In Vitro and In Vivo Characterization of the Dopamine D4 Receptor, Serotonin 5-HT2A Receptor and Alpha-1 Adrenoceptor Antagonist (R)-(1)-2-Amino-4-(4-Fluorophenyl)- 5-[1-[4-(4-Fluorophenyl)-4-Oxobutyl]Pyrrolidin-3-yl]Thiazole (NRA0045)

(R)-(1)-2-Amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)-4oxobutyl]pyrrolidin-3-yl]thiazole (NRA0045), a novel thiazole derivative, has high affinities for the human cloned dopamine D4.2, D4.4 and D4.7 receptors, with Ki values of 2.54, 0.55 and 0.54 nM, respectively. NRA0045 is approximately 91-fold more potent at the dopamine D4.2 receptor, compared with human cloned dopamine D2L receptor. NRA0045 also has high affinities for the serotonin (5-HT)2A receptor (Ki 5 1.92 nM) and alpha-1 adrenoceptor (Ki 5 1.40 nM) but weak affinities (IC50 values are approximately 1 mM) for six other neurotransmitter receptors (adenosine1, 5-HT1A, 5-HT1C, dopamine transporter, a2A and a2A) and negligible affinities (IC50 values are over 10 25 M) for 42 other receptors, including neurotransmitters and hormones, ion channels and second messenger systems. Locomotor hyperactivity induced by methamphetamine (1 mg/kg i.p.) in mice was dose-dependently antagonized by NRA0045 (ED50 5 0.5 mg/kg i.p. and 1.9 mg/kg p.o., respectively). Methamphetamine (10 mg/kg i.p.)-induced stereotyped behavior in mice was dose-dependently antagonized by NRA0045, whereas NRA0045 did not exceed 50% inhibition even at the highest dose given (30 mg/kg i.p.). Catalepsy was dose-dependently and significantly induced by NRA0045 in rats, whereas NRA0045 did not exceed 50% induction even at the highest dose given (30 mg/kg i.p.). Thus NRA0045 blocks behaviors associated with activation of the mesolimbic/mesocortical dopaminergic neurons more selectively than behaviors associated with nigrostriatal dopaminergic neurons. In rats, tryptamineinduced clonic seizure, a 5-HT2 receptor-mediated behavior, was also dose-dependently inhibited by NRA0045 (ED50 5 1.7 mg/kg i.p.). Norepinephrine-induced lethality is regarded as being induced through the alpha-1 adrenoceptor. NRA0045 dose-dependently antagonized norepinephrine-induced lethality in rats (ED50 5 0.2 mg/kg i.p.). Thus NRA0045 may have a unique antipsychotic activity with regard to dopamine D4 and 5-HT2A receptors and alpha-1 adrenoceptor antagonistic activities, without producing the extrapyramidal side effects. Brain dopamine synapses are considered to be overactive in schizophrenics (Seeman, 1992). This overactivity may stem from either an excess release of dopamine or overactivity of dopamine receptors. Much evidence for the hypothesis of dopamine overactivity in schizophrenics relies on findings that neuroleptics block dopamine D2 receptors in direct relation to their clinical antipsychotic potencies (Seeman et al., 1975; Seeman, 1992; 1995). Recently, however, molecular biological approaches suggest that the cloned dopamine receptors (D1–D5) can be divided into two groups that correspond to the dopamine D1 and D2 receptor classification that had previously been identified pharmacologically (Mansour and Watson, 1995). The dopamine D1 and D5 receptors have a dopamine D1-like pharmacology, whereas the dopamine D2, D3 and D4 receptors have a dopamine D2-like pharmacological profile (Mansour and Watson, 1995). Among these five Received for publication November 11, 1996. ABBREVIATIONS: ANOVA, analysis of variance; MAP, methamphetamine; NRA0045, (R)-(1)-2-amino-4-(4-fluorophenyl)-5-[1-[4-(4-fluorophenyl)4-oxobutyl]pyrrolidin-3-yl]thiazole; NE, norepinephrine; 5-HT, serotonin; PET, positron emission tomography; SPET, simple PET; mRNA, messenger ribonucleic acid; cDNA, complementary deoxyribonucleic acid; RT-PCR, reverse transcription-polymerase chain reaction; NMDA, Nmethyl-D-aspartate; PCP, phencyclidine; MK-801, (1)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine. 0022-3565/97/2821-0056$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 1 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:56–63, 1997 56 at A PE T Jornals on M ay 6, 2017 jpet.asjournals.org D ow nladed from cloned dopamine receptor subtypes, the dopamine D4 receptor had an interesting anatomical distribution and pharmacological profile. The distribution of dopamine D4 mRNA was observed at a higher density in the frontal cortex and mesolimbic system than in the primary motor area—specifically, the nigrostriatal pathway (Van Tol et al., 1991; O’Malley et al., 1992). The observed higher dopamine D4 mRNA density in the cerebral cortex (particularly