European Journal of Pharmacology 441 (2002) 137 – 140
`
`www.elsevier.com/locate/ejphar
`
`Short communication
`
`The antipsychotic aripiprazole is a potent, partial agonist
`at the human 5-HT1A receptor
`
`Shaun Jordan a,*, Vuk Koprivica a, Ruoyan Chen a, Katsura Tottori b,
`Tetsuro Kikuchi b, C. Anthony Altar a
`
`aNeuroscience Department, Maryland Research Laboratories, Otsuka Maryland Research Institute, 9900 Medical Center Drive, Rockville, MD 20850, USA
`bCNS Research Group, Research Institute of Pharmacological and Therapeutical Development, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno,
`Kawauchi-cho, Tokushima 771-0192, Japan
`
`Received 3 January 2002; received in revised form 6 March 2002; accepted 12 March 2002
`
`Abstract
`
`Aripiprazole, 7-{4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy}-3,4-dihydro-2(1H)-quinolinone, a novel antipsychotic with partial
`agonist activity at dopamine D2 receptors, bound with high affinity to recombinant human 5-HT1A receptors (h5-HT1A) in Chinese hamster
`ovary cell membranes and displayed potent, partial agonism at 5-HT1A receptors in a guanosine-5V-O-(3-[35S]thio)-triphosphate
`([35S]GTPgS)-binding assay that was blocked completely by a selective 5-HT1A receptor antagonist. An interaction with 5-HT1A
`receptors may contribute to the overall efficacy of aripiprazole against symptoms of schizophrenia, including anxiety, depression, cognitive
`and negative symptoms, and to its favorable side-effect profile. Combined with previous studies demonstrating the potent partial agonism of
`aripiprazole at dopamine D2 receptors, this study suggests aripiprazole is the first dopamine – serotonin system stabilizer. D 2002 Elsevier
`Science B.V All rights reserved.
`
`Keywords: Aripiprazole (OPC-14597); Partial agonist; Schizophrenia; 5-HT (5-hydroxytryptomine, seretonin); Dopamine
`
`1. Introduction
`
`Aripiprazole, 7-{4-[4-(2,3-dichlorophenyl)-1-piperazi-
`nyl]butyloxy}-3,4-dihydro-2(1H)-quinolinone,
`is a novel
`antipsychotic with a mechanism of action that differs from
`all currently marketed typical and atypical antipsychotics.
`Biochemically, aripiprazole is a partial agonist at members
`of the D2 family of dopamine receptors (Inoue et al., 1996;
`Burris et al., 2000). In vivo, aripiprazole displays dopamine
`D2 receptor antagonist effects in models of dopaminergic
`hyperactivity (e.g. blockade of apomorphine-induced stereo-
`typy) and dopamine D2 receptor agonist activity in a model
`of dopaminergic hypoactivity (blockade of increased dop-
`amine synthesis in reserpine-treated rats) (Kikuchi et al.,
`1995). Limited preclinical evidence indicates that aripipra-
`zole has activity at several serotonin receptors of clinical
`relevance to schizophrenia including antagonism at 5-HT2A
`(Kikuchi et al., unpublished observations). Aripiprazole is
`
`* Corresponding author. Tel.: +1-240-683-3306; fax: +1-301-721-7306.
`E-mail address: shaunj@otsuka.com (S. Jordan).
`
`an efficacious treatment for the positive and negative
`symptoms of schizophrenia; however, it does not induce
`significant extrapyramidal symptoms or elevate serum pro-
`lactin, and has a low propensity to produce weight gain,
`sedation or prolongation of QTc interval on electrocardio-
`gram (Carson et al., 2000; Kane and Ingenito, 2000).
`While it has long been established that antipsychotic
`drug activity is a direct correlate of dopamine D2 receptor-
`binding affinity (Seeman et al., 1976), recent attention has
`focused on the 5-HT1A receptor as a therapeutic target for
`the development of improved antipsychotic drugs (Meltzer,
`1999; Millan, 2000). Several clinically effective antipsy-
`chotics bind in vitro with moderate to high affinity to cloned
`human 5-HT1A receptors (Richelson and Souder, 2000), and
`ziprasidone and clozapine behave as partial agonists at
`cloned human 5-HT1A receptors (Newman-Tancredi et al.,
`1998). In the present study, a guanosine-5V-O-(3-[35S]thio)-
`triphosphate ([35S]GTPgS)-binding assay was used to deter-
`mine the potency and relative intrinsic activity of aripipra-
`zole at the human 5-HT1A receptor (h5-HT1A) expressed
`stably in recombinant Chinese hamster ovary (CHO) cell
`membranes. A competitive radioligand-binding assay was
`
`0014-2999/02/$ - see front matter D 2002 Elsevier Science B.V All rights reserved.
