Clozapine: Selective Labeling of Sites Resembling 5HT6 Serotonin Receptors May Reflect Psychoactive Profile
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Clozapine: Selective Labeling of Sites Resembling 5HT6 Serotonin Receptors May Reflect Psychoactive Profile Charles E. Glatt, Adele M. Snowman, David R. Sibley, and Solomon H. Snyder Departments of Neuroscience, Pharmacology, and Molecular Sciences, and Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A., and Experimental Therapeutics Branch, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, U.S.A. ABSTRACT Background: Clozapine, the classic atypical neuroleptic, receptors consistent with the drug's anticholinergic exerts therapeutic actions in schizophrenic patients un- actions. The drug competition profile of the second responsive to most neuroleptics. Clozapine interacts with site most closely resembles 5HT6 serotonin recep- numerous neurotransmitter receptors, and selective ac- tors, though serotonin itself displays low affinity. tions at novel subtypes of dopamine and serotonin re- [3H]Clozapine binding levels are similar in all brain ceptors have been proposed to explain clozapine's regions examined with no concentration in the cor- unique psychotropic effects. To identify sites with which pus striatum. clozapine preferentially interacts in a therapeutic setting, Conclusions: Besides muscarinic receptors, clozapine we have characterized clozapine binding to brain mem- primarily labels sites with properties resembling 5HT6 branes. serotonin receptors. If this is also the site with which Materials and Methods: [3H]Clozapine binding was clozapine principally interacts in intact human brain, it examined in rat brain membranes as well as cloned- may account for the unique beneficial actions of cloza- expressed 5-HT6 serotonin receptors. pine and other atypical neuroleptics, and provide a mo- Results: [3H]Clozapine binds with low nanomolar lecular target for developing new, safer, and more effec- affinity to two distinct sites. One reflects muscarinic tive agents. INTRODUCTION ifested by more recently developed atypical Clozapine is an important therapeutic agent in neuroleptics, including risperidone, olanz- treating schizophrenia. Though developed as a epine, seroquel, and sertindole (2). neuroleptic, it is unique in its therapeutic pro- Therapeutic actions of conventional neuro- file and may provide major benefits to patients leptics correlate closely with their potencies in who are resistant to other neuroleptics (1,2). blocking dopamine D2 receptors, which pre- Even in patients who respond to conventional sumably explains their therapeutic and EPS neuroleptics, clozapine may be more effica- actions (3-5). Imaging D2 receptors in humans cious (2). Clozapine appears to relieve negative by positron emission tomography reveals less symptoms, including apathy and emotional occupancy of D2 receptors by atypical than withdrawal, that resist conventional neurolep- conventional neuroleptics, when administered tics and displays a very low incidence of extra- at clinically effective doses (6). The lesser inci- pyramidal side effects (EPS). Diminished EPS dence of EPS associated with the atypical drugs and greater therapeutic efficacy are also man- may also reflect their greater anticholinergic potencies, as muscarinic anticholinergic drugs Address correspondence and reprint requests to: Solomon H. Snyder, Johns Hopkins University School of Medicine, are well known to relieve Parkinsonian, EPS 725 N. Wolfe Street, Baltimore, MD 21205, U.S.A. symptoms (7,8). 398 Copyright © 1995, Molecular Medicine, 1076-1551/95/$10.50/0 Molecular Medicine, Volume 1, Number 4, May 1995 398-406 C. E. Glatt et al.: Clozapine Binding Sites 399 To explain the greater antischizophrenic ac- Preparation of Membranes from 5HT6 tions of atypical neuroleptics, researchers have Receptor Expressing Cells evaluated effects of these drugs at novel sites. Thus, Human embryonic kidney 293 cells stably expressing clozapine displays uniquely high affinity for dopa- rat 5HT6 receptors were grown in D-modified Eagle's mine D4 receptors (9). Serotonin 5HT2 receptors medium with 10% FBS, 1 mM sodium pyruvate, and have also been implicated. Ritanserin, a selective G418 at 300 ,ug/ml. Cells were grown to confluence, 5HT2 antagonist, decreases EPS elicited by haloper- rinsed with 5 ml 0.5 mM EDTA in phosphate buff- idol (10). Mianserin, also a 5HT2 antagonist, re- ered saline (PBS). Cells were then washed for 5 min lieves negative symptoms in schizophrenics receiv- with an additional 5 ml EDTA/PBS. Cells were re- ing conventional neuroleptics (11). Compared leased by this treatment and treated as above for with conventional neuroleptics, atypical drugs tend brain membranes. Final membrane concentration to have higher affinities for 5HT2 than D2 receptors was -10.0 mg/ml protein. These cells express ap- (12). However, there are exceptions such as chlor- proximately 800 fmol/mg protein of 5HT6 receptor promazine and amoxapine, both more potent at binding activity (14). 