in the frontal lobe) and in the mesolimbic area has been recognized as an important characteristic of this receptor, because this is a CNS area of direct interest in schizophrenia (Tamminga et al., 1992; Weinberger, 1988). The dopamine D4 receptor has a high affinity for the antipsychotic clozapine (Van Tol et al., 1991), which has potent antipsychotic actions and a very low incidence of extrapyramidal motor side effects (Wagstaff and Bryson, 1995). PET and SPET studies have demonstrated that a good clinical response to clozapine occurs despite low dopamine D2 occupancy (Brucke et al., 1992; Farde et al., 1992; Pilowsky et al., 1992), which strongly suggests that the action of clozapine is not mediated by dopamine D2 receptor blockade. Conversely, in up to 30% of schizophrenics, there is a maximal dopamine D2 receptor blockade (Pilowsky et al., 1993). Clozapine at 100 to 200 nM blocks dopamine D2 receptors, in contrast with the therapeutic concentration of 10 to 20 nM found in the spinal fluid of clozapine-treated patients (Seeman, 1992). However, cloned dopamine D4 receptors can be blocked with clozapine concentrations (10–20 nM) that are found in the spinal fluid or water phase of plasma from clozapine-treated patients (Seeman, 1995; Seeman and Van Tol, 1994). In addition, the existence of dopamine D4-like sites and their elevation in schizophrenia have been reported (Seeman et al., 1993; Murray et al., 1995; Sumiyoshi et al., 1995). Kerwin and Collier (1996) reported that both haloperidol and clozapine mediate antipsychotic efficacy at dopamine D4 receptors and that the additional selectivity and affinity of haloperidol at dopamine D2 receptors are responsible for the neurological side effects. Thus clozapine’s clinical efficacy for schizophrenics was hypothesized to be associated with its dopamine D4 preference, and a dopamine D4 antagonist has the potential to be an effective antipsychotic agent lacking the extrapyramidal side effects. Dopamine D4 ligands such as 5-(4-chlorophenyl)-4-methyl3-(1-(2-phenylethyl)piperidin-4-yl)isoxazole (Rowley et al., 1996), 3-[[4-(4-chlorophenyl)piperazin-1-yl]-methyl]-1H-pyrrolo[2,3-b]pyridine (Kulagowski et al., 1996), (S)-(2)-4-[4-[2(isochroman-1-yl)ethyl]-piperazin-1-yl]benzonesulfonamide (TenBrink et al., 1996), JL18 (Liegeois et al., 1995), 2-naphthoate esters (Boyfield et al., 1996) and YM-43611 (Hidaka et al., 1996; Ohmori et al., 1996) have been described. The dopamine D4 receptor antagonistic activities and potencies of these compounds, however, have not been demonstrated in in vitro and in vivo functional studies. We report here the receptor binding and neuropharmacological activities of a novel dopamine D4 receptor antagonist, RA0045 (fig. 1). Materials and Methods Animals. Male ICR mice (20–35 g, Charles River, Atsugi, Japan) were housed 10 per cage. Male Wistar rats (150–260 g, Charles River, Atsugi, Japan) were housed three per cage and used for behavioral experiments and neurochemical studies. Male Hartley guinea pigs (150–200 g, Charles River, Atsugi, Japan) were housed three per cage and used for neurochemical studies. Animals were maintained under a 12-hr light/dark cycle (lights on 7:00 A.M.) in a temperatureand humidity-controlled holding room. Food and water were available ad libitum. All studies reported here have been reviewed by the Taisho Pharmaceutical Co., Ltd. Animal Care Committee and have met the Japanese Experimental Animal Research Association standards as defined in the Guidelines for Animal Experiments (1987). Human dopamine D4.2 receptor expression construct. The human dopamine D4.2 cDNA was cloned by RT-PCR. Total RNA was prepared from human neuroblastoma SK-N-MC cells by means of the acid guanidine-phenol/chloroform method described by Chomczynski and Sacchi (1987), and cDNA was synthesized using reverse transcriptase (SuperscriptII, BRL, Gaithersburg, MD, USA). The oligonucleotide primers used in the RT-PCR were 59-CGGAATTCCCGGGCGCGCCATGGGGAACCG-39 (sense) and 59-AAGGTACCTACAAAAGCGCCCTCCCCATCTCCTTG-39 (antisense). The PCR conditions were 1 min at 98°C, 1 min at 63°C and 4 min at 74°C for 35 cycles. The amplified cDNA, including the entire coding region of human dopamine D4.2 (an open reading frame encoding 387 amino acid residues) was then cloned into the expression vector pcDLDPE derived from pcDLSRa296 (Takebe et al., 1988). In this plasmid, the PstI site of pcDLSRa296 was converted to an EcoRI site by ligation of an EcoRI linker to its blunting termini, and the PstI-EcoRI short