No Evidence for Binding of Clozapine, Olanzapine And/Or Haloperidol to Selected Receptors Involved in Body Weight Regulation

Home , Cholecystokinin receptor, Endothelin receptor, Histamine H1 receptor

No evidence for binding of clozapine, olanzapine and/or haloperidol to selected receptors involved in body weight regulation

FM Theisen1, M Haberhausen1, The underlying mechanisms of antipsychotic (AP)-induced weight gain are 2 2 unknown, but both central and peripheral AP target receptors could MA Firnges , P Gregory , potentially be involved. This study used radioligand binding assays to 3 1 JH Reinders , H Remschmidt , compare the binding affinities of clozapine, olanzapine and haloperidol for J Hebebrand4 and J Antel2 candidate receptors potentially involved in AP-induced weight gain. Selected candidates derived from known pathways involved in body weight 1Clinical Research Group, Department of Child regulation included receptors classified as anorexigenic (bombesin receptor and Adolescent Psychiatry, University of subtype 3, calcitonin gene-related peptide receptor, cholecystokinin Marburg, Marburg, Germany; 2Solvay Pharmaceuticals Research Laboratories, receptor, melanocortin-4 receptor, neurotensin receptor 1) or orexigenic Hannover, Germany; 3Solvay Pharmaceuticals (cannabinoid receptor 1, galanin 1 receptor, melanin-concentrating hor- Research Laboratories, CP Weesp, The mone receptor (MCHR), neuropeptide Y1 receptor) as well as receptors 4 Netherlands and Department of Child and involved in physiological actions related to digestion and fluid homeostasis Adolescent Psychiatry, University of Essen, Essen, Germany (angiotensin II type 1 receptor, bradykinin B2 receptor, endothelin receptor, neurokinin 1 receptor, vasoactive intestinal polypeptide receptor 1). Correspondence: Clozapine, olanzapine and haloperidol exhibited negligible affinities to all Dr FM Theisen, Clinical Research Group, of these receptors except for the MCHR (Ki ¼ 501 nM; haloperidol). With Department of Child and Adolescent Psychiatry, University of Marburg, Hans-Sachs- respect to other candidates from (neuro)transmitter systems already Str. 6, D-35033 Marburg, Germany. suggested to be involved in AP-induced weight gain, the binding profile of E-mail: [email protected] olanzapine resembled that of clozapine, with high affinity (Kio10 nM) for serotonin (5-HT) 5-HT2A, 5-HT2C and 5-HT6, muscarinic M1 and histamine H1 receptors. In contrast, the binding profile of haloperidol was substantially different (high affinity only for the dopamine D1 receptor). In conclusion, we have not identified a novel binding site of the two investigated atypical AP that could contribute to the induced weight gain. The Pharmacogenomics Journal (2007) 7, 275–281; doi:10.1038/sj.tpj.6500418; published online 19 September 2006

Keywords: antipsychotics; body weight gain; binding profile; neurotransmitter; neurohor- mones; genetics

Introduction

Weight gain occurs in up to 50% of patients under chronic administration of antipsychotics (AP).1 Weight gain not only impairs physical health but also reduces treatment compliance and may cause adverse psychosocial conse- quences. Although there is generally marked variability in incidence and severity Received 19 January 2006; revised 19 July 2006; accepted 24 July 2006; published online of weight gain among the various AP, especially the atypical AP clozapine and 19 September 2006 olanzapine are associated with this side effect.1–3 The underlying mechanisms are Candidate receptors for antipsychotic-induced weight change FM Theisen et al 276

