D3 partial agonist and 5-HT1A agonist actions. I. Neurochemical effects

Neuropharmacology 39 (2000) 1197–1210 www.elsevier.com/locate/neuropharm

PD 158771, a potential antipsychotic agent with D2/D3 partial agonist and 5-HT1A agonist actions. I. Neurochemical effects H.C. Akunne a,*, K.T. Zoski a, M.D. Davis a, L.W. Cooke a, L.T. Meltzer a, S.Z. Whetzel a, Y.H. Shih a, D.J. Wustrow b, L.D. Wise b, R.G. MacKenzie a, L.M. Georgic a, T.G. Heffner a, T.A. Pugsley a a

Neuroscience Therapeutics, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, USA b Chemistry, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105, USA Accepted 20 October 1999

Abstract The neurochemical effects of a novel dopamine (DA) D2-like and serotonin (5-HT) 5-HT1A agonist, PD 158771, are described. PD 158771 exhibited affinities for human D2L, D3 and D4.2 receptors expressed in Chinese hamster ovary (CHO)-K1 cells with Ki (nM) values of 5.2, 13.7 and 34.8 respectively. PD 158771 showed high affinity for cloned human 5-HT1A (Ki =2.6 nM) and rat hippocampal 5-HT1A receptors (Ki =3.5 nM). Weaker affinities were observed at alpha 1-adrenergic (Ki =43 nM), histamine H1 (IC50 =30 nM), 5-HT2A (Ki =24.5 nM) and sigma (σ) ⫺1 binding sites (Ki =24.5 nM). In measures of in vitro functional activity, PD 158771 stimulated [3H]thymidine uptake in CHO p-5 cells transfected with hD3 receptors with a maximal effect of 23% relative to quinpirole. In hD2L, the corresponding value was 60% with an EC50 of 29 nM, again indicating partial DA agonist action of PD 158771. In vivo, PD 158771 produced a dose-related decrease in DA synthesis in the striatum and mesolimbic regions of rat brain treated with gamma-butyrolactone (GBL), indicating a DA autoreceptor agonist action. In animals not treated with GBL, PD 158771 produced a dose-related decrease in DA synthesis and extracellular DA. A decrease in 5-HT synthesis in several brain areas was observed consistent with an agonist response. Further support for DA autoreceptor agonist action is that PD 158771 produced a partial inhibition of the firing of substantia nigra zona compacta DA neurons, an effect reversed by haloperidol. In conclusion, PD 158771 exhibited affinities for DA and 5-HT receptors, appears to possess DA and 5-HT agonist actions; and it could provide improved antipsychotic profile with minimal side effects.  2000 Elsevier Science Ltd. All rights reserved. Keywords: DA receptors; Antipsychotic; Thymidine uptake; Autoreceptor; Haloperidol; Agonist

1. Introduction Psychosis is a mental disorder that remains one of the most desired for improved treatment. A number of schizophrenics are refractory to current available antiAbbreviations: DA, dopamine; 5-HT, serotonin; GBL, gamma-butyrolactone; NE, norepinephrine; DOPA, L-dihydroxyphenylalanine; EPS, extrapyramidal side effects; 8-OH-DPAT, 8-hydroxy-2-(di-npropylamino) tetralin; SDZ 208-912, N-[(8-α)-2-chloro-6-methylergoline-8-yl]-2,2-dimethylpropanamide; CI-1007, R-(+)-1,2,3,6-tetrahydro-4-phenyl-1[(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine maleate; PEI, polyethylenimine; GTP, guanosine 5⬘-triphosphate; Gpp(NH)p, guanyl 5⬘,6⬘-imidodiphosphate. * Corresponding author. Tel.: +734-622-2830; fax: +734-622-2855. E-mail address: [email protected] (H.C. Akunne).

psychotics. Moreover, conventional antipsychotics, mostly DA D2 agents, cause extrapyramidal side effects (EPS) which present a problem for the patients and often limits compliance. Antagonism of D2 receptors in DA-rich mesolimbic and mesocortical brain areas has been related to the antipsychotic efficacy of neuroleptics whereas antagonism of D2 receptors in striatum has been thought to be associated with the EPS side effects (Seeman et al., 1976). Therefore, the rationale for the use of DA D2 antagonists was based primarily on the fact that dysfunctions of the dopaminergic pathways are implicated in schizophrenia. In addition, DA D3 and D4 receptors, because of their preferential distribution in the limbic region as compared to the striatal regions of brain, are also an interesting

0028-3908/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 9 9 ) 0 0 2 2 4 - 5