segment was deleted. Cell culture and transfection. COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 mg/ml streptomycin, in a CO2 incubator at 37°C. Full-length cDNA clones of human D4.2 ligated into pcDLDPE were transfected into COS-7 cells, using the Lipofectin (BRL, Gaithersburg, MD) procedure (Felgner et al., 1987). The cells were harvested after 72 hr by centrifugation at 400 3 g. The cell pellet was washed with phosphate-buffered saline and stored at 280°C until use. Membrane preparation. The cell pellet was homogenized with 50 mM Tris-HCl buffer containing 5 mM EDTA (pH 7.4) using an ultra-turrax T25 homogenizer (IKA-LabortechniK, Staufen, Germany), and centrifuged in a Hitachi 55P-72 centrifuge at 48,000 3 g for 20 min at 4°C. The supernatant was discarded, and the pellet obtained was rehomogenized with 50 mM Tris-HCl buffer containing 5 mM EDTA (pH 7.4) and re-centrifuged at 48,000 3 g for 20 min at 4°C. The final pellet was suspended in 50 mM Tris-HCl buffer containing 5 mM EDTA, 1.5 mM CaCl2, 5 mM KCl and 120 mM NaCl (pH 7.4) at a protein concentration of 0.3 mg/ml and was used as the membrane preparation. Protein concentration was determined by the method of Lowry et al., (1951) using the Folin phenol reagent. Receptor binding assays. The binding assay for dopamine D4 receptor was performed according to Van Tol et al. (1991). The membranes (0.5 ml) were incubated with [H]spiperone (0.5 nM) for 120 min at 27°C. NRA0045 or dopamine receptor-related compounds were included in the reaction mixture, simultaneously. The reaction was terminated by rapid filtration through Whatman GF/B glassfiber filters presoaked with 0.3% polyethyleneimine, after which the Fig. 1. Chemical structure of NRA0045. 1997 NRA0045 and Dopamine D4 Receptor 57 at A PE T Jornals on M ay 6, 2017 jpet.asjournals.org D ow nladed from filters were washed with 3 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4) three times. Nonspecific binding was determined in the presence of 10 mM haloperidol. Specific binding was defined by subtracting the nonspecific binding from the binding in the absence of haloperidol. For these steps, we used a multiple cell harvester M-24R (Brandel Biomedical Research and Development Laboratories, Inc., Gaithersburg, MD). Aquazol-2 scintillator (New England Nuclear, Wilmington, DE) (7 ml) was added, and filter-bound radioactivity was quantified in a liquid scintillation spectrometer (Beckman LS6000TA). Dopamine D2 receptor binding assay was performed using [H]spiperone (0.2 nM) and human cloned dopamine D2L in 50 mM Tris-HCl buffer containing 10 mM MgCl2 and 1 mM EDTA (pH 7.4) for 60 min at 27°C according to Lahti et al. (1993). Nonspecific binding was determined in the presence of 10 mM haloperidol. Selective bindings for several receptors, except for both dopamine D4 and D2, were carried out according to established protocols, and the methods are summarized in table 1. Spontaneous locomotor activity in mice. The animals were housed individually in transparent acrylic cages (47 3 28.5 3 29.5 cm), and spontaneous locomotor activity was recorded every 5 min for 60 min, using a SCANET apparatus (Neuroscience Inc., Tokyo, Japan) placed in a sound-proof box. Animals were given i.p. NRA0045 (0.3–3 mg/kg), clozapine (1–10 mg/kg), haloperidol (0.1–1 mg/kg), chlorpromazine (0.3–3 mg/kg) or an appropriate vehicle (10 ml/kg) and placed in individual cages 30 min later. Six groups of five mice for the vehicle and each of three dosages of drugs were used to generate dose-response reactions. The total count of vehicle-treated control group was defined as 100%, and the percent inhibition of each treatment group was calculated and ED50 values determined. MAP-induced locomotor hyperactivity in mice. The animals were housed individually in transparent acrylic cages (47 3 28.5 3 30 cm) and acclimatized for 90 min with a SCANET apparatus placed in a sound-proof box. Animals were given i.p. and p.o. NRA0045 (0.3–3 mg/kg i.p. and 1–10 mg/kg p.o.), clozapine (0.3–3 mg/kg i.p. and 1–10 mg/kg p.o.), haloperidol (0.1–1 mg/kg i.p. and 0.1–1 mg/kg p.o.), chlorpromazine (0.3–3 mg/kg i.p. and 1–10 mg/kg p.o.) or an appropriate vehicle (10 ml/kg) 15 min before the i.p. administration of MAP (1 mg/kg). Fifteen minutes later, locomotor activity was recorded every 5 min for 30 min using a SCANET apparatus placed in a sound-proof box. Six group of five mice for the vehicle and each of three dosages of drugs, were used to generate dose-response reactions. The total count for the