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Home , Clorotepine, Fluperlapine, Iloperidone

Extended Radioligand Binding Profile of : A Broad Spectrum // Antagonist for the Management of Psychotic Disorders Hans Otto Kalkman, Ph.D., Natarajan Subramanian, Ph.D., and Daniel Hoyer, Ph.D.

Iloperidone is a novel psychotropic compound currently 5-HT1B, 5-HT2C and 5-HT6 receptors. The affinity for all other ␣ undergoing Phase III trials. Its affinity for human dopamine receptors was below 100 nM, including norepinephrine 2A, ␣ ␤ ␤ and 5-HT2A and 5-HT2C receptors has been reported 2B, 1, and 2, M1–M5, H1, dopamine previously (Kongsamut et al. 1996). This report presents D1 and D5, CCKA and CCKB, 5-HT7, dopamine and the affinity of iloperidone for a largely extended number of norepinephrine transporters. Thus, iloperidone targets a human neurotransmitter receptors. In a few instances selective set of dopamine, norepinephrine and serotonin human receptors were not available and receptor studies receptor subtypes. The affinity for this particular set of were performed on tissues from laboratory animals. The receptors indicates that iloperidone has the potential to be a present data, supplemented with those of Kongsamut et al. broad spectrum , with efficacy against Ͻ (1996), indicate that iloperidone displays high affinity (KI positive, negative, depressive and cognitive symptoms of ␣ 10 nM) for norepinephrine 1-adrenoceptors, dopamine D3 , and a low propensity to induce side effects. and serotonin 5-HT2A receptors. Intermediate affinity (10– [Neuropsychopharmacology 25:904–914, 2001] ␣ 100 nM) was found for norepinephrine -adrenoceptors, © 2001 American College of Neuropsychopharmacology. dopamine D2A and D4 receptors and serotonin 5-HT1A, Published by Elsevier Science Inc.

KEY WORDS: Receptor binding profile; Psychotic disorders; The prototypical classical neuroleptic agent Iloperidone; Adrenergic receptors; Dopamine receptors; is an effective antipsychotic compound (Leucht et al. Serotonin receptors 1999) which, unfortunately, also induces severe ex- trapyramidal side effects (EPS) and hyperprolactine- From the Pharma, Basel, Switzerland. mia. The term “” was originally Address correspondence to: Dr. Hans Kalkman, Novartis introduced to describe compounds, such as , Pharma, WSJ-360-405, CH-4002 Basel, Switzerland, Tel.: ϩ 41 61 324 which not only suppressed psychotic symptoms, but 4304, Fax: ϩ 41 61 324 5474, E-Mail: [email protected] tis.com differed from haloperidol by having a low tendency to Received October 25, 2000; revised April 9, 2001; accepted April induce EPS and increase plasma prolactin levels. It is 24, 2001. postulated that dopamine D2 receptor blockade is the Online publication: 5/8/01 at www.acnp.org/citations/Npp 050801115. mechanism by which antipsychotic activity is achieved (Creese et al. 1976; Seeman et al. 1976). However, since

NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6 © 2001 American College of Neuropsychopharmacology Published by Elsevier Science Inc. 0893-133X/01/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0893-133X(01)00285-8

NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6 Extended Binding Profile of Iloperidone 905