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target for new antipsychotic drugs (Bouthenet et al., 1991). For example, clozapine, which shows improvement in both the positive and negative symptoms of schizophrenia with a low incidence of EPS, exhibits high affinity binding to D4 receptors (Seeman and Van Tol, 1994). In addition, low intrinsic activity DA agonists, such as SDZ 208-912, have been reported to be efficacious in schizophrenia (Coward et al., 1990; Naber et al., 1992); and (-)-3PPP, a partial DA agonist, showed a significant decrease in psychotic symptoms (Lahti et al., 1998). Furthermore, DA partial agonists are selective for presynaptic autoreceptors and this is thought to be a reflection of their relatively low intrinsic activity (Carlsson, 1983). This concept may explain the improvement in schizophrenia treatment observed after low doses of apomorphine and N-propyl-norapomorphine (Hollister et al., 1980; Taminga et al., 1986). Agonists to 5-HT1A receptors, 8-hydroxy-2-(di-npropylamino)tetralin (8-OH-DPAT) and ipsapirone, have been shown to antagonize neuroleptic-induced catalepsy (Broekkamp et al., 1988; Wadenberg and Ahlenius, 1991). Furthermore, low dose administration of 8-OH-DPAT produces a suppression of avoidance behavior (a rodent model for antipsychotic action), an effect that disappears with increasing doses. It appears that an agent possessing partial agonist activity in the DA and 5-HT systems might be of benefit in the treatment of psychotic disorders. Specific interactions between the catecholaminergic and serotoninergic systems in mediating neurolepticinduced effects and extrapyramidal dysfunction have been reported (Broekkamp et al., 1988; Millan et al., 1995; Wadenberg et al., 1994; Wadenberg and Ahlenius, 1991). A number of theories have postulated that clozapine’s 5-HT2A antagonist activity plays a role in its distinct therapeutic profile (Meltzer, 1989; Meltzer and Nash, 1991). This has led to a new class of 5-HT2A/DA receptor antagonists (for example, ritanserin) for the treatment of schizophrenia that show equivalent efficacy to currently available therapeutic agents but produce less EPS. Furthermore, interaction between a 5-HT1A agonist and a typical DA D2 antagonist was reported to enhance antipsychotic-like effects and reduce catalepsy in rats (Wadenberg and Ahlenius, 1991). PD 158771 (trans-{4-[2-(4-phenyl-piperazin-1-yl)ethyl]-cyclohexyl}-pyrimidin-2-yl-amine) was synthesized (Wustrow et al., 1998) as a potential atypical (i.e. low EPS liability) antipsychotic agent. This report focuses on the biochemical and electrophysiological profile of PD 158771, which exhibited partial agonist actions at DA D2, D3 receptors and agonist action for 5HT1A receptors. The behavioral profile showing activities consistent with antipsychotic efficacy are presented in an accompanying paper (Corbin et al., accompanying manuscript) (Fig. 1).

Fig. 1. Structure of PD 158771: trans-{4-[2-(4-phenyl-piperazin-1yl) -ethyl]-cyclohexyl}-pyrimidin-2-yl-amine.

2. Materials and methods 2.1. Drugs All radioactive compounds were purchased from various commercial sources. The following compounds were gifts from the respective companies: haloperidol (McNeil Pharmaceuticals, Spring House, PA), SCH23390 (Schering Corporation, Kenilworth, NJ), SDZ 208-912 (Sandoz Inc., East Hanover, NJ) and apomorphine HCl (Merck and Co., Rahway, NJ). Gamma-butyrolactone (GBL), 3-hydroxybenzyl-hydrazine dihydrochloride (NSD 1015) (Sigma Chemical Co., St. Louis, MO), 8-OH-DPAT and quinpirole HCl (Research Biochemicals Int., Natick, MA) were purchased. Talipexole (BHT90), EMD 38362, PD 158771 and CI-1007 were synthesized at the Department of Chemistry, Parke-Davis Pharmaceutical Research (Ann Arbor, MI). Tissue culture reagents were obtained from Gibco (Grand Island, NY). Rat brain 5-HT1A, 5-HT6 and 5-HT7 cloned receptors were purchased from Receptor Biology, Inc. (Baltimore, MD). PD 158771 is a white crystalline solid with a solubility of 0.008 mg/ml at pH 7.3. For in vitro studies, the compound was dissolved in 1% dimethyl sulfoxide (DMSO), and for in vivo studies, PD 158771 was dissolved in physiological solution. 2.2. Animals Male Long-Evans rats (180–250 g) were used for the neurochemical studies. In the microdialysis and electrophysiology experiments, Sprague-Dawley rats (250–350 g) were used. All animals were maintained under controlled environmental conditions with a 12:12 h lightdark cycle in accordance with guidelines established by the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 80-23, revised 1978); and American Association for the Accreditation of Laboratory Animal care. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques, when available. Free access to water and food (pellets) was provided. Frozen brain tissues

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from rat hippocampal (for 5-HT1A receptors), rat cortical (for alpha 1-adrenergic receptors) and rat striatal (for DA D1 receptors) regions were purchased from Analytical Biological Services Inc. (ABS, Wilmington, DE). 2.3. Tissue culture Transfection of human DA receptors and tissue culture preparation were carried out as previously published (Akunne et al., 1995; Chio et al., 1994; MacKenzie et al., 1994). CHO-K1 cells expressing the human D4.2 receptor were also prepared as previously described (Shih et al., 1997). M1, M2, M3, M4 and M5 human cDNA transfected into CHO cells were also prepared by the standard cell culture technique and membranes appropriately harvested for the studies. 2.4. Membrane preparation 2.4.1. Cells Confluent cell cultures were harvested by replacement of medium with cold phosphate-buffered saline (PBS) containing 0.05% EDTA followed by centrifugation at 1000g for 2 min. Pellets obtained were suspended in an appropriate volume of ice-cold TE buffer (25 mM TrisHCl, 1 mM EDTA, pH 7.4) and centrifuged at 20 000g for 15 min at 4°C. The final pellets obtained were homogenized with a Polytron (Brinkman Instruments, Westbury, NY) at setting 6, for 5 s, for use in radioligand binding assays or stored at ⫺80°C. Protein content for the membranes and tissues for use in the assays ranged from 15–61.5 µg of protein per assay tube. 2.4.2. Membranes Frozen tissue membranes were placed on ice to thaw slowly on the day of experiment. Tissues were also processed as with harvested cells described previously. The pellet from tissue for DA D1 assay was resuspended as above and the homogenate was incubated at 37°C for 10 min, then set on ice for 45 min. This was then centrifuged as previously described. The final pellets were resuspended in the appropriate volume of Tris-HCl buffer (50 mM, pH 7.7 at 20°C) for subsequent use in each binding assay. Protein was determined by the Bradford assay (1976) using the microplate reader (BIORAD) for analysis. Experimental compounds were made up as stock solutions in DMSO. The final concentration of 0.1% DMSO used in the incubation mixture had no effect on specific binding of any of the receptor radioligand binding studies. Every observation was carried out in triplicate. 2.5. Receptor binding studies 3

2.5.1. [ H]Spiperone binding to CHO-K1 cells Frozen aliquots of membrane preparations from CHO cells expressing the human DA D2L, D3 or D4.2 receptors