vehicle-treated control group was defined as 100%, and the percent inhibition of each group was calculated and ED50 values determined. MAP-induced stereotyped behavior in mice. The animals were placed individually in transparent acrylic cages (24 3 17.6 3 12 cm) and allowed a minimum of 60 min to acclimatize to the new environment. The mice were given i.p. NRA0045 (3–30 mg/kg), clozapine (3–30 mg/kg), haloperidol (0.3–3 mg/kg), chlorpromazine (1–10 mg/kg) or an appropriate vehicle (10 ml/kg) 30 min before the i.p. administration of MAP (10 mg/kg). Ten minutes later, stereotyped behavior was scored every 10 min for 80 min, using the following scoring system: 0, normal behavior; 1, exploratory behavior and discontinuous sniffing; 2, continuous sniffing; 3, continuous sniffing, discontinuous licking, biting or gnawing; 4, continuous liking, biting and gnawing (Okuyama et al., 1993). Eight mice for the vehicle and each of three dosages of drugs were used to generate dose-response reactions. The total score for the vehicle-treated control group was defined as 100%, and the percent inhibition of each treatment group was calculated and ED50 values determined. Induction of catalepsy in rats. The animals were placed individually in transparent acrylic cages (36 3 30 3 17 cm) and allowed a minimum of 60 min to acclimatize to the new environment. The rats were given i.p. NRA0045 (3–30 mg/kg), clozapine (3–30 mg/kg), haloperidol (0.1–1 mg/kg), chlorpromazine (0.3–3 mg/kg) or an appropriate vehicle (10 ml/kg). Thirty minutes later, catalepsy was scored every 30 min for 90 min, using the following system: 0.5 (maximum score: 1), a posture with the right and left forelimbs on the right and left platforms 3 cm high was kept for 10 sec; 1 (maximum score: 2), a posture with the right and left forelimbs on the right and left platforms 9 cm high, kept for 10 sec (2 points were given only when the posture with both forelimbs on the platforms was kept for 30 sec) (Okuyama et al., 1993). Six rats for the vehicle and each of three dosages of drugs were used to generate dose-response reactions. The maximum score (18 points) was defined as 100%, and the percent induction of each group was calculated and ED50 values determined. Tryptamine-induced clonic seizure in rats. The animals were placed individually in clear acrylic cages (31 3 36 3 17.5 cm) and allowed a minimum of 45 min to acclimatize to the new environment. The rats were given i.p. NRA0045 (0.1–3 mg/kg), clozapine (1–10 mg/kg), haloperidol (0.3–3 mg/kg), chlorpromazine (1–10 mg/kg) or an appropriate vehicle (10 ml/kg) 30 min before the i.v. administration of tryptamine (20 mg/kg). The duration of tryptamine-induced clonic seizure was monitored. Six rats for the vehicle and each of 3 to 4 dosages of drugs were used to generate dose-response reactions. The duration for the vehicle-treated control group was defined as 100%, and the percent inhibition of each group was calculated and ED50 values determined. NE-induced lethality in rats. The animals were placed individually in a clear acrylic cage (36 3 30 3 17 cm) and allowed a minimum of 60 min to acclimatize to the new environment. The rats were given i.p. NRA0045 (0.03–1 mg/kg), clozapine (0.3–10 mg/kg), haloperidol (0.3–10 mg/kg), chlorpromazine (0.1–3 mg/kg) or an appropriate vehicle (10 ml/kg). Thirty minutes later, NE (1.25 mg/kg) was administered i.v. Inhibition of NE-induced lethality was judged to be positive unless death had occurred 30 min after NE administration. Ten rats for the vehicle and each of four dosages of drugs were used to generate dose-response reactions. The vehicle-treated control group was defined as 100%, and the percent inhibition of each treatment group was calculated and ED50 values determined. Statistical analysis. For determination of the equilibrium dissociation constant (Kd), saturation binding data were analyzed by Scatchard plot analysis, and the Kd values were calculated using a computer program, sp123, developed by Dr. H. Ono of the University of Tokyo for PC-9801 (NEC, Tokyo, Japan) personal computer. In the competition binding assay, the concentration of test compound that caused 50% inhibition of specific radiolabeled ligand binding (IC50 values) was determined from each concentration-response curve. After determination of IC50 values using the Marquardt-Levenberg nonlinear least-squares curve-fitting procedure of the MicroCal ORIGIN program (MicroCal, Northampton, MA) running on a Microsoft Windows 3.1, and Ki values for each test com-