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`0022-3565/02/3021-381–389$7.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 302:381–389, 2002
`
`Vol. 302, No. 1
`33175/990838
`Printed in U.S.A.
`
`Aripiprazole, a Novel Antipsychotic, Is a High-Affinity Partial
`Agonist at Human Dopamine D2 Receptors
`
`KEVIN D. BURRIS,1 THADDEUS F. MOLSKI, CEN XU, ELAINE RYAN, KATSURA TOTTORI, TETSURO KIKUCHI,
`FRANK D. YOCCA, AND PERRY B. MOLINOFF1
`Neuroscience Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut (K.D.B., T.F.M., C.X., E.R.,
`F.D.Y., P.B.M.); and CNS Research Group, Research Institute of Pharmacological and Therapeutic Development, Otsuka Pharmaceutical Co.
`Ltd., Tokushima, Japan (K.T., T.K.)
`Received January 15, 2002; accepted March 27, 2002
`
`ABSTRACT
`Aripiprazole is the first next-generation atypical antipsychotic
`with a mechanism of action that differs from currently marketed
`typical and atypical antipsychotics. Aripiprazole displays prop-
`erties of an agonist and antagonist in animal models of dopa-
`minergic hypoactivity and hyperactivity,
`respectively. This
`study examined the interactions of aripiprazole with a single
`population of human D2 receptors to clarify further its pharma-
`cologic properties. In membranes prepared from Chinese ham-
`ster ovary cells that express recombinant D2L receptors, aripi-
`prazole bound with high affinity to both the G protein-coupled
`and uncoupled states of receptors. Aripiprazole potently acti-
`vated D2 receptor-mediated inhibition of cAMP accumulation.
`Partial
`receptor
`inactivation using the alkylating agent
`N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) sig-
`nificantly reduced the maximum effect of aripiprazole on inhi-
`
`bition of cAMP accumulation. This effect was seen with con-
`centrations of EEDQ that did not alter the maximal inhibitory
`effect of dopamine. Consistent with the expected effects of a
`partial agonist,
`increasing concentrations of aripiprazole
`blocked the action of dopamine with maximal blockade equal
`to the agonist effect of aripiprazole alone. The efficacy of aripi-
`prazole relative to that of dopamine varied from 25% in cells
`that lacked spare receptors for dopamine to 90% in cells with
`receptor reserve. These results, together with previous studies
`demonstrating partial agonist activity at serotonin 5-hydroxy-
`tryptamine (5-HT)1A receptors and antagonist activity at
`5-HT2A receptors, support the identification of aripiprazole as a
`dopamine-serotonin system stabilizer. The receptor activity
`profile may underlie the unique activity of aripiprazole in ani-
`mals and its antipsychotic activity in humans.
`
`7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]-
`Aripiprazole,
`butyloxy}-3,4-dihydro-2(1H)-quinolinone, is the first next-
`generation atypical antipsychotic that is active against pos-
`itive and negative symptoms of schizophrenia (Petrie et al.,
`1997; Saha et al., 2001), has a low propensity for extrapyra-
`midal side effects (Petrie et al., 1997; Saha et al., 2001),
`causes minimal weight gain or sedation (Petrie at al., 1997;
`Carson et al., 2002), and produces no elevation in serum
`prolactin levels (Petrie et al., 1997; Saha et al., 2001) or
`prolongation of QTc interval on ECG (Kane et al., 2000;
`
`This work was funded by Bristol-Myers Squibb Company and Otsuka Phar-
`maceutical Co. Ltd. Results of this study were presented at the XXIInd Colle-
`gium Internationale Neuropsychopharmacologicum Congress, July 9 –11,
`2000, Brussels, Belgium and the VIIIth International Congress on Schizophre-
`nia Research, April 28 –May 2, 2001, Whistler, BC, Canada.
`1Present address: Palatin Technologies, 175 May Street, Suite 500, Edison,
`NJ 08837.
`Article, publication date, and citation information can be found at
`http://jpet.aspetjournals.org.
`DOI: 10.1124/jpet.102.033175.
`
`Carson et al., 2002). The mechanism of action of aripiprazole
`differs from that of currently marketed typical and atypical
`antipsychotics. Previous preclinical studies have provided
`evidence that aripiprazole is a dopamine-serotonin system
`stabilizer with potent partial agonist activity at dopamine D2
`and 5-HT1A receptors and antagonist activity at 5-HT2A
`receptors (Inoue et al., 1996; Jordan et al., 2001; T. Kikuchi,
`unpublished observations).