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were used within one month of being stored at ⫺80°C. The assay was essentially as reported previously (Akunne et al., 1995) with minor modifications. Briefly, 50 µl of [3H]Spiperone (0.2 nM for D2 and D4.2 or 0.5 nM for D3, final concentration), were added to polypropylene microtubes (Marsh Biomedical Products, Inc., Rochester, NY) along with 50 µl of either test drug (eleven different concentrations), nonspecific or AB (assay buffer, 25 mM Tris-HCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM EDTA, 120 mM NaCl, and 6 mM MgCl2, pH 7.4 at 37°C), and 400 µl of membrane preparation for a total volume of 500 µl. The incubation time was 60 min. The nonspecific ligand used was 1 µM haloperidol yielding about 10% of total binding. Incubation was terminated by rapid filtration which was followed by four washes (4 ml of AB) for each assay. Filtration was carried out using a Brandel MB-48R (Biomedical Research and Development, Inc., Gaithersburg, MD) and in some cases the filters were soaked in 0.5% polyethylenimine (PEI, Sigma Chemical Company, St. Louis, MO) for at least 1 h. A Brandel MLR96 cell harvester was also used where appropriate. Following the addition of liquid scintillation cocktail, radioactivity remaining was counted with either a Beckman LS 6800 liquid scintillation counter (for 48-well filters) or a Wallac 1205 Betaplate (for 96-well filters) at efficiency of 45 and 50% respectively. 2.5.2. [3H]N-0437 binding to CHO-K1 cells For DA D2A (agonist) binding using membranes from CHO-K1 cells transfected with the cDNA for DA D2 receptor, [3H] N-0437 (1.0 nM, final concentration) was used in the presence of 1 µM (+)butaclamol as nonspecific ligand which represented about 15% of the total binding. The standard protocol presented above for DA binding was modified to exclude sodium in the buffer (assay buffer: 25 mM Tris-HCl, 1mM EDTA and 6 mM MgCl2 (TEM buffer), pH 7.4 at 37°C). The total incubation volume was also 500 µl. 2.5.3. [3H]8-OH-DPAT binding to SF9 cells Competition binding was conducted in membranes prepared from SF9 cells transfected with the cDNA for 5-HT1A receptors using 0.3 nM (final concentration) of [3H]8-OH-DPAT. The standard protocol described by Peroutka (1985) was slightly modified. The buffer consisted of 50 mM Tris-HCl, 0.1% ascorbic acid, 0.5 mM EDTA and 10 µM pargyline at pH 7.7. Incubation time was 30 min at 25°C and nonspecific binding was defined as the amount of [3H]8-OH-DPAT binding in the presence of 1 µM 8-OH-DPAT. This represented about 15% of total binding. 2.5.4. [3H]SCH 23390 binding to rat striatal membranes The affinity of PD 158771 to DA D1 receptor was determined using rat striatal membranes. Membrane

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weight averaged 3 mg original tissue/ml and 0.3 nM, final concentration of the [3H]SCH 23390 was used. The buffer consisted of 25 mM Tris-HCl, 5 mM KCl, 1.5 mM CaCl2, 1 mM EDTA, 120 mM NaCl, and 6 mM MgCl2, pH 7.4 at 37°C. Incubation was for 15 min at 37°C and nonspecific binding was defined as the amount of [3H]SCH 23390 binding in the presence of 10 µM SCH 23390. This protocol was a modification of that described by Billard et al. (1984). 2.5.5. [3H]Ketanserin binding to rat cortical membranes The rapid filtration protocol described by Leysen et al. (1982) was used to characterize the 5-HT2A receptor. The assay buffer consisted of 50 mM Tris-HCl, 10 µM pargyline, 4 mM CaCl2 and 0.1% ascorbic acid, pH 7.7. [3H]Ketanserin final concentration was 1 nM, at 5 mg original tissue concentration per ml. Incubation time was for 30 min at 25°C and nonspecific binding was defined as the amount of [3H]ketanserin in the presence of 10 µM methysergide which represented about 20% of total binding. 2.5.6. [3H]LSD binding to rat 5-HT6 and 5-HT7 receptors expressed in human embryonic HEK 293 cells Minor modifications of the rapid filtration method described by Monsma et al. (1993) were used in the present study. Frozen membranes suspended in 10 mM Tris HCl, 2 mM EDTA, pH 7.2 were thawed rapidly and diluted with assay buffer which consisted of 50 mM Tris HCl, 10 mM MgCl2 and 0.5 mM EDTA, pH 7.4. This was homogenized with a Polytron at setting 5 for 5 s. Pellets obtained were then suspended in assay buffer at tissue concentration of 61.5 µg (5-HT6) and 15.89 µg (5-HT7). This membrane preparation (100 µl) was then added to assay tubes with or without drug, 100 µl of [3H] LSD (final concentrations, 5 and 4 nM for 5-HT6 and 5-HT7, respectively) and 100 µl of 1 µM 5-HT to define nonspecific binding. The total volume was 1 ml for each tube. After incubating for 60 min at 37°C, assay was terminated by rapid filtration through Whatman GF/C glass fiber filters (soaked for 1 h in 0.5% PEI) on a Brandel MB-48R cell harvester, followed by 3 washes of 5 ml ice-cold 50 mM Tris HCl, pH 7.4. Individual filter discs containing the bound ligand were placed in counting vials with 5 ml of scintillation fluid (Ready Gel, Beckman Instrument Inc., Fullerton, CA) and counted using a Beckman LS-6800 liquid scintillation counter at 45% efficiency. 2.5.7. [3H]NMS binding to muscarinic receptors expressed in CHO cells Membrane from CHO cells stably transfected with human muscarinic receptor subtypes M1, M2, M3, M4 and M5 and processed as described before, were used.

