selective 5-ht6 receptor ligands: progress in the development of a novel pharmacological approach to...

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Pharmacology & Therapeutics 117 (2008) 207–231www.elsevier.com/locate/pharmthera

Associate editor: A. G. Ramage

Selective 5-HT6 receptor ligands: Progress in the development of a novelpharmacological approach to the treatment of obesity and

related metabolic disorders

D.J. Heal a,⁎, S.L. Smith a, A. Fisas b, X. Codony b, H. Buschmann b

a RenaSci Consultancy Ltd, BioCity, Nottingham, NG1 1GF, UKb Laboratorios Dr Esteve S.A., Av Mare de Deu de Monserrat 221, 08041 Barcelona, Spain

Abstract

The increasing global prevalence of obesity unequivocally demonstrates that neither behavioural (diet and exercise) nor pharmacologicalapproaches to this health problem are working. In this area of high unmet clinical need, the 5-HT6 receptor has generated enormous interestamongst academic and pharmaceutical industry scientists as a molecular target for the development of a new generation of safe and more effectiveanti-obesity drugs. In this review, we have described the major developments that have occurred in the fields of the medicinal chemistry andpharmacology of 5-HT6 ligands, with particular emphasis on their potential application as novel anti-obesity drugs. The last 5 years havewitnessed an increasing understanding of the 5-HT6 receptor and its structural requirements that has produced an explosion in the number anddiversity of novel, highly selective 5-HT6 receptor agonists, partial agonists and antagonists that have been designed and synthesized. In animalmodels, 5-HT6 receptor ligands of all functional types have been shown to decrease food intake when given acutely and chronically, to evokeprofound and sustained weight-loss in obese animals, and concomitantly to improve a number of cardio-metabolic risk factors. Comparator studiesin obese animal models, which are highly predictive of clinical outcomes, indicate that 5-HT6 ligands may have the potential to be moreefficacious in the treatment of obesity than the current generation of anti-obesity drugs.© 2007 Elsevier Inc. All rights reserved.

Keywords: 5-HT6 ligands; Metabolic disorders; Obesity; Rodent models; Treatment

Abbreviations: BMI, body mass index; CB1, cannabinoid receptor type 1; CoMFA, comparative molecular field analysis; CoMSIA, comparative molecular similarityindices analysis; DIO, dietary-induced obese; HDL, high density lipoprotein; IDF, International Diabetes Federation; HQSAR, hologram quantitative structure–activity relationship; IOTF, International Obesity Task Force; LDL, low density lipoprotein; NCEP-ATP III, National Cholesterol Education Program AdultTreatment Panel III; PPH, primary pulmonary hypertension; QSAR, quantitative structure–activity relationship; RT-PCR, reverse transcription polymerase chainreaction; SNRI, serotonin-noradrenaline reuptake inhibitor; SSRI, selective serotonin reuptake inhibitor; UKPDS, UK Prospective Diabetes Study Group; VLDL,very low density lipoprotein.

Contents

⁎ CorrespondingE-mail addres

0163-7258/$ - seedoi:10.1016/j.phar

1. The obesity epidemic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2082. An overview of the pharmacotherapy of obesity with particular reference to serotonergic approaches 2093. Distribution of 5-HT6 receptors in the central nervous system . . . . . . . . . . . . . . . . . . . 2104. New developments in the chemistry of 5-HT6 receptor ligands . . . . . . . . . . . . . . . . . . . 2115. Receptor binding and functional characterisation of 5-HT6 ligands in vitro. . . . . . . . . . . . . 2196. Effects of various 5-HT6 receptor ligands on satiety, food intake and body weight . . . . . . . . . 2227. The impact of 5-HT6 receptor-mediated weight-loss on obesity-related cardio-metabolic risk factors 226

author. Tel.: +44 115 912 4260; fax: +44 115 912 4263.s: [email protected] (D.J. Heal).

front matter © 2007 Elsevier Inc. All rights reserved.mthera.2007.08.006

mailto:[email protected]

http://dx.doi.org/10.1016/j.pharmthera.2007.08.006

Fig. 1. The increasperiod between 19Force (IOTF) webs

208 D.J. Heal et al. / Pharmacology & Therapeutics 117 (2008) 207–231

8. Potential side-effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2279. Future prospects for 5-HT6 receptor ligands in the treatment of obesity . . . . . . . . . . . . . . 227References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

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1. The obesity epidemic

Amongst healthcare experts around the world, there is now aconsensus that the global epidemic of obesity will be one of theleading causes of morbidity and mortality for this and futuregenerations unless the inexorable rise in the prevalence ofthis disorder is reversed. The estimate of the current globalprevalence of obesity provided by the International ObesityTask Force (IOTF, 2007) states that there are over 300 millionpeople worldwide who would be classified as clinically obese,ie having a body mass index (BMI=weight in kg/height in m2)of ≥30. Taking the UK as an example, the data presented inFig. 1 show that until 1990 levels of adult obesity remainedrelatively low and constant at 5–10% of the population;however, in the subsequent 15 years, the prevalence of obesityin both men and women has more than doubled. What is morealarming is that obesity is rapidly becoming a serious problemin children and the data for the period 2000–2006 indicate that

ing prevalence of adult and childhood obesity over the 25-year80 and 2005. Data taken from the International Obesity Taskite (IOTF, 2007).

childhood obesity is estimated to afflict 20–25% of males and25–30% of females (Fig. 1). The health consequences of theobesity epidemic are now so serious that some experts believe itwill reverse the inter-generational increase in life expectancywith this generation of children now being first who can expectto live significantly less long than their parents (Daniels, 2006).The metabolic disturbances that occur in obesity, andparticularly central adiposity, are drivers of other life-threaten-ing metabolic disorders including dyslipidaemia (elevatedplasma concentrations of triglycerides and low densitycholesterol and decreased plasma concentrations of high densitycholesterol) hypertension, proinflammatory atherogenesis, pre-diabetes (insulin resistance and impaired glucose tolerance) andType II diabetes (Mokdad et al., 2001; Pi-Sunyer, 2002;National Cholesterol Education Program Adult Treatment PanelIII [NCEP,ATP,III]). The co-existence of this cluster of cardio-metabolic risk factors in subjects was initially described asSyndrome X (Reaven, 1988), but it is now more commonlyreferred to as the Metabolic Syndrome (Alberti & Zimmet,1998; NCEP-ATP III, 2002; International Diabetes Federation[IDF], 2005). All of the above metabolic disorders have beendemonstrated to increase the risk of serious macrovascularevents, including myocardial infarction, stroke and angina (UKProspective Diabetes Study Group UKPDS 38, 1998; Montaniet al., 2002; Beckman et al., 2002; NCEP-ATP III, 2002; Flacket al., 2003; Rashid & Leonardi-Bee, 2003; Law et al., 2003;UKPDS 66, 2004). In addition to the morbidity and mortalityrisks associated with cardio- and cerebrovascular events, obe-sity is also a predisposing factor in ≤30% of cases of cancer ofthe colon, breast, kidney and digestive tract (IOTF, 2007). Aswell as the life-threatening consequences of obesity, there areother chronically debilitating conditions associated with thismetabolic disorder including sleep apnoea, aggravated arthritis,gout, gallstones, and not least, low self-esteem and an increasedincidence of affective disorders in obese individuals (Goodman& Whitaker, 2002; Wardle et al., 2002; Ackard et al., 2003;Simon et al., 2006).

The most plausible explanation for the rapid increase inobesity is that it is being driven by a combination of genetic,social and environmental factors. The “thrifty genotype” hasensured the survival of humans during alternating periods ofglut and famine. However, this thrifty genotype is not suited toan environment where there is an almost unrestricted access toan abundance of low cost calorie-dense foods, ie a modernWestern diet, combined with a relatively sedentary lifestyledominated by office work and automobiles (Prentice & Jebb,1995; Deedwania, 2004; Ello-Martin et al., 2005). Although asignificant proportion of people manage very successfully tomaintain a healthy body weight by careful dietary selection anda reasonable level of physical exercise, the percentages shownin Fig. 1 demonstrate that for many others this route has notresulted in the desired healthy outcome. For some of the obese

209D.J. Heal et al. / Pharmacology & Therapeutics 117 (2008) 207–231

population, pharmacotherapy can provide the requisite adjunc-tive support to diet, exercise and lifestyle modification that willdeliver clinically beneficial (≥5%) weight reduction. Althoughperfect body image is now an established aspiration for agrowing proportion of people, particularly in many Westernizedpopulations where abnormally low BMIs are regarded asfashionably desirable, the cosmetic aspects of pharmacotherapyare not the subject of this review which is focused exclusivelyon the drug treatment of obesity for medical purposes.

2. An overview of the pharmacotherapy ofobesity with particular reference to serotonergic approaches

A list of approved anti-obesity drugs together with the drugcandidates in mid- and late-stage clinical development is shownin Table 1. Current drug treatment of obesity is dominated bythree drugs, ie phentermine, sibutramine and orlistat, with afourth drug, ie rimonabant, having entered some markets in2006 and 2007. These four drugs have quite differentpharmacological mechanisms. Phentermine is an off-patent,amphetamine-like β-phenylethylamine that suppresses appetiteby releasing dopamine and noradrenaline in the brain;sibutramine (Meridia®, Reductil®) is a serotonin and noradre-naline reuptake inhibitor (SNRI) that reduces food intake byenhancing satiety (Heal & Cheetham, 1997; McNeely & Goa,1998); rimonabant (Acomplia®) is a cannabinoid CB1 receptorantagonist that reduces food intake by a central mechanism, butin addition, also improves plasma lipid profiles and glycaemiccontrol by a peripheral action (Bensaid et al., 2003; Vickers &Kennett, 2005; Juan-Picó et al., 2006). Finally, orlistat (Xe-nical®), which is the only drug with a peripheral anti-obesitymechanism, impairs the absorption of fat from food by theinhibition of gastric and pancreatic lipases (McNeely &

Table 1Current and future drugs to treat obesity

Compound Mode of action Status Company

Phentermine Sympathomimetic Registered(generic)

SmithKline-French

Sibutramine SNRI Registered Knoll/AbbottOrlistat Lipase inhibitor Registered RocheRimonabant CB1 antagonist Registered Eu Sanofi-AventisMK-0364 CB1 antagonist Phase 3 MerckCP-945,598 CB1 antagonist Phase 3 PfizerSLV-319 CB1 antagonist Phase 2 Solvay/BMSCetilistat Lipase inhibitor Phase 2 AlizymeP57 Hoodia-gardonii extract Phase 2 PhytopharmAOD 9604 hGrowth hormone

fragmentPhase 2 Metabolic

Lorcaserin 5-HT2C agonist Phase 2 ArenaVabicaserin 5-HT2C agonist Phase 2 WyethBVT 933 5-HT2C agonist Phase 2 Biovitrum/GSKATHX-105 5-HT2C agonist Phase 2 AthersysAVE-1625 CB1 antagonist Phase 2 Sanofi-AventisQnexa Topiramate+phentermine Phase 2 VivusHMR 1426 Inhibitor of gastric

emptying (?)Phase 2 Sanofi-Aventis

GI 181771 CCK-A antagonist Phase 2 GSK

SNRI = serotonin–noradrenaline reuptake inhibitor. CCK-A = cholecystokinintype A.

