Clozapine: Selective Labeling of Sites Resembling 5HT6 Receptors May Reflect Psychoactive Profile

Charles E. Glatt, Adele M. Snowman, David R. Sibley, and Solomon H. Snyder Departments of Neuroscience, , 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: , the classic atypical neuroleptic, receptors consistent with the 's 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 receptors, and selective ac- tors, though serotonin itself displays low affinity. tions at novel subtypes of 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 , olanz- treating . Though developed as a epine, seroquel, and (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 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 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. , 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). , 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 and , 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 , 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 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 1,000 0.6

Adrenergic a 1 0 E41 n >10,000 OA0 0 n Muscarinic 0a0 Cholinergic 0.2 Scopolamine 0.8

Atropine 1.0 nn-V.U I ...... Butyrophenones -1010 i04 10 10-7 o10, Concentration of Scopolamine (nM) 400 FIG. 1. Inhibition of [3HIclozapine binding to 30 rat brain membranes by scopolamine Shown is a typical displacement curve for [3H]cloza- 20 pine bound to rat brain membranes. The data repre- 30 sent the mean percent of maximum specific binding (defined with 1 ,tM clozapine). The experiment was 30 replicated three times. 20 Tricyclic ,tM, almost 1000 times higher than its affinity for histamine HI receptors. The potent a-I adreno- 30 ceptor antagonist prazosin has an IC50 greater 150 than 10 mM. Similarly, agents active at sigma, 30 GABA, and glutamate receptors display low af- finity for [3H]clozapine sites. 10 Clozapine is well known to influence dopa- 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). For instance, (+) has a Ki value in the as of a displacing curve of unlabeled clozapine low nanomolar range for the D1 receptor but a Ki of competing for [3H]clozapine binding reveals a sin- 1 ,uM for [3H]clozapine binding. D2, D3, and D4 gle binding site with a Kd of 6.7 nM and a Bmax of receptors have a fairly similar pharmacologic pro- 240 fmol/mg protein (Fig. 2b). Since dopamine file. One major exception is that D4 receptors dis- receptors are most highly concentrated in the cor- play a low affinity for most neuroleptics but rela- pus striatum, we compared [3H]clozapine binding tively high affinity for clozapine (9). Thus, the in the striatum, hippocampus, and cerebral cortex potency of clozapine at D4 receptors is consistent (Table 2). The K(d's are similar in all areas, suggest- with its potency at [3H]clozapine binding sites. ing similar binding sites, and the Bmax values are However, there are other major differences. For roughly equal, suggesting a regional distribution of instance, spiperone displays subnanomolar affinity [3H]clozapine binding that does not fit with a clas- for D2, D3, and D4 receptors but has a Ki of 30 nM sical dopamine D1 or D2 receptor. at [3H]clozapine sites. Further, displays The relative potencies of numerous drugs in low nanomolar potency at D2, D3, and D4 sites, but, inhibiting [3H]clozapine binding provide sugges- at 10 ,tM concentration, it fails to affect [3H]cloza- tions about the types of receptors that are most pine sites. Finally, the Ki of dopamine for [3H]cloza- likely involved (Table 1). Triprolidine, a classic pine sites is 5 mM, more than 1000 times greater histamine Hi antagonist, displays an IC50 of 1 than its 1i for D2, D and D receptors. C. E. Glatt et al.: Clozapine Binding Sites 401

TABLE 2. Regional distribution of [3HJclozapine binding -0 Eu Region Kd (nM) Bmax (pmol/mg) I-I Cerebral cortex 6.7 0.24 r._Cu Striatum 7.3 0.17 Nm 0 Hippocampus 4.3 0.11

Cu Rats were killed by decapitation, their brains rapidly re- PC moved and dissected on ice. Regions were homogenized 0 separately and pellets were resuspended to approximately the same protein concentration ( 15 mg/ml). Binding was assessed at varying [3H]clozapine concentrations. Ki and Bmax values were determined by Scatchard analysis. Data are the means of three determinations with results varying less than 15%.