For the binding assay, aliquots of cell membranes (10– 40 µg) were added to 2 ml of buffer containing appropriate concentrations of test compounds and 0.1 nM [3H]NMS. Nonspecific binding was determined using 1 µM atropine. Incubations were carried out for 120 min at 25°C with constant shaking. These were terminated by rapid vacuum filtration through GF/B Whatman filters using Brandel cell harvester. Filters were washed 3 times with 5 ml of ice-cold buffer, placed in vials with 10 ml of Beckman Ready Gel scintillant, and then allowed to sit overnight before counting. Additional receptor binding studies were undertaken to determine the selectivity of PD 158771 (Pugsley et al., 1995a). Inhibition of binding of [3H]ligand to each receptor (final concentration), brain area, nonspecific agents (final concentration) and methods were carried out as follows: alpha 1-adrenergic, [3H]prazosin (0.1 nM), rat cortex and phentolamine (1 µM) and alpha 2adrenergic, [3H]MK-912 (0.5 nM), rat cortex and yohimbine (10 µM). 2.6. Mitogenesis assay The effect of PD 158771 on [3H]thymidine uptake was carried out as described previously (Pugsley et al., 1995b). CHO p-5 cells transfected with hD2L and hD3 cDNA were seeded into 96-well plates at a density of about 5×105 cells/well. These were grown for 2 days at 37°C in an incubator with alpha minimum essential medium (αMEM) and 10% fetal calf serum containing penicillin (100 U/ml) and streptomycin (100 µg/ml). The wells were then rinsed with serum-free media and 90 µl of fresh media were added along with 10 µl of test agents (quinpirole, apomorphine, PD 158771) in vehicle or vehicle alone. After culture for 16–17 h, [3H]thymidine (0.25 µCi/well) was added for 4 h. Cells were trypsinized and harvested onto filtermats using a Brandel MLR96 cell harvester. Liquid scintillant was added and radioactivity remaining was counted on the Wallac 1205 Betaplate liquid scintillation counter. 2.7. Biogenic amine studies Interactions with presynaptically located striatal D2like receptors were determined with gamma butyrolactone (GBL) which was used to reduce neuronal activity within the nigrostriatal and ventral tegmental mesolimbic pathways (Walters and Roth, 1976). In this model, test compounds were administered at 1 h with GBL (750 mg/kg, i.p.) and 3-hydroxybenzyl-hydrazine dihydrochloride (NSD 1015, 100 mg/kg, i.p.) dosed 30 min before animals were sacrificed. NSD-1015 was used to inhibit L-aromatic amino acid decarboxylase activity to allow measurement of L-3, 4-dihydroxyphenylalanine (DOPA) synthesis. In another set of studies, the synthesis rate of rat brain

H.C. Akunne et al. / Neuropharmacology 39 (2000) 1197–1210

catecholamines and 5-HT was estimated by measuring the accumulation of DOPA and 5-hydroxytryptophan (5HTP) following treatment with NSD-1015. This test is usually a reflection of drug actions at both pre- and postsynaptic neurotransmitter sites (Carlsson, 1983). Test compounds were administered at 1 h and NSD 1015 (100 mg/kg. i. p.) 30 min before animals were sacrificed. In all experiments, rats were sacrificed by decapitation. The brains were rapidly removed and placed on an ice-cold plate for dissections of the striatum, nucleus accumbens plus olfactory tubercle (mesolimbic region), brainstem and cortex. Tissue samples were then used for assay or were frozen on dry ice and stored at ⫺70°C for no more than a month before use. Concentrations of DOPA and 5-HTP in tissue samples were determined concurrently by using HPLC with electrochemical detection (Pugsley et al., 1995a,b). 2.8. Microdialysis Measurements of extracellular DA, DOPAC, 5-HT, 5HIAA of rat striatum and nucleus accumbens were determined following treatment with PD 158771 and the protocol was described before (Pugsley et al., 1995a). Briefly, male adult rats were anesthetized with 1.5 mg/kg (i.p.) urethane and mounted in a stereotaxic frame (Kopf). Anesthesia was assessed by the absence of corneal reflexes. Others have also shown that anesthesized animals can provide reliable data and that steady state amine levels can be achieved (Auerbach et al., 1995). In this study, the core body temperatures of the rats were maintained at 37°C for the duration of the experiment. Verification of probe placement was confirmed via postmortem microtome tissue sectioning. The recovery rate with the dialysis probe was determined to be approximately 15%. Data are reported as percent change from baseline values, which were mean of the three samples proceeding treatment. 2.9. Electrophysiology: neuronal firing Adult male rats were anesthetized with chloral hydrate (400 mg/kg, i. p.) and mounted in a stereotaxic apparatus as described previously (Meltzer et al., 1995). The body temperature was maintained at 36.5–37.5°C throughout the experiment. A burr hole was drilled in the skull over the substantia nigra (A9) neurons. Experiments were always conducted with neurons that met specific electrophysiological characteristics as midbrain DA neurons (Grace and Bunney, 1983). The activity of each DA neuron was monitored for 5 min before drug injection. Baseline firing rate was calculated by averaging the rate over the 2 min before the first drug injection. Drugs were injected via a cannulated tail vein at 2 min intervals, with each dose doubling the preceding cumulative total. Drug effects were determined by calculating the 60 s

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period of maximal change in rate after injection. This was expressed as a percentage change from the base-line firing rate. At the end of each experiment, the recording site was marked by passing ⫺25 µA of current through the electrode for a minimum of 10 min, which resulted in the deposition of blue dye at the pipette tip. The brain was removed and fixed in 10% buffered formalin. Serial frozen sections, 75 µm thick, were cut and examined under a light microscope for location of the dye spot. This confirmed that all recording sites were located within the substantia nigra zona compacta. 2.10. Data analysis For the receptor binding results, data from saturation plots were analyzed by Scatchard (1949) transformation of binding data using the LIGAND program (Munson and Rodbard, 1980). The inhibition constant (Ki) was also estimated using the iterative curve-fitting LIGAND program. ED50 or EC50 values from functional studies were calculated from log dose-effects by nonlinear regression with a two-parameter (slope, EC50/ED50) sigmoidal curve-fitting program. For the neurochemical studies, ED50 were also calculated from log-dose effect functions and statistical comparisons were made with the use of analysis of variance followed by Newman-Keuls test was performed to determine any significant differences between treatments.