Benfield, 1998). Thus, with the exception of sibutramine,which is an SNRI, none of these drugs reduces food intake and/or increases energy expenditure by a pharmacological mechan-ism involving 5-HT.

In responding patients (usually ∼60 to 70% of those treated),these anti-obesity drugs provide a moderate degree of weight-loss generally of the order of 5–10 kg (McNeely & Goa, 1998;McNeely & Benfield, 1998; Haddock et al., 2002; Vickers &Kennett, 2005), although when used in specialist obesity clinicswhere there is a high degree of lifestyle support, the weight-lossis likely to be much greater (Wadden et al., 2005). However,none of these drugs delivers the overall degree of weight-lossdesired by patients and physicians, and each has its ownburden of side-effects, eg abuse liability and hypertensionwith phentermine, increased blood pressure and heart rate withsibutramine, gastro-intestinal disturbance with orlistat andnausea and mood disturbances with rimonabant (McNeely &Benfield, 1998; McNeely & Goa, 1998; Pi-Sunyer et al., 2006;Scheen et al., 2006). Thus, there is still a significant unmetclinical need for the development and introduction of novelanti-obesity drugs that have greater efficacy and a reducedadverse event burden than the existing agents, includingrimonabant, which has recently been refused regulatory ap-proval in the USA.

Drug candidates in mid- (Phase 2) to late-stage (Phase 3)clinical development are also shown in Table 1. This list includesa range of CB1 receptor antagonists from Merck, Pfizer, Solvay/BMS together with the follow-up compounds from Sanofi-Aventis, ie AVE-1625. Compounds with a variety of otherpharmacological mechanisms are currently being evaluated forthe treatment of obesity, but the main thrust from a serotonergicperspective is the 5-HT2C agonist approach, which will bediscussed when we review the status of serotonergic anti-obesitydrugs.

In this section of the review, we will provide a brief overviewof the history of serotonergic anti-obesity drugs as well asproviding information on which serotonergic drug targets arebeing pursued in the search for safer andmore effective therapiesfor this metabolic disorder. All of the compounds in thispharmacological class have a central mechanism of action. Thehypothalamus is responsible for the regulation of metabolicbalance, ie it controls both food intake and energy expenditure,and is accepted to be the likely site of action for all of these drugs,because they have all been shown to reduce food intake inanimals by enhancing satiety (Halford et al., 1998; Svartengrenet al., 2003; Gannon et al., 2006).

The amphetamines, ie DL-amphetamine (Benzedrine®) andD-amphetamine (Dexedrine®), were the first centrally-actingdrugs for the treatment of obesity. In the 1950's/60's, the use ofthese drugs in obesity was widespread until their diversion andabuse led to a major reduction in the prescribing of thesepsychostimulants as appetite suppressants. The pharmaceuticalindustry responded to this challenge by synthesizing a range ofβ-phenyethylamine congeners with a markedly lower potentialfor abuse; phentermine and DL-fenfluramine were the mostimportant products of this Research and Development endea-vour. The bulky ρ-trifluoromethyl substitution on the phenyl


ring in DL- and D-fenfluramine prevents these compounds fromacting as substrates for the dopamine reuptake transporter,thereby eliminating the psychostimulant dopamine-releasingcomponent (Rothman et al., 2001). Although these drugs,especially D-fenfluramine, were initially believed to act asselective 5-HT-releasing agents and reuptake inhibitors, laterresearch revealed that they were able to release noradrenaline aswell as 5-HT in vitro (Heal et al., 1998; Rothman et al., 2001)and in vivo (Rothman et al., 2003). In fact, Rothman et al.(2003) reported that in vivo D-fenfluramine and its activemetabolite, D-norfenfluramine, were non-selective monoaminereleasing agents. Racemic DL-fenfluramine (Pondamin®), andlatterly D-fenfluramine (Redux®), were moderately effective inproducing weight-loss in obese patients (Guy-Grand et al., 1989;Haddock et al., 2002), but they were not as effective as laterintroductions like sibutramine (Hanotin et al., 1998). However,when used together with phentermine as “fen/phen”, the efficacyof this drug combination was exceptionally powerful (Weintraubet al., 1984, 1992). The reign of D-fenfluramine was relativelyshort following reports first of a greater incidence of primarypulmonary hypertension (PPH) in patients, who had been takingthis 5-HT-releasing agent (Abenhaim et al., 1996), and latterly,cardiac valve damage (Connolly et al., 1997). Both drugs werevoluntarily withdrawn from the market by the manufacturersin 1997.

The SSRI, fluoxetine, which reduces food intake in rodentsby enhancing satiety (Halford et al., 1998), has also beenevaluated for its potential to treat obesity in humans. In clinicaltrials, in obese subjects, fluoxetine was found to producereasonably good weight-loss at 8 weeks (Levine et al., 1989);however, in a longer 1-year, Phase 3 trial, the weight-lossproduced by this SSRI was found to reach a nadir at ∼6 monthsand, thereafter, tolerance to its weight-loss action developed toan extent that the patient group on fluoxetine weighed almostexactly the same at the end of the trial as the group givenplacebo (Goldstein et al., 1994). Since it is now accepted thatdrug treatment of obesity requires long-term therapy, this failureto maintain efficacy at 12 months effectively halted the devel-opment of fluoxetine in obesity.

Table 2Serotonergic approaches to the treatment of obesity

Compound Mode of action Status Company

Fenfluramine 5-HT releasingagent

Withdrawn 1997 Servier

Dexfenfluramine 5-HT releasingagent

Withdrawn 1997 Servier

Fluoxetine SSRI Abandoned for lackof efficacy in Phase 3

Lilly

Lorcaserin 5-HT2C agonist Phase 2 ArenaVabicaserin 5-HT2C agonist Phase 2 WyethATHX-105 5-HT2C agonist Phase 2 AthersysBVT 74316 5-HT6 antagonist Phase 1 BiovitrumPRX-07034 5-HT6 antagonist Phase 1 EpixE-6837 5-HT6 agonist Preclinical EsteveV 17627 5-HT2C agonist Preclinical VernalisSUVN 503/SUVN 504 5-HT6 antagonists Research Suven

SSRI = serotonin-selective reuptake inhibitor.

More recently, experiments performed in rodents have indi-cated that D-fenfluramine-induced hypophagia is a 5-HT2C

receptor-mediated response (Vickers et al., 1999, 2001) andseveral companies have sought to develop selective 5-HT2C

agonists as clinical candidates for obesity. The best knowncompounds are listed in Table 2. Arena Pharmaceuticals hasrecently reported Phase 2 clinical results for their 5-HT2C

agonist, lorcaserin. In a 12-week, Phase 2B trial, the mosteffective dose of lorcaserin, ie 10 mg bid, produced a meanplacebo-subtracted, weight-loss of ∼2.5 kg (Smith et al., 2006).Consistent with the hypothesis that the anti-obesity action ofD-fenfluramine is indirectly mediated by activation of 5-HT2C

receptors, the degree of weight-loss observed with lorcaserinwas similar to that reported for D-fenfluramine at 12 weeks byHanotin et al. (1998). However, it is important to note that inthis head-to-head study of D-fenfluramine and sibutramine, theweight-loss produced by sibutramine was significantly greater(Hanotin et al., 1998). In summary, therefore, these prelimin-ary results suggest that selective 5-HT2C agonists are unlikelyto deliver greater efficacy to treat obesity than their progenitor,D-fenfluramine, and in our opinion, are unlikely to addressthe clinical need for anti-obesity drugs that are significantlymore effective than those currently available. In terms of drug-induced side-effects, the echo-cardiographic results from thisstudy showed no evidence of heart valve damage with lorcaserintreatment (Smith et al., 2006) that had previously been observedwith D-fenfluramine (Connolly et al., 1997) suggesting that thisdirect agonist approach may have circumvented this seriouscardiac adverse event. On the other hand, since PPH is anextremely rare adverse event, it is unlikely that a potentialassociation between PPH and 5-HT2C agonist therapy can bediscounted until any drug has been widely prescribed post-approval. Moreover, 5-HT2A receptor agonism has beenimplicated in the actions of serotonergic hallucinogens(Smith et al., 1998; Nelson et al., 1999), so it is essentialthat any 5-HT2C agonist is highly differentiated with respect toits 5-HT2C/5-HT2A receptor selectivity; in this regard,lorcaserin has only ∼15-fold 5-HT2C/5-HT2A selectivity(Smith et al., 2006).

From this overview, it is clear that serotonergic drugs havenot thus far proven to be particularly effective as anti-obesitytherapies and attempts to exploit known mechanisms linked toD-fenfluramine do not appear to have the potential to deliversignificantly greater weight-loss than their antecedent. Recently,however, new impetus has been injected into anti-obesity drugresearch with the unexpected finding that 5-HT6 receptorligands may offer the potential for greater efficacy in the treat-ment of obesity than existing serotonergic pharmacologicalapproaches.

3. Distribution of 5-HT6

receptors in the central nervous system

The 5-HT6 G-protein coupled receptor was first cloned fromrat striatal tissue using RT-PCR in the early 1990's (Monsmaet al., 1993; Ruat et al., 1993). Consistent with the structure ofthis family of receptors, the 5-HT6 receptor is a seven


transmembrane-spanning protein of about 440 amino acids andthe human and rat receptors have an 89% sequence homology(Kohen et al., 1996). The 5-HT6 receptor is highly unusual for amember of the serotonin receptor family because its distributionis almost exclusively within the central nervous system (CNS;Monsma et al., 1993; Ruat et al., 1993). The mouse amino acidsequence shows 97% homology with the rat 5-HT6 amino acidsequence and 89% similarity to the human sequence (Hirst et al.,2003). With regard to the serotonin receptor family, the 5-HT6

receptor shows the most sequence homology with 5HT2

receptors (Setola & Roth, 2003). Characterisation of the receptorin transfected cell lines demonstrated that it was positivelycoupled to adenylyl cyclase (Monsma et al., 1993; Ruat et al.,1993; Max et al., 1995).