.5'u .2 fifty times more potent at clozapine binding sites A than at 5HT1C receptors. In addition, mesul- PC is more than 300 times as potent at SHT1c sites than at [3H]clozapine sites. The SHT1E site is 0 also excluded, as ergotamine has a 60-fold higher C c) affinity for [3H] clozapine sites than for the cloned 5HTlE receptor. The 5HT2 receptor can likewise be excluded, since it has a dramatically higher affinity for ritanserin than [3H]clozapine binding sites. The 5HT7 receptor does not fit, )Oa Clozapine (nM) because 's affinity for SHT7 receptors is 50 times greater than for [3H]clozapine sites. FIG. 2. (a) Saturation binding plot of The 5HT6 receptor displays high affinity for a [3HJclozapine to rat brain membranes and variety of psychotropic drugs (14, 35). We utilized (b) displacement curve and Scatchard analysis for unlabeled clozapine competing with cell lines expressing molecularly cloned rat 5HT6 [3HJclozapine. receptors and in our laboratory compared drug Data in Panel a were plotted using Cricketgraph. affinities for [3H]clozapine, [3H] 5HT, and [3H]LSD Shown are total (----@---- ), nonspecific (----LO----), binding to the expressed 5HT6 receptors with and specific (----U----) binding in pmol/mg protein [3H]clozapine binding in rat cerebral cortex mem- as well as Scatchard analysis of this data. In Panel b, branes (Table 5). The sirnilarities in the pharmacol- data are presented as fraction of total specific bind- sites ing defined by 1 ,tM unlabeled clozapine. The exper- ogy of the 5HT6 receptor and [3H]clozapine iment was replicated three times. are striking. Tricyclic structures, including tricyclic antidepressants and phenothiazines, display nano- molar potencies at 5HT6 receptors and [3H]cloza- A substantial number of serotonin receptor pine sites. Some structures related to , such as subtypes have been differentiated by bind- methiothepin, are extremely potent at 5HT6 recep- ing studies. Molecular cloning has led to the tors and clozapine sites, whereas mesulergine, demonstration of an even greater heterogeneity which is in the same structural class, is about 100 so that many of the sites labeled in brain mem- times less potent at both sites. For some drugs, branes by ligand binding actually represent a affinities vary depending on whether the ligand is mixture of 5HT receptor subtypes. To compare [3H]clozapine, [3H] 5HT, or [3H]LSD. However, drug potencies at 5HT receptor subtypes with none of these differences are marked. One notable [3H]clozapine binding sites, we have summa- exception is 5HT itself whose Ki at [3LH]clozapine rized reports from the literature on molecularly sites is 0.1-0.2 mM (data not shown) whereas it cloned and expressed receptors (Table 4). displays a 0.15 ,tM 1i at 5HT6 receptors. [3H]jClozapine sites in brain are not likely to in- The relatively low potency of SHT itself at volve SHT1c receptors, as spiperone is almost [3H]clozapine binding sites remains a puzzle. Po- 402 Molecular Medicine, Volume 1, Number 4, May 1995

TABLE 3. Comparison of drug affinities for [3HJclozapine binding sites in rat brain and cloned dopamine receptors Ligand D1 tmab D2 Mc D3 nMc D4 nMd [3H]Clozapine

SCH 23390 0.11 1,000 (+) Butaclamol 0.90 0.83 40 1,000 190 > 1,000 148 Spiperone 220 0.07 0.61 0.05 30 210 24 20 4.1 50,000 Dopamine 2,500 474 25 28 500,000 203 0.45 9.8 5.1 400 5.3 7.4 340 200 0.3 9.5 6,000 Thioridazine 3.3 7.8 12 30 Chlorpromazine - 2.8 6.1 37 20 Raclopride - 1.8 3.5 237 >10,000 - 2.1 >10,000 Fluphenazine - 46 20 Trifluoperazine 3 30 Clozapine 56 180 9 10