`PII: S 0 0 1 4 - 2 9 9 9 ( 0 2 ) 0 1 5 3 2 - 7
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`
`also performed with hydrogen-3 8-hydroxy-2-(di-n-propy-
`lamino)tetralin ([3H]8-OH-DPAT) to evaluate the binding
`affinity of aripiprazole at this recombinant h5-HT1A recep-
`tor.
`
`2. Materials and methods
`
`[35S]GTPgS (1200 Ci/mmol), [3H]8-OH-DPAT (124.9
`Ci/mmol) and CHO cell membranes stably expressing the
`h5-HT1A receptor (CHO-h5-HT1A, receptor expression
`(Bmax) = 1.0 pmol/mg membrane protein) were purchased
`from NEN Life Science Products (Boston, MA). Aripipra-
`zole and ziprasidone were synthesized by Otsuka Pharma-
`ceutical (Tokushima, Japan). Risperidone and GDP were
`purchased from Sigma (St. Louis, MO) and all other
`reference compounds were obtained from RBI (Natick,
`MA).
`Estimates of potency (pEC50) and relative intrinsic activ-
`ity (Emax, maximal drug effect on basal [35S]GTPgS binding
`to CHO-h5-HT1A membranes expressed as a percentage of
`the effect of 10 AM serotonin (5-HT)) were obtained for
`aripiprazole, 5-HT, (+)-8-OH-DPAT, ()-8-OH-DPAT, zip-
`
`rasidone, buspirone, risperidone, clozapine and WAY-
`100635 (N-[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-
`N-2-pyridinylcyclohexanecarboxamide) by incubating each
`drug at 10 different concentrations for 60 min at 22 jC with
`CHO-h5-HT1A membranes (10 Ag protein), mixed with
`buffer (25 mM Tris – HCl, 50 mM NaCl, 5 mM MgCl2,
`0.1 mM EGTA, pH = 7.4) containing GDP (1 AM) and
`[35S]GTPgS (0.1 nM). The same [35S]GTPgS-binding assay
`was also used to determine the inhibitory potency (pIC50) of
`tested at
`the selective 5-HT1A antagonist WAY-100635,
`0.01, 0.1, 1, 10, 50, 100, 500, 1000, 5000 and 10,000 nM
`concentrations, against a 10 AM concentration each of
`aripiprazole, 5-HT, ziprasidone and clozapine.
`All incubations were performed in triplicate, proceeded
`for 60 min at room temperature, and were terminated by
`rapid filtration through Whatman GF/B filter paper pre-
`soaked in 50 mM Tris – HCl, pH = 7.4, using a Brandel
`ice-cold washes with the same
`buffer. Radioactive counts were detected by liquid scintilla-
`tion counting (Clingamma, LKB/Wallach).
`The h5-HT1A receptor-binding affinity of aripiprazole
`was determined against the 5-HT1A-selective radioligand,
`[3H]8-OH-DPAT. The h5-HT1A membranes (15 – 20 Ag pro-
`tein) were incubated for 60 min at 20 jC in a buffer (50 mM
`Tris – HCl, 10 mM MgSO4, 0.5 mM EDTA, 0.1% ascorbic
`acid, pH = 7.4) containing 1 nM [3H]8-OH,-DPAT and
`vehicle or aripiprazole at the same concentrations used in
`the [35S]GTPgS assays. The binding assays were terminated
`by rapid filtration through Whatman GF/B filter paper
`presoaked in 50 mM Tris – HCl, pH = 7.4, and washed once
`in 4 ml of the same buffer. Nonspecific binding was defined
`in the presence of 10 AM (+)-8-OH-DPAT. The binding af-
`finity of aripiprazole for h5-HT1A receptors (Ki) was calcu-
`
`harvester and 4 3 ml
`
`lated by the equation, Ki=(IC50)/(1+([[3H]8-OH-DPAT]/Kd),
`where the Kd for [3H]8-OH-DPAT at h5-HT1A = 0.69 nM
`(NEN Life Science Products) using nonlinear regression
`analysis.
`All estimates of h5-HT1A-binding affinity, potency, rel-
`ative intrinsic activity and inhibitory potency were deter-
`mined by nonlinear regression analysis of each binding
`isotherm using GraphPad Prism version 3.00 for Windows
`(GraphPad Software, San Diego, CA).
