Binding of antipsychotic drugs to human brain receptors

Life Sciences 68 (2000) 29–39

Binding of antipsychotic drugs to human brain receptors Focus on newer generation compounds Elliott Richelson*, Terry Souder Departments of Psychiatry and Psychology, and Pharmacology, Mayo Foundation for Medical Education and Research and Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL 32224, USA Received 4 May 2000; accepted 14 June 2000

Abstract Using radioligand binding assays and post-mortem normal human brain tissue, we obtained equilibrium dissociation constants (Kds) for nine new antipsychotic drugs (iloperidone, melperone, olanzapine, ORG 5222, quetiapine, risperidone, sertindole, ziprasidone, and zotepine), one metabolite of a new drug (9-OH-risperidone), and three older antipsychotics (clozapine, haloperidol, and pimozide) at nine different receptors (a1-adrenergic, a2-adrenergic, dopamine D2, histamine H1, muscarinic, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors). Iloperidone was the most potent drug at the two adrenergic receptors. ORG 5222 was the most potent drug at dopamine D2 and 5-HT2c receptors, while ziprasidone was the most potent compound at three serotonergic receptors (5-HT1A, 5-HT1D, and 5-HT2A). At the remaining two receptors, olanzapine was the most potent drug at the histamine H1 receptor (Kd50.087 nM); clozapine at the muscarinic receptor (Kd59 nM). Certain therapeutic and adverse effects, as well as certain drug interactions can be predicted from a drug’s potency for blocking a specific receptor. These data can provide guidelines for the clinician in the choice of antipsychotic drug. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Histamine H1 receptor; Muscarinic receptor; a1-Adrenoceptor; a2-Adrenoceptor; Dopamine D2 receptor; Serotonin 5-HT1A receptor; Serotonin 5-HT2A receptor

Introduction Antipsychotic drugs are antagonists of many neurotransmitter receptors in human brain [1–3]. The potency of this blockade can be used to predict the likelihood of adverse side effects and drug interactions in clinical practice [4]. In addition, relative affinities for dopamine D2 and serotonin 5-HT2A receptors may predict whether an antipsychotic drug is atypical [5]. * Corresponding author. Tel.: (904) 953-2439; fax: (904) 953-2482. E-mail address: [email protected] (E. Richelson) 0024-3205/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 9 1 1 -5

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Figure 1. Structures of some new antipsychotic drugs.

Since we last reported the results of this type of study, several new antipsychotic drugs have been approved for use in the United States or are close to being approved. Among those are iloperidone, olanzapine, ORG 5222, and ziprasidone (Fig. 1). Along with clozapine, these drugs have been classified as atypical neuroleptics, a term that refers to their propensity to have a low incidence of extrapyramidal side effects. In addition, although others have studied these compounds at brain receptors [6], none has used human brain as the source of receptors. Therefore, we wanted to find the binding potencies of these and some additional compounds at several different receptors in human brain. Thus, we evaluated twelve antipsychotic drugs and a metabolite of one at a1-adrenergic, a2-adrenergic, dopamine D2, histamine H1, muscarinic, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors. Methods and materials Tissue preparation Normal brain tissue was obtained at the time of autopsy and stored at 280 8C until it was homogenized in 10 volumes of ice-cold 50 mM NaKPO4 buffer, pH 5 7.4, using a Brinkmann homogenizer, model PT 10/30 (10 sec, setting 6). The homogenate was then spun at 38 000 g for 10 min in a Beckman model J2-21 centrifuge. The pellets were resuspended in fresh NaKPO4 buffer and centrifuged again at 38 000 g. The final pellets were resuspended in NaKPO4 and diluted to a concentration of 10 mg wet weight per ml and stored at 280 8C until just before assay. For use in the radioligand binding assay, adequate amounts of tissue homogenates were thawed and spun as before. The pellets were washed and resuspended in fresh 50 mM Tris-HCl buffer. The volume for the resuspension varied with the receptor being

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Table 1 Parameters used in radioligand binding assaysa Estimation of Non-Ppecific Bound

Radioligand Receptor a1-adrenergic a2-adrenergic dopamine D2 histamine H1 muscarinic serotonin 5-HT1A serotonin 5-HT1D serotonin 5-HT2A serotonin 5-HT2C

[3H]-Compound prazosin rauwolscine spiperone pyrilamine QNBb 8-OH-DPATc 5-carboxamidotryptamine (5-CT) ketanserin serotonin