All numbers represent Ki values from the literature as noted or [3H]clozapine to rat brain membranes. Values for drug potencies at [3H]clozapine binding sites are means of two to three determinations that varied less than 20%. aDeary et al. (16) bZhou et al. (17) cSokoloff et al. (18) dVan Tol et al. (9)

tencies of and other in cology similar to that we have seen for the competing for [3H]antagonists are often relatively cloned 5HT6 receptor. It is possible that other weak and vary considerably depending on the li- sites, such as the D4 receptor, may be labeled by gand employed. Antagonist ligands such as [3H]clozapine but would escape detection be- [3H]clozapine may fix the conformation of the re- cause of low abundance in brain or because of ceptor in an antagonist preferring conformation overlapping pharmacology as occurs with multi- that diminishes affinity of the . Alterna- ple serotonin receptors. tively, sites labeled by [3H]clozapine may not be One notable feature of [3H]dozapine binding is 5HT6 receptors but instead a distinct receptor with its equal distribution in the cortex, hippocampus, and similar drug specificity. striatum. In one published study of doned 5HT6 re- ceptors, mRNA levels were roughly equal in these three regions (33). While Sibley and collaborators DISCUSSION (14) initially described high levels of 5HT6 mRNA in The main finding of this study is abundant, high- the corpus stiatum with negligible levels in cortex and affinity binding of [3H] clozapine to rat brain hippocampus, reexamination using in situ hybridiza- membranes. Our results are in agreement with tion indicates substantially higher levels of 5HT6 re- other studies of [3H]clozapine binding showing ceptor mRNA in cortex and hippocampus than first two high-affinity binding sites (34, 35). The first reported (D. R. Sibley and J. E. Lachowitz, unpub- is a muscarinic cholinergic receptor which has lished observations). Thus, both on the basis of drug low nanomolar affinity and represents the ma- specificity and regional distribution, the [3H]dlozapine jority -60% of [3H]clozapine binding in the binding sites not involving muscarinic receptors re- brain. The second site demonstrates a pharma- semble the 5HT6 receptor. ~ t ~ ~ .... = II> :i" ... ~. ~ o N ~

tn 403 r:- - n ~ g et Sites al.: ~. C. E. Glatt Clozapine~ Binding

0

0 o 0 o0Or0 two 0 0 \0 0 N O 0 of C)0 = -_ . o6 5.3 9.1 9.8 o4 8.2 24.6 1,000 5,000 o N 00 81.9 123 (nM) 0

_ A >1,000 200,000 values [3HIClozapine

U-' mean 0 0 are ,n 100

m un 00 0 U-' 00 0 00 sites Co 0 ue '0 C\ -_ t- o 0

1.000 co 15 35-52 6 \0 5HT/ 4.0 0.3-8.8 9.9-20

0.6-1.8 cf_ 265-37,500 13.4 21.1 binding

k 00 N M.-- N 3.2 5B 0u t 3.2 4.4 Xo 15.8 4- - 126 , 398 v-, ._ l.584 receptors 5HT [3Hlclozapine for

k 0 0 A 0\ C\ 5A

0 3.9 3.9 I,' °. 0 0 63 IC) co serotonin (nM) (nM) (nM) 700 251 251 A >10,0 5HT

r affinities 6o i^ A >10,000

._ (U F 0 Lt Drug cloned 0 0 *H0 o0 Lt~ 'P.4oi U-' 0.5 and 0I

(nM) - 0 0 5HTj O 1.0-5.0 1.0-5.0 5.0 0.4 0.2 A00>1.000 4,, 250 *5-Po 0 membranes. brain

i 0 N'i 0 0 0 0 VI\ C' 4..t 0 1F 0 0 ON rat

V brain (U 0 1- 0 0

3,162 r0

in C' Le\ (nM) rat

- 50-55 67-79 70-125 5HT A >1,000 >1.000 9.9-20 C~ A 6.3-14 "A to U, \0N 04

sites co -Z Po I' 0 0 1.O,h w 0

._binding 1.41E INo 6.7 8.14 o H . °° '_ 0 2,300 ._ (nM) 300 \0600 W. A >1.000 m >1.000 .5 binding (U M5HT -o ,*-% 0> Ul -, 1 3.0 [3Hlclozapine 7.2 ro oo,c 2.7 N H ,- 0 NIN ,N28.5 ri I 957