`Like many antipsychotics, aripiprazole binds with high
`affinity to members of the D2 family of dopamine receptors
`(Kikuchi et al., 1995; Lawler et al., 1999). Whereas currently
`marketed antipsychotics are believed to exert their effects
`through antagonism of D2 (and possibly 5-HT2) receptors
`(see Miyamoto et al., 2000 for a recent review), aripiprazole
`may exert its effects through partial agonism at D2 receptors.
`In multiple studies in vivo, aripiprazole has been shown to
`have potent agonist activity at dopamine autoreceptors. For
`example, aripiprazole decreases ␥-butyrolactone- and reser-
`
`ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); BSA, bovine serum albumin; CHO, Chinese hamster ovary; DMSO, dimethyl sulfoxide;
`EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; HEK, human embryonic kidney; [125I]7-OH-PIPAT, R-(⫹)-trans-7-hydroxy-2-(N-n-pro-
`pyl-N-3⬘-iodo-2⬘-propenyl)aminotetralin; S(⫺)-3-PPP, S(⫺)-3-(3-hydroxyphenyl)-N-n-propylpiperidine, preclamol; GMP-PNP, 5⬘-guanylylimido-
`diphosphate; OPC-4392, 7-(3-[4-(2,3-dimethylphenyl)piperazinyl]propoxy)-2-(1H)-quinolinone.
`
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`382
`
`Burris et al.
`
`pine-induced DOPA accumulation (Kikuchi et al., 1995), con-
`sistent with a decrease in presynaptic tyrosine hydroxylase
`activity. The inhibitory effect of aripiprazole on ␥-butyrolac-
`tone-induced DOPA accumulation is blocked by the D2 re-
`ceptor antagonist haloperidol. Administration of aripiprazole
`to laboratory rats results in decreased extracellular levels of
`dopamine in the striatum and frontal cortex suggestive of
`decreased release of dopamine (Semba et al., 1995). Finally,
`the ability of aripiprazole to decrease spontaneous firing of
`dopaminergic neurons in the ventral tegmentum by activa-
`tion of D2 autoreceptors was shown by extracellular record-
`ing in vivo (Momiyama et al., 1996).
`Whereas results of the above studies are consistent with
`agonist activity of aripiprazole at D2 receptors, in other in vivo
`studies, aripiprazole displays properties of a D2 receptor antag-
`onist. For example, aripiprazole blocks apomorphine-induced
`stereotypy and locomotor activity and does not produce stereo-
`typy or increased locomotion when administered alone (Kikuchi
`et al., 1995). Consistent with blockade of dopamine receptors
`coupled to the inhibition of prolactin release, administration of
`aripiprazole to male rats results in a 2-fold increase in levels of
`serum prolactin (Inoue et al., 1996).
`In vivo, partial agonists may act predominantly as agonists
`or antagonists depending upon the level of endogenous re-
`ceptor activation. Partial agonist activity of aripiprazole at
`D2 receptors may explain its antagonist properties in animal
`models of dopaminergic hyperactivity (e.g., blockade of apo-
`morphine-induced stereotypy) and agonist activity in an an-
`imal model of dopaminergic hypoactivity (blockade of in-
`creased dopamine synthesis in reserpine-treated rats)
`(Kikuchi et al., 1995). A variety of efficacy values for aripi-
`prazole at D2-like receptors have been reported using differ-
`ent in vitro preparations where endogenous dopaminergic
`tone is eliminated. In slices of rat pituitary, aripiprazole
`decreased spontaneous release of prolactin with an effect
`approximately 50% that of talipexole, a D2 receptor agonist.
`Consistent with a partial agonist effect, aripiprazole moder-
`ately blocked the effect of talipexole (Inoue et al., 1996).
`Likewise, in C6 cells that express recombinant rat D2L re-
`ceptors linked to the inhibition of cAMP accumulation, aripi-
`
`prazole displayed modest agonist activity with a maximum
`effect 30% that of dopamine (Lawler et al., 1999). In contrast,
`in CHO cells that express transfected rat D2L receptors,
`aripiprazole completely blocked the ability of dopamine to
`inhibit forskolin-stimulated accumulation of cAMP while
`having no efficacy alone, consistent with antagonist activity
`at D2 receptors (Lawler et al., 1999). Similarly, in rat striatal
`membranes, increased GTPase activity stimulated by the D2
`receptor agonist quinpirole is completely blocked by aripipra-
`zole. However, aripiprazole alone does not stimulate GTPase
`activity (Inoue et al., 1997).