3. Results 3.1. Receptor binding studies PD 158771 bound to a number of DA and 5-HT receptor subtypes. A summary of its affinities for various systems in the CNS is presented in Table 1 and Fig. 2. As expected of a D2 agonist, PD 158771 exhibited higher affinity for D2L with agonist radioligand, [3H]N-0437 at 5.2 nM than an affinity of 42 nM for D2L receptors labeled with DA antagonist ligand, [3H]spiperone (Table 1). PD 158771 also exhibited affinities for D3 and D4 receptors expressed in CHO-K1 cells with respective Ki values for displacement of [3H]spiperone of 13.7±3.7 nM and 35±3.7 nM. In all cases, PD 158771 displaced [3H]spiperone in a monophasic fashion in agreement with the slope factors which were close to unity. In contrast to its high affinity for D2-like receptors, PD 158771 had only weak affinity for rat striatal D1 receptor (Ki =2596 nM). The affinities of PD 158771 for 5-HT1A receptors using rat hippocampus and SF9 cells transfected with the h5-HT1A receptor were comparable at 3.5 and 2.6 nM, respectively. PD 158771 also bound to 5-HT2 receptors in rat cortex and to 5-HT7 receptors (in HEK 293 cells) with Ki values of 29 and 119 nM, respectively. PD

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Table 1 Receptor binding profile of PD 158771a Receptor

[3H]Ligand

Tissue/cells

Ki (nM ± S.E.M.)

hDA D2L hDA D3 hDA D4.2 hDA D2L DA D1 5-HT1A

N-0437 Spiperone Spiperone Spiperone SCH 23390 8-OH-DPAT

5.15±1.37 13.7±3.66 34.8±3.76 42.0±8.41 2596±357 3.5±0.8

h5-HT1A 5-HT2A r5-HT6 r5-HT7 Alpha 1 Alpha 2 hMuscarinic hMuscarinic hMuscarinic hMuscarinic hMuscarinic

8-OH-DPAT Ketanserin LSD LSD Prazosin MK-912 NMS NMS NMS NMS NMS

CHO K1 cells CHO K1 cells CHO K1 cells CHO K1 cells Rat Striatum Rat hippocampus SF9 Rat cortex HEK 293 cells HEK 293 cells Rat brain Rat brain CHO K1 cells CHO K1 cells CHO K1 cells CHO K1 cells CHO K1 cells

M1 M2 M3 M4 M5

2.6±0.5 24.5±3.6 ⬎10000b 119±18 43±9.9 194±24 548±30 1016±99 4980±153 1240±120 1027±298

a In all experiments, at least nine different concentrations of each compound was studied. Values represent means of 3 to 4 separate experiments. Experiments were carried out as described in the “Materials and methods” section. b IC50 ⬎10000 nM (⬍50% inhibition). h = Cloned human receptor. r = cloned rat receptor.

Fig. 2. Representative competition of PD 158771 for radioligand binding at D2L, D3 and 5-HT1A receptors. Membranes from CHO-K1 transfected with D2L and D3 receptors and SF9 cells transfected with 5-HT1A were incubated with [3H]spiperone (0.2 nM for D2L receptors and 0.5 nM for D3 receptors) or 0.83 nM of [3H]8-OH-DPAT for 5HT1A receptors and varying concentrations of PD 158771. The percent specific bound is plotted, and values shown are for triplicate determinations of binding for a single experiment (S.E.M. less than 10%).

158771 had no activity for 5-HT6 receptors expressed in HEK 293 cells. It exhibited affinities for rat brain alpha 1- (Ki =43 nM) and alpha 2-adrenergic receptors (Ki =194 nM) but showed weak affinity for the human muscarinic M1 receptors (Ki =548 nM) with little or no affin-

ities for other cloned human muscarinic receptors (M2, M3, M4 and M5) (Table 1). Further studies with PD 158771 conducted by Panlabs, Inc. (Seattle, WA) confirmed the weak affinities of PD 158771 for rat whole brain alpha 1-, alpha 2-adrenergic and muscarinic M1 receptors as reported (Table 1). In addition, PanLabs data on PD 158771 showed binding affinities to histamine H1 (Ki =10 nM), s-1 receptor (Ki =8 nM), and sodium channel-2 binding sites (Ki =2691 nM) with no activity for a number of other neurotransmitter/neuromodulator sites that included angiotensin AT1, bradykinin B2, cholecystokininA, cholecystokininB, endothelin ETA and ETB, galanin, histamine H3, kainate, leukotriene B4, neurokinin NK1, neuropeptide Y2, NMDA, phencyclidine, 5-HT3 and thromboxane. The compound was also inactive in selected tests for inhibition of various enzymes that included calcineurin, calpain, EGF tyrosine kinase, nitric oxide (constitutive and inducible), protein kinase Calpha and Cbeta. 3.2. Mitogenesis assay Additional studies were carried out to identify agonist or antagonist effects of PD 158771 at D2L and D3 receptors expressed in CHO-p5 cells. Agonist stimulation of D2L and D3 receptors results in stimulation of [3H]thymidine uptake (Chio et al., 1994). PD 158771 stimulated [3H]thymidine uptake in CHO-p5 cells expressing either hD2L (EC50, 29 nM) and hD3 receptors (maximal effect of 23%) (Table 2, Fig. 3). The reference agents, quinpirole and apomorphine, exhibited 100% maximal effect. In comparison, PD 158771, like CI-1007 and SDZ 208912 (Pugsley et al., 1995a) expressed partial agonist action at both receptors with maximal effects of 60, 66 and 19% respectively. Quinpirole, which was used as the reference agent, exhibited EC50 value of 5 nM for D2L receptors.