unknown, but genetic factors are seemingly involved.4 In actions related to digestion and fluid homeostasis (such as clinical studies, clozapine has been associated with incre- angiotensin II type 1 receptor, bradykinin B2 receptor, ments in appetite, carbohydrate craving5 and binge eating endothelin receptor, neurokinin 1 receptor, vasoactive episodes,6–8 suggesting a change in energy intake. Currently, intestinal polypeptide receptor 1)34–38 could be involved in the effects of AP on energy expenditure have been less well weight changes observed under treatment with AP. studied. In prospective clinical studies, olanzapine-induced In the present article, we have attempted to investigate weight gain was associated with an increase in caloric the potential involvement of several central and peripheral intake,9 whereas resting energy expenditure did not change receptors in AP-induced weight gain by using commercially over the observation periods of four9 and 1210 weeks. In available radioligand binding assays (CEREP, 86600 Celle l‘ contrast, an observation in three patients treated with Evescault, France). It was hypothesized that the differential clozapine revealed a reduction of resting metabolic rates binding profiles of typical and atypical AP may contribute by 10.3–16.0% within 1 month.11 to differences demonstrated with respect to weight gain. We Because AP bind to many neurotransmitter receptors in therefore explored the binding affinities of clozapine and human brain, the multiple therapeutic and adverse effects olanzapine in comparison with those of haloperidol, a of various AP may depend on the combination of receptors typical AP that causes relatively low weight gain.2,3 Based on occupied.12 The effect of genetic variation of diverse the CEREP offer, we chose (1) receptors of diverse periph- candidate receptor genes implicated in weight gain has erally and centrally acting hormones and peptides derived been investigated.13–20 For example, the À759C/T poly- from known pathways involved in body weight regulation morphism of the serotonin (5-HT) 5-HT2C receptor gene was and related to digestion and fluid homeostasis; (2) receptors studied in patients with weight gain upon use of cloza- of other candidates from (neuro)transmitter systems already pine14,18–21 or other AP.17,22,23 However, as yet unknown suggested to be involved in AP-induced weight gain. pharmacodynamic (including both central and peripheral AP target receptors) and pharmacokinetic (i.e. enzymes that metabolize AP) factors may be involved in AP-induced Materials and methods weight gain. So far, research into this side effect was based on the binding profiles of AP and has thus primarily focused Radioligand binding assays were performed at CEREP, on classical neurotransmitter receptors such as dopaminer- Poitiers, France and included (1) 14 potential candidates gic, serotonergic, histaminergic, muscarinic and/or adrener- known from pathways involved in body weight regulation gic receptors; the role of peripherally and centrally acting or related to digestion and fluid homeostasis (see Table 1) as hormones and peptides involved in body weight regulation well as (2) 17 candidates of the serotonergic, dopaminergic, 1,3,13,15,16 is largely unknown. muscarinic, histaminergic and adrenergic transmitter sys- Factors implicated in the susceptibility to clozapine- or tems (see Table 2). 6,24–26 olanzapine-related weight gain include leptin and The binding assay methods are summarized in Supple- 26–28 ghrelin. We found no evidence for a direct peripheral mentary Table 1. Following incubation, the membranes or effect of clozapine on fat-cell formation or production of cells in suspension were rapidly filtered under vacuum 29 leptin and other fat-cell-derived factors. The increase in through glass fiber filters (GF/B, Packard or Filtermat A or B, serum leptin levels under treatment with AP is most likely Wallac (now PerkinElmer, Inc., Boston, MA, USA)). Filters secondary to weight gain and seemingly not mechanistically were then washed several times with an ice-cold buffer using 25 involved. Additionally, other changes in biological para- a cell harvester (Packard or Tomtec). Bound radioactivity meters related to body weight, such as tumor necrosis factor- was measured with a scintillation counter (Topcout, Packard alpha (TNF-alpha), soluble TNF receptors and interleukin-6 or Betaplate, Wallac) using a liquid scintillation cocktail 30 (IL-6) may be involved in AP-induced weight gain. The (Microscint 0, Packard) or a solid scintillant (MeltiLex B/HS, rapidly evolving knowledge derived from molecular obesity Wallac). research benefits the identification of peripherally and centrally acting hormones and peptides involved in body weight regulation and/or fluid homeostasis. The investiga- Experimental protocols tion of such peptides and receptors involved in body weight The three test compounds (clozapine, haloperidol HCL, regulation may be a promising approach to investigate the olanzapine) were dissolved at 1 mM in dimethylsulfoxide mechanisms potentially underlying AP-induced weight (DMSO) and shipped to CEREP. The compounds were first gain. Candidates derived from known pathways involved tested in all assays at 1 mM. If the compounds caused more in body weight regulation include receptors classified as than 50% inhibition of the specific binding at this anorexigenic (such as bombesin receptor subtype 3, calcito- concentration, they were further tested at 0.1 mM. If they nin gene-related peptide receptor, cholecystokinin receptor, again inhibited more than 50% of the specific binding at melanocortin-4 receptor, neurotensin receptor 1)31,32 or this concentration, they were finally tested at 0.01 mM. Each orexigenic (such as cannabinoid receptor 1, galanin 1 determination was made in duplicate. In each experiment, receptor, melanin-concentrating hormone receptor the respective reference compound was tested at a minimum (MCHR), neuropeptide Y1 receptor).31,33 Additionally, it of eight concentrations in duplicate to obtain a competition could be speculated that receptors involved in physiological curve in order to validate the experiment.