In the rat brain, the highest levels of 5-HT6 receptor mRNAare present in the striatum, olfactory tubercle, nucleusaccumbens, hippocampus, cortex, cerebellum, hypothalamusand the amygdala (Monsma et al., 1993; Ruat et al., 1993; Wardet al., 1995). The highest density of mRNA within thehippocampus was found to be in the dentate gyrus, CA1,CA2 and CA3 regions (Ruat et al., 1993; Ward et al., 1995). Thelocalization of 5-HT6 receptor protein has also been determinedusing immunohistochemical or radiolabelling techniques.Receptor presence was found in the olfactory tubercle, cortex,nucleus accumbens, striatum, hippocampus, cerebellum, thala-mus, substantia nigra, superficial layer of the superiorcolliculus, motor trigeminal nucleus and facial nucleus and inthe hypothalamus (Gérard et al., 1997; Hamon et al., 1999; Foneet al., 2002; Roberts et al., 2002), which is the brain regionresponsible for the regulation of food intake and energyexpenditure, and is an important site for the action ofcentrally-acting anti-obesity drugs. Hence, there is a very goodcorrelation between the distributions of 5-HT6 receptor mRNAand protein. However, levels of both 5-HT6 receptor mRNAand protein were found to be relatively low in mouse brain,and despite a high degree of receptor homology across the rat,human and mouse, important differences exist between themouse 5-HT6 receptor subtype and the rat and humanhomologues of this receptor (Hirst et al., 2003). Thus, the5-HT6 receptor is not expressed at high levels in the basalganglia of the mouse, unlike the human or rat, and in thisspecies it is expressed at much lower levels in a number ofother brain regions also (Hirst et al., 2003). Furthermore, site-directed mutagenesis experiments performed by Hirst andcolleagues (2003) revealed that the binding pocket whereantagonists and agonists bind is quite different in the mouse5-HT6 receptor compared with the agonist and antagonistdocking sites present in the human or rat 5-HT6 receptorsubtypes. Together, these inconsistencies indicate that whilstthe rat is a good surrogate species to predict the pharmacol-ogy of 5-HT6 receptor ligands in humans, much more cautionneeds to be exercised when evaluating data that have beengenerated using mice.

Light and electron microscopic techniques revealed that rat5-HT6 receptor immunoreactivity was associated only withneurones (Hamon et al., 1999) and was present in the dendriticprocesses of cells in the hippocampus (Gérard et al., 1997).

Hamon et al. (1999) found that receptor immunoreactivity waslocalized in the strata oriens and radiatum of the CA1 region andthe molecular layer of the dentate gyrus oriens. However, thepyramidal and granular cell layers did not show any immuno-labelling. Taken together with the presence of 5-HT6 mRNA inthe latter areas (Ward et al., 1995), this finding indicated that5-HT6 receptors may be transported from the pyramidal andgranule cell bodies to dendritic areas.

When serotonergic neurones in the brains of rats wereablated with 5,7-dihydroxytryptamine, this did not affect thelevels of 5-HT6 receptor mRNA in the hippocampus, striatumand nucleus accumbens (Gérard et al., 1996), suggesting that it isnot present as an autoreceptor on serotonergic nerve terminals.Lesioning of the nigro-striatal pathway with 6-hydroxydopa-mine also did not alter the level of the 5-HT6 antagonist, [

125I]-SB258585, binding in any of the brain regions examined,suggesting that 5-HT6 receptors are not located on dopaminergicneurones (Roberts et al., 2002). Similarly, lesioning of thecholinergic system with the immunotoxin, 192-IgG-saporin, didnot alter the density of 5-HT6 receptor mRNAor protein, arguingagainst these receptors being located on cholinergic neurones(Marcos et al., 2006). Neurones which express dendritic 5-HT6

receptors have been shown to innervate glutamic aciddecarboxylase-positive cells. Using a dual-labelling immuno-histochemical technique, 5-HT6 receptors were co-localizedwith GABAergic neurones in the hippocampus in greater than20% of 5-HT6 immunoreactive neurones (Fone et al., 2002).

The human 5-HT6 receptor was cloned by Kohen andcolleagues in 1996 and they found the highest expression ofmRNA to be in the caudate nucleus, followed by thehippocampus and amygdala. Low expression levels werefound in the thalamus, subthalamic nuclei and substantianigra. East et al. (2002) found that the relative distribution ofthe radiolabelled 5-HT6 antagonist, SB-258585, in the striatum,hippocampus and cortex was similar to that reported in the rat. Inaddition, the distribution was in agreement with 5-HT6 receptormRNA and determined by in situ hybridisation (East et al.,2002).

4. New developments in thechemistry of 5-HT6 receptor ligands

A range of different antipsychotic and antidepressant drugsshow reasonable affinity for the 5-HT6 receptor (Monsma et al.,1993; Kohen et al., 2001; Slassi et al., 2002; Davies et al.,2005). Agents that bind at human 5-HT6 receptors with Ki

valuesb50 nM include 5-methoxytryptamine, bromocriptine,octoclothepin, and the neuroleptics, clozapine, olanzapine,loxapine, chlorpromazine and fluphenazine (Unsworth &Molinoff, 1994; Davies et al., 2005; Holenz et al., 2005).

Soon after its discovery in 1993, the 5-HT6 receptor becamean attractive target for medicinal chemistry, since it wasestablished that numerous tricyclic antipsychotics and anti-depressants showed high affinity for that receptor (Johanssonet al., 2005). Since then, many new 5-HT6 ligands have beensynthesized, amongst which, various selective agents have beenidentified (Johansson et al., 2005). Fig. 2 shows the impressive

Fig. 2. Number of publications and patents in the 5-HT6 field between the years 1993 and 2006. Data taken from the SciFinder database searching the key word 5-HT6,April 2007.


increase in the number of publications and patents in the 5-HT6

receptor field, demonstrating a high interest in industrial andacademic research.

Historically, the first ligands for the 5-HT6 receptor werefound in the late 1990's by high-throughput screening of com-pound libraries at Roche and GlaxoSmithKline, resulting in theantagonist tool compounds, Ro 04-6790 (1) and Ro 63-0563(2) (Sleight et al., 1998), as well as in the first clinical devel-opment candidate, the phenyl–piperazine 5-HT6 receptor anta-gonist, SB-271046 (3)(Bromidge et al., 1999; Fig. 3). Thiscompound was entered into a Phase I human trial, but wasdiscontinued most probably due to insufficient blood-brainbarrier penetration. At about the same time, Richard A. Glennonfrom Virginia Commonwealth University, one of the pioneers of5-HT6 receptor ligand synthesis, developed the first indole-based structures, starting from the endogenous ligand, serotonin(Glennon et al., 1999; Lee et al., 2000; Slassi et al., 2002).

Fig. 3. Structures of early lead structures and tool compounds; Ro-04-6

The concept of a pharmacophore is one of the most basicideas in medicinal chemistry. It is commonly understood as aspatial orientation of various functional groups or features of amolecule, which are required for activity at a biomoleculartarget (Van Drie, 2003; Gunner et al., 2006). Pharmacophoredevelopment can be approached from two perspectives:calculations involving ligands only (a ligand-based approach),or an approach which also includes information about thestructure of a receptor binding site (a receptor-based approach).However, only three of the recently published studies havedescribed pharmacophore models for 5-HT6 receptor antago-nists. An excellent overview of pharmacophore models,developed for different subtypes of 5-HT receptors belongingto the GPCR family, was recently presented by Bojarski (2006).

By using a ‘deconstruction–reconstruction’ strategy, Glen-non and coworkers were able to identify first the ‘minimum’pharmacophore requirements for efficient 5-HT6 receptor

790 (1), Ro-63-0563 (2), SB-271046 (3), EMDT (4), MS-245 (5).


binding, and then proceeded consecutively to ‘introduce’chemical groups into the ‘minimum’ binding structure in orderto guarantee selectivity and functionality whilst maintaininghigh affinity (Glennon, 2003). His work culminated in thesynthesis of a large variety of different indole-derived, selective5-HT6 receptor binding structures, eg the agonist, EMDT (4) andthe antagonist, MS-245 (5) (Glennon et al., 2000) (Fig. 3).

Glennon was the first researcher to discover the importance ofa sulfonamide motif within the indole-type structures (originallyintroduced as N-protective group; Glennon et al., 2000) forefficient 5-HT6 receptor binding and antagonism (Tsai et al.,2000).

Since the publication of Glennon on the first structure–activity relationship of 5-HT-like structures in 1999 (see part 1:Glennon et al., 1999) and 2003 (see part 2: Glennon, 2003),significant contributions to an understanding of the pharmaco-phore have been made, either by receptor- or ligand-basedmodelling.

The first receptor-based pharmacophore modelling studieswere described by Pullagurla et al. (2003) and also by Hirst et al.(2003). Pullagurla et al. (2004) published a pharmacophoremodel based on homology modelling with the bovine rhodopsincrystal structure. Automated docking studies suggested twodifferent binding sites for indole-based ligands, one beingattributed to agonist, the other one to antagonist functionality.These binding site proposals were consistent with site-directedmutagenesis studies performed in earlier investigations (Boesset al., 1997, 1998; Hirst et al., 2003).

In 2004, two papers from a South Korean research groupwere published, establishing hologram quantitative structure–activity relationship (HQSAR) (Doddareddy et al., 2004a) andcomparative molecular field analysis/comparative molecularsimilarity indices analysis (CoMFA/CoMSIA) 3D QSAR

Fig. 4. A hypothetical framework model of 5-HT6 receptor ligands. Model proposestructural requirements for efficient binding. Motifs I and IV (pink colour) represenrepresent a double acceptor and a proton donor moiety, respectively (Y=C or N; Z

(Doddareddy et al., 2004b) studies based on different trainingsets of 5-HT6 receptor antagonists (mono- or bicyclic aryl–piperazines and indoles, respectively) and the lowest energyconformation of the model of tryptamine. With regard to stericand electrostatic contributions, nearly the same regions weretraced by CoMFA and CoMSIA contour maps. Stericallyfavored regions were found on an arylsulfonyl group, whereaslarge substituents were unacceptable at the amine side-chain.