z C\ or

U'A5HT [3Hlclozapine noted 0 0 0

(U o~~~~r as at 0 oo 0

c,d,e 0 oU 0 00o~~~~00 0

1B o~~~~~~- Io~~~~~~~~~~~~~~~~~~~ - (nM) (nM) 13 -416-48 m >10,000 >10,000 A>10,000 A A 4.2 5HT 436-1.823 465 cu literature 20%. affinities VE s 0 the 5- 0 0_ than - 0 ,b drug Q

oUfrom less a \0 0 7a

1a 1- C-' eN of I _ (nM) Co N 63-134 Er, o o) values 5HT 400 j

0 0 varying 0.06 0.27-1.66 K 0(o i12,100-23,000 (21) (26) (25) (29) (27) al. (22) . (28) ~ al. Comparison (19) al. (20) represent (31) (32) (23) al. al. et (24) (30) et al. al. et al. al. 4. et et al. C'Z -~ al. o- et et determinations al. P-4 al.

et 400e et .4.- DPAT

> et Q D ~3J i E et

';4 bick et Ev et

LUr ' .o numbers three gom

00 Albert Clozapine 5HT Spiperone kMatthes hMcAllister nShen mRuatetal. IRoth Ligand iAmlaiky 8-0H All to Mesulergine Ketanserin Ritanserin Methiothepin b Ergotamine aFargin dAdham eVoigt 'Maroteaux TABLE jMeltzer 9Z 'Roth 404 Molecular Medicine, Volume 1, Number 4, May 1995

TABLE 5. Comparison of drug affinities for [3HJclozapine binding sites in rat brain membranes and cloned 5HT6 receptors [3HIClozapine K1 (nM) [3H]5HT (nM) [3HILSD (nM) 5HT6 Clone Cortex 5HT6 Clone

Clozapine 10 10 20 13 Methiothepin 9 7 0.4 2 20 15 5 8 Dihydroergotamine 25 40 5 13 150 80 54 150 250 36 100 15 56 65 Nortriptyline 400 30 148 Amitriptyline 100 10 82 70 4,000 5,000 1,700 Ritanserin 1,000 150 16 44 Mesulergine 2,000 1,000 1,700 Thioridazine 300 25 Ketanserin 1,000 80 Ergotamine 10 12 Risperidone >1,000 100 950 1,000 Methysergide 800 1,000

Ki values are means of two to three determinations that varied less than 20%.

Compared with conventional neuroleptics, slow Alo but not Ag cells could explain the low- clozapine and other atypical neuroleptics display ered EPS incidence associated with clozapine. two unique properties, a low incidence of EPS Metabolic measures showing greater dopamine and greater therapeutic efficacy for negative and release in the corpus striatum following atypical possibly even for positive symptoms of schizo- than conventional neuroleptics support the neu- phrenics. This enhanced efficacy was first shown rophysiologic data (36,40). A link to 5HT2 recep- in patients resistant to conventional neuroleptics tors comes from observations that combinations (1), but may hold for nonresistant patients as of the 5HT2 selective drug ritanserin and the D2 well (1,36). Properties unique to the atypical antagonist haloperidol elicit striatal dopamine drugs may thus clarify neural mechanisms me- metabolic patterns resembling clozapine (41). diating negative symptoms as well as modulating Similarly, administration of ritanserin alleviates EPS. Influences on 5HT receptors appear rele- EPS provoked by haloperidol (10). The similarity vant to clozapine actions, as clozapine displays of 5HT6 and 5HT2 receptors suggests that phar- very great potency at most 5HT receptors, and macologic data implicating 5HT2 receptors in ac- 5HT receptors modulate dopamine systems in- tions of atypical neuroleptics would apply also to volved in EPS. Thus, chronic administration of 5HT6 receptors. Interestingly, ritanserin's affinity conventional neuroleptics decreases firing rates for 5HT6 receptors varies depending on the li- of the Alo dopamine cells that project to the gand employed with Ki values of 16 nM with and the Ag cells of the substantia [3H] 5HT and 1000 with [3H] clozapine. At nigra that project to the corpus striatum. Pre- [3H] clozapine binding sites in cortical mem- sumably, the slowed firing rate of the dopamine branes, ritanserin has a 1i of 150 nM. cells accentuates the effects of direct dopamine Notably, while clozapine has low nanomolar receptor blockade. The observations that clozapine affinity for numerous serotonin as well as other (37) and other atypical neuroleptics (13,38,39) receptors, the predominant receptors labeled by C. E. Glatt et al.: Clozapine Binding Sites 405