`The purpose of this study was to clarify the functional
`activity of aripiprazole at D2 receptors and to demonstrate
`how partial agonism in conjunction with modulation of com-
`ponents of the signal transduction pathway may explain the
`range of activities of aripiprazole at D2 receptors.
`
`Experimental Procedures
`Materials. [125I]7-OH-PIPAT (2200 Ci/mmol) was purchased from
`PerkinElmer Life Sciences (Boston, MA). [3H]Spiperone was pur-
`chased from Amersham Biosciences (Piscataway, NJ). Haloperidol,
`(⫹)-butaclamol hydrochloride, S(⫺)-PPP, terguride, quinpirole hy-
`drochloride, Tris, EDTA, BSA, and polyethylenimine were purchased
`from Sigma-Aldrich (St. Louis, MO). 5⬘-guanylylimidodiphosphate
`was purchased from Calbiochem (San Diego, CA). Tissue culture
`plates (100 ⫻ 20-mm) were purchased from Corning Glassworks
`(Corning, NY). F-12 Nutrient Mixture (Ham), fetal bovine serum,
`and G418 sulfate were purchased from Invitrogen (Carlsbad, CA).
`Tissue Culture. CHO cells that express human recombinant D2L
`receptors (CHO-D2L) have been previously described (Filtz et al.,
`1993). Cells were grown at 37°C in 5% CO2 as a monolayer in
`medium consisting of F-12 supplemented with 10% fetal bovine
`serum and G418 sulfate (500 ␮g/ml).
`Radioligand Binding Assays. Cells were rinsed twice with
`phosphate-buffered saline (155 mM NaCl, 3.3 mM Na2HPO4, and 1.1
`mM KH2PO4, pH 7.4), and incubated for 5 to 10 min at 4°C in
`hypotonic lysis buffer consisting of 10 mM Tris (pH 7.4) and 5 mM
`EDTA. Cells were transferred from plates to polypropylene tubes
`(16 ⫻ 100 mm), homogenized, and centrifuged at 32,000g for 20 min.
`Pellets were resuspended in buffer consisting of 50 mM Tris (pH 7.7
`at 26°C) and 1 mM EDTA, then stored at ⫺80°C until needed. On the
`
`Fig. 1. Cells were incubated for 10 min with 10
`␮M forskolin and 100 ␮M 3-isobutyl-1-methylxan-
`thine in the absence or presence of increasing
`concentrations of dopamine or aripiprazole. cAMP
`was measured using the Biotrak cAMP Direct
`Screening Assay System (Amersham Biosciences).
`Accumulation of cAMP stimulated by forskolin
`ranged from 3 to 11 pmol/well. Data were fit to a
`four-parameter logistic equation using GraphPad
`Prism version 3.0 (GraphPad Software, San Di-
`ego, CA). The data shown are the mean ⫾ S.E.M.,
`n ⫽ 8 (CHO-D2L) and 5 (HEK-293-D2L; inset)
`experiments.
`
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`day of an experiment, homogenates were thawed, resuspended by
`homogenization, and centrifuged at 32,000g for 20 min. Following
`centrifugation, supernatants were discarded, and remaining pellets
`were resuspended in buffers as detailed below. Binding of [3H]spip-
`erone was carried out in buffer containing 50 mM Tris (pH 7.4 at
`25°C), 100 ␮M GMP-PNP, and 1% DMSO. Homogenates (2–3 ␮g of
`protein/tube) were incubated with [3H]spiperone (10 –1000 pM) for
`90 min at 25°C. Binding of [125I]7-OH-PIPAT was carried out in
`buffer consisting of 50 mM Tris (pH 7.7 at 25°C), 2 mM MgCl2, 0.1%
`BSA, 0.025 mN HCl, and 1% DMSO. Homogenates (10 ␮g of protein/
`tube) were incubated with [125I]7-OH-PIPAT (200 pM) for 60 min at
`37°C. Assays were stopped by addition of cold wash buffer (50 mM
`Tris, [3H]spiperone or 20 mM Tris, [125I]7-OH-PIPAT). Filtration
`over glass fiber filters (Whatman GF/B; Whatman, Clifton, NJ) pre-
`viously soaked in 0.05% polyethylenimine (for [3H]spiperone bind-
`ing) or 20 mM Tris (for [125I]7-OH-PIPAT binding) was carried out
`using a Brandel cell harvester (Brandel Inc., Gaithersburg, MD).