In a parallel study, the same authors described HQSARmodels, developed for two training sets of 5-HT6 receptorantagonists (Doddareddy et al., 2004a). The first consisted ofstructurally similar arylsulfonamide derivatives, obtained frompapers published by Bromidge et al. (1999), whilst the othercontained compounds with a diversified structure, discussed inGlennon's review (Glennon, 2003). Both those models werepositively validated by external test sets. The contribution mapfor the second training set showed major, pharmacologicallyimportant fragments, ie two nitrogen atoms (one from thepiperazine ring and the other of sulfonamide function), whereasa more detailed contribution of individual atoms to the overallactivity could be seen on the map created for the first set.

Apart from the above-cited 3D QSAR studies on 5-HT6

receptor antagonists, Holenz et al. (2006) recently developed a2D hypothetical framework model for designing potent 5-HT6

receptor binding agents. Based on a medicinal chemistry-guidedanalysis of reference compounds, a simplified qualitativepharmacophore framework model was established.

The main components of a 5-HT6 ligand are depicted inFig. 4. They comprise two hydrophobic areas (squares I and IV),the core one (square IV) being mostly an indole or indole-likecore or a mono- or bicyclic aryl motif. The other one (square I) isopen to diverse hydrophobic structural elements, which quiteoften are dominated by the commercial availability of sulfonyl

d by Holenz et al. (2005, 2006). Shown are the most common groups and thet hydrophobic areas, whereas motif II (blue color) and motif III (green color)=N or C).


chlorides. Favorable motifs include phenyl, naphthyl, ben-zothiophene, imidazo[2,1-b]thiazole, and also p-aminophenyl,as suggested from the modeling study of Pullagurla et al. (2004).In addition, a protonizable nitrogen (proton donor, in themajority of cases a tertiary aliphatic amine function; see circleIII) (Fig. 4) and usually a double hydrogen bond acceptor (circleII) (Fig. 4) are amongst the four key requirements. The hydrogenbond acceptor is nearly always identical to a sulfonamide or asulfonyl function. In some cases also lactames or even a benzylfunction have been reported (Harris et al., 2004; Kolanos et al.,2005), indicating that this H bond acceptor function can beomitted under certain circumstances. The geographical orienta-tion of the four key motifs within the proposed frameworkallows for prediction (and optimization) of novel 5-HT6 receptorligands.

Later, López-Rodríguez et al. (2005) came to a similarconclusion on the basis of a more elaborate 3D pharmacophoremodel, using Catalyst software and a training set of 45 5-HT6

receptor antagonists. By matching their results with acomputational 5-HT6 receptor model, which again was basedon the bovine rhodopsin crystal structure, the exact interactionsof the four motifs in the pharmacophore model with amino acidsin the transmembrane domain of the receptor were predicted.The structural features essential for affinity were also discussedin relation to potential interactions with the respective residuesin the binding pocket of a computational model of the 5-HT6

receptor. The schematic representation of connections betweenthe pharmacophoric elements and the corresponding aminoacids predicted to be involved in the ligand–receptor interac-tions was also proposed and is schematically shown in Fig. 5.

Given the postulated stringent pharmacophore requirementsdescribed above, it becomes obvious that there is a stronggeneral resemblance of nearly all reported 5-HT6 ligands.Chemically, they can be clustered into two main groups, one ofwhich comprises the indole and indole-like (mainly generatedby scaffold hopping) sub-classes derived from the endogenousligand serotonin, the other main group comprises the mono- andbi- and tricyclic aryl–piperazine sub-classes, analogs from thefirst high-throughput screen hits found by GlaxoSmithKline. Afifth sub-class comprises miscellaneous core structures andbears at least an arylsulfonyl motif as common feature. In Fig. 6general formulae of the different categories of 5-HT6 receptorligands classified by Holenz et al. (2006) are shown.

An overview of 5-HT6 ligands covering the literature untilmid-2005 is reviewed by Holenz et al. (2006). Following thisstructural classification, examples from the patent literature areshown in Fig. 7, which were published from mid-2005 untilApril 2007.

The earlier indole-type 5-HT6 ligand structures very muchresemble serotonin, with the sulfonyl function attached to theindole-N1. Compounds (6)–(10) in Fig. 7 represent newerexamples of this sub-class representing the first generation of5-HT6 receptor ligands.

In order to enhance the freedom-to-operate (with respect topatentability) within the indole-type structural class as well as tounderstand in more detail the conformational requirements ofthe four key motifs for high-affinity ligands, the principle of

‘scaffold hopping’ has been widely employed. Scaffold hoppingis a well-known medicinal chemistry concept using 2D or 3Dmodeling tools in order to substitute a core scaffold known toproduce high-affinity ligands by another one leading to similargeographical orientation of the substituents (these beingpharmacophore key motifs). This process results in unprece-dented biomimetic compounds, which also have potentiallyhigh binding affinities. Thus, using similar substitution patternsfor the key motifs, several bicyclic and polycyclic systems havebeen synthesized in order to generate biomimetic analogsof indole-type ligands. Compounds (11)–(21) (Fig. 7) areexamples of the newer generation of this sub-class of 5-HT6

receptor ligands. 1H-pyrrolo[2,3-b]pyridine (cf. (11)), 3,3-dimethyl-2,3-dihydro-1H-indole (cf. (12)), chromane (cf.(13)), 1,2,3,4-tetrahydro-naphthalene (cf. (14) and (15)),benzofurans (cf. (18)), and 1H-indazole (cf. (13), (14), (19),(20), and (21)) are used as indole mimetics (Fig. 7). Compound(11) is described as an agonist, whereas all other structures inFig. 7 are antagonists.

Historically, the research group of GlaxoSmithKline washeavily involved in pioneering the monocyclic aryl–piperazineligands as an early structural class of 5-HT6 receptor ligandsfound by high-throughput screening. Chemically, the firstoptimization efforts were performed mainly at the sulfonamidemotif introducing a range of commercial sulfonylchlorides, andat the free piperazine nitrogen. One main optimization focuswas to improve blood-brain barrier penetration (MedicinalChemistry- XVIIIth International Symposium, 2004) of theirlead compound, the Phase I candidate, SB-271046 (3) (Fig. 3).A newer example of this sub-class is compound (22) (Fig. 7)without any sulfonyl or sulfonamide moiety. This compoundclass was identified by a virtual screening campaign.

Similar to the phenyl–piperazine ligands are bi- and tricyclicaryl–piperazine structures such as compounds (23)–(30) (Fig.7), with appropriately attached arylsulfonyl groups showingpromising 5-HT6 receptor binding affinities. The first represen-tatives were reported in 1999 by Roche (Stadler et al., 1999).Since then, nearly unlimited possibilities to attach the piperazineand the arylsulfonyl motifs to bi- and tricylic (partial) aromaticsystems have been realized by many research groups, resultingin high-affinity ligands such as compounds (24) or (27) (Fig. 7).

Compounds (31)–(35) in Fig. 7 represent a collection ofnewer compounds with different core structures, but all bearingan arylsulfonyl motif. Compounds (36)–(39) (Fig. 7) are alsodisclosed as selective 5-HT6 receptor ligands, but not bearingany of the structural features used for the previous classification.

In Fig. 8, some newer pharmacological tool compounds,preclinical candidates as well as clinical candidates have beensummarised. Data on the 5-HT6 receptor ligand, E-6837 (40)(Fig. 8), has been published as an active compound causinggradual weight-loss in dietary-induced obese rats (DIO rats;Fisas et al., 2006). SB-737050A and SB-773812 are non-selective 5-HT6 receptor ligands that also have affinity for otherreceptors such as 5-HT2A, 5-HT2C, D2 and D3. They areantipsychotic drugs under clinical development having reachedPhase 2. FMPD and BGS-20761 are examples of non-selectivepreclinical tool compounds with antipsychotic properties.

Fig. 5. Schematic representation of the pharmacophore model of 5-HT6 receptor antagonists. Hydrophobic site (HYD), hydrogen bond acceptor group (HBA),aromatic ring (AR) and positive ionizable (PI). Shown are the most common functional groups observed in highly active compounds, and the predicted amino acids inthe transmembrane domain of the 5-HT6 receptor involved in the interactions with the ligand (López-Rodríguez et al., 2005).

Fig. 6. Classification of 5-HT6 receptor ligands. Data taken from Holenz et al. (2006).


Fig. 7. Examples of 5-HT6 ligands described in the patent literature from mid-2005 until April 2007. (Ki or pKi values given as reported in the references).


Fig. 7 (continued ).


Fig. 7 (continued ).


To our best knowledge, eight compounds that are selective5-HT6 receptor ligands have entered clinical trials (Filla et al.,2002; Ahmed et al., 2003; Cole et al., 2003; Saegis, 2005; Coleet al., 2005a, 2005b; Piñeiro-Núñez et al., 2005); their chemicalstructures, functionalities and therapeutic targets are sum-marised in Table 3. Six of them are considered as being ‘underactive development’. As described before, for the first 5-HT6

receptor ligand receptor Phase 1 compound, SB-271046 (cf 3)(Fig. 3), clinical development was discontinued, probably dueto low blood-brain barrier penetration (Medicinal Chemistry-XVIIIth International Symposium, 2004). The Wyeth agonist,WAY-181187 (SAX-187), was investigated in Phase 1 clinicaltrials targeting anxiety and generalized anxiety disorders (Coleet al., 2003; Cole et al., 2005b), but no recent developments

have been reported by the company. Most advanced of thecompounds is the Phase 2 5-HT6 receptor antagonist fromGlaxoSmithKline, SB-742457, with a therapeutic indication ofcognitive dysfunction associated with Alzheimer's disease(Ahmed et al., 2003) and the Lilly 5-HT6 receptor antagonist,LY-483518 or SGS-518 (Filla et al., 2002; Piñeiro-Núñez et al.,2005), licensed to Saegis Pharmaceuticals for development incognitive impairment associated with schizophrenia (Saegis,2005). The 5-HT6 receptor antagonists, PRX-07034 and BVT-74316, are both in Phase 1 trials for the treatment of obesity.PRX-07034 from Epix Pharmaceuticals is also under investiga-tion for the treatment of cognitive impairment in Alzheimer'sdisease or schizophrenia. SYN-114, a 5-HT6 receptor antago-nist from Synosia Therapeutics which they have acquired under

Fig. 8. Pharmacological tool compounds, preclinical candidates and clinical candidates with high affinity for the 5-HT6 receptor.


license from Roche, is in Phase 1 clinical development forcognitive disorders. The structure of SYN-114 has not yet beendisclosed. Finally, Wyeth has reported SAM-315 (5-HT6

receptor antagonist) under active development in clinicalPhase 1 for the treatment of Alzheimer's type dementia.