Table 2 EC50 values and maximal effect for stimulation of [3H]thymidine uptake into CHO-p5 cells relative to full agonist, quinpirolea Test agent

EC50, nM (D2L)

*ME (%) EC50, nM (D3)

ME (%)

Quinpirole PD 158771 SDZ 208-912 CI-1007 Apomorphine

5±1 29±6 0.6±0.2 1.3±0.5 26±7

100±0 60±6 19±7 66±9 99±5

100±0 23±7 14±4 84±8 100±5

2.6±1 2.8±1.3 ND 4.5±1.2 84±8

*ME = maximal effect. ND = Not Determined. Experiments were conducted as described in the “Materials and methods” section. Values represent the mean ± S.E.M. of three experiments. Quinpirole was used as the reference agonist (D2/D3). Intrinsic activity was expressed relative to quinpirole (100% at 1 µM). a

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Fig. 3. Representative curves for stimulation of [3H]thymidine uptake by PD 158771. Values represent mean ± S.E.M. of three experiments each with triplicate observations for each concentration point. Intrinsic activity was expressed relative to quinpirole (100%).

3.3. Biogenic amine studies The ability of PD 158771 to activate functionally defined DA autoreceptors was demonstrated in the GBLinduced DOPA accumulation test. PD 158771 dosedependently (1.0–30 mg/kg, p.o.) decreased DOPA accumulation in various rat brain regions with ED50 values of 14 and 12.5 mg/kg for the striatal and mesolimbic systems, respectively (Table 3). A comparison with several reference compounds revealed that PD 158771 Table 3 Reversal of the GBL-induced DOPA accumulation by PD 158771 in rat striatum and mesolimbic regiona Treatment

Striatum No GBL GBL PD 158771 PD 158771 PD 158771 PD 158771 Mesolimbic No GBL GBL PD 158771 PD 158771 PD 158771 PD 158771

Dose (mg/kg)

– – 1 3 10 30 region – – 1 3 10 30

DOPA

% Reversal

1517±122 4245±192 4237±352 3475±508 3003±157b 2578±76b

– – 0.2 28 45 61

1030±88 1715±59 1680±74 1628±123 1397±79b 1228±95b

– – 5 12 46 71

ED50 (mg/kg)

was less potent than CI-1007, EMD 38362 and several other DA agonists (Table 4). The effect of PD 158771 was significantly different from that of apomorphine. The submaximal effects of PD 158771 in striatum were consistent with a partial DA agonist compound. In support of the above findings, PD 158771 dosedependently decreased DOPA accumulation in the DAenriched striatum at 10 and 30 mg/kg (i. p.) and in mesolimbic brain regions at 3, 10 and 30 mg/kg (i.p.) without GBL pretreatment (Table 5). PD 158771 had no significant effect on DOPA accumulation in the norepinephrine (NE)-enriched frontal cortex and brainstem regions. Similarly, PD 158771 (0.3–30 mg/kg, i. p.) decreased 5HT synthesis (as assessed by the accumulation of 5HTP) in various brain regions in a dose-dependent manner (Table 6). PD 158771 significantly decreased 5-HT Table 4 Reversal of the GBL-induced DOPA accumulation by PD 158771 and various DA agonists in rat striatuma

14.0

12.5

Test compound ED50 (mg/kg, i.p.)

Maximal effect (% reversal)

PD 158771 CI-1007 Talipexole Apomorphine EMD 38362 SDZ 912

61±5 % @ 30 mg/kg 59±5% @ 30 mg/kg 95±3% @ 0.3 mg/kg 93±8% @ 4 mg/kg 87±4% @ 30 mg/kg 6±2% @ 10 mg/kg

a

a

Mesolimbic region — Nucleus accumbens and olfactory tubercle. PD 158771 was administered (p.o.) 30 mins before combined treatment with GBL (750 mg/kg, i.p.) and NSD 1015 (100 mg/kg, i.p.) given 30 mins before animals were sacrificed. Each value is mean ± S.E.M. of at least 4 animals. ED50 values were determined graphically. b P ⬍0.05 versus control group (GBL, no drug).

7.0 3.0 0.02 0.24 5.4 ⬎10

PD 158771 was compared with other reference agents for partial agonist property. Drugs were administered (i.p.) 30 mins before combined treatment with GBL (750 mg/kg, i.p.) and NSD 1015 (100 mg/kg, i.p.) given 30 mins before animals were sacrificed. Each value is mean ± S.E.M. of at least 4 animals. ED50 values were determined graphically and maximal effect (% reversal) from control (GBL) was also determined.

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Table 5 Effects of PD 158771 on DA synthesis (DOPA accumulation) in various rat brain regionsa Treatment

Control PD 158771 PD 158771 PD 158771 PD 158771

Dose (mg/kg, i.p.)

– 0.3 3 10 30

DOPA (% Control ± S.E.M.) Striatum

Mesolimbic

Frontal cortex

Brainstem

100±1 93±8 115±7 79±5b 54±5c

100±2 100±5 82±2b 71±3c 63±8c

100±5 88±6 98±6 102±4 107±6

100±4 – 96±7 100±9 94±11

a PD 158771 was administered 30 min before NSD 1015 (100 mg/kg, i.p.). Animals were sacrificed 30 min following NSD 1015 treatment. Each value is mean of 4 to 12 animals and is expressed as percent of control values that were (ng/g ± S.E.M.): striatum, 1221±100; mesolimbic, 926±94; frontal cortex, 126±10; brainstem and 143±6. The ED50 values were extrapolated as 6.5 and 7.5 mg/kg (i. p.) for the striatum and mesolimbic regions respectively. b P ⬍0.01. c P ⬍0.05, versus control group.

