0022.3565/95/2751.01O1$03.OO/0 THE Jouai.

OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTKS Copyright 10 1995 by The American Society for Pharmacology and .JPET 275:101-113, 1995

vol.
Experimental Therapeutics

Printed

275, No. 1 in U.S.A.

Ziprasidone Dopamine
T. F. SEEGER, Research for P. A.

(CP88,O59): and Serotonin

SEYMOUR, A. W.

A New Antipsychotic Receptor Antagonist

S. H. ZORN, D. W. SCHULZ, L

with Combined Activity

A. LEBEL, S. McLEAN,

SCHMIDT,

V.

GUANOWSKY,

H.
Division,

R.

HOWARD,
Inc., 26, 1995 Groton,

J.

A.
CT

LOWE

III and

J.

HEYM

Central Accepted

Pfizer June

publication

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

ABSTRACT
6-fold higher potency than for blockade of d-amphetamine(CP-88,059) is a combined 5-HT (serotonin) and hyperactivity, a measure of central dopamine D2 rereceptor antagonist which exhibits potent effects induced in preclinical assays predictive of antipsychotic activity. Whereas ceptor antagonism. Ziprasidone also as high affinity h for the 5-HT1A, 5-HT1 D and 5-HT2C receptor subtypes, which may the compound is a dopamine antagonist in vitro and in vivo, its further enhance its therapeutic potential. The prediction of anmost potent action is antagonism of 5-HT2A receptors, where efficacy without severe motor side effects is supits affinity is an order of magnitude greater than that observed tipsychotic ported by the relatively weak potency of ziprasidone to produce for dopamine D2 sites. Laboratory and clinical findings have led catalepsy in animals, contrasted with potent its antagonism of to a hypothesis that antagonism of 5-HT2A receptors in the conditioned avoidance responding and dopamine agonist-inbrain limits the undesirable motor side effects associated with duced locomotor activation and stereotypy. The compound is dopamine receptor blockade and improves efficacy against the in animals at doses producing effective dopamine negative symptoms of schizophrenia. Ziprasidone possesses well tolerated an in vitro 5-HT2A/dopamine D2 receptor affinity ratio higher antagonism in the brain. Ziprasidone should be a valuable addition to the treatment of psychotic disorders. than any clinically available antipsychotic agent. vivo, ziprasiIn done antagonizes 5-HT2A receptor-induced head twitch with

Ziprasidone dopamine

promise in clinical trials, interest in compounds that can mainstay of psychosis without causing significant EPS has grown. four decades. control agents have Clozapine is the prototype of such agents, and probably long been considered less than optimal in terms of incidence would be in wider clinical usage were it not for a high mciand degree of therapeutic response. Specifically, a substan- dence (1-2%) of agranulocytosis, a potentially fatal blood tial number of schizophrenics are refractory to currently disorder (Lieberman et at. , 1989). Clozapine also causes conavailable antipsychotics and, for the majority in whom siderable resedation and has a relatively high rate of seizure sponse is considered adequate, there is usually far more induction compared to other antipsychotics. Nevertheless, improvement in positive symptoms (hallucinations, deluclozapine has shown that DA receptor antagonism and reDA D2 receptor antagonists treatment for psychotic disorders However, despite their undeniable have been over the benefit, the last these
sions and disordered thinking) than for negative or deficit sultant antipsychotic efficacy can occur with a very low mci-

(blunted affect, social withdrawal and anergia) dence of movement disorders (Claghorn et at. , 1987). In ad(Ortiz and Gershon, 1986). In addition, conventional antip- dition, clinical studie8 have demonstrated the efficacy of sychotics cause a high incidence of EPS, which are often very clozapine in antipsychotic drug refractory schizophrenics and troubling and debilitating to patients and thereby limit comsuggest that it is more effective than traditional antipsychotpliance (van Putten, 1974; Tarsy, 1983). ics in treating negative symptoms (Kane at. , 1988). et In recent years, a number of drugs have been heralded asSome investigators have postulated that the drugs atypical antipsychotics, generally defined at the clinical 5-HT2A receptor antagonist efficacy plays a major role in level as being efficacious with minimal EPS liability. producing its distinct therapeutic profile (Meltzer at., et Whereas many of these drugs have failed to fulfill their 1989; Deutch et at. , 1991). For example, examination of data
Received for publication November 29, 1994.

symptoms

for a inverse

number of correlation

clinically between

tested the

antipsychotics ratio of affinity

reveals 5-HT2A for

an

ABBREVIATIONS:

DA,

dopamine;

EPS,

extrapyramidal

side

effects; -HT, 5

5-hydroxytryptamine

(serotonin);

CHO,

Chinese

hamster

ovary;

Gpp[NH]p, 5-guanylyl-imidodiphosphate; cAMP, cyclic AMP; HEPES, methylxanthine; IP, inositol phosphate; HPLC, high performance movanillic acid; ANOVA, analysis of variance; P1, phosphatidylinositol.

4-(2-hydroxyethyl)-1 liquid chromatography;

-piperazineethanesulfonic acid; IBMX, 3-isobutyl-1 DOPAC, 3,4-dihydroxyphenylaceticacid; HVA, ho-

101

102 relative Seeger,

Seeger

at al.

Vol. 275

to DA D2 receptors and EPS liability (Schmidt and Haloperidol (MW 376) was purchased from Research Biochemicals International (Natick, MA).All radioactive ligands were purchased 1986; Altar et at. , 1986). Open clinical trials with the from New England Nuclear (Boston, MA) or Amersham (Arlington 5-HT2A antagonist ritanserin have demonstrated both a reHeights, IL). Apomorphine hydrochloride and quipazine dimaleate duction in EPS and negative symptom scores after utilizing were purchased from Sigma Chemical Co. (St. Louis, MO). d-Amthis drug as an adjunct treatment inschizophrenics mainphetamine was synthesized at Pfizer Inc., Central Research Divitamed on conventional neuroleptics (Bersani et al. , 1986; sion. Reyntjens et at. , 1986). Clinical experience with risperidone, Drug administration. Except where otherwise noted, ziprasia newly marketed antipsychotic incorporating 5-HT2A and done (which is poorly soluble in water) and risperidone were susDA D2 receptor antagonist activity (Leysen et at. , 1988), has pended in a vehicle consisting of 5% Emuiphor (an emulsifying shown that, within a narrow dose range (4-6 mg), efficacy agent), is 5% ethanol and 90% saline, and were administered p.o. by achieved with lower EPS liability than conventional treatgavage. Haloperidol was dissolved in water acidified by acetic acid (pH = 2.0-3.0) for p.o. administration. Compounds were given in a ments such as haloperidol (Mesotten et at. , 1989; Bersani et volume of 2 mI/kg. d-Amphetamine sulfate was dissolved saline in at. , 1990). A recent direct double-blind comparison between and given s.c. at a dose of 1.4 mg/kg in a volume of 1 mI/kg. Apomorrisperidone and clozapine found no significant differences in phine hydrochloride was dissolved in 0.1% ascorbic acid and was efficacy between the two agents (Heinrich et at. , 1994). Although there have been numerous other theories regarding dimaleate was dissolved in distilled water and given s.c. at a dose of the unique clinical proffle of clozapine, including its interac4 mg/kg in a volume of 4 mI/kg. tions with other DA,5-HT, muscarinic and noradrenergic Receptor binding studies. Binding assays on membranes from receptor subtypes (Baldessarini and Frankenburg, 1991), rat, a guinea pig, pig, bovine, human brain or membranes derived from new class ofatypical antipsychotics, the mixed 5-HT2AIDA cell lines were performed according to standard procedures. LTKD2 receptor antagonists, has arisen largely on the strength ofcells expressing the human DA receptor were obtainedrom f Dr. Olivier Civelli (Oregon Health Sciences University, Portland, clinical results with ritanserin and risperidone. OR). CHO cells expressing the human DA D3 receptor were obtained Herein we describe the biochemical, neurochemical and from Dr. J. C. Schwartz (Institut National deSante la et de la behavioral characterization of ziprasidone (CP-88,059; 5-12Recherche M#{233}dicale, Paris, France). Tissues or cell lines of interest (4-(3-benzisothiazolyl)-piperazinyl)ethyl}-6-chloro1,3were homogenized in various buffer solutions (details are given in
dihydro-2H-indol-2-one hydrochloride hydrate) (fig. 1), which given s.c. at a dose of 0.75 mg/kg in a volume of 1 mI/kg. Quipazine