clozapine also interacts with D2 receptors it is likely that resemblance to clozapine, it was noted that iloperidone ␣ ␣ there are additional properties embedded in the clozap- possesses high affinity for norepinephrine 1- and 2C ine molecule that explain its “atypicality.” One influen- adrenergic receptors. tial theory was put forward by Meltzer et al. (1989), who suggested that about a 10-fold higher affinity for serotonin 5-HT2A over D2 receptors could be the key METHODS factor in achieving improved clinical response and ab- sence of EPS. This hypothesis has led to the develop- The radioligand receptor binding assays are listed in ment of new with the desired 5-HT2A/D2 Table 1. affinity ratio, although each of these compounds also binds to additional receptor sites. Several of these novel Materials antipsychotic compounds indeed display clear benefits in terms of side-effect profile. Interestingly, clozapine is Radioligands were purchased from NEN Life Science active in schizophrenic patients who are treatment- Products, USA, except for 3H-RX821002 and 3H-Me- refractory to other antipsychotics, including the more sulergine, which were obtained from Amersham Phar- 125 recent 5-HT2A/D2 receptor antagonists (Wahlbeck et al. macia Biotech Ltd, UK, and I-GTI which was obtained 1999; Conley et al. 1999; Taylor and Duncan-McConnell from ANAWA, Switzerland. Iloperidone was synthe- 2000). The elucidation of the multiple receptor interac- sized by Hoechst Marion Roussel. Unless specified oth- tions of clozapine suggested that other neurotransmit- erwise, all other chemicals were of reagent grade and ob- ter systems may also play a role in the efficacy and tol- tained through standard commercial sources. erability of that molecule. Particularly, the anti- adrenergic effects of clozapine are now receiving in- Membrane Preparation creased attention (Nutt 1994; Litman et al. 1996; Hertel et al. 1999). Iloperidone is a new psychotropic agent Total rat brain (minus cerebellum) membranes were currently undergoing Phase III trials for the treatment purchased from Analytical Biological Services (ABS) of psychotic disorders (Figure 1). Iloperidone was se- and prepared according to the following customer- lected from a large series of piperidinyl-benzisoxazoles supplied protocol. Male Wistar albino rats (250–300 g) because it showed a 300-fold greater potency in a test were decapitated, the brains removed, the cerebral cor- for limbic activity (inhibition of -induced tices dissected out and the rest of the brains homoge- climbing) than in a test for nigrostriatal activity (inhibi- nized in 10 volumes of ice-cold 50 mM Tris-HCl buffer, tion of apomorphine-induced stereotypy) (Strupcze- pH 7.7, for 30 s. The homogenate was centrifuged at wski et al. 1995). The large difference of potency of ilo- 1000 g for 10 minutes, the supernatant collected and peridone in these tests is expected to result in an centrifuged at 35,000 g for 10 minutes. The pellet was improved ratio of therapeutic effect to EPS liability resuspended in buffer and washed by four further 10- compared with standard antipsychotics. min centrifugations at 35,000 g. The final pellet was re- Previous studies have investigated the receptor suspended in buffer (2 mL/brain) and aliquots (2 mL) binding profile of iloperidone with rat receptors (Sze- were frozen and stored at -80ЊC. Prior to use, the mem- wczak et al. 1995) and a limited number of human ho- brane suspension was thawed quickly at 37ЊC, centri- mologues of dopamine and 5-HT receptor subtypes fuged at 35,000 g for 10 minutes, washed once by sus- (Kongsamut et al. 1996). These experiments demon- pension in the assay buffer and recentrifuged. The final stated that iloperidone displays the desired 5-HT2A/D2 pellet was resuspended and homogenized in the assay affinity ratio. The aim of the present study was to deter- buffer to give the desired membrane concentration. mine the receptor affinity profile of iloperidone at a Guinea pig brain (minus cerebellum) membranes wider range of human neurotransmitter receptors. In were also purchased from ABS and prepared according to the above protocol for rat brain membranes. The guinea pigs used were male Dunkin-Hartley with a 250–300 g body weight. Calf brains were obtained from the local slaughterhouse and the caudate dissected over ice. Membranes were pre- pared as described previously (Bruinvels et al. 1992). Cell membranes from Chinese hamster ovary (CHO) ␣ ␣ ␣ cell lines expressing recombinant 2A, 2B, 2C, and CRF2␣, and cell membranes from human embryonal kidney (HEK) 293 expressing recombinant D2A and 5-HT3, were prepared in the Nervous System Depart- Figure 1. Chemical structure of iloperidone. ment, Novartis Pharma, Basel, Switzerland. They were

906 H.O. Kalkman et al. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6

Table 1. Affinity Profile of Iloperidone for Human Receptors

a Receptor Radioligand Cell Internal reference, KI (nM) Iloperidone K1 (nM)