`
`3. Results
`
`Fig. 1 shows the effects of aripiprazole and reference
`drugs on basal [35S]GTPgS binding to the h5-HT1A receptor
`expressed in CHO cell membranes. Aripiprazole stimulated
`[35S]GTPgS binding to the h5-HT1A receptor with a potent
`(pEC50 = 8.67 F 0.16), partial agonist (Emax = 68.1% of the
`
`Fig. 1. Drug effects upon basal [35S]GTPgS binding to CHO cell mem-
`branes expressing the h5-HT1A receptor. All data points are means F S.E.M.
`of triplicate determinations from a single representative experiment and are
`expressed as a percentage of the stimulatory effect of 10 AM 5-HT on basal
`[35S]GTPgS binding.
`
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`139
`
`Table 1
`Functional parameter estimates for aripiprazole and reference drugs in a
`[35S]GTPgS-binding assay using CHO cell membranes expressing the h5-
`HT1A receptor
`
`Agonist
`
`5-HT
`(+)-8-OH-DPAT
`
`( )-8-OH-DPAT
`
`Ziprasidone
`Buspirone
`Aripiprazole
`Clozapine
`Risperidone
`
`F SEM
`pEC50
`8.43 F 0.16
`8.37 F 0.08
`8.21 F 0.06
`8.70 F 0.08
`7.48 F 0.13
`8.67 F 0.16
`6.14 F 0.13
`6.66 F 0.16
`
`Emax(%)
`
`98.4
`98.5
`82.9
`79.0
`74.2
`68.1
`64.7
`25.1
`
`R2
`
`0.99
`0.99
`0.99
`0.99
`0.99
`0.99
`0.98
`0.99
`
`HT1A [35S]GTPgS-binding assay are similar to previously
`published estimates for these drugs (Lejeune et al., 1997;
`Newman-Tancredi et al., 1996, 1998; Pauwels et al., 1997).
`Thus, the present h5-HT1A [35S]GTPgS-binding assay was
`able to identify correctly drugs with varying degrees of
`intrinsic agonist efficacy and potency. The h5-HT1A selective
`nature of the present assay was further demonstrated by the
`ability of the selective 5-HT1A receptor antagonist WAY-
`100635 to inhibit fully 5-HT, aripiprazole, ziprasidone and
`clozapine-induced increases in [35S]GTPgS binding. In this
`respect, WAY-100635 was a 50- to 100-fold more potent
`inhibitor of clozapine than either 5-HT, aripiprazole or
`ziprasidone. A likely explanation for this is that 5-HT,
`aripiprazole and ziprasidone were far more potent in stim-
`ulating the h5-HT1A receptor in the present system than was
`clozapine. Thus, higher concentrations of WAY-100635 were
`required to block 10 AM concentrations of the more potent
`drugs, than the less potent clozapine.
`Most neuropharmacological evidence is consistent with
`aripiprazole being a potent, partial agonist at the dopamine
`D2 receptor (Inoue et al., 1996; Burris et al., 2000). The
`present data, as well as the ability of aripiprazole to
`stimulate [35S]GTPgS binding in rat hippocampal mem-
`branes (S. Jordan, unpublished observations) and suppress
`5-HT metabolism in vivo (Jordan et al., 2001), provide the
`first evidence that aripiprazole is also a potent, partial
`agonist at 5-HT1A receptors. It is interesting to note that
`the h5-HT1A receptor agonist potency of aripiprazole, zipra-
`sidone and clozapine in the present [35S]GTPgS-binding
`assay are similar to their respective published binding
`affinities for h5-HT1A receptors (Schotte et al., 1996;
`Richelson and Souder, 2000).
`A variety of preclinical data has suggested that the 5-
`HT1A receptor is a therapeutic target for the development of
`improved antipsychotic drugs (Meltzer, 1999; Millan,
`2000). A putative association has been postulated between
`partial agonist activity at 5HT1A receptors and improve-
`ments in anxiety, depression, cognitive and negative symp-
`toms, and decreased extrapyramidal symptom liability
`(Millan, 2000). In animal studies, 5-HT1A receptor agonists
`can diminish catalepsy, a model of extrapyramidal symp-
`toms in humans, induced by dopamine D2 receptor antag-
`onists (McMillen et al., 1988; Andersen and Kilpatrick,
`1996; Prinssen et al., 1999), whereas elevations in serum
`prolactin produced by dopamine D2 receptor antagonists
`can be attenuated by partial 5HT1A receptor agonists (Nash
`and Meltzer, 1989).