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Table 1 Selected candidate receptors for body weight gaina

Receptor Receptor Endogenous ligand Function according to the agonistic physiological (symbol) action bb3 Bombesin receptor subtype 3 Bombesin Anorexigen CGRP Calcitonin gene-related peptide Calcitonin gene-related peptide Anorexigen receptor (CGRP) CCKA (CCK1) Cholecystokinin receptor Cholecystokinin (CCK) Anorexigen MC4 Melanocortin-4 receptor (MC4-R) a-MSH Anorexigen NT1 (NTS1) Neurotensin receptor 1 Neurotensin Anorexigen CB1 Cannabinoid receptor 1 Cannabinoids Orexigen GAL1 Galanin 1 receptor Galanin Orexigen MCH Melanin-concentrating hormone Melanin-concentrating hormone Orexigen receptor (MCH) Y1 Neuropeptide Y1 receptor Neuropeptide Y (NPY) Orexigen m AT1 Angiotensin II type 1 receptor Angiotensin II (AngII) Vasoconstrictor, thirst and salt appetite, numerous physiological actions including m synthesis and secretion of aldosterone and conservation of fluids and solvents (BKB) B2 Bradykinin B2 receptor Bradykinin Bradykinin acts as a potent vasodilator throughout the body and circulates in increased levels during digestion ETB Endothelin receptor subtype B Endothelin (ET) Vasoconstrictor, activation of glycogenolysis in hepatocytes, modulation of ovarian function, presumably relevant in the central nervous system control of fluid and electrolyte homeostasis NK1 Neurokinin 1 (NK1) receptor Substance P Substance P participates in the inflammatory response of the bladder; NK1 receptor antagonists may prevent chemotherapy- induced nausea and vomiting m m m VIP1 (VPAC1) Vasoactive intestinal peptide Vasoactive intestinal peptide (VIP) digestion, secretion of intestinal juice, k receptor 1 HCO3, H+ secretion m ¼ increase; k ¼ decrease. aInformation obtained from references.31–38

Analysis and expression of results performed. Because findings across studies vary significantly The specific radioligand binding to the receptors is defined in methodology, with the use of different receptor/tissue as the difference between total and nonspecific binding sources, radioligands, or in vitro assay conditions, we 40–59 determined in the presence of an unlabelled ligand. IC50 converted the binding data from the literature into values (concentration causing a half-maximal inhibition of semiquantitative categories (no or low affinity, moderate control specific binding) and Hill coefficients (nH) were affinity, high affinity; see Table 2) to provide better determined for the reference compounds by nonlinear comparability. regression analysis of their competition curves. These parameters were obtained by Hill equation curve fitting.

The IC50 values obtained for the reference compounds are Results within accepted limits of historic averages obtained 70.5 log unit. The IC50 values were converted to the affinity The radioreceptor binding affinities of clozapine, olanzapine constants (Ki values) by application of the Cheng–Prusoff and haloperidol for the candidates derived from known 31–38 equation: Ki ¼ IC50/[1 þ L/Kd], where L is the concentration pathways involved in body weight regulation (Table 1) of radiolabelled ligand (agonist or antagonist) and Kd is its were all negligible (Kio1000 nM), with the exception of the 39 affinity for the receptor of the membrane preparation. MCHR (Ki ¼ 501 nM; haloperidol). The radioreceptor binding affinities of clozapine, olanza-

Comparison to binding data derived from the literature pine and haloperidol for the dopamine (D1,D5), serotonin In order to compare the results of our study to binding data (5-HT1A, 5-HT2A, 5-HT2C, 5-HT3, 5-HT6, 5-HT7), muscarinic derived from the literature, a review of published binding (M1,M2,M3,M4,M5) and histamine (H1,H2,H3) receptors, data that integrates data from different sources, including and the norepinephrine (b3) adrenoceptor were comparable Medline/PubMed and websites such as the ‘Information with results derived from the literature (Table 2).40–59The system for G protein-coupled receptors (GPCRs)’,40 was binding profile of olanzapine resembled that of clozapine,

The Pharmacogenomics Journal Candidate receptors for antipsychotic-induced weight change FM Theisen et al 278

with high affinity (Kio10 nM) for serotonin 5-HT2A, 5-HT2C 3

H and 5-HT6, muscarinic M1 and histamine H1 receptors, and 1000 (+) 1000 (+) 1000 (+) 4 4 4 moderate affinity (Kio100 nM) for dopamine (D1,D5) and muscarinic (M4,M5) receptors. In contrast, haloperidol had 2

H no or low affinity for all these receptors except the 1000 (+) 1000 (+) dopamine (D1,D5)-binding sites. 4 4 1 H similar in different tissues. Discussion