Human Brain Tissue Used

zFinal Conc. (nM)

Compound

Final Conc. (nM)

Brain Region

mg wet wt/tube

0.1 0.4 0.2 0.6 0.1 0.4 0.5

prazosin rauwolscine spiperone pyrilamine QNB 8-OH-DPAT 5-CT

10 25 50 250 2.5 50 100

FC FC CN FC CN FC SN

7.5 10.0 1.5 15 0.7 12.5 5.0

0.3 4.0

ketanserin Serotonin

FC FC

2.5 20.0

25 1000

Incubation of assay mixture for the 5HT1A and 5HT2C receptors was 30 min at 37 8C and for the 5HT1D receptor, 60 min 25 8C. All other assays were incubated at 37 8C for 60 min. b quinuclidinyl benzilate. c 8-hydroxy-2-(di-n-propylamino)tetralin. a

studied to provide the final tissue concentration indicated in Table 1. For 5HT1A assays the tissue homogenate of human cortex was processed further as described before [7]. For 5HT1D assays, human substantia nigra tissue homogenates were prepared further as previously described [8]. For 5HT2C assays, the tissue homogenate for human frontal cortex was processed further into microsomal fractions as previously described [9]. Radioligand binding assays Assays were done on the Beckman Biomek 1000 workstation outfitted with a side arm loader [10]. Nine different receptors were studied using modifications of previously reported methods [1,2,8,11–13]. Table 1 lists the radioligands and conditions for the different receptors. The incubation conditions for the 5HT1A and 5HT2C receptors were 30 min at 37 8C, while the 5HT1D receptor was 60 min at 25 8C. All other assays were incubated at 37 8C for 60 min. After incubation the samples were filtered under vacuum onto Wallac BetaPlate A filter mats using a Tomtec Harvester 96. The tubes and filters were rinsed with 4 3 2 ml icecold 0.9% NaCl. The filters were then counted using a Wallac BetaPlate 1205 scintillation counter. Specific binding to the receptor was calculated as the difference between the total binding (zero unlabeled ligand) and nonspecific binding (excess unlabeled ligand). Data analysis The data were analyzed using the LIGAND program [14] to calculate equilibrium dissociation constants (Kd). The program has been modified by us to provide the Hill coefficient. Geometric mean of the Kd [15] and its standard error [16] were calculated. Unless noted otherwise, we present mean values from at least three independent experiments, each done in duplicate.

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Source of materials The radiochemicals [3H]quinuclidinyl benzilate (QNB) (56.1 Ci/mmol), [3H]pyrilamine (29.4 Ci/mmol), [3H]prazosin (78.1 Ci/mmol), [3H]rauwolscine (77.0 Ci/mmol), [3H]8-hydroxy2-(di-n-propylamino)tetralin (8-OH-DPAT) (137 Ci/mmol), [3H]spiperone (83 Ci/mmol), [3H]5-CT (83.1 Ci/mmol), [3H]serotonin (114 Ci/mmol), and [3H]ketanserin (84.5Ci/mmol) were purchased from New England Nuclear (Boston, MA, USA). The following compounds were generously provided by the manufacturers: risperidone, 9-OH-risperidone, ketanserin tartrate (Janssen Pharmaceutica, Piscataway, NJ, USA); olanzapine (Eli Lilly and Co., Indianapolis, IN, USA); ziprasidone, prazosin HCl (Pfizer Central Research, Inc., Groton, CT, USA). The following compounds were purchased: 5-hydroxytryptamine, creatinine sulfate complex, 8-hydroxy-2-(di-n-propylamino)tetralin (8-hydroxy-DPAT) hydrobromide, and pyrilamine maleate (Sigma Chemical Co., St. Louis, MO, USA); rauwolscine HCl (Indofine Chemical Co., Inc., Somerville, NJ, USA); quinuclidinylbenzilate (QNB) and spiperone (Research Biochemicals Inc., Natick, MA. USA).