`The present study demonstrated that aripiprazole is a
`high affinity, potent, partial agonist at the h5-HT1A receptor
`expressed in CHO cell membranes. In the context of
`previous studies demonstrating that aripiprazole is a partial
`agonist at dopamine D2 receptors (Inoue et al., 1996; Burris
`et al., 2000) and an antagonist at 5-HT2A receptors (Kikuchi
`et al., unpublished observations), the results of the present
`study suggest that aripiprazole may best be described as a
`dopamine – serotonin system stabilizer. The preclinical pro-
`
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`pIC50 F SEM
`6.66 F 0.10
`6.41 F 0.10
`6.75 F 0.09
`8.47 F 0.29
`
`R2
`
`0.99
`0.99
`0.99
`0.87
`
`WAY-100635 inhibition
`10 AM 5-HT
`10 AM Aripiprazole
`10 AM Ziprasidone
`10 AM Clozapine
`Agonist potency (pEC50) and relative intrinsic activity (Emax, maximal drug
`effect on basal [35S]GTPgS binding expressed as a percentage of that
`produced by 10 AM 5-HT) were estimated by nonlinear regression analysis
`of the data shown in Fig. 1. Nonlinear regression was also used to estimate
`the inhibitory potency (pIC50) of WAY-100635, tested at 0.01, 0.1, 1, 10,
`50, 100, 500, 1000, 5000 and 10,000 nM, against 10 AM concentrations
`each of 5-HT, aripiprazole, ziprasidone and clozapine. R2 represents the
`goodness of fit between observed concentration effect data points and
`nonlinear functions derived for each drug or drug combination studied.
`
`effect of 10 AM 5-HT on basal [35S]GTPgS binding) profile
`(Table 1). In comparison, potent, full agonist profiles were
`detected for 5-HT and (+)-8-OH-DPAT, while ( )-8-OH-
`
`DPAT and ziprasidone behaved as potent, partial agonists.
`Less potent, partial h5-HT1A agonist profiles were displayed
`by buspirone, risperidone and clozapine, whereas WAY-
`100635 failed to stimulate basal [35S]GTPgS binding on
`its own. However, WAY-100635 blocked the stimulatory
`effects of 10 AM concentrations of 5-HT, aripiprazole,
`ziprasidone and clozapine on [35S]GTPgS binding to
`CHO-h5-HT1A cell membranes, in a concentration-depend-
`ent manner in each case (Table 1). In this respect, WAY-
`100635 was 100-fold more potent as an inhibitor of cloza-
`pine than of either aripiprazole or ziprasidone. Aripiprazole
`bound with high affinity (Ki = 1.65 nM, 95% confidence
`interval = 1.09 to 2.48 nM; IC50 = 4.03 nM, 95% confidence
`interval = 2.68 to 6.08 nM) to the h5-HT1A receptor in a
`competition-binding assay using [3H]8-OH-DPAT.
`
`4. Discussion
`
`The main finding of the present study is that aripiprazole
`displayed a potent, partial agonist profile in a h5-HT1A
`[35S]GTPgS-binding assay using recombinant CHO cell
`membranes. These data are consistent with the observation
`that aripiprazole binds with high affinity to the h5-HT1A
`receptor. The potency and relative intrinsic activity of zipra-
`sidone and all other reference drugs tested in the current h5-
`
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`S. Jordan et al. / European Journal of Pharmacology 441 (2002) 137–140
`
`file of aripiprazole is supportive of clinical efficacy in the
`treatment of schizophrenia with an excellent safety and
`tolerability profile.
`
`References
`
`Andersen, H.L., Kilpatrick, I.C., 1996. Prevention by ( F )-8-hydroxy-2-
`(di-n-propylamino)tetralin of both catalepsy and the rises in rat striatal
`dopamine metabolism caused by haloperidol. Br. J. Pharmacol. 118,
`421 – 427.
`Burris, K.D., Molski, T.F., Ryan, E., Xu, C., Tottori, K., Kikuchi, T., Yocca,
`F.D., Molinoff, P.B., 2000. Aripiprazole is a high affinity partial agonist
`at human D2 dopamine receptors. Int. J. Neuropsychopharmacol. 3
`(Suppl. 1), S129.
`Carson, W.H., Kane, J.M., Ali, M., Dunbar, G.C., Ingenito, G., 2000.
`Activity of aripiprazole in psychotic disorders: comparison with halo-
`peridol and placebo. Eur. Neuropsychopharmacol. 10 (3), S309.