`Nonspecific binding was defined with 2 ␮M (⫹)-butaclamol.
`Maximum binding (Bmax) and KD values were determined by un-
`weighted linear regression analysis of transformed saturation bind-
`ing data (Scatchard, 1949). Protein concentrations were determined
`by the method of Bradford (1976) with BSA as a standard.
`Accumulation of cAMP. Cells were harvested by successive
`washing and centrifugation in Cell Dissociation Buffer (Invitrogen).
`The final pellet was resuspended in phosphate-buffered saline con-
`taining 0.9 mM CaCl2, 0.5 mM MgCl2, and 0.5% BSA. Approximately
`6 ⫻ 104 cells were added to each well of a 96-well plate. Cells were
`exposed to test compounds for 10 min at 37°C in the presence of 10
`␮M forskolin and 100 ␮M 3-isobutyl-1-methylxanthine. The reaction
`was terminated by the addition of 0.15 N HCl. Accumulation of
`cAMP was measured using the cAMP SPA Direct Screening Assay
`kit (Amersham Biosciences).
`Receptor Inactivation Studies. Cells were collected in Cell
`Dissociation Buffer and centrifuged at 100g for 5 min. Cells were
`resuspended in F-12 media and divided equally into three separate
`tubes containing either vehicle (0.1% DMSO) or 1 ␮M or 10 ␮M
`EEDQ and incubated for 60 min at 37°C in 5% CO2. Following
`treatment with EEDQ, cells were washed once by centrifugation and
`resuspension in F-12 media. Cells were centrifuged, and the pellet
`was resuspended in phosphate-buffered saline containing 0.9 mM
`CaCl2, 0.5 mM MgCl2, and 0.5% BSA. Approximately 6 ⫻ 104 cells
`were added to each well of a 96-well plate, and the effects of agonists
`on forskolin-stimulated accumulation of cAMP were determined as
`above.
`
`Results
`Binding of Agonists and Antagonists to D2 Recep-
`tors. The affinity values of human D2L receptors for agonists
`
`Aripiprazole Is a D2 Receptor Partial Agonist
`
`383
`
`and antagonists were determined for receptors labeled with
`the agonist [125I]7-OH-PIPAT and with the antagonist
`[3H]spiperone under conditions that promote, respectively,
`coupling or uncoupling of receptors to G proteins (Burris et
`al., 1995). Butaclamol and haloperidol, D2 receptor antago-
`nists, bound with slightly higher affinity to the antagonist-
`labeled noncoupled state of D2L receptors than to the G
`protein-coupled state labeled with [125I]7-OH-PIPAT (Table
`1). In contrast, the agonists dopamine and quinpirole bound
`with 34- to 67-fold higher affinity to the G protein-coupled
`state of D2 receptors. The partial agonists S(⫺)-PPP, ter-
`guride, and aripiprazole displayed higher affinity for the G
`protein-coupled state of D2 receptors than for the noncoupled
`state (Table 1). The ratio of affinities for the partial agonists
`was intermediate between those for full agonists and antag-
`onists.
`Stimulation of D2 Receptors by Aripiprazole and
`Other Agonists: Effects of Modulation of Receptor
`Density. Slightly higher affinity for the G protein-coupled
`state compared with the noncoupled state of D2 receptors
`suggest that aripiprazole is a partial agonist at D2 receptors.
`The ability of aripiprazole to stimulate D2 receptors was
`examined directly in CHO cells that express human recom-
`binant D2L receptors coupled to the inhibition of cAMP ac-
`cumulation. The increase in cAMP accumulation induced by
`exposure to 10 ␮M forskolin was potently inhibited by dopa-
`mine (Fig. 1). Haloperidol (1 ␮M), a D2 receptor antagonist,
`completely blocked the inhibition of cAMP accumulation by
`dopamine (data not shown). Similar to the effects of dopa-
`mine, increasing concentrations of aripiprazole potently in-
`hibited the increase in cAMP accumulation stimulated by
`forskolin (Fig. 1). Consistent with the activity of a partial
`agonist, the maximum effect of aripiprazole was approxi-
`mately 85% that of dopamine. The effect was not unique to
`CHO cells, as similar results were obtained in HEK-293 cells
`that express human recombinant D2L cells (Fig. 1, inset).
`The relationship of the density of D2 receptors to the effi-
`cacy of aripiprazole and other partial agonists was examined.