3 Ͼ Adenosine A1 H-DPCPX CHO CPA, 5.37 10,000 3 Ͼ Adenosine A2A H-NECA CHO NECA, 93.3 10,000 125 Ͼ Adenosine A3 I-AB-MECA CHO NECA, 100 10,000 ␣ 3 2A adrenoceptor H-RX821002 CHO RX821002, 0.776 162 ␣ 3 2B adrenoceptor H-RX821002 CHO RX821002, 3.47 162 ␣ 3 2C adrenoceptor H-RX821002 CHO RX821002, 3.09 16.2 ␤ 3 Ͼ 1 adrenoceptor H-CGP12177 Sf9 Propanolol, 9.77 10,000 ␤ 3 Ͼ 2 adrenoceptor H-CGP12177 Sf9 ICI118551, 3.55 10,000 3 Ͼ Cannabinoid1 H-CP55940 HEK 293 WIN55212-2, 708 10,000 3 Ͼ CholecystokininA H-L-354,718 NIH 3T3 CCK 8-sulphated, 6030 10,000 3 CholecystokininB H-L-365,260 NIH 3T3 CCK 8, 17.8 9333 125 Ͼ CRF2␣ I-Sauvagine CHO D-Phe CRF, 33.5 10,000 3 ϩ Dopamine D2A H- HEK 293 ( ), 8.13 21.4 Dopamine transporter 3H-WIN35428 CHO-K1 WIN35428, 25.7 2951 3 5-HT1A H-8-OH DPAT CHO-K1 8-OH DPAT, 0.692 93.1 3 Ͼ 5-HT3 H-GR65630 HEK293 5-HT, 871 10,000 3 5-HT6 H-LSD HeLa Methiothepin, 0.631 63.1 3 5-HT7 H- Sf9 112 Norepinephrine transporter 3H-Nisoxetine MDCK GBR 12909, 813 1479 3 9 9 Ͼ Neurokinin1 H-Sar SP CHO Sar SP, 1.00 10,000 125 ␤ Ͼ Neurokinin2 I-NKA CHO -Ala NKA, 9.77 10,000 125 7 Ͼ Neurokinin3 I-NKB CHO Me Phe NKB, 9.77 10,000 Opiate ␦ 3H-DPDPE CHO-K1 Naloxone, 79.4 Ͼ10,000 Opiate ␬ 3H-Naloxone CHO-K1 Naloxone, 0.631 Ͼ10,000 Opiate ␮ 3H-Naloxone CHO-K1 Naloxone, 0.759 Ͼ10,000 3 Muscarinic M1 H-N-methylscopolamine CHO , 4.68 4898 3 Muscarinic M2 H-N-methylscopolamine CHO Atropine, 11.0 3311 3 Ͼ Muscarinic M3 H-N-methylscopolamine CHO Atropine, 10.7 10,000 3 Muscarinic M4 H-N-methylscopolamine CHO Atropine, 10.0 8318 3 Ͼ Muscarinic M5 H-N-methylscopolamine CHO Atropine, 6.31 10,000

The displacement curves were established with eight concentrations (10-fold dilution steps) of iloperidone. Each concentration was tested in dupli- cate. The KI values are the mean value of three separate experiments. For clarity, SEM-values are omitted since the variation was within 5% of the mean. aWhere an internal reference is given, each assay was performed along with a full dose response curve of an internal reference compound to authenticate the quality.

Radioligand Binding Assays thawed and homogenized just before the assay. Mem- branes expressing recombinant 5-HT7 (Sf9 cells with 96-well Microtiter Plate Filtration Assay. In general, baculovirus expression) were prepared by the Biotech- the membrane preparations were homogenized after nology Dept, Novartis Pharma, Basel, Switzerland. thawing and pretreated as required. The following membrane preparations were pur- Individual radioligand binding assays for different chased from NEN Life Science Products, USA: NIH 3T3 receptors were performed as outlined by Herz et al. cells expressing recombinant CCKA and CCKB; Sf9 cells (1997) and references therein, with minor modifications ␤ ␤ expressing recombinant 1 or 2 receptors (all baculovi- if required. The binding studies were performed in 96- rus expression); HeLa cells expressing recombinant well plates (Falcon) in a total volume of 250 ␮L, consist-

5-HT6 receptors; and CHO cells expressing recombinant ing of the radioligand, drug (iloperidone or reference NK1, NK2, or NK3 receptors. Other membrane prepara- compound) and membrane preparation (cells, rat or tions were purchased from Receptor Biology Inc: MDCK guinea pig brain membranes) diluted in appropriate cells expressing recombinant norepinephrine transporter buffer. Non-specific binding was determined in the

(hrNET); CHO cells expressing adenosine A1, A2A or A3 presence of an appropriate drug specific for the recep- receptors or muscarinic M1, M2, M3, M4 and M5 receptors; tor under study. The plates were incubated at equilib- HEK 293 cells expressing cannabinoid1 receptors; CHO- rium for a specified time, as determined by kinetic ex- K1 cells expressing recombinant dopamine transporter periments, for each receptor assay. Reactions were (hrDAT); and CHO-K1 cells expressing recombinant opi- terminated by flash filtration and inverse transfer to 96- ate ␦, opiate ␮ and opiate ␬ receptors. Membranes from well filter plates (96-well cell harvester, filter plates