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

was discovered in thesearch for an agent which could capture the advantages ofcombined DA D2 and 5-HT2A receptor antagonism. Ziprasidone possesses a high vitro in 5-HT2A/DA D2 receptor affinity ratio, but it also is active a number of other DA,5-HT and noradrenaline receptor subtypes, which may further enhance its efficacy and safety
relative to traditional antipsychotic agents. In PhasecmII

table
membranes

1) using

a Brink.man

Polytron

at

setting rounds

for

20

see,

and

ical studies, ziprasidone has demonstrated particularly with regard to extrapyramidal doses which are associated with efficacy
schizophrenia (Gunn and Harrigan, 1995).

saturating excess of a known receptor of interest (as listed in table After 1). sufficient time to attain equilibrium at the appropriate good tolerability, allowing temperature, incubations were terminated by rapid filtration side effects, at through Whatman GFIB filters using a Brandel cell harvester. The in patients with membranes were washed 3 times with 4-nil aliquots ice-cold of buffer. Membrane-bound ligand was determined by liquid scintillation counting of the filters in Ready-Safe scintillation cocktail (for competitor for the
tritiated ligands) or by direct counting in a gamma counter (for

recovered after multiple centrifugation and resuspension in fresh ing tissue homogenates were added to at tion buffer, various concentrations of test tritiated or iodinated ligand. Nonspecific radioligand binding in the presence of a

were

ice-cold

of separation butTer. The

by result-

test tubes containing incubadrug and the appropriate binding was determined by

251-ligands). For all receptor types, the dissociation constant(Kd) for Animals. Male CD rats (200-350 g), from Charles River Laborathe radioligand was determined previously by saturation analysis in tories (Kingston, NY), served as subjects in most biochemical and all the tissue of interest, and used to calculate apparent K values by behavioral studies. Rats weregroup-housed on a 12-hr lightll2-hr means of the Cheng-Prusoff (1973) equation. dark lighting cycle under standard laboratory conditions for at least GTP effect on DA D2 receptor affinity. Thefunctional nature 1 week before experimentation. For p.o. drug administration, ani- of binding to the DA D2 receptor was evaluated in vitro after the mals were fasted for 18 hr before testing. In some experimente, male method ofCreese et al. (1979). Binding assays wererun as described Hartley guinea pigs from Charles River, pig choroid plexus obtained above using [3H]spiperone to radiolabel DA D2 receptors. The ability from Pel-Freez (Rogers, AK) or bovine brain obtained from a local of a test compound to inhibit this binding was determined in the abattoir (Copaco, Bloomfield, CT) were used for preparation of brain presence or absence of 00 M Gpp[NH]p, 1 a nonhydrolyzable analog tissue homogenates. Human cortex was obtained from the National of GTP, which was added to the incubation. The affinity was deterDisease Research Interchange (Philadelphia, PA). mined from each displacement curveand a ratio ofbinding affinities Drugs. Ziprasidone (MW 467) was synthesized at Pfizer Inc., with and without Gpp[NH]p was constructed. GTP has been shown Central Research Division (Groton, CT). Risperidone (MW 410) was to significantly decrease the affinity of agonists without altering the a gift from the Janssen Research Foundation (Beerse, Belgium). affinity of antagonists (Creese et al., 1979; Sibley et al., 1982).
Measurement ofeffects on neurotransmitter uptake. Monouptake studies were conducted with crude synaptosomal fractions ofhomogenates ofrat caudate (for DA uptake), cortex 5-HT (for uptake) or hypothalamus (for norepinephrine uptake). The concenamine trations

Materials

and

Methods

HT, 5 riM monoamines

Fig. 1. Structure

of ziprasidone.

of concentrations oftest the synaptosomal were incubated for 5mm at 37#{176}C. After incubation, mixtures were ifitered through GFIB glass-fiber filters using a Brandel cell harvester. Radioactivity was determined by liquid scmAfter

of radiolabeled monoamines were [3H]DA or 10 riM [3H]norepinephrine.

and mixtures varying

as follows:

5 aM

[3H]-5-

addition compounds,

1995 TABLE Assay 1

Zlprasldone:

A New Mtlpsychotlc

I 03

condftions
Receptor

for radloligand

binding

studies Tissue
nM Blank Buffer/pH

Ligand concentration [H]SCH23390, 0.6

DA D-1

Rat caudate

(+)-Butaclamol,

1 p.M

50 mM

Tns

HCI,

pH 7.7,

120

mM

NaCI, 5 mM KCI, 1 mM MgCI2, 2 mM CaCI2 DA D-2 H]Spiroperidol,

+

0.2 nM,

Rat

caudate

(+)-Butaclamol,

5 tM

50 mM

Tns

HCI,

100

mM

NaCl,

DA D-3

1 M prazosin + 500 nM cinanserin H]Spiropendol, 0.1 nM

mM
Human D3-transfected (+)-Butaclamol, 2 tM 50 mM

MgCI2,
Tns

pH 7.2
HCI, pH 7.4, 120 mM

CHO cells
DA D-4 H]Spiropendol, 0.1 nM

Human D4-transfected COS-7 cells

Rat cortex

(+)-Butaclamol,

2 gM

NaCI, 2 mM 50 mM NaCI, 2 mM

5 mM KCI, 2 mM MgCI2, CaCI2 Tns HCI, pH 7.4, 120 mM 5 mM KCI, 2 mM MgCI2, CaCI2

5-HT-1A

rH]-8OH-DPAT,
HJMesulergine, D [H]-5-HT, 2 nM nM mesulergine
+

1 .5 nM
1 nM 100 + 100
+

5-HT,

10 .tM

50 mM Tns HCI, 0.1 % ascorbate, 4 mM CaCl2, 10 p.M pargyline,

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

5-HT-2C 5-HT-1

Pig choroid

plexus

5-HT,

10 pM

pH 7.7 50 mM Tns HCI, 0.1 % ascorbate,

4 mM CaCI2, pH 7.7

Bovine

caudate

5-HT,

10 pM

5-HT-2A

nM 8-OH-DPAT rH]Ktansein, 0.4 nM

1 pM prazosin
+

50 mM Tris HCI, 0.1 % ascorbate, 4 mM CaCl2, 10 p.M pargyline, pH 7.7 2 p.M

50 mM Tns HCI, pH 7.7

Rat frontal

cortex

Methysergide,

500

5-HT-3 5-HT-4 5-HT-6 5-HT-7 Alpha-i Alpha-2 adrenergic

nM mepyramine rH]1CS205930 2 nM
(H]GR113808 35 pM

Mouse
Guinea

NG-108
pig

cells

MDL-72222,

10 p.M

20 mM Tns HCI, 154 mM NaCl,

pH 7.5

caudate

rHILSD, rHILSD, adrenergic

2.4 nM 3.7 nM

Rat recombinant
membranes

5-HT, 32 p.M Methiothepin, 10 p.M

50 mM HEPES,
50 mM Tns HCI,

pH 7.5
10 mM MgSO4,

Rat recombinant
membranes

Methiothepin,
Phentolamine,

10 p.M
10 p.M 100 10 p.M

rH]Prazosin, .2 nM H]para-aminoclonidine, 0.13 nM

rHIDHA, [125l]Ovine 2 nM CRH, 40 pM

Rat forebrain Rat cortex Rat whole Rat cortex brain

Noreplnephnne, p.M
Propranolol,

0.5 mM EDTA, 50 mM Tns HCI, 0.5 mM EDTA, 50 mM Tris HCI, 50 mM Tns HCI,
pH 7.7 50 mM Tns HCI,

pH 7.4 10 mM MgSO4, pH 7.4 pH 7.7 0.1% ascorbate,

pH 8.0

Beta adrenergic

CRH

nh CRH,

1 p.M

20 mM PIPES, pH 7.0, 10 mM MgCl2, 2 mM EGTA, 0.04%

BSA

CCK-A

[125I]BH-CCK-8, [125I]BH-CCK-8,

60 pM 50 pM

Guinea Guinea

pig

pancreas

L-364,718, CI-988,

1 p.M 1 p.M 10 p.M

1 mM

50 mM Tris HCI, pH 7.4, 8 mM

MgCl2, 8.3 mM DTT 10 mM HEPES, pH 6.5, MgCI2, 1 30 mM NaCI, EGTA, 0.5% BSA 5 mM 1 mM

CCK-B

pig cortex cortex

pig caudate

Histamine Histamine

H-i H-2

[H]Mepyramine, rH]Tiotidine,

2 nM 2 nM

Human
Guinea

Triprolidine,

50 mM Tns acetate,
50 mM Tns HCI,

pH 7.2
120 mM

Histamine,

pH 7.4,

NaCl, 5 mM KCI, 1 mM MgCl2, 2 mM CaCl2

Histamine Muscannic Neurokinin-i H-3 (a)-HJMethyIhistamine, 1 nM (NK1)