5. Receptor binding and functionalcharacterisation of 5-HT6 ligands in vitro

The use of recombinant systems expressing the 5-HT6

receptor has been reported since the initial discovery of thereceptor (Monsma et al., 1993; Ruat et al., 1993). These systemshave been used for study of the binding properties of 5-HT6

ligands as well as for investigation of their functional actions onsecond messenger pathways. In HEK-293 cells stably trans-fected with the rat 5-HT6 receptor, serotonin elicits a potentstimulation of cAMP activity, which can be blocked byantipsychotic and antidepressant drugs (Monsma et al., 1993).Accordingly, when transiently expressed in transfected COS-7cells, the rat 5-HT6 receptor also appeared to be coupledpositively to cAMP production (Ruat et al., 1993). Later on,Kohen et al. (1996) reported that serotonin was able to increasecAMP levels through its action at the human 5-HT6 receptortransiently transfected in COS-7 cells, further confirming thatthe 5-HT6 receptor was positively coupled to cAMP formation.Analogous to the rat receptor, the human 5-HT6 receptor-

induced cAMP production was antagonized by antipsychotics,which are non-selective ligands of the 5-HT6 receptor. Tofurther study the 5-HT6 receptor and its physiological function,potent and selective ligands for the receptor were required. Thefirst 5-HT6 receptor ligands, Ro 04-6790 and Ro 63-0563, werecharacterised using human 5-HT6 receptors (Sleight et al.,1998). Using HeLa cells stably expressing the recombinanthuman 5-HT6 receptor, both compounds behaved as competi-tive antagonists, inducing a concentration-dependent rightwardshift of the 5-HT-induced cAMP accumulation curve. NeitherRo 04-6790 nor Ro 63-0563 had any agonist or inverse agonistactivity. Subsequently, SB-271046 (Bromidge et al., 1999), oneof the initial ligands used for the in vivo study of the 5-HT6

receptor, was characterised in terms of its in vitro functionality(Routledge et al., 2000). SB-271046 inhibited the 5-HT-inducedstimulation of cAMP, shifting the 5-HT concentration responsecurve to the right in a concentration-dependent manner, with nosuppression of the maximal response, consistent with acompetitive antagonism. Since then, many other 5-HT6 receptorligands (Holenz et al., 2006) have been characterised using thisin vitro approach. Therefore, recombinant expression systemsare well accepted for their ability to differentiate betweenclosely related compounds with respect to their intrinsicefficacy properties at the 5-HT6 receptor. However, it isknown that variations in the systems used (ie chosen cell line,receptor density, G protein combinations) may lead to different

Table 35-HT6 ligands in clinical development

Code name Structure Source Developmentphase a

Status ofdevelopment a

Functionality Therapeuticindication

SB-271046 GlaxoSmithKline Phase 1 Development stopped,probably due to lowblood-brain-barrierpenetration

Antagonist Treatment ofAlzheimer´sdementia

WAY-181187(SAX-187)

Wyeth Phase 1 Phase 1 trials begun inlate 2003. No recentdevelopments have beenreported by the company

Agonist Treatment of acuteanxiety andgeneralizedanxiety disorders

PRX-07034 EpixPharmaceuticals

Phase 1 Under activedevelopment(by October 2006, thefirst Phase 1 study hadbeen completed, anda second Phase I studywas underway,data from the trialwere expected inmid-2007)

Antagonist Treatment of cognitiveimpairment associatedwith Alzheimer´sdisease or schizophreniaTreatment of obesity

BVT-74316 Biovitrum Phase 1 Under active development(by September 2006,Phase 1 trials had begun.Results were anticipatedin the first half of 2007)

Antagonist Treatment ofobesity

SYN-114 Structure not disclosed SynosiaTherapeutics(originallydevelopedat Roche)

Phase 1 Under activedevelopment(January 2007)

Antagonist Treatment ofcognitivedisorders

SAM-315 Wyeth Phase 1 Under activedevelopment(October 2006)

Antagonist Oral treatment ofAlzheimer´s typedementia


Table 3 (continued )

Code name Structure Source Developmentphase a

Status ofdevelopment a

Functionality Therapeuticindication

LY-483518(SGS-518)

Saegis(in October2002, Saegislicensedthe drug fromEly Lilly)

Phase 2a Under active development(in March 2005, a Phase 1study was completed.In December 2005 thecompany completed aPhase 2a trial inschizophrenic patients)

Antagonist Treatment ofcognitiveimpairmentassociated withschizophrenia

SB-742457(GSK-742457)

GlaxoSmithKline Phase 2b Under activedevelopment(in September 2005the compound enteredPhase 2 trials forAlzheimer´s disease,trials were ongoingin March 2007)

Antagonist Treatmentof Alzheimer´stype dementia

a Information taken from the following data bases: Integrity Database, Prous Science (2007) and Investigational Drugs Database IDdb3, Thomson Current Drugs (2007).


functional outputs for the drugs under investigation, rangingfrom antagonism to full agonism at the same receptor (Hoyer &Boddeke, 1993). Moreover, using site-directed mutagenesis ofthe 5-HT6 receptor, a constitutively active receptor wasdescribed (Purohit et al., 2003), that demonstrated clear inverseagonism with some of the previously reported antagonists(Romero et al., 2006). At the constitutively active 5-HT6 humanreceptor, SB-271046 displayed negative efficacy although itwas reported previously as being virtually free of intrinsicactivity (Routledge et al., 2000). Similar negative efficacy waspreviously reported for clozapine (Teitler et al., 2002) andfluphenazine (Purohit et al., 2005). Moreover, depending on thesystem employed, Ro 04-6790 displayed inverse agonism orantagonism (Romero et al., 2006). E-6801 and E-6837 (Holenzet al., 2005), two novel 5-HT6 receptor ligands were reported tobehave either as partial agonists or full agonists (Romeroet al., 2006). Thus, Romero et al. (2006) observed that E-6801and E-6837 were highly potent full agonists of both the wild-type human 5-HT6 receptor expressed in Cos-7 cells and themutant S267K human 5-HT6 receptor (Purohit et al., 2005) alsoexpressed in this cell line. E-6801 was a high affinity, lowefficacy, partial agonist at rat 5-HT6 receptors in the absence offorskolin, but it was a highly potent agonist with similar efficacyto 5-HT in the presence of forskolin. Similarly, E-6837 behavedas an agonist at rat 5-HT6 receptors only in the presence offorskolin. This modulation of agonist-induced cAMP produc-tion has been reported either for wild-type or constitutivelyactive Gs-coupled receptors (Litosch et al., 1982; Alewijnseet al., 1997; Krobert & Levy, 2002). Thus, the use of eitherforskolin or a constitutively active S267K 5-HT6 receptorenhances the resolution to analyze the agonist efficacy of 5-HT6

receptor ligands (Romero et al., 2006). Interestingly, closelyrelated compounds have been described to display differentfunctionalities, with the S-enantiomers generally behaving as

5-HT6 receptor antagonists and R-enantiomers displayingagonist properties (Cole et al., 2005a, 2005b). Also, the bind-ing affinities of the two isomers for a given compound can bequite different, suggesting more than one mode of binding(Abate et al., 2005). Therefore, although the usefulness of thesein vitro systems for screening purposes or even for compoundcharacterisation is well established, in the case of the 5-HT6

receptor, there is a considerable amount of uncertaintyregarding the functionality of many ligands, and consequently,a degree of caution needs to be exercised when attempting todefine pharmacological effects as being agonist-, antagonist- orinverse agonist-mediated.

With this caveat in mind, an increasing body of evidence isemerging to indicate that 5-HT6 receptor agonists andantagonists can evoke identical responses in a number ofbehavioural paradigms. Thus, 5-HT6 receptor antagonists (egPRX-07034) have been demonstrated to be active in bodyweight reduction as has E-6837, a 5-HT6 receptor partial agonist(discussed in detail in Section 6). A similar situation exists in theresearch of cognitive dysfunction. 5-HT6 receptor antagonistshave been demonstrated to be active in several models andconditions (Woolley et al., 2004; Mitchell & Neumaier, 2005).However, one study (Lindner et al., 2003) is inconsistent withthe hypothesis that 5-HT6 receptor antagonists have therapeuticpotential in cognitive disorders. Moreover, it has been reportedthat the 5-HT6 receptor full agonist, E-6801, was active inimprovingmemory in the novel object discrimination test (Fone,2006). The activity of this compound was comparable to that ofthe 5-HT6 receptor antagonist, PRX-07034 (King et al., 2006),which has recently been reported to display a dose-dependenttrend in improvement of cognitive function, associatedwith drugtreatment (http://www.predixpharm.com/products/prx-07034.asp). Other agonists and partial agonists have been tested inthe novel object discrimination test with positive results, further

http://www.predixpharm.com/products/prx-07034.asp

http://www.predixpharm.com/products/prx-07034.asp


confirming the complexity of 5-HT6 receptor pharmacology(unpublished results).

Very recently a similar paradox has emerged in the anti-depressant field in the articles by Wesolowska and Nikiforuk(2007) and Svenningsson et al. (2007). Wesolowska andNikiforuk (2007) observed that the potent 5-HT6 receptorantagonist, SB 399885, decreased the immobility of mice in thetail suspension and forced-swim tests of antidepressant-likeactivity. In this study, SB 399885 was also found to haveanxiolytic-like activity in both the Vogel conflict drinkingmodel and the elevated plus-maze that was approximatelyequivalent to diazepam. In contrast, Svenningsson et al. (2007)observed a significant reduction in the immobility of mice inthe tail suspension test after administration of the 5-HT6

receptor agonist, 2-ethyl-5-methoxy-N,N-dimethyltryptamine(EMDT), which was abolished by the 5-HT6 receptorantagonist, SB 271046, when the latter compound was givenat a dose that did not of itself significantly reduce immobilityin this test.