CHO-K1 cells expressing recombinant 5-HT1A receptors GFC, coated with PAI as necessary, Canberra Packard). were obtained from EuroScreen, SA. The plates were dried for 30 minutes at 56ЊC and sealed NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6 Extended Binding Profile of Iloperidone 907

Table 2. Affinity Profile of Iloperidone for Receptors for Which Human Homologue Was Unavailable

a Receptor, species Radioligand Tissue Internal reference, KI (nM) Iloperidone KI (nM) Nicotine, rat 125I-epibatidine Brain Epibatidine, 0.1 Ͼ10,000 , rat 3H- Brain Diazepam, 4.67 12,882 3 Histamine H1, guinea pig H-Pyrilamine Brain Pyrilamine, 5.01 437 125 5-HT1B/1D, bovine I-GTI Calf caudate 89.1

The displacement curves were established with eight concentrations (10-fold dilution steps) of iloperidone. Each concentration was tested in dupli-

cate. The KI values are the mean value of three separate experiments. For clarity, SEM-values are omitted since the variation was within 5% of the mean. aWhere an internal reference is given, each assay was performed along with a full dose response curve of an internal reference compound to authenticate the quality.

␣ at the bottom with an adhesive sheet (Topseal, Can- 2C adrenoceptors (16.2 nM), human D2A (21.4 nM), hu- ␮ berra Packard). Subsequently, 50 L of scintillation man 5-HT1A (93.1 nM), bovine 5-HT1B/1D (89.1 nM) and fluid (Microscint-20, Canberra Packard) was added to human 5-HT6 receptors (63.1 nM). Low affinity (KI 100– ␣ each well, the plates sealed on top and the radioactivity 1000 nM) was observed at the human 2A adrenoceptor ␣ counted in a 96-well plate counter (Topcount, Canberra (162 nM), human 2B adrenoceptor (162 nM), guinea pig Packard). The displacement curves were established histamine H1 (437 nM) and human 5-HT7 receptors (112 with eight concentrations (10-fold dilution steps) of ilo- nM). In addition, iloperidone had very low affinity (KI peridone. Each concentration was tested in duplicate. 1000–10,000 nM) at the human CCKB receptor, human The KI values are the mean value of three separate ex- dopamine and norepinephrine transporters, and hu- periments. man muscarinic M1, M2, M4 receptors. There was no sig- Ͼ ␮ nificant cross-reactivity (KI 10 M) at all other recep- tors tested. Additional Binding Studies Additional binding studies were performed in accor-

dance with previously described methods: 5-HT1B/1D DISCUSSION and 5-HT7 (Bruinvels et al. 1992; Hoyer et al. 1997). Non-specific binding was determined in the presence of The affinity of iloperidone for a number of receptor ␮ 10 M 5-HT (5-HT1 and 5-HT7 sites). sites considered important for antipsychotic activity has been reported previously (Szewczak et al. 1995; Kongsamut et al. 1996). The highest affinity was ob- Data Analysis ␣ served for the rat 1-adrenoceptor (KI 0.4 nM; Szewczak A standard data reduction algorithm was used to calcu- et al. 1995), while the compound also displayed a high

late percent specific binding in the presence of the test affinity (KI values below 10 nM) at the human recombi- compound as follows: nant 5-HT2A receptor (KI 5.6 nM), the short splice form (2B) of the human recombinant D receptor (K 6.3 nM), ()[]B– NSP ⁄ []T– NSP × 100 2 I and the D3 receptor (KI 7.1 nM; Kongsamut et al. 1996). ϭ where: B binding in the presence of test compound, These authors reported a KI value of iloperidone for the ϭ NSP non-specific binding in the presence of excess long splice form of the human D2 receptor of 13.3 nM, inhibitor, and T ϭ total binding. which is in good agreement with data obtained in the

IC50 values were derived (where feasible) from a present study (21.4 nM). In addition, Kongsamut et al. 4-parameter logistic fit and were converted to KI values (1996) reported a low affinity of iloperidone to receptors using the Cheng-Prusoff equation (Cheng and Prusoff of the D1 family: D1 (KI 216 nM) and D5 (KI 319 nM). The 1973). affinity for the 5-HT2C receptor was reported as 42.8 nM. The entire data analysis was performed by a dedi- The present study extends the radioligand binding cated program linking the raw data to a custom driven profile of iloperidone with a large series of human re-

