitro study. The research approach described in this article is 5HT2CR pathway requires downstream activation of the MC4R. exceptional in its way of approaching a possible solution for The mean percentage change in body weight reached by defective MC4Rs. The study by Rene´ et al.8 tested the use of phentermine plus extended-release topiramate or lorcaserin pharmacological chaperones28,29 to restore cell surface expression was between 4.5 and 10.9% at 1 year of treatment compared and function of mutant MC4R. One of the five pharmacological with 1.2–2.5% in controls.33 chaperones tested restored function of some of the mutant In conclusion, there are currently no effective MC4R agonists for receptors tested. This pharmacological chaperone first has to be clinical treatment of obese humans. Despite there being no tested in animal and human studies, but could be an interesting treatment, research for the defects in the pathways of MC4R are candidate for treatment of a specific subset of patients with MC4R- emerging, which provide insight into mechanisms that could be deficient obesity. used as novel targets for future therapy. From this systematic The second study of importance did not meet the inclusion review, we have come to the conclusion that there can be criteria because it is an in vitro study. Interestingly, the authors potential drugs for the treatment of MC4R-mutated and -obese used 11 naturally occurring MC4R mutations and describe patients in the future, while careful observation of clinical side the differences in pathogenesis between these mutations. In this effects in humans. study by Roubert et al.,30 the IRC-022493- and IRC-022511-ligands studied described good results for these agonists because of increasing potencies as compared with a-MSH in either wild-type CONFLICT OF INTEREST or the mutated human MC4R. This was studied in transfected cells in test tubes containing the mutated MC4-receptors from The authors declare no conflict of interest. 11 different mutated obese patients. This study also developed an excellent way of classifying MC4R mutations. Hereby, different links in the chain-reaction pathway resulting in different mutations REFERENCES on which different drugs can work were described. The 1 Withrow D, Alter DA. The economic burden of obesity worldwide: a systematic classification was made based on the known functional review of the direct costs of obesity. Obes Rev 2011; 12: 131–141. alterations in cells that are the following: intracytoplasmatic 2 Skidmore PM, Yarnell JW. 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& 2014 Macmillan Publishers Limited International Journal of Obesity (2014) 163 – 169