`Exposure of CHO-D2L cells to increasing concentrations of
`EEDQ resulted in a progressive decrease in the density of
`receptors as determined by binding of [3H]spiperone (Fig. 2).
`Exposure of cells to 1 ␮M EEDQ for 1 h resulted in a greater
`than 50% decrease in the density of D2L receptors, whereas
`exposure to 10 ␮M EEDQ resulted in a nearly 80% decrease
`
`TABLE 1
`Affinity values of the G protein-coupled and noncoupled states of D2L receptors for agonists, partial agonists, and antagonists
`Ki values were determined by the method of Cheng and Prusoff (1973) using IC50 values determined by competition for the binding of the agonist [125I]-7-OH-PIPAT or the
`antagonist [3H]spiperone to membranes prepared from CHO cells expressing human recombinant D2L receptors. Binding of [125I]-7-OH-PIPAT was performed under
`conditions favoring formation of the agonist-preferring G protein-coupled state of D2 receptors (Ki high), whereas binding of [3H]spiperone was performed under conditions
`promoting the noncoupled state of D2 receptors (Ki low). IC50 values were obtained by fitting data to a one-site competitive binding curve using GraphPad Prism version 3.0.
`Data are the mean ⫾ S.E.M., n ⫽ 3 to 4 or the mean ⫾ range, n ⫽ 2 experiments.
`
`Agonists
`Quinpirole
`Dopamine
`Partial agonists
`S(⫺)-PPP
`Terguride
`Aripiprizole
`Antagonists
`Butaclamol
`Haloperidol
`
`[125I]-7-OH-PIPAT
`
`9.5 ⫾ 1.5
`17 ⫾ 1.0
`
`56 ⫾ 4.5
`0.16 ⫾ 0.01
`0.34 ⫾ 0.02
`
`0.43 ⫾ 0.09
`0.30 ⫾ 0.06
`
`Ki, nM
`
`[3H]-Spiperone
`
`634 ⫾ 151
`576 ⫾ 192
`
`1034 ⫾ 231
`0.36 ⫾ 0.04
`0.70 ⫾ 0.22
`
`0.16 ⫾ 0.01
`0.16 ⫾ 0.02
`
`Ki Low/Ki High
`
`67
`34
`
`18
`2
`2
`
`0.4
`0.5
`
`
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`Fig. 2. The density of D2L receptors was determined by saturation binding of [3H]spiperone to washed membranes prepared from CHO-D2L cells
`previously incubated in the absence or presence of EEDQ at 37°C for 60 min. Nonspecific binding was determined with 2 ␮M (⫹)-butaclamol. KD and
`Bmax values were determined by fitting data to a one-site binding isotherm.
`
`in the density of receptors (Fig. 2). The affinity of D2 recep-
`tors for [3H]spiperone was not affected by exposure of cells to
`EEDQ.
`In cells exposed to 1 ␮M EEDQ, dopamine inhibited fors-
`kolin-stimulated cAMP accumulation with a 2.5-fold lower
`potency than in cells exposed to vehicle (Fig. 3A). There was
`no significant change in the maximum effect of dopamine,
`consistent with the presence of receptor reserve. The potency
`of dopamine was further reduced in cells exposed to 10 ␮M
`EEDQ (Fig. 3A). Exposure of cells to 10 ␮M EEDQ resulted in
`a small decrease in efficacy. The apparent dissociation con-
`stant (KA) of dopamine was calculated from a double-recip-
`rocal plot of equieffective concentrations of dopamine deter-
`mined in cells incubated in the presence and absence of 10
`␮M EEDQ, according to the method of Furchgott and Bursz-
`tyn (1967). The KA value for dopamine was 178 nM (Fig. 3A,
`inset). In contrast to effects seen with dopamine, the maxi-
`mum effect of aripiprazole was significantly reduced in cells
`exposed to 1 ␮M EEDQ (Fig. 3B). In cells exposed to 10 ␮M
`EEDQ, the maximum effect of aripiprazole was further re-
`duced. The efficacy of aripiprazole relative to dopamine was
`reduced from nearly 90 to 25% upon partial inactivation of
`D2 receptors with 10 ␮M EEDQ (Fig. 3, A and B). A double-
`reciprocal plot of equieffective concentrations of aripiprazole
`determined in cells incubated in the presence and absence of
`1 ␮M EEDQ yielded a KA of 28 nM (Fig. 3B, inset). As seen
`with aripiprazole, the maximum effects of S(⫺)-PPP and
`terguride were progressively reduced in cells exposed to in-
`creasing concentrations of EEDQ (Fig. 4A,B). The KA values
`of S(⫺)-PPP and terguride were 129 and 3.6 nM, respectively
`(Fig. 4, A and B, inset).