Excel 7.0 macro and Graphpad Prism V 2.1. All affini- ceptors. A moderate affinity (KI values between 10 and ␣ ties are expressed as KI values (mol/L). 100 nM) was observed for 2C (KI 16.2 nM), 5-HT1A (93.1 nM) and 5-HT6 (63.1 nM) receptors and bovine 5-HT1B/1D receptors (89.1 nM). Since antipsychotic compounds

RESULTS are dosed until a significant occupation (50–80%) of D2 receptors is obtained (Nordström et al. 1993; Kapur et The derived in vitro receptor binding profile of iloperi- al. 2000), one can assume that therapeutic doses of ilo-

done is included in Table 1 and Table 2. Iloperidone peridone will result in relevant occupancy of D3, ␣ ␣ displayed moderate affinity (KI 10–100 nM) at human 5-HT2A, 5-HT2C, 1 and 2C receptors. Whether 5-HT1A, 908 H.O. Kalkman et al. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6

Figure 2. Bargraph of selected receptor affinities (pKI in nM) of iloperidone. These affinities were measured at recombinant ␣ human receptors, with the exception of the 1-adrenoceptor (rat), the 5-HT1B receptor (bovine) and (guinea pig). The displacement curves were established with eight concentrations of iloperidone. Each concentration was ␣ tested three times. Variation was within 5% of the mean. Data for the 1, the dopamine receptors, 5-HT2A and 5-HT2C recep- tor were reported previously (Kongsamut et al. 1996).

5-HT1B and 5-HT6 receptors will be occupied is less cer- The potential relevance of each individual receptor site tain. At therapeutic doses, in vivo binding of iloperi- is discussed, roughly in order of declining affinity. done at receptors with lesser affinity (e.g. the adenosine family of receptors) would be negligible. The binding Affinity for ␣ Receptors profile is summarized in graphical form in Figure 2. On 1 ␣ the basis of this receptor binding profile one may pre- Three different subtypes of the human 1-adrenoceptor ␣ ␣ ␣ dict some of iloperidone’s clinical properties. ( 1A, 1B, 1D) have been identified by molecular cloning ␣ (Bylund 1992). The affinity of iloperidone to those 1- adrenoceptor subtypes is not yet known. But since ilo- Overall Receptor Binding Affinity ␣ peridone displayed a very high affinity for rat brain 1 Considering the present results in conjunction with adrenoceptors, it probably will have significant affinity ␣ ␣ ␣ those reported by Kongsamut et al. (1996), it is evident for the human 1A, 1B and 1D subtypes. Animal exper- ␣ that iloperidone binds to three classes of monoamine iments provide circumstantial evidence that 1 block- receptor subtypes: dopamine, serotonin and norepi- ade might contribute to antipsychotic activity. For in- nephrine receptors, but not to any other neurotransmit- stance, administration to rats dose-dependently ter receptor class. The moderate to high affinity of ilo- decreased burst firing and regularized the firing pattern ␣ peridone for human D2, 5-HT2A and 1 receptors of ventral tegmental dopamine neurons (Grenhoff and suggest that iloperidone will have antipsychotic activ- Svensson 1993). Disruption of prepulse inhibition by ity (Baldessarini et al. 1992; Willner 1997). Iloperidone’s the psychotomimetic drug, , in rats (a pu- ␣ binding to 5-HT2C and 2C receptors will modify the tative model for the sensory motor gating deficit of ␣ therapeutic response and perhaps add additional thera- schizophrenic patients), is normalized by 1-adrenocep- peutic activity. On the other hand, iloperidone is de- tor blocking antipsychotic compounds (Bakshi and void of affinity for receptor sites which are related to Geyer 1997). These two preclinical experiments suggest ␣ side effects (e.g. muscarine and histamine H1 receptors). that inappropriate 1-adrenoceptor stimulation could NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6 Extended Binding Profile of Iloperidone 909