Results The data for the equilibrium dissociation constants for antipsychotic drugs at nine different receptors are presented in Table 2. The compounds are presented alphabetically. Reference compounds are also presented at the bottom of this table. Hill coefficients for all compounds at all receptors were essentially equal to unity, showing that binding of these drugs obeyed the law of mass action. Alpha1-adrenoceptor To study the a1-adrenoceptor, we used the radioligand [3H]prazosin, which had a Kd 5 0.28 6 0.01 nM (n536).The neuroleptics with the most potent binding at this receptor were iloperidone and ORG 5222 with Kd’s of 0.31 and 1.1 nM, respectively (Table 2). Pimozide and melperone were the least potent competitive antagonists at this receptor (Table 2). Alpha2-adrenoceptor The radioligand, [3H]rauwolscine, which was the most potent compound tested, had a Kd52.560.1 nM (n528). Antipsychotics competitively antagonized this radioligand. Iloperidone was the most potent compound at the a2-adrenoceptor (Kd 5 3.0 nM), with risperidone (Kd 5 8 nM) being the next most potent (Table 2). The metabolite of risperidone, 9-OH-risperidone, had one-tenth the affinity of its parent compound for this receptor. Dopamine D2 receptor Using the human caudate nucleus, we found that [3H]spiperone had a Kd 5 0.2860.02 nM (n519). Spiperone was the most potent antipsychotic drug tested by about 7 fold, with ORG 5222 being next most potent (Kd52.0 nM. Quetiapine was the least potent compound studied (Kd 5 770 nM).

b

a

0.2860.01

6.860.8 1761 0.3160.02 180620 4464 1.160.1 7665 8.160.9 2.760.3 10.1 60.8 3.960.2 2.660.3 7.360.3

2.560.1

15.060.6 6006100 3.060.2 150620 280630 1661 650640 80610 861 80 610 190630 15469 18068

0.2860.02

210630 2.660.5 3.360.1 180620 2063 2.060.3 2964 770630 3.7760.04 2.860.3 2.760.2 2.660.1 861

2.560.1 0.1760.02 0.3660.02 0.3260.02

5-HT1D. 130610 4064 1562 34006100 150620 10.260.7 31006300 560690 3.960.5 1961 2061 2.460.1 80610

5-HT1A

Muscarinic

3.160.5 961 160620 260620 .10000 18006300 12.360.9 60006300 3362 580650 .10000 22006200 0.08760.005 3665 610680 9.360.8 70006300 1562 2563 8006100 8866 1961 14006200 300620 5.260.5 3400063000 190620 3.460.4 8800 6600 480 640 320630 500061000 1050640 4.660.4 2440680 1.960.1 3.360.4 330680 280620

a1-adrenergic a2-adrenergic Dopamine D2 Histamine H1 5-HT2A

5-HT2C

2.660.1 2.960.2

2.5960.01 4.860.4 6163 47006400 0.2060.02 1461 10263 21006200 1.4860.05 4.160.2 0.7760.03 0.2760.03 14.360.1 570640 3164 35006500 0.1560.02 3264 1.21 60.06 48 65 0.1460.01 661 0.1260.01 0.960.1 2.660.1 3.260.3

Values are geometric means6SEM in nanomolar. When SEMs are presented, compounds were tested in at least three independent experiments. Major active metabolite of risperidone.

clozapine haloperidol iloperidone melperone olanzapine ORG 5222 pimozide quetiapine risperidone 9-OH-risperidoneb sertindole ziprasidone zotepine Reference Compoundsc prazosin rauwolscine spiperone pyrilamine QNB 8-OH-DPAT 5-CT ketanserin serotonin

Compound

Table 2 Equilibrium dissociations constants for antipsychotic drugs at human brain receptorsa