Guinea

pig cortex

Thioperamide,
Atropine,

I 0 p.M

50 mM

Tns

HCI,

pH

7.5

rHIQNB, 0.3 nM [125I]BH-Substance

pM

P, 80

Rat forebrain IM-9 cells (human)

CP-99,994,

10 p.M 1 p.M

50 mM Na-K

phosphate,

pH 7.4

Nicotinic

rH]Nicotine,

4 nM

Rat whole Rat forebrain

brain

Carbachol,

1 00 p.M

50 mM Tns HCI, pH 7.4, 1 mM MnCI2, 1 mM MgCl2, 0.02% BSA 50 mM Tns HCI, pH 7.4, 120 mM

NaCI,
Naltrexone, 2 p.M

5 mM KCI, 3 mM CaCI2,
50 mM NaCI,

Mu opiate Sigma Benzodiazepine

rH]Naloxone,

1 nM

1 mM MgCl2 50 mM Tns HCI,

Al

Adenosine

(+)-[H]-3-PPP, 2 nM H]Flunitrazepam, 1 nM H]Cyclohexyladenosine,

1 nM

Rat whole brain Rat cerebellum Rat cortex

Pentazocine, 5 p.M Aiprazolam, 10 p.M /-Phenylisopropyladenosine, 1 0 p.M

pH 7.4 50 mM Tns HCI, pH 7.7 50 mM Tns acetate, pH 7.1

50 mM Tns HCI, pH 7.7

with uptake 0#{176}C at taken as the blank value. uptake was calculated as picomoles per milligram of protein, with IC50 values for test drugs determined from linear regression analysis of the concentration-response curves. K values were calculated using the equation: K = IC5/(1 + [agonistl/[agonist EC50]) (Minneman and Johnson, 1984). Antagonism ofhuman DA D2 receptor modulation of cAMP tillation spectrometry
Neurotransmitter

formation.

CHO

cells

expressing

the

short

form

of the

human

were obtained from Dr. J. R. de Wet (Pfizer Inc., Groton, CT), and were grown to confluence in Dulbeccos minimal essential media (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum, 400 mg/mI ofgeneticin, 2 mM l-glutamine and 10 U/nil of penicillin-streptomycin. Monolayers were disrupted and cells cbslodged with 5 mM EDTA and were resuspended in phosphate-buffered saline buffer containing 5 mM MgC12, 30 mM HEPES and 50 p.M IBMX DA (a phosphodiesterase inhibitor). Cells (-200,000/tube)

D2 receptor

104

Seeger

et al.

Vol. 275

were exposed to 3 p.M forskolin (an adenylate cyclase activator), nM quinpirole (a DA D2 agonist) or forskolin plus quinpirole antagonist for 10 mm. In experiments with antagonists, cells were exposed to antagonists 10mm before agonist challenge. The effect of
ziprasidone in the absence of the agonist quinpirole

was

used

to

judge agonist activity. tion of 6 N perchioric

with the addiacid, and samples were neutralized with 5 N were achieved isocratically using a reverse-phase Waters Nova Pack potassium hydroxide and 2 M Tris buffer. cAMP levels were meaC18 column and a mobile phase consisting ofO. 1 M monobasic sodium sured using a commercially available competitive binding (Amkit phosphate, 1.0 mM disodium EDTA and 1.0 mM sodium octane ersham). ICse values were calculated by linear regression analysis of sulfonic acid. The mobile phase pH wasadjusted to 3.7 to 4.0 with the concentration-response curves. K, values were calculated using concentrated citric acid. Amine content was quantified by a computthe equation: K = ICS(1 -- [agonisti/Eagonist EC50]) (Minneman ing integrator which determined the ratio of each peak area to that and Johnson, 1984). of the internal standard. Ratios were multiplied by the appropriate DA Dl, 5-HT1A and 5-HT1D receptor modulation of adenyscale factor for each substance. Absolute amounts, expressed as late cyclase activity. Washed membranes were prepared from nanograms per gram of protein, were obtained from standard curves freshly dissected rat striatum (DA Dl receptors), guinea pig hipthat had been programmed into the computing integrator. The stapocampus (5-HT1A receptors) or guinea pig substantia nigra (5tistical significance of treatment effects was determined with a onefollowed by Dunnetts t test. HT1D receptors). The membranes were incubated at 30#{176}C a reac- way ANOVA in tion medium containing a-[32P}ATP, described as previously (Schulz Antagonism ofd-amphetamine-induced hyperactivity. Suband Mailman, 1984). To evaluate antagonist activity, samples also cutaneous administration of d-amphetamine produces a dramatic contained 32 p.M DA (DA Dl assay) or 3 p.M forskolin plus 32 nM increase in horizontal locomotor activity in rats, a phenomenon which has been attributed to activation of the mesolimbic DA sys5-HT (5-HT1D assays), depending on the receptor being studied, along with varying concentrations of ziprasidone. The effects of tern, and may therefore ziprovide a rodent model of excessive dopamprasidone in the absence of the endogenous agonists DA and 5-HT inergic activity implicated in schizophrenia (Roberts at., 1975; et were used to judge agonist activity. The reaction was terminatedy b Niemegeers and Janssen, 1979). Ziprasidone was tested its for abiladding 2% sodium dodecyl sulfate. [3P]cAMP was separated from ity to antagonize d-amphetamine-induced hyperactivity as a measure of in vivo dopaminergic antagonist activity. Locomotor activity [32PIATP by using a two column chromatographic procedure with added [3H]CAMP to monitor recovery of the column (Salomon et at., was measured in custom-made Plexiglas chambers (30 X cm cm) 30 1974). The amount of32P]cAMP [ formed was determined by liquid housed in sound-attenuating wooden cabinets. Horizontal locomotor scintillation counting, with results expressed as picomoles per activity was monitored and recorded by a PDP/11 computer and was as the number offloor quadrants crossed in a given time minute per milligram of protein. IC and EC50 values were calcu-measured period. After an overnight habituation period the to chambers, rats lated by linear regression analysis of the concentration-response curves. Apparent K, values were calculated using the equation: K = were administered vehicle or test compounds, followed mm later 60 ICS(1 + [agonistV[agonist EC}) (Minneman and Johnson, 1984). by d-amphetamine sulfate (1.4 mg/kg s.c.) or vehicle. Data were Antagonism of 5-HT2A, 5-HT2C and alpha-i adrenergic rerecorded for the 3-hr duration of the d-amphetamine hyperactivity ceptor-coupled IP accumulation in rat brain slices and choeffect. Treatment group means were compared with ANOVA folrold plexus. [3HIIP accumulation in rat brain cerebral cortical slices lowed by Dunnetts t test, and ID values, defined as the dose that produced a 50% inhibition of the d-amphetamine response, were and choroid plexus was examined by using a modification of a myo-inositol prelabeling technique described previously et(Zorn at., calculated by linear regression analysis of the dose-response data. of apomorphine-induced stereotypy. Adminis1987; Nomura et at., 1987). In brief, rat brain cerebral cortical slices Antagonism ofthe DA receptor agonist apomorphine to rats elicits stereo(0.35 X 0.35 mm) were prepared by using a Mdllwain tissue chopper, tration typy consisting of continuous sniffing, licking or gnawing (Ernst, or whole rat choroid plexus was isolated by dissection and tissues were then incubated for 45 mm at 37#{176}C a modified in Krebs-Ringer 1967; Costall and Naylor, 1974). Inhibition of this response is cornmonly used to evaluate the opaminergic d antagonist properties of buffer (millimolar: NaC1, 118; KC1, 5; CaCl2, 1.3; MgSO4, 1.2; experimental compounds (Niemegeers and Janssen, 1979). Rats NaHCO3, 25; and dextrose, 11.1) oxygenated with 95% 02-5% CO2. were administered test compounds or vehicle and were individually During this time the buffer was changed at 15-mmintervals, with 10 mM LiC1 added before the final preincubation period. The slices were placed into clear Plexiglas cages for observation. Three hours later, hydrochloride (0.75 mg/kg s.c.) was administered to all then preincubated for an additional 30 mm at 37#{176}C with [3H]-myo- apomorphine inositol (1 p.Ci/tube) in the presence of L1C1 and 10 p.M pargyline subjects. Beginning 10 mm later, rats were rated for stereotypy by an (only in the 5-HT2C and 5-HT2A assays). After prelabeling the observer who was blind with respect to treatment, and the rating was repeated at 10-mm intervals for a total of five ratings, by using period, slices/choroid plexus were exposed theto various test agents scale (Costall and Naylor, 1974): 0, no stereotyped for 45 mm under the sameexperimental conditions. Tissues were the following 1, discontinuous sniffing; 2, continuous sniffing; 3, continexposed to antagonists 5 rain before agonist exposure. The effect behavior; of discontinuous biting, gnawing or licking; continuous 4, ziprasidone in the absence of an agonist was used to judge agonist uous sniffing, biting, gnawing or licking with no locomotor activity. activity. The reaction was terminated by the addition chloroof a for each subject were combined to produce a total stereoform/methanol (1:2 v/v) solution, and the water soluble [3H]IPs were Ratings typy score (rating). Treatment groups were compared with Kruskalisolated by a batch technique using a Dowex AG 1 x 8 anion exANOVA followed by Kruskal-Wallis multiple comparisons change resin. EC and IC values were calculated by linear regres- Waffis program, BBN Software Products, Cambridge, MA) and IDse sion analysis of the concentration-response curves. Corrected ICse (RS-1 values (apparent K values) were calculated using the equation: K = values were calculated by linear regression analysis of the doseresponse data. IC5/(l + [agonistj/Iagonist EC]) (Minneman and Johnson, 1984). Antagonism of quipazine-induced head twitches. AthnimsKb and pA2 values were derived from Schild analysis. tration of the direct 5-HT2A and 5-HT3 agonist, quipazine, elicits a Measurement of drug effects on monoamine metabolite 1evhead shaking behavior (head twitch), the pharmacolels. Rats (n = 10 per dose group) were treated with p.o. doses of characteristic ogy of which has been attributed to activation of 5-HT2A receptors ziprasidone (0.32, 1, 3.2 or 10 mg/kg) or risperidone (0.1, 0.56, 1 or 5.6 (Lucid et at., 1984). Thus, ziprasidone was evaluated as a 5-HT2A mg/kg) at 1 hr before sacrifice by decapitation. Whole forebrain was antagonist by its ability to antagonize quipazine-induced head removed immediately, weighed and homogenized in 40 volumes (w/v) twitches. Rats were administered test compounds or vehicle and of HPLC mobile phase containing 30% methanol and 20mg/I of