[3H]clozapine in the present study were musca- 7. Miller RJ, Hiley CR. (1974) Anti-muscarinic rinic cholinergic and 5HT6-like. In part, this may properties of neuroleptics and drug-induced reflect receptor density in that the very low den- . Nature 248: 596-597. sity of D4 receptors in rat brain (9) may have 8. Snyder SH, Greenberg D, Yamamura HI. precluded their labeling. Alternatively, confor- (1974) Antischizophrenic drugs and brain cho- mational alterations in receptors elicited by a linergic receptors. Arch. Gen. Psych. 31: 58-61. radioligand may influence a drug's apparent af- 9. Van Tol HHM, Bunzow JR, Guan H, et al. finity. Accordingly, the best indication of sites (1991) Cloning of the gene for human do- with which the drug will interact in vivo will pamine D4 receptor with high affinity for the come from properties of a radiolabeled drug's clozapine. Nature 350: 610-614. binding in the intact human brain. Whether the 10. Bersani G, Grispini A, Marini S, Pasini A, same sites are labeled by [3H]clozapine in rat Valducci M, Ciani N. (1986) Neuroleptic- brain membranes is unclear. Nonetheless, sites induced extrapyramidal side effects: Clinical labeled by [3H]clozapine, whether reflecting 5HT6 perspectives with ritanserin (R35667), a new or related receptors, may provide a model system selective 5-HT2 receptor blocking agent. for identifying candidate atypical neuroleptics and Curr. Ther. Res. 40: 492-499. clarifying their unique psychoactive properties. 11. Rogue A, Rogue P. (1992) Mianserin in the management of schizophrenia. In: Schizo- phrenia Intl. Conf. Abstr. Vancouver, British ACKNOWLEDGMENTS Columbia, p. 135. Supported by USPHS Grant MH-18501, Research 12. Meltzer HY, Shigehiro M, Lee J. (1989) Clas- Scientist Award DA-00074 to SHS, and a grant of sification of typical and atypical antipsy- the Stanley Foundation Research Awards Pro- chotic drugs on the basis of dopamine D1, D2 gram. We thank H. Meltzer for advice and 0. and serotonin 2 pKi values. J. Pharmacol. Civelli for assistance in initial studies of cloned Exp. Ther. 251: 238-246. dopamine receptors. 13. Stockton ME, Rasmussen K. (1993) A com- parison of olanzepine and clozapine effects on dopamine neuronal activity: An electro- REFERENCES physiological study. Neurosci. Abstr. 19: 383. 1. Kane J, Honigfeld G, Singer J, Meltzer H. 14. Monsma FJ, Shen Y, Ward RP, Hamblin (1988) Clozapine for the treatment-resistant MW, Sibley DR. (1993) Cloning and expres- schizophrenic. Arch. Gen. Psych. 45: 789-796. sion of a novel serotonin receptor with high 2. Meltzer HY. (1988) Clozapine: Clinical advan- affinity for tricyclic psychotropic drugs. Mol. tages and biologic mechanisms. In: Schulz C, Pharmacol. 43: 320-327. Tamminga C, Chase TN, Christensen AV, 15. Cheng YC, Prusoff WH. (1973) Relationship Gerlach J (eds). Schizophrenia: A Scientific Focus. between the inhibition constant (Ki) and the Oxford University Press, New York, pp 302-309. concentration of inhibitor which causes 50 3. Peroutka SJ, Snyder SH. (1980) Relationship percent inhibition (IC50) of an enzymatic re- of neuroleptic drug effects at brain dopa- action. Biochem. Pharm. 22: 3099-3108. mine, serotonin, -, and his- 16. Dearry A, Gingrich JA, Falardeau P, Fremeau tamine receptors to clinical potency. Am. J. T, Bates MD, Caron MG. (1990) Molecular Psychiat. 137: 1518-1522. cloning and expression of the gene for a hu- 4. Richelson E. (1984) Neuroleptic affinities for man D1 . Nature 347: 72-76. human brain receptors and their use in predict- 17. Zhou Q, Grandy DK, Thambi L, Kushner ing adverse effects. J. Clin. Psychiat. 45: 331-336. JA, Van Tol HHM, Cone R, Pribnow D, Salon 5. Seeman P. (1992) Dopamine receptor se- J, Bunzow JR, Civelli, 0. (1990) Cloning and quences. Therapeutic levels of neuroleptics expression of human and rat DI dopamine occupy D2 receptors, clozapine occupies D4. receptors. Nature 347: 76-80. Neuropsychopharmacology 7: 261-284. 18. Sokoloff P, Giros B, Martres M, Bouthenet 6. Farde L, Nordstrom A-L, Wiesel FA, Pauli S, M, Schwartz J. (1990) Molecular cloning Halldin C, Sedvall G. (1992) Positron emis- and characterization of a novel dopamine sion tomographic analysis of central DI and receptor (D3) as a target for neuroleptics. D2 dopamine receptor occupancy in patients Nature 347: 146-151. treated with classical neuroleptics and cloza- 19. Fargin A, Raymond JR, Lohse MJ, Kobilka pine. Arch. Gen. Psych. 49: 538-544. BK, Caron MG, Lefkowitz, RJ. (1988) The 406 Molecular Medicine, Volume 1, Number 4, May 1995