be involved in the pathogenesis of schizophrenia and 1995). Alternative splicing of the D2 receptor produces a ␣ 1-adrenoceptor blockade could thus be a useful phar- long (D2A) and a short (D2B) variant in humans (re- macologic attribute for an antipsychotic compound. viewed by Sokoloff and Schwartz 1995). The D2 family There are however, side effects such as postural hy- of receptors has been strongly linked to both the benefi- ␣ potension that can occur through 1-adrenergic block- cial and side effects associated with antipsychotic ade. Clinical studies have shown that clozapine fre- agents. Attempts to divorce therapeutic benefit from quently causes postural early in the course adverse effects were given great impetus by reports of treatment (Baldessarini and Frankenburg 1991). Ilo- that clozapine bound with high affinity and some selec- peridone has shown similar effects early in treatment tivity to the dopamine D4 over D2 receptor subtypes but tolerance is seen to develop as treatment continues (Van Tol et al. 1991). High affinity antagonists with (data on file). marked selectivity have now been synthesized and, in some cases, examined in schizophrenic patients. No therapeutic efficacy was apparent (Kramer et al. 1997; Affinity for 5-HT2A Receptors Truffinet et al. 1999). These results indicate that D4 re- As indicated in the introductory section, iloperidone ceptor blockade is unlikely to be a major contributor to adheres to the theory put forward by Meltzer et al. antipsychotic activity. Although iloperidone displayed

(1989), in that it has a higher affinity for the 5-HT2A re- relevant affinity for the human D4 receptor (KI 25 nM; ceptor than for the D2 receptor. The theory predicts a Kongsamut et al. 1996), this probably has no therapeu- better tolerability than classical antipsychotic com- tic consequence. pounds. Interestingly, the hallucinogen in- Iloperidone, like most commonly used antipsychot- duced schizophrenia-like in humans which ics, has significant affinity for the dopamine D3 receptor was blocked by the selective 5-HT2A receptor antago- and is likely to interact at these sites at therapeutically nist, (Vollenweider et al. 1998). Effective relevant doses. Selective D3 receptor antagonists have doses of psilocybin significantly decreased [11C]raclo- been described, but these are not yet tested in schizo- pride binding in the striatum, which is indicative for an phrenic patients. Based on the pattern of expression of increase in endogenous dopamine levels in this brain the D3 receptor, this subtype could, however, be rele- structure (Vollenweider et al. 1999). Unexpected vant for antipsychotic activity. The D3 receptor is though, selective 5-HT2A receptor antagonists were not mainly expressed in mesocorticolimbic projection areas very effective in the treatment of psychoses in schizo- such as the medial forebrain bundle, the shell of the nu- phrenia (Truffinet et al. 1999; Hoechst Marion Roussel, cleus accumbens, olfactory tubercle, amygdala and cor- Company Press Release, 1999). Nevertheless, if com- tical structures, but less in the nigrostriatal and tubero- bined with D2 receptor blockade, 5-HT2A blockade infundibular dopamine systems (Bouthenet et al. 1991). might still represent a useful pharmacologic principle. Finally, differences in affinity for the two splice

Morisset et al. (1999) have proposed that central forms of the human D2 receptor have also attracted at- 5-HT2A receptor blockade could represent a mechanism tention (Malmberg et al. 1993; Usiello et al. 2000). for improved cognition. The authors showed that antipsy- Whereas most antipsychotic compounds display equal chotics with high 5-HT2A affinity, in- affinity for both variants, clozapine and , cluding iloperidone, stimulated histamine neuron activity, two compounds with a low propensity to cause EPS, which enhances alertness via H1 receptor activation. bound with higher affinity to the D2B than to the D2A form of the receptor. Usiello et al. (2000) found that the cataleptic effect of the haloperidol Affinity for D2A, D2B, D3 and D4 Receptors was absent in D2A receptor deficient mice. These au- According to their different pharmacologic profile and thors suggested that therapeutic activity could be re- intracellular signaling pathways, dopamine receptors lated to blockade of presynaptic D2B receptors, whereas have traditionally been classified into two major popu- extrapyramidal side effects could be avoided if the lations, designated D1 and D2. Molecular cloning tech- compound would fail to bind to D2A receptors. As pub- niques have identified additional subtypes of dopam- lished by Kongsamut et al. (1996), iloperidone displays ine receptor whose profiles suggest them to be higher affinity for the D2B than for the D2A form which, members of either the D1 or D2 families (Sokoloff and according to the foregoing, would indicate a reduced Schwartz 1995). Thus, the cloned D5 receptor resembles propensity to cause EPS. the classical D1 receptor in being positively coupled to cAMP production and also in terms of sequence homol- Affinity for ␣ and ␣ Adrenoceptors ogy and the absence of introns. On the other hand, the 2A 2C