The

reaction

was terminated

isoproterenol 33 as an internal standard. Homogenates were centriplus fuged at 15,000 x g for 2 mm. The supernatants were ifitered through Mifiex GS-0.22 micron filters. Filtrate was loaded into autosampler vials and levels of DOPAC, HVA, DA, 5-HT and 5-hydroxyindoleacetic acid were measured by HPLC with electrochemical detection (Saller and Salama, 1984). Chromatographic separations

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

1995
individually
minutes

Zipraskione:
placed into clear Plexiglas cages for observation.Sixty administered

A New Antipsychotic
base-line
and

conditions
motor activity

associated
suppressant

with

high
properties

activity
to evaluate

to assess locoits
the possible

later,
subjects

quipazine

dimaleate

(4.0 g/kg m twitches that was

compared

s.c.)

was

to all
this

and
the

a 30-mm

observation

period

was

initiated.
group

period,

was
Treatment

recorded

number by an
means

ofhead observer
were

exhibited blind to
with

During contribution ofgeneral by each subject in the d-amphetamine

treatment. dark
followed

cycle,

rats
and

behavioral antagonism were administered

immediately

depression test. At the vehicle

placed into

to results the

obtained

beginning
or various
activity

ofthe
doses
chambers,

12-hr
of test
which

group

ANOVA

by compounds

Dunnetts t test, and sion analysis of the

Inhibition

ID values dose-response

were determined data.

by linear

regres- were

of

conditioned

avoidance

behavior.

Inhibition

conditioned avoidance responding is another widely used model the characterization of antipsychotic potential, because most, all, clinically used drugs show activity in this assay (Cook Catania, 1964; Kuribara and Tadokoro, 1981). Rats were trained an active avoidance paradigm in Colbourn shuttleboxes housed
standard Colbourn isolation chambers. Training consisted

animals, for the measurement of locomotor activity. Horizontal locomotor activity was measured over a 4-hr ofperiod to assess drug effects during the time period which correfor sponded to measurement ofd-amphetamine antagonism. Treatment if group not means were compared with ANOVA followed by Dunnetts t test, values were calculated by linear regression analysis and and ID in the dose-response data. in
of re-

unfamiliar

to the

of

peated pairings ofa tone and light cue (20-sec intertrial interval 30 x trials) followed 5 sec later by a 1 mA shock, until shock a avoidance In Vitro Biochemical Studies criterion of 80% avoidance was achieved three on successive daily Receptor binding studies. Ziprasidone bound to a numruns. On test days, animals were first given 30 predrug avoidance ber of DA, 5-HT and noradrenergic receptor subtypes. A trials in a morning session. In the afternoon session, animals were summary of its affinity for various receptors in the central administered test compounds or vehicle and were presented with 30 system tissue is presented in table 2. Ziprasidone additional avoidance trials beginning 120 mm later. Mean pro- and nervous postdrug avoidances were compared with repeated measures had high affinity for both5-HT2A (pK = 9.38) and DA D2 ANOVA followed by Newman-Keuls test. ID50 values were calcureceptors (pK1 = 8.32). Its in vitro affinity for DA D2 receplated by linear regression analysis of the dose-response data. tors was roughly equivalent to that of risperidone = (pK Induction ofcatalepsy. Catalepsy is an akinetic state related 8.43) to and 5-fold lower than that observed for haloperidol blockade of dopaminergic neurotransmission in the extrapyramidal (pICk 9.15). Addition of100 p.M Gpp[NH]p to the [3Hlspipmotor system (Sanberg, 1980). Neuroleptics associated with severe erone binding assay did not change the affinity of ziprasiEPS in the clinic are potent cataleptogenic agents in rats (Niemedone, but decreased by 6-fold the affinity of DA, suggesting geers and Janssen, 1979). The abifity ziprasidone of to induce catathat ziprasidone is a DA D2 receptor antagonist. The lepsy was compared with that of risperidone and haloperidol. Rats 5-HT2A/ DA D2 affinity ratio for ziprasidone (11) is the were administered test compounds or vehicle and tested for catahighest that we observed compared any reference to antipsylepsy 120 and 180 mm later. The catalepsy score for each subject was taken as the average of two trials. Compounds were evaluated for chotic that was assayed including risperidone (6.9), clozapine cataleptogenic activity using a modification ofthe procedure of Cos(5.5), chlorpromazine (0.3) and haloperidol (0.016). tall et at. (1975). Testing was accomplished by placing each rat in an Ziprasidone also bound with high affinity to the human DA upright position with its forepaws resting on a horizontal bar (9-mm D2L receptor and to the newly described human A D3 and D diameter) suspended at a height of 10 cm and recording the latency D4 receptors. Thus, ziprasidone had similar affinity at the to remove the forepaws and climb down normal to a posture. The DA D3 receptor (pK = 8.14) and 8-fold lower affinity for maximum time limit was set at 90 sec. A mean immobility score of 20 the DA D4 receptor (pK = 7.50) compared with its affinity for sec was used as the criterion for the presence of catalepsy. An the human DA D2L receptor (pK = 8.41 0.25). By comparextrapolated ED20 dose was calculated by linear regression analison, risperidone and haloperidol had affinities for the DA D3 ysis of the dose-response data.
Inhibition

Results

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

of
on 2

spontaneous

ziprasidone TABLE

spontaneous

locomotor locomotor activity

activity.
was
examined

The

effect under

ofand than

D4

receptors
affinities

that

were
for the

roughly
DA

2- to 3- and
D2 receptor,

5-fold

lower

their

respectively

Receptor

bInding

and

neurotransmltter

uptake
of separate

inhibition
experiments
in

profile
parenthesis.

for ziprasidone, risperidone

pK1

and haloperldol

Values are pKi (-logiC,)

meanS S.E.s. Number

Receptor (Ligand) Ziprasidone DA Di (rH]SCH23390) DA D2 (Hspiperone) 6.28 8.32

Risperidone (3) (6) (3) (3) (5) (4) (6) (6) (3) 6.37 8.43 8.02 8.07 9.27 6.68 7.60 6.76 0.08 0.03 0.18 0.12 0.04 0.07 0.06 0.05

Haloperidol (3) (4) (3) (3) (4) (3) (7) (5) 6.67 9.1 5 8.61 8.48 7.35 5.96 0.15(3) 0.03 (3) 0.05(3) 0.07(3) 0.13 (5) 0.05(3) <5 (3) <5 (3) 8.20 0.1 1 (4)

DA 03 ([l-1]racIopnde) DA D4 (HJspiperone)
5-HT2A (rHjketanserin)

8.14 7.49
9.38

5-HT1A

(rHJ-80H-DPAT)

8.47
8.88 8.69

5-HT2C ([H]mesuIergine) 5-HT1 D (rHJ-5-HT) Alpha-i (H]prazosin) Histamine Hi (H]mepyramine)

0.17 0.04 0.03 0.1 1 0.03 0.05 0.05 0.04 0.07

7.98
7.33

0.03 (3)

9.13
7.73

0.15 (3) 0.09 (3)

pK

6.36

0.09(3)

Neurotransmitter Reuptake Blockade:

Ziprasidone Norepinephnne: 5-HI: DA: 7.30 7.29 6.58 0.01 (4) 0.06 (3)

Risperidone <5 5.88

Halopendol (3) 5.33 5.78

(3) 0.06

0.02 (3)

<5 (3)

5.52

0.1 1 (3) 0.02 (3) 0.04 (3)

106
(table 2).