`KA values for dopamine, aripiprazole, S(⫺)-PPP, and ter-
`guride were used to compare the relative efficacy of agonists
`as a function of occupancy of receptors. A steep hyperbolic
`
`occupancy-effect relationship was seen for dopamine (Fig. 5).
`The response to dopamine was nearly maximal at 20% occu-
`pancy of D2 receptors. Inhibition of cAMP accumulation by
`50% was achieved with occupancy by dopamine of only 2% of
`the receptors. Greater levels of occupancy of receptors by
`terguride, S(⫺)-PPP, and aripiprazole were required for the
`same response (Fig. 5).
`Given the partial agonist effect in cells exposed to 10 ␮M
`EEDQ, the ability of aripiprazole to antagonize the effect of
`dopamine was examined. Dopamine (100 nM)
`inhibited
`cAMP accumulation by approximately 60% (Fig. 6). Consis-
`tent with the action of a partial agonist, increasing concen-
`trations of aripiprazole blocked the effect of dopamine up to
`the maximum agonist effect seen with aripiprazole alone
`(Fig. 6).
`
`Discussion
`A range of efficacy values for aripiprazole at D2 receptors
`has been reported in different cell lines and tissues. The
`present study examined the efficacy of aripiprazole at a sin-
`gle population of human D2 receptors. Multiple factors, in-
`cluding intrinsic activity, receptor density, and coupling effi-
`ciency in the signal transduction cascade, contribute to the
`activity of an agonist in a given system (Kenakin 1997).
`Agonists with low intrinsic activity may show agonist or
`antagonist activity depending upon the sensitivity of the
`method used for detection, the level of basal or endogenous
`receptor activation, and the molecular properties of the sig-
`naling event under investigation (Hoyer and Boddeke, 1993).
`Given the level of complexity, current techniques cannot
`unambiguously determine the intrinsic efficacy of an agonist
`at a receptor (Clarke and Bond, 1998; Kenakin, 1999). One
`approach that may avoid the variables associated with the
`
`
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`Fig. 3. CHO-D2L cells were incubated in the absence or presence of increasing concentrations of EEDQ at 37°C for 60 min. Inhibition of
`forskolin-stimulated cAMP accumulation by dopamine (A) and aripiprazole (B) was determined in washed cells as in Fig. 1. Accumulation of cAMP
`stimulated by forskolin ranged from 3 to 15 pmol/well. In cells exposed to 1 and 10 ␮M EEDQ, the mean ⫾ S.D. level of cAMP accumulation stimulated
`by forskolin was 105 ⫾ 17 and 115 ⫾ 13%, respectively, of that seen with cells exposed to vehicle. The data shown are the mean ⫾ S.E.M., n ⫽ 7
`experiments. Inset, the apparent KA values for dopamine and aripiprazole were determined from a plot of the reciprocals of equieffective concentra-
`tions of drugs from cells incubated in the absence (1/[A]) and presence (1/[A⬘]) of 1 ␮M EEDQ using the equation: KA ⫽ (slope ⫺ 1)/y ⫺ intercept
`(Furchgott and Bursztyn, 1967).
`
`signal transduction cascade involves calculation of the ratio
`of affinity values of agonists for coupled and noncoupled
`states of D2 receptors (Lahti et al., 1992). D2 receptors exist
`in multiple states having high and low affinity for agonists
`(Zahniser and Molinoff, 1978; DeLean et al., 1982). The ag-
`onist-preferring high-affinity state of D2 receptors is thought
`to reflect the active state of receptors and involves the for-
`mation of a ternary complex of agonist, receptor, and G
`protein (Wregget and DeLean, 1984). Whereas antagonists
`bind with equally high affinity to noncoupled and G protein-
`coupled states of D2 receptors, agonists typically display
`higher affinity for the G protein-coupled state.