5HT2 than D2 sites (13). To assess clozapine actions at various receptors, most studies have evaluated clozapine's potency in [3H]Clozapine Binding Assays competing for the binding of radioligands. How- Binding assays were performed in a final volume ever, ligands exert "induced fit", altering receptor of 500 ,l Tris HCI, pH 7.4. Membrane prepara- conformation so that the potency of a drug in tion (0.25 ml) was added to each tube. Com- competing for binding of a radioligand may not pounds for competition were added in a 50-p,l faithfully reflect that drug's propensity to bind to volume. Fifty microliters of a 2% bovine serum the receptor in vivo. Ideally, one would like to albumin (BSA) solution was added to reduce specify the receptors to which clozapine binds nonspecific filter binding. [3H] Clozapine (specific when encountering the human brain in vivo. activity 51.3-89.1 Ci/mmol) in 50 ptl was added To evaluate sites to which clozapine binds to give a final concentration of 1 nM. For all preferentially, we have examined the binding of experiments performed in the presence of sco- [3H]clozapine itself to rat brain membranes. We polamine, 100 p,l was added to give a final con- report labeling of muscarinic cholinergic recep- centration of 10 nM. For the initial experiments tors as well as sites resembling 5HT6 serotonin without scopolamine, 100 gl of distilled water receptors. were used. Nonspecific binding was determined by addition of 1 ,M (final concentration) unla- beled clozapine. Tubes were incubated for 20 MATERIALS AND METHODS min at 37°C. The incubation was terminated by rapid filtration over 0.5% All unlabeled drugs were obtained from Research polyethyleneimine- soaked filters (GF/B) and washed 2x 3 ml with Biochemicals International (Natick, MA, U.S.A.). ice-cold 50 mM NaCl. [3H]Clozapine was generously provided by Dr. S. Hurt NEN-Dupont (Boston, MA, U.S.A.). RESULTS Preparation of Rat Brain Membranes As an initial screen, we evaluated inhibition Sprague-Dawley rats (200-300 g) were killed by of [3H]clozapine binding by agents acting at decapitation. Brains were rapidly removed and sites where clozapine is thought to exert effects specific brain regions dissected. Tissue was ho- (Table 1). Scopolamine and atropine are uniquely mogenized by Polytron in 50 mM Tris HCI, pH potent with IC50 values of 0.8 nM and 1.0 nM, 7.4, at 4°C. Homogenates were centrifuged at respectively. Scopolamine maximally inhibits 48,000 X g for 10 min. Pellets were resuspended about 60% of [3H]clozapine binding at 5 nM (Fig. and rehomogenized in the same buffer and cen- 1). Accordingly, in all subsequent experiments we trifuged a second time at 48,000 X g for 10 min. include 10 nM scopolamine so that hereafter Pellets were resuspended in 50 mM Tris HCI, pH [3H]clozapine binding will refer to binding mea- 7.4, to a concentration of 15 mg/ml. Except in sured in the presence of scopolamine. regional dissection studies, membranes from [3H] Clozapine binding is saturable with a Kd of whole rat brain minus cerebellum were em- 4.5 nM and Bmax of 380 fmol/mg protein (Fig. 2a). ployed for binding assays. Scatchard analysis of [3H]clozapine binding as well 400 Molecular Medicine, Volume 1, Number 4, May 1995 TABLE 1. Drug effect on [3Hlclozapine 1.2- binding in rat brain membranes 1.0 a 0 Drug Ki (nM) a 0.8 Histamine HI n II Triprolidine 1,000 0.6 Adrenergic a 1 0 E41 n Prazosin >10,000 OA0 0 n Muscarinic 0a0 Cholinergic 0.2 Scopolamine 0.8 Atropine 1.0 V.Unn- I .......... Butyrophenones -1010 i04 10 10-7 o10, Concentration of Scopolamine (nM) 400 Pipamperone FIG. 1. Inhibition of [3HIclozapine binding to Spiperone 30 rat brain membranes by scopolamine Phenothiazines Shown is a typical displacement curve for [3H]cloza- Fluphenazine 20 pine bound to rat brain membranes. The data repre- Trifluoperazine 30 sent the mean percent of maximum specific binding (defined with 1 ,tM clozapine). The experiment was Thioridazine 30 replicated three times. Chlorpromazine 20 Tricyclic ,tM, almost 1000 times higher than its affinity for Antidepressants histamine HI receptors. The potent a-I adreno- Nortriptyline 30 ceptor antagonist prazosin has an IC50 greater Imipramine 150 than 10 mM. Similarly, agents active at sigma, Amitriptyline 30 GABA, and glutamate receptors display low af- Ergot Alkaloids finity for [3H]clozapine sites. Ergotamine 10 Clozapine is well known to influence dopa- Dihydroergotamine 20 mine receptors with an IC50 for D1 and D2 recep- tors of 172 nM and 182 nM, respectively (3). Ac- cordingly, we compared the potencies of various Increasing concentrations of the indicated compounds were agents in competing for used to inhibit the binding of 1 nM [3H]clozapine to rat dopamine-related brain membranes. Ki values were determined from the [3H]clozapine binding with their affinities for 'C50's by the method of Cheng and Prusoff (15). Results cloned and expressed D1, D2, D3, and D4 receptors in are the means of at least two experiments run duplicate. (Table 3). [3H]Clozapine does not seem to bind to Dia or Dlb receptors (Dib being the same as D5).