Seegeretal. In contrast to its potent interaction with the dones interaction human vitro by measuring stimulation by DA. the with its Studies the DA ability measuring Dl to receptor block rat was adenylate striatal

Vol. 275 determined cyclase adenylate

DA D2, D3 and lower affinity for

by tum. displacement Its affinity

D4 the
of was

receptors, ziprasidone had DA Dl receptor (pK= 6.28) as

[3H]SCH23390 roughly equivalent binding with in

a
the that and

100-fold in
rat 6.67,

defined

cyclase striaulate
p.M.

activation
at

by
DA

DA
Dl

indicated
receptor.

that
Ziprasidone

ziprasidone
did not

is
stim-

an

antagonist of risperi-

done or haloperidol respectively).

adenylate cyclase activity at concentrations up to 10 However, it was found to block the stimulation of rat adenylate cyclase by DA (32 jiM) in a concentrationUnlike risperidone and haloperidol, ziprasidone had high striatal manner, with pK of 7.03 0.26 a (fig. 2B). Risaffinity for several other 5-HT receptor subtypes. It had a dependent pK peridone had a similar activity at the DA Dl receptor = (pK value of8.47 for the5-HT1A receptor site in the rat ortex, c at 6.85 0.13). which risperidone and haloperidol had values pK of6.68 and In the hippocampus, 5-HT1A receptors are coupled to the 5.96, respectively. At the 5-HT1D receptor in bovine the for this receptor (pK 6.37 =
striatum, ziprasidone had pK a of 8.69, whereas that of inhibition of adenylate cyclase (De Vivo and Maayani, 1986).

risperidone
trations below

was 5-HT2C

6.76,
10 p.M.

and

haloperidol
also

was
had

inactive
very

at
high

As is the concenforskolin-induced affinity guinea pig

case

with

5-HT,

ziprasidone
in adenylate

was
cyclase

found

to
activity

inhibit
in

Ziprasidone

increases

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

hippocampus, indicating that it is an agonist at the 5-HT1A receptor with a pEC50 of 7.86 0. 14 (fig. whereas risperidone had a lower affinity for this receptor 2C). The agonist activity of ziprasidone was blocked by (pK1 = 7.60) and haloperidol was inactive at concentrations 1 p.M WAY100135 [N-tert-butyl-3-(4-(2-methoxyphenyl)piperbelow 10 p.M. azin-i-yl)-2-phenylpropionamide clihydrochloridel, a 5-HT1A Ziprasidone had moderate affinity for atpha-1 the adreno(Fletcher et at., 1993). Risperidone, in contrast, was ceptor, with apK1 of 7.98 against [3Hlprazosin binding. Ris- antagonist found to be inactive at concentrations up to 10 p.M. A similar peridone bound with 14-fold higher affinity = (pK9.13) to assay, using guinea pig substantia nigra, was used to evaluate this receptor, and haloperidol bound with a pK of 8.20. of ziprasidone on the 5-HT1D receptor. Ziprasidone Ziprasidone had moderate affinity for the human histamine the action was found to have no effect on forskolin stimulation of adenyH-i receptor with pK a of 7.33. This was approximately late cyclase at concentrations up to 1 p.M. However, was it able one-half that found for risperidone (pK = 7.73) and 8-fold to potently block the inhibition of forskolin effects by 32 nM more potent than haloperidol (pK 6.36). Ziprasidone = had 5-HT, indicating that it is a 5-HT1D antagonist with a value pK very low affinity (IC50 values > 1 p.M) for alpha-2 or beta adrenoceptors, 5-HT3, 5-HT4, muscariic or nicotinic acetyl- of 9.52 0.22 (fig. 2D). of 5-HT2A, 5-HT2C and alpha! receptor choline, mu or sigma opiate, benzodiazepine, adenosine Al, Antagonism P1 turnover in rat brain slices and choroid neurokinin-i, corticotrophin releasing hormone, cholecysto- coupled plexus. In cerebral cortical slices prepared from rat brain, kinin A and Band histamine H2 and H3 receptors. In preagonist stimulation of5-HT2A receptors results in the hydroliminary studies, ziprasidone also had moderate affinity for lysis of P1 (Conn and Sanders-Bush, 1984, 1985, 1986; Kenthe recently described 5-HT6 and 5-HT7 receptors pK with dali and Nahorski, 1985; Macor et at. , 1992). This system was values of 6.89 and 7.63, respectively. utilized to determine whether ziprasidone is an antagonist of In Vftro Functional Studies 5-HT2A receptor coupled P1 turnover. In this regard, 5-HT at 100 p.M produced a 50 to 75% increase in IP accumulation Antagonism of DA D2 receptor modulation of cAMP basal levels (data not shown). The response to 100 p.M accumulation. Additional studies were carried out to iden- above 5-HT was blocked by the 5-HT2A receptor antagonists kettify agonist or antagonist effects at receptors in which zi(pK1 = 9.13 0.26 nM) and ritanserin (pK= 9.62 prasidone binds with high affinity. Agonist stimulation of anserin DA 0.31). Ziprasidone also antagonized the 5-HT (100 p.M)-stimD2 receptors results in an inhibition of adenylate cyclase II response in a concentration-dependent manner activity (Albert et at. , 1990; Stoofand Kebabian, 1981). Thus, ulated a pK1 of 8.94 0.08. By comparison, risperidone antagCHO cells expressing the short form of the human DA with D2 the 5-HT2A receptor-mediated response with a pK of receptor were utilized to determine whether ziprasidone is onized an 0.18. Ziprasidone competitively antagonized the antagonist of the DA D2 receptor by measuring cAMP accu-9.31 receptor coupled response as indicated by rightward mulation. Forskolin (3.M) produced p a 3- to 5-fold increase in 5-HT2A parallel shifts of the 5-HT dose-response curve for stimulathe level of cAMP accumulation compared to basal values tion of IP accumulation without reducing the maximal re(fig. 2A). This response was potently attenuated by the DA spouse (fig. 3A). A Schild analysis revealed pA a of 8.57 and D2 receptor agonist quinpirole (33 nM), which in was turn a Kb of 2.8 riM for ziprasidone for the 5-HT2A receptor. The blocked by the DA D2 receptor antagonists haloperidoland compound had no direct agonist effect in this system at risperidone at 10 nM. Ziprasidone produced a concentrationup to 100 p.M. dependent antagonism ofthe quinpirole-induced effect with a concentrations In rat brain choroid plexus, 5-HT produces hydrolysis of P1 pK1 = 8.89 0.20. Ziprasidone had no direct agonist effect in by stimulating 5-HT2C receptors (Connt at. , 1986). e This this system at concentrations up to 100 p.M. In comparison, system was utilized to determine whether ziprasidone is an risperidone also inhibited the DA D2 receptor-mediated reantagonist of 5-HT2C receptor coupled P1 turnover. 5-HT at sponse with a pK1 = 8.75 0.04. 100 nM produced a 2-fold increase in IP accumulation above Effects on adenylate cyclase activity mediated by DA basal levels. Consistent with literature reports, the response Dl, 5-HT1A and 5-HT1D receptors. The DA Di dopamine to was blocked completely by the 5-HT receptor antagreceptor is linked to adenylate cyclase activation and uses 5-HT onist mesulergine at 100 nM (Hoyeret al. , 1989). Ziprasidone cAMP as an intracellular second messenger (Stoof and Keantagonized the 5-HT (100 nM)-stimulated IP response in a babian, 1981). The functional characterization of ziprasifor the (formerly 5-HT1C) receptor (pK 8.88), =

1995

Ziprasidone:

A New Antipsychotic

107

A
20
C-.. 0e

B
550
0
)..

.g.

>E
=-

500

15

FORSK

Dopamin.

<a
-

00

E n
<0

g4
E

10

.!a #{176}E
>.., c.