Table 6 Effects of PD 158771 on 5-HT synthesis (5-HTP accumulation) in various rat brain regionsa Treatment

Control PD 158771 PD 158771 PD 158771 PD 158771

Dose (mg/kg)

– 0.3 3 10 30

5-HTP (% Control ± S.E.M.) Striatum

Mesolimbic

Frontal cortex

Brainstem

100±3 92±6 85±2b 55±4c 69±4c

100±2 108±5 80±5b 69±6c 78±3c

100±3 80±11 78±4b 73±3b 58±3c

100±4 – 98±5 87±11 79±7b

a PD 158771 was administered 30 min before NSD 1015 (100 mg/kg, i. p.). Animals were sacrificed 30 min following NSD 1015 treatment. Each value is mean of 4 to 12 animals and is expressed as percent of control values that were (ng/g ± S.E.M.): striatum, 226±27; mesolimbic, 461±39; frontal cortex, 172±23 and brainstem, 156±9. b P ⬍.05, versus control group. c P ⬍.01.

synthesis in rat striatum, mesolimbic and frontal cortex regions at 3, 10 and 30 mg/kg and at 30 mg/kg in the brainstem. A comparison of the effects of 8-OH-DPAT and PD 158771 on 5-HT synthesis in the striatum (Fig. 4) showed maximal effect occurring at 1 to 3 mg/kg (65% and 59% reversal, respectively) for 8-OH-DPAT. For PD 158771, the maximal effect occurred at 10 to 30 mg/kg (55% and 69% reversal, respectively). The estimated ED25 values (dose that decreases 5-HTP by 25%) were 4.5 and 0.24 mg/kg for PD 158771 and 8-OHDPAT, respectively. 3.4. Microdialysis measurements In intracerebral microdialysis, the administration of PD 158771 at 10 mg/kg, i.p. caused a significant decrease (35–50%) in DA overflow in the rat nucleus accumbens and the striatum (Fig. 5a). The effects in nucleus accumbens were evident 20 min after dosing and lasted for up to 120 min post treatment. In the same paradigm, the full DA agonist, apomorphine, caused significant decreases in extracellular DA levels in the stria-

tum and the nucleus accumbens (Fig. 5b). This change was evident 40 min post treatment and lasted for up to 2 h after dosing. PD 158771 (10 mg/kg, i.p.) decreased extracellular 5-HT levels in nucleus accumbens with the effects lasting up to 150 min after dosing (Fig. 6). 3.5. Electrophysiology: neuronal firing Another mechanism by which DA autoreceptor agonists decrease dopaminergic neurotransmission is by inhibiting the firing of brain DA neurons through direct actions on DA D2/D3 receptors located on cell bodies and dendrites. Intravenous administration of PD 158771, in a cumulative dose manner, produced a dose-related and haloperidol reversible inhibition of substantia nigra DA neurons (Fig. 7). PD 158771, had a profile that was similar to that of CI-1007 but differed from that of the high intrinsic activity agonist, apomorphine and the low intrinsic activity agonist SDZ 208-912. Relatively high doses of PD 158771 and CI-1007 produced maximal inhibition of approximately 75%. In contrast, apomorphine produced a near complete inhibition of 96%, while

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Fig. 4. Effect of PD 158771 and the 5-HT1A agonist 8-OH-DPAT on 5-HT synthesis (5-HTP accumulation) in rat striatum. Both compounds were administered 30 min before NSD 1015 (100 mg/kg, i.p.) and animals were sacrificed 30 min after the NSD 1015. Each value is mean of 4–12 determinations and is expressed as a percentage of control values which were 201±20 ng/g ± S.E.M. **P ⬍.01, *P ⬍.05, versus control group.

SDZ 208-912 produced a maximal inhibition of only 27%. The average baseline firing rate of DA neurons in these different groups ranged from 4.5 to 6.2 spikes/s. Thus PD 158771 produced a partial inhibition of these DA neurons. Agonist effects of all compounds were reversed by haloperidol (data not shown).

4. Discussion One of the more promising advancements in schizophrenia research has been the discovery of the antipsychotic, clozapine. Clozapine has shown superior clinical efficacy for treating a percentage of schizophrenics that are non-responsive to conventional antipsychotics. An added advantage of clozapine is the lack of any substantial EPS liability, so prevalent with classical antipsychotics (Meltzer, 1989). However, clozapine has other adverse effects (e.g. granulocytopenia) which necessitate further research for agents with improved benefit/risk ratios. Although the DA system and especially the D2 receptors, have been implicated in the pathophysiology of psychosis, dysregulation of 5-HT receptors activity at 5-HT neurons has also been associated with the pathophysiology of several psychiatric disorders (Meltzer, 1995). Compounds blocking both dopaminergic and serotoninergic systems (e.g. DA D2 and 5-HT2A receptor antagonists such as ritanserin and clozapine) may be superior to classical antipsychotics in the treatment of psychiatric disorders, in that they retain efficacy but with reduced side effects. Another way of modulating dopaminergic