D3 and D4 subtypes most closely resemble the D2 recep- Iloperidone’s next highest affinity is to the norepineph- ␣ tor in being inhibitory on cAMP production and having rine 2C binding site. Clozapine displays nanomolar af- ␣ a similar intron distribution (Sokoloff and Schwartz finity for the 2C adrenoceptor (KI 9.1 nM) and some se- 910 H.O. Kalkman et al. NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6

␣ lectivity relative to the 2A subtype (KI 50 nM) (Schotte LC neurons, enhanced cortical DA and NA in the fron- et al. 1996). It has been speculated that clozapine’s su- tal cortex and thus an effect against negative symptoms perior therapeutic activity is at least partly explained by of schizophrenia. ␣ 2-adrenoceptor blockade (Nutt 1994; Litman et al. Activation of 5-HT2C receptors by or 1996; Hertel et al. 1999). Other therapeutic effects of mCPP suppresses food intake in laboratory animals. For ␣ clozapine may also be related to 2-adrenoceptor block- this reason the blockade of 5-HT2C receptors is suspected ade. In monkeys with MPTP-induced Parkinsonism, to contribute to the hyperphagia and weight gain ob- dyskinetic movements induced by L-dopa were dimin- served with antipsychotic treatment. Chronic treatment ␣ ished by , an 2 antagonist (Henry et al. 1999; of rats with selective 5-HT2C receptor antagonists did not ␣ Grondin et al. 2000). Thus, blockade of 2-adrenergic re- lead to weight gain (Kennett et al. 1997). Also the anti- ceptors could explain the potent antidyskinetic effect of psychotic drug displays relevant 5-HT2C clozapine (Bennett et al. 1994; Pierelli et al. 1998). Exper- blockade (Schotte et al. 1996), but did not induce pro- iments in genetically altered mice show that overex- found weight gain in humans (Allison et al. 1999). Thus, ␣ pression of 2C receptors contributes to behavioral de- although 5-HT2C receptor agonists induce hypophagia, ␣ spair and accordingly, blockade of 2C receptors could the opposite is not necessarily observed with antago- be antidepressive (Sallinen et al. 1999). Other preclinical nists. Also iloperidone, despite its affinity for 5-HT2C re- ␣ studies indicate that overexpression of 2C receptors ceptors, produced, if compared to placebo, minimal worsens spatial recognition and induces anxiety-like weight gain in schizophrenic patients (data on file). behavior (Björklund et al. 1998, 1999). These effect were Selective 5-HT2C receptor antagonists displayed anxi- ␣ reverted by an 2 antagonist, suggesting that blockade olytic activity (Kennett et al. 1996, 1997) and suppressed ␣ of 2C receptors might result in improved cognition and haloperidol-induced catalepsy (Reavill et al. 1999). On anxiolytic activity. On the other hand, also undesired theoretical grounds, 5-HT2C receptor antagonists might ␣ properties may be related to blockade of 2-adrenocep- also be useful for the treatment of Parkinson’s disease ␣ tors. Idazoxan and other 2 antagonists are proconvul- (Fox and Brotchie 1999). Iloperidone’s affinity for 5-HT2C sant in mice (Jackson et al. 1991). Such an effect was not receptors could, therefore help to explain the low pro- ␣ seen in a mutant mice strain that lacked functional 2A pensity to induce catalepsy and the anxiolytic effects receptors (Janumpalli et al. 1998), suggesting that pro- seen in animal studies (Corbett et al. 1993). ␣ convulsant activity is related to blockade of 2A adren- ergic receptors. ␣ Affinity for 5-HT6 Receptors Iloperidone has a low affinity for 2A receptors indi- cating a low propensity to induce convulsions. In con- The relevance of 5-HT6 receptor blockade in the phar- ␣ trast, the affinity of iloperidone for 2C-adrenoceptors macologic profile of antipsychotic compounds remains could be of clinical relevance as it might result in anti- speculative. Roth et al. (1994) reported that clozapine depressant and anxiolytic activity and in improved cog- and several atypical antipsychotic agents (rilapine, nition. , , , and ) had high affinities for the 5-HT6 receptor. It is interesting that selective 5-HT receptor antagonist, Ro Affinity for 5-HT Receptors 6 2C 04-6790 in rats increased neurotransmission