:#{176}-

450

.:

400-

s
0

<.9: C.)
0

E
V

FOASK + QUINPIROLE
I I I I 1

0,9:
<

350

BASAL
0

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

-9

-8

-7

.6

.5

-9

-8

-7

.6

-5

-4

LOG
+

Forskolln

[Zlprasldone] M + 33 nM Quinplrolo

LOG (Zlprasldone)M + 32 iM Dopamin.

C
800
.1.

FOR
00 <.

5K 2000

FORSK

700

o.
.!a .!o
600 1800

E on .! >E
V .(

_9:

500

0 10 LMDPAT

o.
V

1600
32

nM
0
.1.

5-HT

400

-a

.8

-7

.6

-5

-4

-10

-9

-8

-7

.6

.5

LOG

[Zlprasldon.] + Forskolln

M
+

LOG [Zlprasldon#{149}J M Forskolln + 32 nM 5-HI

Fig. 2. Ziprasidone effects on adenylate cyclase measures. Each point blockade of quinpirole (33 nM) inhibition of forskolin (FORSK)-stimulated receptor (average pK = 8.89 0.20; n = 3). In this representative experiment,

represents.the ean m
cAMP

S.E.M. of three replications. A, ziprasidone accumulation in CHO cells expressing the human DA basal CAMP accumulation was 0.19 pmol of cAMP per tube. 2.27

B, ziprasidone

inhibition

of DA Di

receptor-mediated

adenylate

cyclase

activity

in the rat striatum.

This representative

experiment

shows

that

ziprasidone inhibits DA-stimulated (32 p.M) adenylate cyclase activity in a concentration-dependent manner with average pKvalue of 7.03 0.26, n = 6. C, ziprasidone inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampus, indicating that it acts as a 5-HT1A agonist (average pEC = 7.86 0.14, n = 6). In this representative experiment, basal adenylate cyclase activity was 150 3 pmol/mln/mg of protein. D, ziprasidone blockade of 5-HT(32 nM) inhibition of FORSK-stimulated adenylate cyclase activity in guinea pig substantia nigra mediated by 5-HT1 D receptors (average pK = 9.52 0.22, n = 5). The challenge concentration (32 nM) 5-HI is about 6-fold above its IC for inhibiting of FORSK-stimulated adenylate cyclase; thus, the EC for ziprasidones reversal of this effect is about 7-fold above its pK1 value. In this representative experiment, basal adenylate cyclase activity was 2114 pmoVmin/mg 0 of protein. DPAT, 2-(di-N-propylamino)tetralln.

concentration-dependent manner with apK of 8.0 0.1. tent with a previous study (Nomura et at., 1987). Ziprasidone Risperidone also inhibited the 5-HT2C-mediated response, inhibited the norepinephrine (100 p.M)-stimulated IP rebut was 8-fold less potent (pK 7.1 0. 1) than = ziprasidone sponse in a concentration-dependent manner with apK of (fig. 3B). Ziprasidone had no direct agonist effect in this 8.10 0.07. Risperidone also inhibited the alpha-i adrenosystem at concentrations up to 100 p.M. ceptor response (pK = 8.43 0.08), but was about 2-fold In cortical slices prepared from rat brain, agonist stimula- more potent than ziprasidone (fig. 3C). tion ofatpha-l adrenoceptors also results in the hydrolysis of Measurement ofneurotransmitter uptake. Reuptake P1 (Minneman and Johnson, 1984; Nomura et at., 1987). of 5-lIT, DA and norepinephrine into nerve terminals is the Norepinephrine at 100 p.M produces a 3- to 5-fold increase in principal mechanism for terminating the synaptic actions of IP accumulation above basal levels. The response to norepi- these monoamine neurotransmitters. The ability of ziprasinephrine was blocked completely by atpha-1 the adrenergic done to inhibit the uptake of these monoamines into nerve receptor antagonist prazosin with pK a value = 8.62, consisterminals was examined in synaptosomal preparations of

108

Seeger

et al.

Vol. 275

A
2100

a.
0

whole brain. Ziprasidone blocker of neurotransmitter ble 2). It was active

sites, showing with pK less values activity for the

at
at

was found reuptake norepinephrine

and the DA 7.29,

to be a moderately into synaptosomes and 5-HT

respectively, site
=

potent (tareuptake
although

g
a B a
U

of 7.30

reuptake

(pK 6.58).

These

activities and

approximate
tricyclic

the norepinephrine
and

activity and

profile
amitriptyline

that uptake,
found to

was

determined (pK = 7.62 respectively).

antidepressant, haloperidol

7.18
Risperidone

for

5-HT
were

be

a.
I

inactive at all three tions below 1 p.M.

monoamine

reuptake

sites

at

concentra-

.
A
.01
.1 1

5HT ALONE ZIPRASIDONE

ZIPRASIDONE

30

nU
100 riM

In Vivo
nists

Neurochemical

Studies

Measurement
10 100 1000 10000

of
the

monoamine rates
doses of

metabolites.
concentration of DA

DA and
in the and

antago-

[Serotonln],

jiM

increase

forebrain

metabolites

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

by

increasing
and

DA
caused HVA at

turnover
dose-dependent po.

(Anden
increases 3.2 mg/kg

Stock,
level above

1973).
of (fig.

Ziprasidone

DOPAC

I!
E8

4A). Levels at the highest dose tested (10 mg/kg p.o.) were 220% of control for DOPAC and 160% of control for HVA. No significant changes in the level of DA, 5-HT or 5-hydroxyindoleacetic acid were found at any dose up to 10 mg/kg p.o. The ziprasidone-induced increases in DA metabolite levels, although significant, were less than those seen for risperidone, which caused significant increases in DOPAC HVA and at p.o. doses of 0.56 mg/kg and above, and maximal increases of 460 and 270% of control for DOPAC and HVA, respectively, at a 5.6-mg/kg p.o. dose (fig. 4B). Haloperidol, at a p.o. dose of 1 mg/kg, induced significant increases of 295% of control for DOPAC and 385% of control levels HVAof (data not shown).
Concentration,

nil

Behavioral
Antagonism ity.

Studies activity crossings Ziprasian

haloper-

C
100

U 10

of d-amphetamine-induced hyperactivd-Amphetamine increased horizontal locomotor from less than 50 to approximately 500 quadrant when measured over a 3-hr period (data not shown). done dose-dependently antagonized this response, with

m50
00

value

of

1.5

mg/kg

p.o.

(fig.

5).

Risperidone

and

40

idol also dose-dependently antagonized motor activation, with ID50 values P.O., respectively. Antagonism of apomorphine-induced The 0.75-mg/kg dose of apomorphine
cally produced a total stereotypy score

d-amphetamine of 0.44 and

0. 14

locomg/kg

used

stereotypy. in this typitest

12 and 15 of

20

between

A ZIPRASIDONE

RISPERIDONE

a maximum dependently
apomorphine,
100 1000 10000

of 20. blocked
with an

Figure the
ID50

6 shows stereotypy
value of 2.4

that ziprasidone response produced

mg/kg p.o. (table

doseby
3).

and haloperidol also dose-dependently blocked Concentration, nU stereotypy, with ID50 values of 1.8 and 0.25 respectively. Fig. 3. Ziprasidone and nspendone effects on IP turnover mediated 5-HT2A, 5-HT2C and alpha-i receptors. A, ziprasidone inhibition of Antagonism of quipazine-induced head twitches. 5-HT2A receptor-coupled P1 turnover in rat brain cerebral corticalThe 4.0-mg/kg dose ofquipazine produced an average of 10 to slices. Concentration-response curves for 5-HT were generated in the 15 head-twitch responses in the 30-mm observation period. presence of fixed concentrations of ziprasidone. Each point represents dose-dependently inhibited quipazine-induced the mean S.E.M. of three replicate data points. Data represent Ziprasidone disintegrations per minute of accumulated [H]IP. Basal rH]lP accu- head twitches (fig. 7), with an ID50 value of 0.27 mg/kg p.o. mulation was 1010 18 dpm. B, inhibition by ziprasidone and nsperidone of 5-HT2C receptor-coupled rHIIP accumulation in rat brain

10

Risperidone apomorphine mg/kg p.o., by

choroid

receptor-coupled rHJIP accumulation in rat brain cerebral plexus. Each point represents the mean percentage of the of alpha-i response observed with 100 nM 5-HT for three to six values derived cortical slices. Each point represents the percentage of the response observed with 100 p.M norepinephrine and is the mean S.E.M. of from separate experiments, each performed in triplicate. The coefficient of variation for each point was less

concentration

was 100 nM. C, inhibition

than or equal by ziprasidone

to 20%. The 5-HT three to performed and nsperidone

four data in triplicate.

points

derived

from

separate

experiments,

each

1995

Ziprasldone:

A New

Antipsychotic

I 09

A
200 -00 >

DOPAC
HVA

C 0 .0

4 1
0

....---....-...