Aside from the simplest, but intriguing, possibility that 5-HT6

receptors in the CNS are tonically modulated in such a way thatthey respond identically to both agonists and antagonists, it isappropriate to explore some other explanations for thesefindings that would fit more comfortably with our understandingof the pharmacology of receptor agonists and antagonists. It hasbeen hypothesized that 5-HT6 receptor antagonists influencefeeding behaviour and cognition by blocking the tonic 5-HTactivation of 5-HT6 receptors located on GABAergic interneur-ones in the hypothalamus and in the medial septum, respectively,leading to disinhibition of pro-opiomelanocortin-containingneurones or cholinergic neurones (Woolley et al., 2004).However, other explanations may be proposed for the conflict-ing in vivo results obtained using partial or full agonists. First,the most plausible hypothesis would be a direct action of 5-HT6

receptor agonists on a neuronal population other than GABA-ergic interneurones, ie cholinergic or glutamatergic, for whichthe presence of 5-HT6 receptors has been suggested. Second, theactivation of the rat 5-HT6 receptor transfected into HEK293cells results in a rapid receptor desensitization, without any lossin receptor number, affinity or binding capacity (Max et al.,1995). The authors suggested that 5-HT6 receptor desensitiza-tion occurs by protein kinase A-mediated receptor proteinphosphorylation causing a decrease in the efficacy of receptor/cAMP coupling (Max et al., 1995). However, the 5-HT6 receptoragonist,WAY-466, induced a greater elevation in cortical GABAlevels after chronic administration than that induced by acutetreatment (Schechter et al., 2004). Therefore, in contrast to the invitro desensitization, it appears that in vivo the 5-HT6 receptor isnot so rapidly desensitized. This desensitization was suggestedas a possible explanation for the activity of the 5-HT6 receptorpartial agonist, E-6837, on body weight reduction (Fisas et al.,2006). Third, although more speculative, it could be that 5-HT6

receptor has more than one subtype, which have not so far beenidentified. It is well known that alternate mRNA splicing ofseveral 5-HT receptor genes exists. This phenomenon has alsobeen described for the human and rat 5-HT7 receptor (Heidmannet al., 1997; Jasper et al., 1997), and in fact, splice variants of the

5-HT6 receptor have been described in human brain (Olsen et al.,1999). Co-expression of the variant 5-HT6 transcript and the fulllength 5-HT6 transcript was observed in several brain areas.However, transient transfection of a 5-HT6 variant construct intoCos-7 cells demonstrated that a truncated receptor wastranslocated to the membrane, but appeared non-functional(Olsen et al., 1999). Finally, 5-HT has low affinity for the 5-HT6

receptor (pKi =6.96±0.18) (Romero et al., 2006). The implica-tions of this are not well understood, but it could suggest that theactivity of 5-HT6 receptor ligands is highly dependent on thebaseline levels of the endogenous ligand and the intrinsic level ofactivity of the receptor system. Poorly coupled 5-HT6 receptorsystems may lead to antagonism, whilst agonist properties couldpredominate in efficiently coupled systems (Fisas et al., 2006).

In conclusion, although basic pharmacology is slowly beingelucidated, 5-HT6 receptor research has still to take advantageof the plethora of selective ligands now available, eitheragonists, partial agonists, antagonists or inverse agonists. Abetter pharmacological characterisation of the receptor, mainlyat the in vivo level, is urgently needed.

6. Effects of various 5-HT6 receptorligands on satiety, food intake and body weight

Initial experiments to characterise the function of 5-HT6

receptors in the CNS did not yield any signal to indicate thatmodulating them would have a role to play in the regulation offood intake or body weight. Bourson et al. (1995), Yoshiokaet al. (1998) and Hamon et al. (1999) all administered antisenseoligonucleotides directed against the 5-HT6 receptor via theintracerebroventricular (i.c.v.) route and whilst they allreported a reduction of LSD radioligand labelling of 5-HT6

receptors in the brain, no effect of these procedures on foodintake or body weight was noted in these studies. In 1998,Sleight et al. (1998) described the first pharmacologicalexperiments to be performed using small-molecule, 5-HT6

receptor antagonists. Ro 04-6790 and Ro 63-0563 aremoderately potent 5-HT6 receptor antagonists with affinitiesof 55 nM and 45 nM for the rat 5-HT6 receptor subtype and12 nM and 15 nM for the human 5-HT6 receptor. Although stillrelatively poor in this regard, Ro 04-6790 nevertheless hadbetter CNS penetration than Ro 63-0563, and consequently, theformer was chosen for the in vivo experiments that theseresearchers performed in rats. These studies were designed tomirror the experimental protocol that they had employed intheir earlier antisense investigation (Bourson et al., 1995),which used a 4-day sub-chronic dosing schedule. Although onday-5 of Ro 04-6790 administration, Sleight and colleaguesreported that the rats displayed the behavioural syndrome ofyawning, stretching and chewing that was identical to theone they previously observed after i.c.v. administration of the5-HT6 receptor antisense oligonucleotides, there was no men-tion of any effects of this 5-HT6 receptor antagonist on eitherfood intake or body weight (Sleight et al., 1998). However, it isimportant to note that the manuscript does not mention themeasurement of either food intake or body weight as part ofexperimental protocol, and in the light of the previous failure of


5-HT6 antisense treatment to affect either parameter, there wasno scientific rationale to prompt the measurement of thesevariables. In contrast to these findings, researchers in ProfessorKevin Fone's group reported that i.c.v. injection of 5-HT6

antisense oligonucleotides decreased feeding behaviour in rats(Bentley et al., 1997; Woolley et al., 2001) and body weight(Woolley et al., 2001). Moreover, Woolley et al. (2001) alsoexplored the behavioural and pharmacological effects of thesmall-molecule, 5-HT6 receptor antagonist, Ro 04-6790, andobserved that, at a high dose of 30 mg/kg i.p., this antagonistalso significantly attenuated body weight-gain in growing ratswhen given daily for 3 days. In addition, the rate of weightregained after withdrawal from treatment was extremely slow.In 2000, Tecott and Brennan published a US patent coveringthe 5-HT6 receptor knock-out mouse strain, and in collabora-tion with scientists at Biovitrum, performed the first experi-ments to define the potential of the 5-HT6 receptor as a viabletarget for the discovery and development of novel anti-obesitydrugs. Caldirola (2003) reported that the 5-HT6 receptorknock-out mouse was resistant to dietary-induced obesitywhen maintained on a high-fat diet.

When overviewing these studies with hindsight, it is pro-bable that the early investigations did not observe any effect of5-HT6 antisense oligonucleotide injection on food intake orbody weight because the degree of receptor ablation wasrelatively small, ie 25–30% (Bourson et al., 1995; Yoshiokaet al., 1998; Hamon et al., 1999). This explanation is alsosupported by two additional observations. First, Woolley et al.(2001) reported that body weight was not altered by sub-chronicadministration of 10 mg/kg Ro 04-6790; only the higher doseof 30 mg/kg was effective. Sleight et al. (1998) had pre-viously estimated that Ro 04-6790 at 30 mg/kg would occupy∼70% of 5-HT6 receptor sites in the CNS. Second, Caldirola(2003) observed profound effects on food intake and bodyweight in 5-HT6 receptor knock-out mice after these receptorshad been totally eradicated. In turn, this has led to a widespreadview that 5-HT6 receptor antagonists will evoke hypophagiaand weight-loss only under conditions of very high receptoroccupancy. Caldirola (2003) also presented the preliminarypharmacology results for Biovitrum's high affinity, small-molecule, 5-HT6 receptor antagonist, ie BVT 5182 (5-HT6

Ki =0.2 nM), showing that when given acutely this compounddose-dependently reduced the food intake of ob/ob mice byenhancing satiety, and when given repeatedly, BVT 5182 pro-duced a sustained reduction in food intake and weight-loss inDIO mice. In subsequent communications at scientific sympo-sia, the weight-loss evoked by BVT 5182 in DIO mice wasshown to be accompanied by a reduction in visceral adiposity,and plasma leptin and insulin concentrations (Svartengren et al.,2003). Importantly, in view of the report that the mouse 5-HT6

receptor is substantially different from the homologues in eitherthe rat or humans (Hirst et al., 2003), BVT 5182 was also shownto produce a profound and sustained reduction in food intake,body weight in DIO rats, together with decreases in visceraladiposity and plasma leptin concentrations (Svartengren et al.,2003). Svartengren et al. (2004) enlarged on these findings byreporting that the reductions of food intake in rats produced

by moderate acute doses of BVT 5182 were not mediated bynausea/malaise or by changes in the behavioural activityof animals. These authors demonstrated that GlaxoSmithKline's5-HT6 receptor antagonist, SB 271046, also dose-dependentlyattenuated food consumption in normal rats when given acutely(Svartengren et al., 2004).

Following the lead from Biovitrum, several other pharma-ceutical and biotech companies have now synthesized their ownselective, small-molecule, 5-HT6 ligands and have testedthem in rodent models of obesity. PRX-07034 is a high affinity(5-HT6 Ki =4 nM) 5-HT6 receptor antagonist with 65 to N300-fold selectivity versus other 5-HT receptor subtypes, and withthe exception of the dopamine D3 receptor (Ki =71 nM), it has≥100-fold selectivity over 52 other G-protein-coupled recep-tors, ion channels and transporters (Gannon et al., 2006). Inacute studies, PRX-07034 inhibited food intake in normal leanand DIO female rats (Gannon et al., 2006). Saccharin con-sumption experiments and a behavioural analysis of meal pat-terns revealed no aversive effect of PRX-07034 and ahypophagic mechanism that was consistent with an enhance-ment of satiety (Gannon et al., 2006).