Recent research has shown that the 5-HT2C antagonist, (Bentley et al. 1999). Similarly, clozapine, which has rel- SB206,553 dose-dependently increased the firing rate of evant 5-HT6 receptor affinity (Glatt et al. 1995; Schotte VTA and locus coeruleus (LC) adrenergic neurons in et al. 1996), increased extracellular levels of acetylcho- rats (Gobert et al. 2000). These authors also reported line in rat prefrontal cortex (Parada et al. 1997). It is that the 5-HT2C antagonist dose-dependently increase therefore conceivable that clozapine increases extracel- levels of dopamine (DA) and noradrenaline (NA) but lular levels of via blockade of 5-HT6 re- not serotonin in the frontal cortex. Clozapine has been ceptors. Blockade of 5-HT6 receptors could thus, via in- shown to preferentially increase dopamine release in creased cholinergic neurotransmission, contribute to the medial prefrontal cortex (Moghaddam and Bunney improved cognitive function. The present results show

1990). Since clozapine is a potent 5-HT2C antagonist that iloperidone has moderate affinity for 5-HT6 recep- (Schotte et al. 1996), the effects of clozapine on dopa- tors, while the affinity for muscarine receptors is low. mine release in the prefrontal cortex might be due, at These two features in combination suggest that iloperi- least partly, to blockade of 5-HT2C. Dopamine hypo- done could display efficacy against neurocognitive def- function in cortical dopamine projection has been sug- icits in patients with schizophrenia. However, it is not gested to be responsible for negative symptomatology certain whether therapeutic doses of iloperidone will be (Davis et al. 1991). Iloperidone shows moderate affinity high enough to obtain significant occupancy of the to the 5-HT2C receptors (KI 42.8 nM; Kongsamut et al. 5-HT6 receptor, as its affinity measured in the present 1996) which might lead to disinhibition of the VTA and experiments is rather moderate (KI 63.1 nM). NEUROPSYCHOPHARMACOLOGY 2001–VOL. 25, NO. 6 Extended Binding Profile of Iloperidone 911

Affinity for 5-HT1A Receptors CONCLUSION The same argument holds for the 5-HT receptor since 1A Iloperidone is characterized by a broad spectrum of do- the affinity of iloperidone for human 5-HT receptors 1A pamine, norepinephrine and serotonin antagonism. amounted to 93.1 nM, only. In a cellular assay, iloperi- Thus, as with other new antipsychotics, iloperidone has done produced a concentration-dependent surmount- a high affinity for 5-HT receptors and ␣ adrenergic able antagonism against the 5-HT receptor agonist, 2A 1 1A receptors and moderate affinity for D receptors, indi- 8-OH-DPAT (mean [S.E.M.] pK 7.69 [0.18]; data on 2 B cating antipsychotic efficacy, with a reduced propensity file). to induce EPS. Favourable properties are also suggested Post-mortem studies of patients with schizophrenia by its additional receptor profile. The moderate D re- have revealed increased numbers of 5-HT receptors in 2 1A ceptor affinity is balanced by comparable affinity for the prefrontal cortex (Hashimoto et al. 1993; Burnet et ␣ adrenoceptors, and 5-HT , suggesting potential im- al. 1997). Intrinsic activation of these receptors hyper- 2C 2C provements in cognition and negative symptoms. polarizes the neurons and reduces the output of their Moreover, blockade of ␣ adrenoceptors might trans- neurotransmitter glutamate. Loss and/or hypoactivity 2C late into and anxiolytic activity. Low af- of cortical glutamatergic neurons has been postulated finity for histamine H receptors suggests that iloperi- to underlie the cognitive impairment in Alzheimer’s 1 done has limited propensity to induce weight gain. disease (Francis et al. 1993). Therefore, the normaliza- Extremely low activity at cholinergic receptors suggests tion of glutamate output by a 5-HT antagonist such as 1A that side effects associated with agents iloperidone might help to ameliorate cognitive impair- such as dry mouth, blurred vision and increased fre- ment in patients with schizophrenia. quency of micturition will be avoided. Due to the low ␣ affinity of iloperidone for 2A receptors proconvulsive activity is not expected. Affinity for Muscarinic Receptors This broad receptor binding profile indicates that ilo- The low or negligible affinity of iloperidone for muscar- peridone has the potential to be an effective and well- inic receptors indicates that it may have a low propen- tolerated agent in the treatment of psychotic disorders. sity to cause side effects such as dry mouth, blurred vi- Indeed, preliminary studies have confirmed the favour- sion, increased frequency of micturition or other able efficacy and tolerability of iloperidone in patients anticholinergic effects. with schizophrenia (Davidson et al. 1994; Borison et al. 1996; Cutler et al. 1996).

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