.t0 .0
0

150
C
**

0 100

#{149}O)
...C 0

A -#{149}0-S

ZIPRASIDONE HALOPERIDOL
RISPERIDONE

0.

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

.01 0.1 1 10

.1

10

100

Dose p.o.)
Fig.

(mg/kg,

p.o.)

Ziprasidone

Dose

(mg/kg,

5. Antagonism of d-amphetarnlne-induced hyperactivity by zinsperidone and haloperidol. Oral dose-response curves of antagonism of the increase inage-floor c crossings produced bydamphetamine sulfate administration (1 .4 mg/kg s.c.) after 1 -hr pretreatment r < .oi vs. amphetamine + vehicle control).

prasidone,

B
100
350
0

-ci---300
0)
..-.

DOPAC HVA

0) 0)

**

C 0

03

250
** **

.0 C

.0
0)

0) 0)
....C

200L.

0) 0)

150-0-100
.01 .1 10

E
0)

ZIPRASIDONE HALOPERIDOL
RISPERIDONE

0.

100

0.1

10

Dose

(mg/kg,

p.o.)

Fig. 6. Antagonism of apomorphine-induced stereotypy by ziprasidone, nspendone and haloperidol. Oral dose-response curves of antagonism of the stereotypic behavior elicited by apomorphine hydroFig. 4. Ziprasidone and risperidone effects on DA metabolite levels in administration (0.75 mg/kg s.c.) after 3-hr pretreatment (P < rat forebrain. Levels of DA metabolites in forebrain, rat sacrificed 1 hr chloride .05; P < .01 vs. apomorphine + vehicle control). after treatment with various p.o. oses of ziprasidone d or nspendone. Values in nanograms per gram of tissue are the mean S.E.M. of measurements from i 0 animals. A, dose-response data for ziprasidone. 3). This is in contrast to ziprasidone and risperidone, where Vehicle-treated animals had DOPAC levels of 71 .73.4 and HVA

Rlsperidone

Dose

(mg/kg,

p.o.)

was greater than levels of 67.3 3.0 ng/g of tissue. CP < .01 vs. vehicle control). B, there dose-response data for nsperidone. Vehicle-treated animals had values for head-twitch DOPAC levels of 64.8 2.5 and HVA levels of 66.1 4.5 ng/g of tissue spontaneous locomotion r*P < .01 vs. vehicle control).

a 30-fold separation between the ID50 antagonism and doses that decreased or produced weak catalepsy. Moreover, ziprasidone and risperidone also were more potent in the quipazine antagonism assay than in the amphetamine apomorphine antagonism assays, whereas the reverse (table 3), and a similar result was obtained with risperidone, and was true for haloperidol. but with a more potent ID50 value of 0.04 mg/kg Interp.o. estingly, haloperidol also was active in this assay, with an Inhibition ofconditioned avoidance behavior. Figure ID50 value of 0.29 mg/kg p.o., despite its relatively weak 8 shows that ziprasidone reduced the percentage of successaffinity for the 5-HT2A receptor. The activity of haloperidol flil avoidance responses, with an value of 2.6 mg/kg p.o. in this assay may be due in part general to motor suppres(table 3). Risperidone and haloperidol, in comparison, had ID50 values of 1.6 and 0.93 mg/kg p.o., respectively. sion, because there was little separation between the doses Induction of catalepsy. Our criterion defining a weak that inhibited head twitch and those that reduced spontanestate (immobility for 20 sec or ED2O) was obous locomotor activity or produced signs of catalepsy (tablecataleptic

110
TABLE 3

Seegeretal.
100

Vol. 275

Summary
Values

of the behavioral
are9 5% CL Test

effects of ziprasidone
Ziprasidone

In rats
Haloperidol
0) C 80

in parenthesis

Rispendone

Amphetamine locomotor activity ID (mg/kg) Apomorphine stereotypy

i .53 (1 .06-2.45) 2.43 (1 .46-3.95) 2.59 (0.42-4.98) 0.27 (0.12-0.42)

8.85 (5.26-19.05) 12.07 (9.69-1 5.06)

0.44 (0.23-0.72) 1 .80 (1 .18-2.85) 1 .58 (0.57-4.42) 0.04 (0.03-0.06) 1 .24

(0.85-1 .74)

0.14 (0.10-0.20) 0.25 (0.19-0.33) 0.93 (0.49-1.74) 0.29 (0.18-0.55) 0.68

(0.27-1.41)

0 C 0. 60

00 CO

2.
.0

lD (mg/kg) Conditioned ance

lD (mg/kg)

avoid-

.E
.0

40 0) 0

o5

Quipazine head twitch ID (mg/kg)

Spontaneous locomotor activity

20

a,
-#{149}0--

ZIPRASIDONE

HALOPERIDOL
RISPERIDONE

.
.1 1

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

ID

(mg/kg) #{149} 5.67

(3.67-8.76)

Catalepsy ED2 (mg/kg)

0.79
(0.43-1.46) Fig. 8.

10

00

Dose
Inhibition of conditioned

(mg/kg,
avoidance

p.o.)
behavior by ziprasidone, of antagonism pretreatment. All points

nsperidone
of conditioned

and halopendol.
avoidance

Oral dose-response curves

responding after 2-hr

except the lowest dose for each drug are significantly associated vehicle controls at P .05. <
C 0 .0 U C

different

from the

70

A
-0--

ZIPRASIDONE HALOPERIDOL RISPERIDONE

60

10 C

0 ZIPRASIDONE HALOPERIDOL
RISPERIDONE
1

50
40

.01

.1

0.
0 0 0

11)

30

Dose

(mg/kg,

p.o.)

20

Fig. 7. Antagonism of quipazine-induced head twitches by ziprasidone, risperidone and halopendol. Oral dose-response curves anof tagonism of quipazine head twitch after 180-mm pretreatment < (P .05; P < .01 vs. quipazine + vehicle control).

C.)
10 a. .1 1 10 100

served at an extrapolated haloperidol and risperidone after 0.79 and 5.7 mg/kg Inhibition of spontaneous
cle-treated rats typically

crossings
cording amine

during
chambers antagonism

the first for the

p.o. dose of 12 mg/kg. In contrast, Dose (mg/kg, p.o.) achieved this degree catalepsy of p.o., respectively (fig. 9; table 3). Fig. 9. Induction of catalepsy by ziprasidone, nsperidone and haloperfor induction of minimal catalepsy, which was locomotor activity. Vehi- idol. Oral dose-response defined as the maintenance of an abnormal upright posture for greater exhibit approximately 200 quadrant than 20 sec (ED20 4 hr after placement into the refirst time. This observation period Discussion

corresponded

data collection period in the d-amphetprocedure. Figure 10 shows the effect ziprasidone on spontaneous locomotor activity. Locomotor activity was significantly decreased by ziprasidone with ID50 value of 8.9 mg/kg p.o. By comparison, risperidone and

Antipsychotic drug research over the last decade has been of driven by the need for an agent which matches the therapeutic benefits of clozapine, but is free of serious toxicity. Clozaan pine demonstrates superior clinical efficacy with minimal EPS liability, and its use does not lead to the development of haloperidol inhibited the response with ID50 values of 1.24 tardive dyskinesia. Moreover, it is effective in many patients and 0.68 mg/kg p.o. respectively. Compared its to antiamwho are unresponsive to onventional c antipsychotics (Kane phetamine potency, ziprasidone is 6 times less potent et at , 1988). al. However, a potentially lethal blood dyscrasia inhibiting spontaneous locomotor activity. The observed dii- (agranulocytosis) limits its use to patients refractory to other ferences calculated for ri8peridone and haloperidol are 3- and medications. Numerous theories have been advanced to explain this atypical clinical profile, ranging from the musca5-fold, respectively.

to the

1995
100

Ziprasidone: havior
sponses. sponding,

A New Antipsychotic for antagonizing conditioned

dependent upon

III
DA agonist avoidance reintact central

than

are
a learned

required
behavior

re-

Ziprasidone

alsoinhibits

C 0 .0
C

80

a a

dopaminergic that ziprasidone

function (White et at., possesses potent vivo

1992).
DA D2

These
receptor

data predict positive

show
antagonist

60

properties ziprasidone
toms of
a a

in

will
schizophrenia.