5 AP-induced weight gain is a clinically relevant side effect. M 1000 (+) 251 + 794 + The mechanisms by which weight gain occurs are unknown. 4 Both central and peripheral AP target receptors (pharmaco-

4 dynamic level) and enzymes that metabolize AP (pharma- M

1000 (+) cokinetic level) may be involved in AP-induced weight 4 gain.13,15,16 This study primarily aimed at investigating the binding affinities of the atypical AP clozapine and olanza- 3

M pine in comparison with the typical AP haloperidol for 1000 (+)

4 selected candidate receptors for AP-induced weight gain. First, we investigated selected candidates derived from the 2

M known pathways involved in body weight regulation. These 1000 (+)

4 included receptors for ligands with anorexigenic and orexigenic action as well as receptors involved in physiolo-

1 gical actions related to digestion and fluid homeostasis (see M 1000 (+) Table 1). With the exception of the MCHR, to which low 4 affinity binding of haloperidol was detected (Ki ¼ 501 nM),

3 clozapine, olanzapine and haloperidol exhibited negligible b 1000 (+) 3.16 +++ 251 + 100 + 16 ++ 20 ++ 5.01 +++ 1000 (+) 7.94 +++ 100 + 100 + 20 ++ 25 ++ 3.98 +++ 1000 (+) affinities to all other receptors. The results suggest that these 4 4 4 candidate receptors do not play a significant part in the 7 direct pharmacological action of clozapine, olanzapine and ). 5-HT

M haloperidol. However, research into specific physiological 6

10 n roles of neuropeptides has remained elusive after several o i 5-HT K 1000 (+) 400 + decades of research, but transmitter-like, trophic and

4 modulatory actions are seen for most peptides. Thus,

3 because of multiple interactions, an indirect involvement of these central and peripheral AP target receptor systems in 5-HT 1000 (+)

4 AP-induced weight gain cannot be excluded. Finally, it remains possible that AP bind to distinct receptors involved 2C

); +++, high affinity ( in weight regulation, which were not investigated in the M 1000 (+) 5-HT current study. 4

100 n Second, we investigated candidates from (neuro)transmitter 2A o i values) of haloperidol, clozapine and olanzapine for dopamine, serotonin, muscarinic and histamine receptor subtypes K systems already suggested to be involved in AP-induced weight i 5-HT K gain (see Table 2). With respect to these receptors, the binding profile of olanzapine resembled that of clozapine, 1A with high affinity for serotonin 5-HT2A and 5-HT6, muscari- 1000 (+) 2.0 +++ 5.01 +++ 160 + 6.3 +++ 400 + 1000 (+) 158 + 5-HT nic M and histamine H receptors, and moderate affinity 4 4 1 1 for dopamine (D1,D5) and muscarinic (M4,M5) receptors. In 5 Origin/tissue is not specified here because the receptor-binding affinities for haloperidol, clozapine and olanzapine, respectively, were quite D

adrenoceptor subtype contrast, the binding profile of haloperidol was substantially 3 ); ++, moderate affinity ( 40–59 b different, with no or low affinity for the latter receptors M 1 D ++ ++++ ++++ + ++ +(+) ++ ++(+) + +++ (+) +++ ++ + +++except +++ ++ (+) ++(+) the + +++ (+) dopamine ++ (+) +++ (+) +(+) (D ++(+)1,D +++ (+) ++5)-binding ++(+) ++(+) (+) ++ ++(+) sites. (+) ++(+) +++ Because (+) +++ (+) cloza- +++ + (+) (+) + (+) (+) Dopaminergic Serotonergic Adrenergic Acetylcholine Muscarinic Histaminergic

100 n pine, olanzapine and haloperidol all have moderate to high 4 i K

a a a affinity for the dopamine (D1,D5) sites, these receptors are unlikely to contribute to the differences in weight gain observed between these drugs.2 Hence, the different binding Radioreceptor-binding affinities ( Literature Literature Literature profiles of clozapine and olanzapine in comparison to

haloperidol makes the serotonin 5-HT2A, 5-HT2C and 5- HT6, muscarinic M1 and histamine H1 receptors promising Data obtained from references. and the norepinephrine Clozapine Current studyOlanzapine 40 ++ Current study 79 ++ 20 ++ 20 100 ++ + 7.94 +++ 7.94 +++ 398 + 7.94 +++ 126 + Table 2 Haloperidol Current study 6.3 +++ 25 ++ +, no ora low affinity ( candidates for AP-induced weight gain. An extensive