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Histamine H1 receptor We used [3H]pyrilamine to study the histamine H1 receptor of human brain frontal cortex. For pyrilamine (mepyramine) its Kd found in 35 independent experiments was 2.560.1 nM. By far the most potent compound was olanzapine, with Kd 5 87 pM. In fact, olanzapine was the most potent compound that we have tested at the histamine H1 of any class of compounds [11]. Muscarinic receptor For studying the muscarinic receptor, we used the human caudate nucleus and the radioligand [3H]quinuclidinyl benzilate (Kd 5 0.17 nM, n 5 18). QNB is a non-selective muscarinic antagonist, having about equal affinity for the five subtypes of muscarinic receptors [17]. As observed previously by us and others [1,18,19], clozapine was the most with Kd 5 9 nM. The next most potent was olanzapine, which is structurally very similar to clozapine. 5HT1A receptor For this receptor the radioligand chosen was [3H]8-hydroxy-DPAT. This compound was the most potent with a Kd 5 0.3660.02 nM (n524). [3H]8-hyrdoxy-DPAT was competitively antagonized by antipsychotics, with ziprasidone being the most potent, having a Kd (1.9 nM) similar to that of the anxiolytic buspirone (Kd53.8 nM) [20]. Ziprasidone may be a partial agonist at this receptor, based on studies with the recombinant human receptor expressed in Chinese hamster ovary cells [21]. 5HT1D receptor For this receptor we used as the radioligand [3H]5-carboxamidotryptamine ([3H]5-CT). This compound was the most potent at this receptor with a Kd 5 0.3260.02 nM (n58). [3H]5-CT was competitively antagonized by antipsychotics, with ziprasidone again being the most potent (Kd 52.4 nM). Risperidone was nearly as potent, with a Kd 53.9 nM. 5HT2A receptor The 5HT2A receptor antagonist [3H]ketanserin had a Kd 5 2.660.1 nM (n 5 29). [3H]ketanserin was competitively antagonized by antipsychotics, with ziprasidone again being the most potent with a Kd (0.12 nM) more than 20 fold lower than that of ketanserin. However, the affinities of sertindole and risperidone at this receptor were nearly identical to that of ziprasidone (Table 2). 5HT2C receptor For this receptor we used as the radioligand [3H]serotonin, which had a Kd 52.960.2 nM (n514). [3H]serotonin was competitively antagonized by antipsychotics, with ORG 5222 being the most potent (Kd 50.27 nM). The next most potent was ziprasidone, with 3 fold lower affinity at this receptor (Kd 50.9 nM) than that of ORG 5222.

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Discussion In this study we obtained data for a series of antipsychotic drugs and one metabolite of one of these compounds at nine different receptor types in human brain tissue. These receptors included the a1- and a2-adrenergic, dopamine D2, histamine H1, muscarinic acetylcholine, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors. We previously reported results for two of these antipsychotic drugs at some of these receptors [1,2]. Our results from the present study compare well with those from the earlier studies, except for three values. The present results showed clozapine to be more potent at the a2-adrenergic and serotonin 5-HT1A receptors, and haloperidol to be more potent at the histamine H1 receptor than we previously reported. We do not know the reason for these differences. However, our present Kd’s for clozapine are more consistent with results from other laboratories for the a2-adrenergic [22] and serotonin 5-HT1A receptors [23]. Additionally, Schotte et al. [6] obtained inhibitor constants for 10 compounds and 6 human receptors in common with our study. For the 5-HT2C receptor (formerly named the 5-HT1C receptor), we compared the data for rat receptor from Roth et al.[24], who had just 5 compounds in common with our study. However, in all this other work, molecularly cloned human (or rat) receptors expressed in cells were the source of the membranal preparations. We did a linear regression analysis of the log of our data versus the log of their data for the drugs and receptors in common. For the Schotte et al. data, all correlations were significant, with the rank order for the level of significance as follows: D2 (versus the average of their D2S and D2L data, R50.99, P,0.0001). 5-HT1D (versus their 5-HT1Da data, R50.92, P50.0001).5-HT2A (R50.90, P50.0004)55-HT1A (R50.90, P50004).a2-adrenoceptors (R50.85, P50.002 vs.a2C-adrenoceptors; R50.75, P50.012 vs.a2A-adrenoceptors; and R50.69, P50.027 vs. a2B-adrenoceptors).histamine H1 (R50.65, P50.043). For the Roth et al. data with the inclusion of pimozide, which was much weaker in their studies, R50.89, P50.044. However, without pimozide, R50.999, P50.0004. These results validate our work and theirs. Particularly important is the correlation with the 5-HT2C data. This is because we used [3H]5-HT as the radioligand in the frontal cortex, where many other types of 5-HT receptors exist. With the molecularly cloned receptors experiments are more readily performed. In addition, except for the possibility of the presence of endogenously expressed receptors in the host cells, one can be more certain of the specificity of binding of a radioligand when working with the cloned receptors, than when working with homogenates of human brain tissue. On the other hand, there exists the possibility, due to differences in post-translational processing of proteins or the coupling of receptors to components of the membrane, that binding characteristics with cloned receptors are different from those for the endogenous receptors. Our analysis seems to suggest otherwise, however. All the drugs studied, except haloperidol, are presently classified as atypical neuroleptics. On clinical criteria [5], an atypical neuroleptic has antipsychotic efficacy, with minimal extrapyramidal side effects, and does not cause tardive dyskinesia. In the absence of long term data, which would answer the question about tardive dyskinesia, another criterion to define an atypical neuroleptic would be that it causes a small elevation of serum prolactin levels (although risperidone, which is classified as atypical, produces relatively large increases in serum