`
`Comparisons of affinity values were used to predict the
`efficacy of aripiprazole and some known agonists, partial
`agonists, and antagonists at D2 receptors. In CHO cells ex-
`pressing human D2L receptors, the full agonists dopamine
`and quinpirole bound with greater than 30-fold higher affin-
`ity to the G protein-coupled state of D2 receptors than to the
`noncoupled state. The partial agonists, terguride and S(⫺)-
`PPP, although displaying less of a difference between affinity
`values for the different states, had higher affinity for the G
`protein-coupled state of D2 receptors. In contrast, the antag-
`onists butaclamol and haloperidol bound with higher affinity
`to the noncoupled state. Consistent with the properties of a
`
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`Fig. 4. CHO-D2L cells were incubated in the absence or presence of increasing concentrations of EEDQ at 37°C for 60 min. Inhibition of
`forskolin-stimulated cAMP accumulation by S(⫺)-PPP (A) and terguride (B) was determined in washed cells as in Fig. 1. The data shown are the
`mean ⫾ S.E.M., n ⫽ 4 experiments. For experiments with S(⫺)-PPP, accumulation of cAMP stimulated by forskolin ranged from 3 to 11 pmol/well.
`In cells exposed to 1 and 10 ␮M EEDQ, the mean ⫾ S.D. level of cAMP accumulation stimulated by forskolin was 98 ⫾ 19 and 111 ⫾ 12%, respectively,
`of that seen with cells exposed to vehicle. For experiments with terguride, accumulation of cAMP stimulated by forskolin ranged from 3 to 15 pmol/well.
`In cells exposed to 1 and 10 ␮M EEDQ, the mean ⫾ S.D. level of cAMP accumulation stimulated by forskolin was 113 ⫾ 10 and 121 ⫾ 11%, respectively,
`of that seen with cells exposed to vehicle. Inset, the apparent KA values for S(⫺)-PPP and terguride were determined from a plot of the reciprocals of
`equieffective concentrations of drugs from cells incubated in the absence (1/[A]) and presence (1/[A⬘]) of 1 ␮M EEDQ using the equation: KA ⫽ (slope ⫺
`1)/y ⫺ intercept (Furchgott and Bursztyn, 1967).
`
`partial agonist, aripiprazole bound with 2-fold higher affinity
`to the G protein-coupled state of D2 receptors.
`Aripiprazole was a partial agonist at human D2L receptors
`coupled to the inhibition of forskolin-stimulated cAMP accu-
`mulation. Results with terguride and S(⫺)-PPP were consis-
`tent with previous reports that these compounds are partial
`agonists at D2 receptors (Clark et al., 1984; Kehr, 1984; Lahti
`et al., 1992). For a given receptor-effector system, the density
`of receptors plays an important role in determining the po-
`tency and maximum efficacy of agonists (Kenakin, 1999). The
`alkylating agent EEDQ, an irreversible antagonist of dopa-
`mine receptors (Hamblin and Creese, 1983), has been used
`
`previously as a tool to modulate the density of D2L receptors
`in transfected cells (Filtz et al., 1994). Exposure of CHO-D2L
`cells to increasing concentrations of EEDQ resulted in a
`decrease in the density of receptors measured by [3H]spiper-
`one. The efficacy of aripiprazole in CHO-D2L cells ranged
`from 25 to 90% that of dopamine, depending on the density of
`receptors. In preliminary studies, the efficacy of dopamine,
`aripiprazole, (⫺)-3-PPP, terguride, and OPC-4392 were com-
`pared in clonal cell lines that express high (11–18 pmol/mg)
`and low (0.3– 0.9 pmol/mg) densities of D2L and D2S recep-
`tors. Aripiprazole was a partial agonist at both D2S and D2L
`receptors, and density-dependent efficacy comparable to the
`
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`Aripiprazole Is a D2 Receptor Partial Agonist
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`387
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`Fig. 5. The percentage of receptor occu-
`pancy was determined for each concen-
`tration of agonist used in Figs. 3 and 4
`using the equation: % occupancy ⫽ [ago-
`nist]/([agonist] ⫹ KA).
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`studies with EEDQ was seen (Y. Tadori, T. Miwa, K. Tottori,
`K. D. Burris, P. B. Molinoff, F. D. Yocca, and T. Kikuchi,
`unpublished observations).