The Pharmacogenomics Journal Candidate receptors for antipsychotic-induced weight change FM Theisen et al 279

presentation of published receptor binding properties is receptor gene is of interest because the À759C allele is beyond the scope of this study. However, on the basis of our functionally active.64 review integrating data from different sources, we did not In conclusion, the current results suggest that the selected find significant differences between our binding data and candidate receptors derived from known pathways involved results derived from other studies. Certainly, caution is in body weight regulation (Table 1) do not play a significant warranted in comparing findings across studies that vary part in the direct pharmacological action of clozapine, significantly in methodology and it is important to note olanzapine and haloperidol. With respect to other candi- that some inconsistencies exist in the receptor-binding data dates from (neuro)transmitter systems already suggested to reported in the literature. Nonetheless, the research done be involved in AP-induced weight gain (Table 2), our results thus far has yielded certain consistent general properties confirm binding data from the literature indicating an that are useful to consider at a clinical level. involvement of the serotonin 5-HT2A, 5-HT2C and 5-HT6, The differing action of AP on the serotonergic, dopami- muscarinic M1 and/or histamine H1 receptors in this side nergic, cholinergic, g-amino butyric acid (GABA)-ergic, effect. So far, research into AP-induced weight gain has histaminergic and other neurotransmitter systems has been primarily focused on neurotransmitter receptors. Much less hypothesized to be involved in weight gain.1,60–62 In their is known about other receptor systems and AP effects. We clinical investigation of weight changes under the AP believe that the systematic investigation of the role of clozapine, olanzapine, risperidone, sertindole and haloper- peripherally and centrally acting hormones and peptides idol, Wirshing et al.3 found, based on the known receptor- involved in body weight regulation may be a promising binding properties of these drugs, an exponential associa- approach to investigate the mechanisms potentially under- tion between the histamine H1 receptor affinity and the lying AP-induced weight gain. In this context, the rapidly weight gain is observed in the patients. The serotonin 5-HT2 evolving knowledge derived from molecular obesity re- receptor, another plausible candidate for this side effect, was search may benefit the identification of potential candidates not associated with weight gain. Kroeze et al.59 investigated for this side effect. Further studies are necessary to under- the binding properties of 17 typical and atypical AP stand the implication and the possible cross-talk between (including clozapine, olanzapine and haloperidol) towards the diverse transmitter systems potentially involved in AP- 12 neurotransmitter receptors and correlated the results induced weight gain. with short-term weight gain data (derived from Allison 2 et al. ). They found that the receptor affinities of histamine Acknowledgments H1, adrenaline a1A, serotonin 5-HT2C and 5-HT6 receptors, respectively, were significantly correlated with weight gain, We acknowledge the contribution of Peter Eickelmann. This study whereas the eight other receptor’s affinities were not was supported by the ‘Deutsche Forschungsgemeinschaft’ (DFG; Re 471/11-2; Th 707/1-1) and the ‘Bundesministerium fu¨r Bildung und (adrenaline a2A, a2B and a2C; serotonin 5-HT1A, 5-HT2A and Forschung’ (BMBF: National Genome Research Network 01GS0118 5-HT7; dopamine D2; muscarinic M3). Although there are some differences with respect to the methods and the and NGFN2: 01GS0482). receptors investigated, these findings are widely in line with our results indicating an association of the histamine H and 1 Duality of Interest the serotonin 5-HT2C and 5-HT6 receptors with AP-induced 2 short-term weight gain, while the muscarinic M3 receptor None declared. does not seem to play a major role in this side effect. There are some pharmacological clues, which suggest that the serotonergic and histaminergic systems may play a role References in clozapine-induced weight gain.13,15,16 Studies involving 1 Baptista T. Body weight gain induced by antipsychotic drugs: polymorphisms of the serotonin 5-HT1A, 5-HT2A,5- 13,63 63 mechanisms and management. Acta Psychiatr Scand 1999; 100: 3–16. HT2C, 5-HT6 receptor genes, 5-HT transporter gene, 13 2 Allison DB, Mentore JL, Heo M, Chandler LP, Cappelleri JC, Infante MC and the histamine H1 and H2 receptor genes have failed et al. Antipsychotic-induced weight gain: a comprehensive research to demonstrate genotype effects on clozapine-induced synthesis. Am J Psychiatry 1999; 156: 1686–1696. weight gain. The finding of a significant association 3 Wirshing DA, Wirshing WC, Kysar L, Berisford MA, Goldstein D, Pashdag between a À759C/T polymorphism of the 5-HT receptor J et al. Novel antipsychotics: comparison of weight gain liabilities. 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