genomic clone G-21 which resembles the A, Monsma FJ, Shen Y, Meltzer HY, Sibley 13- sequence encodes the DR. (1994) Binding of Typical and Atypical 5HT-IA receptor. Nature 335: 358-360. antipsychotic Agents to 5-Hydroxytryptamine-6 20. Albert PR, Zhou Q, Van Tol HHM, Bunzow and 5-Hydroxtryptamine-7 Receptors. J. Phar- JR, Civelli 0. (1990) Cloning, functional ex- macol. Exp. Ther. 268: 1403-1410. pression, and mRNA tissue distribution of 31. Ruat M, Traiffort E, Leurs R, Tardivel-Lacombe the rat 5-hydroxytryptamine LA receptor J, Diaz J, Arrang J, Schwartz J. (1993) Molec- gene. J. Biol. Chem. 265: 5825-5832. ular cloning, characterization, and localization 21. Maroteaux L, Saudo F, Amlaiky N, Boschert U, of a high-affinity serotonin receptor (5HT-7) Plassat JL, Hen R. (1992) Mouse 5HTlB sero- activating cAMP formation. Proc. Natl. Acad. Sci. tonin receptor: Cloning, functional expression, U.S.A. 90: 8547-8551. and localization in motor control centers. Proc. 32. Shen Y, Monsma FJ, Metcalf MA, Jose PA, Ham- Natl. Acad. Sci. U.S.A. 89: 3020-3024. blin MW, Sibley DR. (1993) Molecuar cloning 22. Adham N, Romanienko P. Hartig P, Wein- and expression of a 5-hydroxytryptanine 7 shank RL, Branchek T. (1991) The rat 5-hy- serotonin receptor subtype. J. Bio. Chem. 268: broxytryptamine LB receptor is the species 18200-18204. homolog of the human 5-hydroxytryptamine 33. Ruat M, Traiffort E, Arrang J, et al. (1993) A ID,B receptor. Mol. Pharmacol. 41: 1-7. novel rat serotonin (5-HT6) receptor: Molec- 23. Voigt MM, Laurie DJ, Seeburg PH, Bach A. ular cloning, localization and stimulation of (1991) Molecular cloning and characteriza- cAMP accumulation. Biochem. Biophys. Res. tion of a rat brain cDNA encoding a 5-hy- Commun. 193: 268-276. droxytryptamine LB receptor. EMBO J. 10: 34. Hauser D, Closse A. 3H-clozapine binding to 4017-4023. rat brain membranes. Life Sci. 23: 557-562. 24. Roth BL, Ciaranello RD, Meltzer HY. (1992) 35. Flamez A, De Backer J, Wiczak N, Vauqelin Binding of typical and G, De Keyser J. (1994) [3H]Clozapine is not agents to transiently expressed 5-HT 1C re- a suitable ligand for labelling of D4 dopa- ceptors. JPET. 260: 1361-1365. mine receptors in post mortem brain. Neuro- 25. Zgombick JM, Schecter LE, Macchi M, Hartig sci. Lett. 175: 17-20. P, Branchek TA, Weishank RL. (1992) Human gene S31 encodes the pharmacologically de- 36. Meltzer HY. (1991) The fined serotonin 5-hydroxytryptamine IE re- of novel antipsychotic drugs. Schizophrenia ceptor. Mol. Pharmacol. 