`Inoue, T., Domae, M., Yamada, K., Furukawa, T., 1996. Effects of the novel
`antipsychotic agent 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]buty-
`loxy)-3,4-dihydro-2(1H)-quinolinone (OPC-14597) on prolactin release
`from the rat anterior pituitary gland. J. Pharmacol. Exp. Ther. 277,
`137 – 143.
`Jordan, S., Koprivica, V., Dunn, R., Tottori, K., Kikuchi, T., Altar, C.A.,
`2001. In vivo effects of aripiprazole on dopaminergic and serotonergic
`function in rat prefrontal cortex and striatum. Soc. Neurosci. Abstr. 27,
`875.3.
`Kane, J., Ingenito, G., 2000. Activity of aripiprazole in psychotic disorders:
`comparison with haloperidol and placebo. Int. J. Neuropsychopharma-
`col. 3 (Suppl. 1), S124.
`Kikuchi, T., Tottori, K., Uwahodo, Y., Hirose, T., Miwa, T., Oshiro, Y.,
`Morita, S., 1995. 7-(4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]buty-
`loxy)-3,4-dihydro-2(1H)-quinolinone (OPC-14597), a new putative
`antipsychotic drug with both presynaptic dopamine autoreceptor ago-
`nistic activity and postsynaptic D2 receptor antagonistic activity. J.
`Pharmacol. Exp. Ther. 274, 329 – 336.
`Lejeune, F., Newman-Tancredi, A., Audinot, V., Millan, M.J., 1997. Inter-
`
`actions of (+)- and ( )-8- and 7-hydroxy-2-(di-n-propylamino)tetralin
`
`at human (h)D3, hD2 and h serotonin1A receptors and their modulation
`of the activity of serotoninergic and dopaminergic neurones in rats. J.
`Pharmacol. Exp. Ther. 280 (3), 1241 – 1249.
`McMillen, B.A., Scott, S.M., Davanzo, E.A., 1988. Reversal of neuroleptic-
`induced catalepsy by novel aryl-piperazine anxiolytic drugs. J Pharm.
`Pharmacol. 40, 885 – 887.
`Meltzer, H.Y., 1999. The role of serotonin in antipsychotic drug action.
`Neuropsychopharmacology 21 (Suppl. 2), 106S – 115S.
`Millan, M.J., 2000. Improving the treatment of schizophrenia: focus on
`serotonin (5-HT)1A receptors. J. Pharmacol. Exp. Ther. 295, 853 – 861.
`Nash, J.F., Meltzer, H.Y., 1989. Effect of gepirone and ipsapirone on the
`stimulated and unstimulated secretion of prolactin in the rat. J. Pharma-
`col. Exp. Ther. 249, 236 – 241.
`Newman-Tancredi, A., Chaput, C., Verriele, L., Millan, M.J., 1996. Cloza-
`pine is a partial agonist at cloned, human serotonin 5-HT1A receptors.
`Neuropharmacology 35, 119 – 121.
`Newman-Tancredi, A., Gavaudan, S., Conte, C., Chaput, C., Touzard, M.,
`Verriele, L., Audinot, V., Millan, M.J., 1998. Agonist and antagonist
`actions of antipsychotic agents at 5-HT1A receptors. Eur. J. Pharmacol.
`355, 245 – 256.
`Pauwels, P.J., Tardif, S., Wurch, T., Colpaert, F.C., 1997. Stimulated [35S]
`GTP gamma S binding by 5-HT1A receptor agonists in recombinant
`cell lines. Modulation of apparent activity by G-protein activation state.
`Naunyn-Schmiedeberg’s Arch. Pharmacol. 356, 551 – 561.
`Prinssen, E.P., Kleven, M.S., Koek, W., 1999. Interactions between neuro-
`leptics and 5-HT(1A) ligands in preclinical behavioral models for anti-
`psychotic and extrapyramidal effects. Psychopharmacology 144, 20 –
`29.
`Richelson, E., Souder, T., 2000. Binding of antipsychotic drugs to human
`brain receptors focus on newer generation compounds. Life Sci. 68,
`29 – 39.
`Schotte, A., Janssen, P.F., Gommeren, W., Luyten, W.H., Van Gompel, P.,
`Lesage, A.S., De Loore, K., Leysen, J.E., 1996. Risperidone compared
`with new and reference antipsychotic drugs: in vitro and in vivo recep-
`tor binding. Psychopharmacology 124, 57 – 73.
`Seeman, P., Lee, T., Chau-Wong, M., Wong, K., 1976. Antipsychotic drug
`doses and neuroleptic/dopamine receptors. Nature 261, 717 – 719.
`
`4 of 4
`
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