Possible Adverse Effects

Possible Therapeutic Effects

Unknown

Blockade of the antihypertensive effects of clonidine hydrochloride, guanabenz acetate, and methyldopa

Potentiation of the antihypertensive effect of prazosin, terazosin, doxazosin, and labetalol Postural hypotension, dizziness Reflex tachycardia

a2-adrenoceptors

Unknown

a1-adrenoceptors Histamine H1

Extrapyramidal movement disorders: dystonia, Parkinsonism, akathisia, tardive dyskinesia, rabbit syndrome Endocrine effects: prolactin elevation (galactorrhea, gynecomastia, menstrual changes, sexual dysfunction in males)

Sedation Drowsiness Weight gain Potentiation of central depressant drugs

Amelioration of the Sedation positive signs and symptoms of psychosis

Dopamine D2

Table 3 Possible therapeutic and adverse effects of receptor blockade by antipsychotic drugs

Blurred vision Attack or exacerbation of narrow angle glaucoma Dry mouth Sinus tachycardia Constipation Urinary retention Memory dysfunction

Mitigation of extrapyramidal side effects

Muscarinic

5-HT1D

5-HT2A

Cognition Unknown Amelioration of enhancement the negative Augmentation of signs and antidepressants symptoms of psychosis Mitigation of extrapyramidal side effects Alleviation of depression Reduction of anxiety Promotion of deep sleep Unknown Unknown Unknown

5-HT1A

Weight gain

Anxiolysis Prophylaxis of migraine headaches

5-HT2C

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prolactin levels [25]). Because of the apparent efficacy of clozapine for treating the negative signs and symptoms (e.g., social withdrawal, flattened affect) of schizophrenia [26], the additional criterion of efficacy in treating the negative features of schizophrenia is sometimes added to the definition of an atypical neuroleptic. However, not all studies show a robust effect of clozapine in treating these negative features [27,28]. In the early stages of drug development, when little or no clinical data are available, preclinical studies showing no or weak potential to cause catalepsy is also a criterion to classify an antipsychotic drug as atypical [29]. In the past fifteen years, several basic research findings have provided hypotheses about what makes a neuroleptic atypical. This research includes radioligand binding studies of antipsychotic drugs at dopamine, serotonin [29–32], and muscarinic [17] receptors, second messenger synthesis at muscarinic receptors [33,34], and electrophysiological studies with rats treated chronically with drugs [35,36]. From this basic research comes the reasonable conclusion that no single hypothesis explains why a particular drug behaves as an atypical antipsychotic. That is, a drug may be an atypical antipsychotic drug by having one (or more) of several different properties. These include agonism at dopamine D1 receptors [37] and muscarinic m4 receptors [33,34]; antagonism at a2-adrenoceptors [38], as well as at dopamine D4 [30], serotonin 5-HT6 [32,39], and serotonin 5-HT7 [39] receptors. However, particular interest has been given to the relative affinity of a drug for serotonin 5-HT2A receptors compared to that for dopamine D2 receptors [29,40]. Specifically, many drugs behave as atypical neuroleptics, when the ratio of the log of their affinity (1/Kd) for the 5-HT2A receptor to that for the dopamine D2 receptor is greater than1. For the antipsychotic drugs listed in Table 2, this criterion is met for all drugs except the typical neuroleptic haloperidol. Among the atypical compounds, the highest ratio is for clozapine (1.29) and the lowest is for melperone (1.04). Additionally, receptor blockade by antipsychotic drugs may be of further clinical relevance, since some drugs that potently block these receptors may cause certain adverse effects and potential drug interactions in patients [4]. Table 3 lists the possible clinical relevance of blocking many of these receptors. For example, histamine H1 receptor blockade may cause somnolence and stimulation of appetite, which can lead to weight gain. Olanzapine, which is a drug with the highest affinity of any we have studied at this receptor, frequently causes these side effects in patients [41]. Therefore, clinicians can use these receptor binding data to minimize or avoid certain adverse effects and certain drug interactions in their patients (Table 3). Acknowledgments This work was supported by Mayo Foundation for Medical Education and Research and Pfizer Inc. We thank the University of Maryland Brain and Tissue Banks for Developmental Disorders and the Harvard Brain Tissue Resource Center, which is supported in part by PHS grant number MH/NS 31862, for providing some normal human brain tissue. References 1. Richelson E, Nelson A. Antagonism by neuroleptics of neurotransmitter receptors of normal human brain in vitro. European Journal of Pharmacology 1984;103:197–204.

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