`Inactivation of a fraction of D2 receptors with EEDQ re-
`sulted in a rightward shift in the dose-response curve for
`dopamine but no change in maximum effect. This suggests a
`nonlinear relationship between occupancy of receptors and
`the response to dopamine. Classically, this has been ascribed
`to the presence of receptor reserve in the tissue (Nickerson,
`1956). In contrast, the maximum effects of terguride, S(⫺)-
`PPP, and aripiprazole were significantly reduced, with pro-
`gressive alkylation indicating a lack of receptor reserve for
`these partial agonists. Occupancy-response curves for the
`partial agonists were less steep and were right-shifted com-
`pared with that seen with dopamine. Interestingly, a nonlin-
`ear relationship between occupancy and response appeared
`to exist for all three partial agonists. Although partial ago-
`nists would be expected to yield linear occupancy-response
`relationships, nonlinear relationships have been reported
`(Kenakin and Beek, 1984; Kenakin, 1997).
`The relative efficacy of aripiprazole was higher in the
`present study than in some previous reports. In C6 cells
`expressing rat D2L receptors, the efficacy of aripiprazole for
`inhibition of isoproterenol-stimulated cAMP accumulation
`was approximately 30% that of dopamine (Lawler et al.,
`1999). In the same study, aripiprazole did not significantly
`inhibit cAMP accumulation in CHO cells expressing rat D2L
`receptors. The lack of efficacy may be due to a lack of receptor
`reserve or differences in the coupling of receptors in the cell
`lines. In contrast to the nearly complete inhibition of forsko-
`lin-stimulated cAMP accumulation by dopamine in the
`present study, dopamine inhibited the response to forskolin
`by only 50% in CHO cells that expressed transfected rat D2L
`receptors (Lawler et al., 1999). Receptor reserve has been
`reported for the effects of agonists at D2 receptors in rat
`pituitary (Meller et al., 1991). Similar to the present results,
`aripiprazole is a partial agonist at D2 receptors linked to
`inhibition of prolactin release in slices of rat pituitary (Inoue
`et al., 1996).
`Differences in receptor reserve may play a role in the
`
`varying efficacy of aripiprazole at pre- and postsynaptic D2
`receptors. Receptor reserve has been reported for the effects
`of agonists at presynaptic D2 receptors (Meller et al., 1987)
`but not for responses mediated by postsynaptic D2 receptors
`(Meller et al., 1988). Meller et al. (1987) hypothesized that
`enhanced sensitivity of presynaptic D2 receptor signal trans-
`duction (compared with postsynaptic) may underlie the re-
`ported autoreceptor selectivity of D2 receptor partial ago-
`nists. The present study demonstrated the effects of
`modulating the density of receptors on the efficiency of signal
`transduction. In the presence of receptor reserve for dopa-
`mine, aripiprazole was an efficacious agonist. In its absence,
`the relative efficacy of aripiprazole was low, and a predomi-
`nant antagonist effect was seen. Likewise, although aripipra-
`zole is a potent partial agonist at all D2 receptors, it functions
`with greater efficacy at presynaptic receptors with substan-
`tial receptor reserve and with lower efficacy at postsynaptic
`D2 receptors with less receptor reserve.
`The molecular mechanisms underlying differences in sen-
`sitivity at pre- and postsynaptic D2 receptors are not known.
`The present studies demonstrate the dependence of efficacy
`on the density of receptors for a single effector system and
`receptor population. At high levels of receptor expression, a
`significant amount of receptor reserve exists for full agonists,
`and partial agonists display high relative efficacy values. In
`the absence of receptor reserve, partial agonists have the
`properties of antagonists. In addition to their stoichiometry,
`the molecular identity of the signaling molecules can modu-
`late the relative efficacies of agonists. The molecular identity
`of the subtypes of D2-like receptors that mediate pre- and
`postsynaptic actions of agonists have not been determined.
`The cataleptic effects of haloperidol reportedly are absent or
`attenuated in D2L receptor-deficient transgenic mice (Usiello
`et al., 2000; Wang et al., 2000). In the same studies, D2
`receptor-mediated inhibition of dopamine release, locomotor
`activity, and the firing rate of dopaminergic neurons in the
`substantia nigra were similar to that seen in wild-type ani-
`mals, suggesting that D2S receptors can function as autore-
`ceptors.
`Many subtypes of G protein-coupled receptors activate a
`
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`Burris et al.
`
`Fig. 6. CHO-D2L cells were incubated in
`the presence of 10 ␮M EEDQ to eliminate
`receptor reserve. Inhibition of forskolin-
`stimulated cAMP accumulation by aripi-
`prazole in the absence and presence of
`100 nM dopamine was determined in
`washed cells as in Fig. 1. Accumulation of
`cAMP stimulated by forskolin ranged
`from 9 to 12 pmol/well. The data shown
`are the mean ⫾ range of two experiments.
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