after have

p.o. administration, clinical efficacy

Indeed, in a recent

and against
positron

that sympwas
with

emission

40

tomography
creased in a

study, D2 receptor

the

binding
manner

of being

[C]raclopride
by ziprasidone,

de-

dose-dependent

82%
20 -0-

DA

occupancy

achieved

after
observed

a single
with

A C
.1 1

ZIPRASIDONE HALOPERIDOL RISPERIDONE

P.O. dose of 60 The highest

mg (Bench et at. , 1993). affinity receptor interaction

10

100

ziprasidone is its antagonism of the 5-HT2A receptor. Its ratio of in vitro 5-HT2.A/ DA D2 receptor affinity (11) is greater than any clinically proven antipsychotic that we have
examined, being twice that seen with the atypical antipsy-

Downloaded from jpet.aspetjournals.org at Health Sciences & Human Services Lib on May 20, 2011

Dose

(mg/kg,

p.o.)

chotic
for the

clozapine
most

and
widely

680
utilized

times is

more
antipsychotic,

than

the

ratio

derived
The

Fig. 10. Inhibition of spontaneous locomotor nsperidone and haloperidol. Oral dose-response in spontaneous locomotor activity in a novel period (P < .01 vs. vehicle control).

activity by ziprasidone, curves for decreases magnitude environment over a 4-hr reported

haloperidol.

of that
with

this the

rinic its

anticholinergic affinity for the

potency
newly

of clozapine
described DA D4

to,

most

recently,
(van et

related 1989). receptor

Tolpetitive
cortex is

known

Functional antagonist
antagonism

because it has been is significantly coratypical antipsychotics (Meltzeret at., classification of ziprasidone as a 5-HT2A is supported by its potent vitro comin
ofthe effect of5-HT on P1 turnover in rat

ratio 5-HT2A/

important, DA D2 ratio

receptor

at., 1991). based on

and blockade of central serotonergic hyperactivity inby quipazine in vivo. Like risperidone, ziprasidone clozapines greater affinity for 5-HT2A receptors duced antagonizes quipazine-induced head twitches after p.o. adrelative to its DA D2 affinity (Meltzer et at., 1989; for review, ministration. Perhaps more important than in its vitro see Deutch et at., 1991). Although the causal mechanism is DA D2 affinity ratio in predicting atypical atunclear, concomitant antagonism of 5-HT2A receptors is 5-HT2A/ believed to reduce the motor side effects induced by antagonism tributes is its in vivo selectivity ratio. The in vivo potency of in blocking head twitch is 6-fold higher than its of DA D2 receptors in the basal ganglia.This premise has ziprasidone potency for antagonism of d-amphetamine-induced hyperacengendered an active area ofpharmaceutical research resulttivity. These data suggest that significant 5-HT2A receptor ing in the emergence of a new class of antipsychotics, the mixed 5-HT2A/ DA D2 receptor antagonists, to which we now blockade will be achieved at low doses, and that the resultant benefits will accompany whatever level of DA D2 blockade is add a new member, ziprasidone. to achieve antipsychotic efficacy. DA D2 receptor antagonism has been positively correlated required differs substantially from risperidone by its with clinical improvement in schizophrenia (Peroutka and Ziprasidone interaction with other serotonergic receptors (5Snyder, 1980), in contrast to other receptors whose role potent in HT2C, 5-HT1A and 5-HT1D). Its affinities for these sites are psychosis remains speculative. Ziprasidone is a potent D2 to or greater than its affinity for the DA D2 receptor. receptor antagonist in vitro and in vivo. Its affinity is com- equal significant occupancy ofthese serotonergic receptors parable to that of risperidone, and 6-fold lower than that Because of will occur at concentrations sufficient to control positive haloperidol. Functional DA receptor antagonism is demonstrated in a variety of assays. Ziprasidone potently reverses symptoms by DA D2 blockade, these interactions may yield clinical benefits for ziprasidone. For example, adthe action of a DA D2 agonist, quinpirole, on forskolin-in- additional of a 5-HT2C agonist, meta-chlorophenylpiperaduced stimulation of cAMP accumulation in cells expressing ministration zine been found to exacerbate positive symptoms in the human DA D2 receptor. In addition, ziprasidone in- has schizophrenics (Iqbal et at. , 1991; Krystal et al. , 1993), sugcreases DA metabolite levels afterystemic s administration, that a 5-HT2C antagonist might add to therapeutic as is characteristic of other known DA receptor antagonists, gesting One hypothesis which has received support
although to a lesser According extent to than Deutch is seen and with haloperidol (1991), efficacy. and pine a for It the has been proposed receptor that may the high affinity to its of clozaassociates 5-HT2C contribute atypical

risperidone.

clinical profile (Canton et at., 1990). The affinity of ziprasirelatively small increase in DA turnover compared to halofor the 5-HT2C receptor (pK 8.88) = is 5-fold greater peridol is characteristic of atypical antipsychotics such done as clozapine. Results from behavioral studies show that ziprasi-than that reported for clozapine, and 18-fold greater than that for risperidone. Ziprasidone potently blocks increases in done, like haloperidol and risperidone, antagonizes d-amphetamine-induced hyperactivity and apomorphine-induced P1 turnover in the rat choroid plexus, a tissue enriched in stereotypy; assays which reflect in vivo DA D2 receptor block5-HT2C receptors, confirming that the compound is a 5-HT2C receptor antagonist. Moreover, consistent with the ade and which have been commonly utilized as predictors of results, ziprasidone is a more potent functional anantipsychotic efficacy. That the effect of ziprasidone in binding the tagonist at the 5-HT2C receptor than risperidone. amphetamine assay is not due to general behavioral depression is supported by the finding that 6-fold higher doses of At the 5-HT1A receptor, ziprasidone is a potent agonist.
ziprasidone are required to suppress spontaneous motor The beimplications of this for the treatment of schizophrenia

112

Seegeretal.

Vol.

275

is a combined antagonist of 5-HT2A and DA D2 recepare uncertain, but findings in animal models suggest some done potential benefits. Agonists at the 5-HT1A receptor such as tors, a profile believed to reduce EPS liability and to improve negative symptoms in schizophrenia. It does not share strucbuspirone are known to have both anxiolytic and antidepressant activity in some patients (Rickels and Schweizer, 1993). tural similarity to clozapine and, therefore, is not expected to Moreover, Wadenberg and Ahienius (1991) have shown thatbe associated with a significant risk for agranulocytosis. Moreover, unlike clozapine, ziprasidone is devoid of antimusthe combination of raclopride, a DA D2 antagonist, with carinic activity. Compared to risperidone, ziprasidone has a 8-hydroxy-2-(di-N-propylamino)tetralin, a selective 5-HT1A wider array of serotonergic effects and alpha less adrenergic agonist, caused additive effects in rats on suppression of conditioned avoidance behavior, although completely pre- antagonist activity. Ziprasidone is well tolerated in animals venting catalepsy caused by raclopride alone. Thus, at doses the producing effective blockade of dopaminergic behav5-HT1A agonist property ofziprasidone may contribute its to iors, and has a favorable separation between activities preof efficacy and side effect liability. Based on this high threshold for inducing catalepsy and may, by analogy, dictive preclinical proffle and the results of ongoing clinical trials, it translate into lower clinical EPS liability. Further support is that ziprasidone will be an effective and wellcomes from the work of Casey (1994), who found that 8-hy- expected tolerated treatment for schizophrenia, with potential for droxy-2-(di-N-propylamino)tetralin reverses haloperidol-inmeasurable benefit on symptoms of psychotic depression, duced dystonia in monkeys, a model of neuroleptic-induced anxiety and other related disorders. EPS in humans (Rupniak et at. , 1986). In addition, among clinically available antipsychotic agents, only clozapine has appreciable affinity for the 5-HT1A receptor (Wander et at., Acknowledgments 1987; Mason and Reynolds, 1992). The agonist potency of The authors thank Frank Ewing, Carol Fox, Jerome Furman, ziprasidone at the 5-HT1A receptor clearly differentiates it Weldon Homer, Elisa Jackson, Celeste Johnson, Patrick Maloney, from risperidone, which is essentially inactive 5-HT1A as a Jean Morrone, Michael Newman and Gwen Robinson for their excelagonist. Another distinguishing feature of ziprasidone is an- technical lent assistance. The authors also appreciate helpful discussions with Dr. B. Kenneth Koe. We grateful are to Dr. Jeffrey de Wet tagonism at the 5-HT1D receptor, with an affinity several and Pratt for providing cells expressing the human DA D2 times greater than that found for the DA D2 receptor. Re- Caroline and D4 receptors. cently, Briley and Moret (1993) have proposed that 5-HT1D
antagonists might act as antidepressants, by virtue of their
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HASKINS,

J. T., Jos,

D., MANSELL,

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