42: 180-185. Bull. 17: 263-287. 26. McAllister G, Charlesworth A, Snodin C, 37. Chiodo LA, Bunney BS. (1985) Possible MS, Noble AJ, Middlemiss DN. (1992) mechanisms by which repeated clozapine Molecular cloning of a serotonin receptor from administration differentially affects the ac- human brain (5HT IE): a fifth 5HTl-like sub- tivity of two subpopulations of midbrain do- type. Proc. Natl. Acad. Sci. U.S.A. 89: 5517-5521. pamine neurons. J. Neurosci. 5: 2539-2544. 27. Amlaiky N, Ramboz S, Boschert U, Plassat J, 38. Skarsfeldt T, Perregaard J. (1990) Sertindole, Hen R. (1992) Isolation of a mouse "5HT1E- a new neuroleptic with extreme selectivity like" serotonin receptor expressed predomi- on A10 versus A9 dopamine neurons in the nantly in hippocampus. J. Biol. Chem. 267: rat. Eur. J. Pharmacol. 182: 613-614. 19761-19764. 39. Goldstein JM, Litwin LC, Sutton EB, Malick 28. Meltzer HY, Shigehiro M, Lee J. (1989) Clas- JB. (1993) Seroquel: Electrophysiological pro- sification of typical and atypical antipsychotic file of a potential atypical antipsychotic. Psycho- drugs on the basis of dopamine D-1, D-2 and pharmacology 112: 292-299. serotonin 2 pKi values. JPET 251: 238-246. 40. Ichikawa J, Meltzer HY. (1991) Differential ef- 29. Matthes H, Boschert U, Amlaiky N, Gailhe, fects of repeated treatment with haloperidol and Plassa, J, Muscatelli, Mattei M, Hen R. (1993) dozapine on dopamine release and metabolism Mouse 5-hydroxytryptamine 5A and 5-hy- in the striatum and the nudeus accumbens. J. droxytryptamine SB receptors define a new Pharmacol. Exp. Ther. 256: 348-357. family of serotonin receptors: Cloning, func- 41. Gudelsky FA, Nwajei EE, DeFife K, Nash JF. tional expression, and chromosomal localiza- (1992) Interaction of amfonelic acid with tion. Mol. Pharmacol. 43: 313-319. antipsychotic drugs on neu- 30. Roth BL, Craigo SC, Choudhary MS, Uluer rons. Synapse 12: 304-311.

Contributed by S. H. Snyder on March 14, 1995.