In this overview, it has been stated that 5-HT6 receptorligands reduce food intake by a mechanism that is consistentwith an enhancement of satiety. Since it is very easy to suppressfeeding in animals using drugs by a variety of mechanisms thatare not clinically acceptable, eg compound-induced activation,sedation, stereotypy, nausea/malaise or taste aversion, it isuseful to explore the effects of the 5-HT6 ligands on feedingbehaviour in some detail. Svartengren et al. (2004) reported thatBVT-5182 (3 and 10 mg/kg s.c.) dose-dependently decreasedthe food intake of obese, ob/ob mice and non-obese rats with nosignificant effect on locomotor activity indicating that its effectswere not due to sedation, behavioural activation or stereotypy.Satiety (the feeling of fullness that leads to the termination of ameal) can be analysed by studying the meal patterns of rodents.BVT-5182 (5 and 15 mg/kg s.c.) had no effect on the latency toeat (a measure of hunger), but it did reduce the number of mealsconsumed and increased the time interval between them; bothresults being indicative that this compound reduced food intakeby enhancing satiety. Rodents lack an emetic reflex and theirmechanism for expelling agents that cause nausea and/or gastro-intestinal malaise, eg lithium chloride, is to consume largequantities of inert substances like kaolin. BVT-5182 (10 mg/kgs.c.), which significantly reduced the cumulative food intake ofnon-obese rats over 6 h and 12 h, did not induce kaolinconsumption in these animals, thereby demonstrating this effectwas not due to the induction of nausea and/or gastro-intestinalmalaise (Svartengren et al., 2004). PRX-07034 (10 mg/kg i.p.),which significantly decreases food intake in non-obese and DIOrats, did not condition an aversion to saccharine consumption ormoderate sodium ingestion after sodium depletion (inducedby furosemide injection plus access to a sodium-free diet),demonstrating that its hypophagic action was not due toconditioned taste aversion. After mild food deprivation, whenrats are given access to food they show a characteristic pattern offeeding, grooming and resting behaviours which is termed the“satiety sequence” (Antin et al., 1975). In a time-sampled variant


of this paradigm, PRX-07034 (10 and 30 mg/kg i.p.)significantly reduced the food intake of non-obese rats andaccelerated and increased post-satiety resting (without causingsedation), but did not alter the levels of activity or groomingcompared with the vehicle-treated controls (Gannon et al.,2006). This pattern of responses is consistent with this 5-HT6

receptor antagonist decreasing food intake by enhancing satiety.In the case of E-6837, which is paradoxically a 5-HT6 receptorpartial agonist, Fisas et al. (2006) reported that at 30 mg/kg p.o.,this compound caused marked hypophagia in non-obese andDIO rats with no effect on locomotor activity. At doses≤60 mg/kg p.o., E-6837 also did not evoke conditioned taste aversionor induce kaolin consumption in non-obese rats (Fisas et al.,2006). Together, these results demonstrate the hypophagic effectof E-6837 is also mediated by satiety enhancement. In summary,therefore, there is substantial evidence from a number of sourcesto support the argument that 5-HT receptor ligands, whetherantagonists or agonists, decrease food intake when given acutelyand this effect is mediated by the clinically acceptablemechanism of the enhancement of satiety.

The mature female rat made obese by giving it free access toa highly palatable, Western, simplified cafeteria diet (DIO rat) isan excellent model of human obesity and many of its co-morbidcomplications (Heal & Jagger, 2005). Moreover, the predictivevalidity of this paradigm for the relative ability to reduce bodyweight and adiposity has been validated using a range of drugswith established efficacy for the treatment of human obesity,including phentermine, sibutramine, orlistat and rimonabant(Jackson et al., 2004, 2005; Heal & Jagger, 2005). In this modelof human obesity, intraperitoneal injection of PRX-07034(10 mg/kg bid) caused a moderate, but sustained, decrease in therats' food consumption that was maintained over the 5 weeks oftreatment (Gannon et al., 2006; Shacham et al., 2006). In turn,this decrease in food intake resulted in a gradual reduction inbody weight that was still diverging from the vehicle-treatedcontrols at the point when administration of PRX-07034 was

Fig. 9. Effect of chronic administration of the 5-HT6 receptor partial agonist, E-6837, oFisas et al. (2006). Results are means (adjusted for differences between treatment grouthe statistical model), n=9–10. Multiple comparisons against the vehicle-treated conare denoted by ⁎pb0.05 (p levels have only been given at one level for clarity).

stopped (Gannon et al., 2006; Shacham et al., 2006). Overall,the mean difference in body weight between the groups of DIOrats treated with PRX-07034 and vehicle was an impressive−12.7%. Moreover, the weight curves indicated that thisreduction was not the maximum possible and continuedtreatment with PRX-07034 would have led to greater weight-loss (Gannon et al., 2006; Shacham et al., 2006). Although inthis initial study, reduced food intake and weight-loss wereobserved only after intraperitoneal injection of PRX-07034, ina subsequent experiment in high-fat fed, obese, male rats,PRX-07034 (30 and 100 mg/kg p.o., bid) was shown todecrease body weight relative to the vehicle controls by 3.7%and 11.8% at the lower and higher doses, respectively, and atthe higher dose, to decrease food intake throughout the 28-daytreatment period.

From the above description, it is clear that 5-HT6 receptorantagonists decrease food intake by enhancing satiety and evokeprofound weight-loss in rodent models of obesity. Such effectsare generally robust having been replicated using a variety ofselective compounds and in several different laboratories. Also,in earlier sections of this review, we have described variousligands that are either partial or full agonists of the rat and/orhuman 5-HT6 receptor homologues; E-6837 and E-6801 areexamples of such ligands (Romero et al., 2006). An intriguingconfounder in this field is that 5-HT6 partial agonists do notincrease food consumption and exacerbate obesity in suchrodent models; on the contrary, they cause hypophagia andreduce adiposity like 5-HT6 receptor antagonists. Thus, E-6837is a partial agonist of the rat 5-HT6 receptor and a full agonist ofthe cloned human 5-HT6 subtype (Romero et al., 2006).Recently, Fisas et al. (2006) used DIO female rats to comparethe effects of E-6837 on food consumption and bodyweight withthose of the reference anti-obesity drug, sibutramine. As shownin Fig. 9, E-6837 does not maximally reduce food intake untilday-3, unlike sibutramine that almost totally abolishes foodintake on day-1 of treatment. This delay in onset of the

n daily food intake in dietary-induced obese, femaleWistar rats. Data taken fromps at baseline [average of days −6 to 0])±SEM (calculated from the residuals oftrol group were by Dunnett's test. Significant differences from the control group

Fig. 10. Effect of chronic administration of the 5-HT6 receptor partial agonist, E-6837, on body weight in dietary-induced obese, female Wistar rats. Data taken fromFisas et al. (2006). Results are means (adjusted for differences between treatment groups at baseline (Day 1))±SEM (calculated from the residuals of the statisticalmodel), n=9–10. Multiple comparisons against the vehicle-treated control group were by Dunnett's test. Significant differences from the control group are denoted by⁎pb0.05 (p levels have only been given at one level for clarity).


maximum hypophagic effect of E-6837 may be explained by theobservation that 4 h after acute oral administration of E-6837(30 mg/kg), this compound occupied only 28% of 5-HT6

receptors present in rat caudate putamen (data on file,laboratorios Dr Esteve). However, the reduction in food intakeseen with sibutramine returns to control values relativelyquickly, but reduced consumption is maintained for consider-ably longer with E-6837 (Fig. 9). When cumulative weekly foodintakes were calculated, E-6837 significantly decreased con-sumption during the first, second and third weeks of treatment,whereas sibutramine significantly reduced food intake onlyduring week-1 (Fisas et al., 2006). This difference in the foodreduction profiles of E-6837 and sibutramine is reflected in therate and duration of active weight-loss on treatment. As shown inFig. 10, E-6837 caused gradual weight-loss in DIO rats that had

Fig. 11. Ability of various 5-HT6 ligands to produce weight-loss in the DIO rat modelaFisas et al. (2006), bGannon et al. (2006), cData on file, RenaSci, dJackson et al. (2

not plateaued after 28 days of compound administration. Incontrast, sibutramine producedmost of its weight-loss during thefirst 2 weeks, and thereafter, the body weight curve of thesibutramine-treated rats paralleled that of the vehicle-treatedcontrols (Fig. 10). The weight-loss versus control after 28 daysof administration was 11.0% for sibutramine and 15.7% forE-6837.

The finding that 5-HT6 partial agonists and antagonistsproduce identical pharmacological effects in animal models isnot restricted to their anti-obesity actions. An identicalphenomenon has also been reported in the cognition fieldwhere 5-HT6 receptor agonists as well as antagonists have beenshown to improve performance of the novel object recognitiontask (Woolley et al., 2003; King et al., 2004; Vickers et al.,2004; Fone, 2006). This paradox is also not explained by

of human obesity in comparison to marketed anti-obesity drugs. Data taken from:004), eJackson et al. (2005).

Table 4Effects of weight-loss induced by 5-HT6 ligands on various cardio-metabolicrisk factors

5-HT6 receptor ligand

BVT 5182(antagonist)

PRX-07034(antagonist)

E-6837(partial agonist)

Adiposity ↓ a,b ↓ a ↓ a

Visceral adiposity ↓ a ↓ a NDPlasma leptin ↓ a,b ↓ a ↓ a

Plasma lipids ±FFAs b ND ±FFAs a

±TGs b ±TGs a

±Glycerol a

Glycaemic control ND ↓ Glucose a ±Glucose a

↓ Insulin a ±Insulin b

↑ OGTT

BVT 5182 — data from Svartengren et al. (2003, 2004).PRX-07034 — data from Gannon et al. (2006) and Shacham et al. (2006).E-6837 — data from Fisas et al. (2006).Experiments performed in: a = DIO rats; b = DIO mice.↓ = decrease; ↑ = increased insulin sensitivity; ± = no change; ND = notdetermined.FFAs = free fatty acids; TGs = triglycerides; OGTT = oral glucose tolerance test.


artefacts generated by the use of different obesity modelsbecause PRX-07034 (a 5-HT6 receptor antagonist) and E-6837(a rat 5-HT6 receptor partial agonist) have both been shown toreduce food intake and adiposity in the female DIO rat model inRenaSci's laboratories (Figs. 9–11; Fisas et al., 2006; Gannonet al., 2006). The technical difficulty of defining the absolutefunctionality of compounds, especially 5-HT6 receptor ligands,with cloned receptors stably transfected in cell lines has beendiscussed extensively in Section 5 of this review. Thus, it maybe that the characterisation of some ligands as either partialagonists (or antagonists) may not hold true for native receptorsin their physiological environment, and as a consequence, thisapparent pharmacological anomaly may be no more than anartefact of the in vitro characterisation of these 5-HT6 receptorligands. On the other hand, it has been suggested that the 5-HT6

receptor may rapidly down-regulate in response to agoniststimulation, or alternatively, serotonergic tone at these receptorsmay be high, leading to a predominantly antagonist action of 5-HT6partial agonists (Fisas et al., 2006). At this time, they are merelyhypotheses and there are no definitive answers to this question.However, a resolution of the issue must be a priority in the 5-HT6receptor field in order to focus research onto the most appropriatecompounds, ie agonists, partial agonists or antagonists, to developas anti-obesity drugs or cognitive enhancers.

In Fig. 11, the weight-losses after 28 days administration ofthe 5-HT6 receptor ligands, ie E-6837 and PRX-07034, arecompared with those previously obtained for a range ofreference anti-obesity drugs with known efficacy in man, iephentermine, sibutramine, orlistat and rimonabant. Encoura-gingly, the results predict that, providing these drugs are safeand well tolerated in humans, the 5-HT6 receptor ligands mayhave the potential to be more efficacious as anti-obesity drugsthan existing compounds.

7. The impact of 5-HT6 receptor-mediatedweight-loss on obesity-related cardio-metabolic risk factors

It is now well accepted that just as obesity is a major cau-sative factor in the development of insulin resistance, impairedglucose tolerance, Type 2 diabetes, hypertension, dyslipidaemiaand a range of other metabolic disorders (Mokdad et al., 2001;Pi-Sunyer, 2002; Kumanyika et al., 2002), moderate, intentionalweight-loss has been shown to have a positive effect on mor-bidity and mortality (Williamson et al., 1995; Williamson,1997; Williamson et al., 1999, 2000; Gregg et al., 2003). Al-though these findings relate to intentional weight-loss throughdiet, exercise and lifestyle modification, the beneficial impact ofanti-obesity drug therapy on cardio-metabolic status, and byinference, on morbidity and mortality is driven by the premisethat drug-induced weight-loss will be accompanied by com-mensurate improvements in metabolic risk factors, ie visceraladiposity, increased glycaemic control, beneficial changes inplasma lipid profiles, reduced plasma uric acid concentrationsand decreased blood pressure. Obese rats and mice are notsuitable models for studying all the above endpoints, particu-larly plasma lipoprotein fractions (HDL-cholesterol, LDL-cholesterol, and VLDL-cholesterol), but they are excellent

predictive indicators for improvements in several other cardio-metabolic risk factors. In addition to demonstrating that severalof these 5-HT6 receptor antagonists and partial agonistsdecreased food intake and body weight in obese rodents,many of these investigations also incorporated measurements ofcardio-metabolic risk factors; a summary of the major findingswith these 5-HT6 receptors ligands in shown in Table 4.Although full body composition analyses have not beenreported for all of these 5-HT6 receptor ligands, Fisas et al.(2006) have shown that weight-loss produced by E-6837 is dueto a selective reduction in body fat with no changes in eitherwater or protein content, indicating that this compound is notcausing weight-loss through the clinically unacceptable routesof dehydration or cachexia. Where fat pads have been weighed,decreases in visceral fat depots have been observed (Table 4)and this finding is of particular clinical relevance becausevisceral adiposity in man is a major driver of cardio-metabolicrisk (Mokdad et al., 2001; Pi-Sunyer, 2002; National CholesterolEducation Program Adult Treatment Panel III [NCEP,ATP,III],2002; Kumanyika et al., 2002; Bloomgarden, 2005; Kolovouet al., 2005). Consistent with the observation that plasma leptinconcentrations correlate with white adipose tissue mass(Hamann & Matthaei, 1996; Morio et al., 1999), the reductionof adiposity in either DIO rats or DIO mice produced by each ofthese 5-HT6 receptor ligands is accompanied by a significant fallin the plasma level of this hormone (Table 4). Improvements inglycaemic control were also observed with all of these 5-HT6

ligands, whether via decreases in the plasma concentrations ofinsulin and glucose or via improved insulin sensitivity in an oralglucose tolerance test (Table 4). As stated above, indices ofimprovements in obesity-related lipid disturbances are difficultto demonstrate in rats and mice. In these experiments, althoughthe animals were clearly obese, they nevertheless had onlyminimal abnormalities in their plasma lipid profiles. Not surpri-singly, therefore, no reductions in plasma triglycerides, free fattyacids and glycerol were observed (Table 4).


Overall, the results from these experiments in polygenicanimal models of human obesity predict that the weight-lossevoked by the 5-HT6 receptor ligands will be translated to areduced cardio-metabolic risk profile and to the improvement ofobesity-related metabolic diseases, particularly Type 2 diabetes.

8. Potential side-effects

The fact that the 5-HT6 receptor is located almost exclusivelywithin the CNS (Monsma et al., 1993; Ruat et al., 1993) has madeit a popular target for drug discovery because of its perceived lowliability for the induction of drug-mediated peripheral side-effects. Whilst this hypothesis will undoubtedly hold true for adirect action of 5-HT6 receptor agonists and antagonists onperipheral organ systems, it does not preclude peripheral side-effects translated via a central action of these compounds. Assuch, it would be unwise to be too complacent about the potentialof these drugs to evoke peripheral adverse events, especially asexamples exist to show that the central actions of serotonergicdrugs alter peripheral organ function, eg 5-HT1A agonists evokebradycardia and hypertension via a central mechanism (seeRamage, 1990). Almost no information has so far appeared inthe scientific literature to indicate whether 5-HT6 ligands willexert indirect effects on peripheral organ systems. However,there are now several 5-HT6 receptor antagonist and agonists inclinical development (see Table 3), and as part of the preclinicalsafety and toxicology evaluation prior to human exposure, theacute and sub-chronic actions of these drugs will have beenevaluated in some detail. Since the drugs have progressed intoclinical development, the findings cannot have revealed anyserious findings on this score.

When considering centrally-mediated adverse events, theexperiments performed in rodents have revealed that both 5-HT6

receptor agonists and antagonists markedly reduce food intakewithout evoking sedation, behavioural activation, nausea/malaise or taste aversion (Svartengren et al., 2003, 2004; Fisaset al., 2006; Gannon et al., 2006). In fact, 5-HT6 ligands havebeen shown to have no locomotor effects at pharmacologicallyeffective doses, eg BVT.5182≤10 mg/kg s.c., SB 271046≤10 mg/kg s.c. (Svartengren et al., 2004), PRX-07034≤30 mg/kg i.p. (Gannon et al., 2006) and E-6837≤30 mg/kg p.o. (Fisaset al., 2006). Recently, it has also been reported that another5-HT6 receptor antagonist, ie SB 399885, did not impairmotor coordination of mice and rats in the rotor-rod test atdoses ≤30 mg/kg i.p. (Wesolowska & Nikiforuk, 2007). It iswell known that 5-HT6 receptor antagonists improve severalaspects of cognitive function (see Woolley et al., 2004;Mitchell & Neumaier, 2005 for reviews), and consequently,these drugs could evoke cognitive side-effects when used inthe treatment of obesity. However, the actions of the 5-HT6

receptor antagonists are pro-cognitive, and as such, their side-effects are predicted to be neutral and perhaps even beneficial.Furthermore, following the recent report that 5-HT6 receptoragonists have pro-cognitive effects that are similar inmagnitude to those of the 5-HT6 receptor antagonists (Fone,2006), if these compounds evoke cognitive side-effects theyare also likely to be beneficial rather than deleterious. Con-

sistent with these predictions, PRX-07034 significantlyimproved cognitive function and showed preliminary evidenceof weight-loss in obese, healthy subjects during a 28-day in-patient Phase 1 trial (www.epixmed.com/products/prx-07034.asp). In this clinical trial, PRX-07034 was well tolerated byhuman volunteers up to a dose of 600 mg, which for a potent,CNS-active drug indicates a very benign side-effect profile(www.epixmed.com/products/prx-07034.asp). The only otherpotential CNS side-effects to have been revealed by preclinicalresearch are related to depression and anxiety. Once again,however, the data appear to be contradictory. Svenningssonet al. (2007) reported that the 5-HT6 receptor antagonist, SB271046, prevented the antidepressant effects of fluoxetine inthe mouse tail suspension test, whilst having no effect in itsown right. However, the 5-HT6 receptor agonist, EMDT, hadan antidepressant-like effect to induce immobility in thisparadigm. In direct contradiction to these findings, Weso-lowska and Nikiforuk (2007) reported that another GlaxoS-mithKline 5-HT6 receptor antagonist, SB 399885, had anantidepressant-like effect in both the tail suspension test andthe forced-swim tests. In addition, these authors reported thatSB 399885 also displayed anxiolytic-like activity in both theVogel conflict drinking and the elevated plus-maze tests. Onthis basis, it is impossible to make any firm predictions, butonce again, since 5-HT6 receptor agonists and antagonistsappear to produce antidepressant- and anxiolytic-like effectsin animal models, it would seem unlikely, therefore, that thesecompounds will have a negative effect on mood when given tohuman subjects.

9. Future prospects for 5-HT6

receptor ligands in the treatment of obesity

Taking an overview of the data provided in this review, it isevident that the 5-HT6 receptor has emerged as a highlyinteresting molecular target for drug development in a numberof therapeutic indications; the most important being obesity andcognitive impairment in Alzheimer's disease and schizophrenia.This hypothesis is evidenced by the innovation that has beenapplied by both academic and industrial medicinal chemists tothe design and synthesis of an increasingly large and diversearray of selective, high affinity 5-HT6 receptor ligands, newpatent applications and publications in the scientific literature.One as yet unresolved paradox relating to the development ofnovel clinical candidates for the treatment of obesity or cog-nitive impairment is whether these therapeutic indications willbe best served by agonists or antagonists of the 5-HT6 receptor.A position that is not helped by the observation that equivalentanti-obesity and pro-cognitive potency and efficacy can bedelivered in animal models by both 5-HT6 receptor partialagonists and antagonists. Whilst some explanations have beenput forward in this review and in other articles, a resolution ofthis question is now a scientific priority. In the obesity in-dication, there is clearly a clinical need for more effective, but atthe same time safe, new pharmacological strategies to deal withthis growing health problem. Results obtained in animal modelsare highly predictive of clinical outcomes in obesity treatment.

http://www.epixmed.com/products/prx-07034.asp




These data show that a range of 5-HT6 ligands, both agonistsand antagonists, produce weight-loss with concomitant im-provements in cardio-metabolic risk factors that are at least asgood as the current generation of anti-obesity drugs and is ahighly encouraging signal that 5-HT6 compounds may ulti-mately provide a significant step forward in anti-obesity phar-macotherapy. With several 5-HT6 ligands already in clinicaldevelopment, answers to this critical question are almost certainto be forthcoming within the next 2–5 years.

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