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and serotoninergic activity is by selective activation of DA D2-like and 5-HT1A autoreceptors that result in decreases in the synthesis and release of DA and 5-HT, respectively (Meltzer, 1989). The potential involvement of 5-HT1A receptors is supported by recent studies showing that clozapine’s action may be related to its partial agonist action at 5-HT1A receptor (Newman-Tancredi et al., 1996; Rollema et al., 1997). In this study, some of the pharmacological effects of PD 158771, a relatively selective partial D2/D3 and 5-HT1A agonist is described as a possible agent to treat psychosis, with a reduced propensity to produce EPS, that is commonly observed with classical antagonists. In radioligand binding studies, PD 158771 exhibited high affinity binding for human DA D2L, D3 and D4 receptors labeled with [3H]spiperone. A higher affinity was achieved when the D2L receptors were labeled with DA agonist [3H]N-0437, suggesting that PD 158771 binds preferentially to the D2 high affinity agonist binding site. Functional efficacy of PD 158771 was studied using [3H]thymidine uptake in DA hD2L and hD3 CHOp5 cells. These studies showed that PD 158771 exhibited partial agonist action at hD2L and hD3 receptors. PD 158771 had maximal effect of only 60% at DA D2L when compared to the full agonist actions of quinpirole (100%) and apomorphine (99%). Besides affinity for D2like and 5-HT1A receptors, PD 158771 also displayed moderate affinities for histamine-H1, alpha 1-adrenergic, s and 5-HT2A receptors, the significance of which is discussed below. No significant affinity for a large number of other receptors was found. Evidence has accumulated that D2 as well as D3 receptors can exist both presynaptically (Meller et al., 1993; Pugsley et al., 1995a) and postsynaptically (Waters et al., 1993). The in vitro data indicating that PD 158771 is partial agonist at DA D2L and D3 receptors is supported by a variety of in vivo neurochemical, electrophysiological and behavioral studies. The ability of DA agonists to reverse the GBL-induced increase in DA synthesis is an important biochemical method for studying interactions with nerve terminal DA autoreceptors (Walters and Roth, 1976). GBL blocks impulse flow in the ventral tegmental-limbic and nigrostriatal neuronal pathways, thereby inducing an increase in DA synthesis. These autoreceptors are normally activated by DA released into the synaptic cleft, and in this study were stimulated by the partial DA agonist PD 158771. The partial agonist nature of PD 158771 at DA autoreceptors is indicated by the incomplete antagonism of the GBLinduced increase in DA synthesis. In comparison the full DA agonist apomorphine caused an almost 100% reversal in the GBL test. The stimulation of DA autoreceptors is also supported by electrophysiological studies indicating partial inhibition of A9 dopaminergic neuronal firing. This is also supported by behavioral studies which show monophasic decline in spontaneous locomotor activity

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Fig. 5. (a) Comparison of the effects of PD 158771 (10 mg/kg, i.p.) on DA release from the nucleus accumbens and the striatum as determined by microdialysis technique. Each value is mean of 4 determinations, and is expressed as a percentage of control values. Statistical significance is indicated between the two brain regions by comparing time-matched values, **P ⬍.01, *P ⬍.05. (b) Comparison of the effects of 2 mg/kg (s.c.) apomorphine (APO) on DA release from the nucleus accumbens versus the striatum. Each value is mean of 4 determinations and is expressed as a percentage of control values which is vehicle injection to each brain region. Statistical significance is indicated for DA by **P ⬍.01, *P ⬍.05, with respect to control values.

over a wide dose range with no stimulation observed at high doses as seen with classical full DA agonists (Corbin et al., accompanying paper). Further support for the DA partial agonist action of PD 158771 was obtained with the microdialysis studies that showed decreases in extracellular levels of DA, results consistent with activation of presynaptic D2 and/or D3 autoreceptors. These responses were less than the changes seen with the full agonist, apomorphine. In the microdialysis paradigm,

decreases in amine levels were long lasting and may be of benefit towards the clinical efficacy of the compound. PD 158771 also displayed reasonable affinity for D4.2 receptors. However, there is no clear evidence that DA D4 receptors are involved in the direct control of the activity of dopaminergic neurons (O’Hara et al., 1996). PD 158771 exhibited high affinity to both cloned human and rat hippocampal 5-HT1A receptors. The hippocampus as well as the septum, amygdaloid and the

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Fig. 6. The effect of PD 158771 (10 mg/kg, i.p.) on intracerebral microdialysis measurements of 5-HT overflow in the nucleus accumbens region of anesthetized rats. Data are expressed as a % of baseline overflow and each value is mean of 3 three animals. **P ⬍.01, *P ⬍.05 with respect to control values.

Fig. 7. A comparison of effects of cumulative i.v. dose-response curves of PD 158771 and reference DA agonists on the firing rate of substantia nigra DA neurons in chloral hydrate anesthetized rats. Each point is the mean ± S.E.M. of from 3 to 15 neurons.

raphe nuclei are brain areas that contain high densities of 5-HT1 sites, most of which belong to the 5-HT1A subtype (Meltzer, 1989). Some of these regions function as centers for modulating emotional behavior through the limbic system. Presynaptic 5-HT1A receptors, located on the cell body of serotonergic neurons, are responsible for feedback inhibition of the activity of these neurons. Activation of 5-HT1A autoreceptors results in suppression of firing of 5-HT neurons and reductions in 5-HT turnover (Hjorth and Magnusson, 1988). In the present studies, PD 158771 was evaluated for activation of 5-HT1A

receptors on serotonergic cell bodies by measuring changes in 5-HT synthesis (5-HTP accumulation) in rat striatum, mesolimbic, frontal cortex and brainstem regions. In this respect, PD 158771 was about 10-fold less potent than 8-OH-DPAT in decreasing 5-HT synthesis in rat striatum. This lower potency may reflect, in part, the 5-fold lower receptor binding affinity of PD 158771 at 5-HT1A receptor (Ki =3.5 nM) when compared to 8-OH-DPAT (Ki =0.67 nM). PD 158771 also decreased extracellular levels of 5-HT in nucleus accumbens, results compatible with 5-HT1A agonist action. Moderate or partial actions at 5-HT1A receptor can lead to decreases in the synthesis and release of 5-HT, and less activation of 5-HT1A postsynaptic serotonergic receptors. These data, together with the relatively weaker hypothermic effect induced by PD 158771 when compared to the full agonist, 8-OH-DPAT (unpublished data), suggest but does not prove that PD 158771 is a partial agonist at 5-HT1A receptors. The implication of this effect for schizophrenia is not clear. However, findings in animals suggest some potential benefit. Agonists or partial agonists at 5-HT1A receptors are known to have anxiolytic activity (Borison et al., 1990; DeVry, 1995). It has also been reported that a DA D2 antagonist combined with the full 5-HT1A agonist 8-OH-DPAT caused additive effects in suppressing rat conditioned avoidance and at the same time blocked the observed catalepsy (Broekkamp et al., 1988; Wadenberg et al., 1994; Wadenberg and Ahlenius, 1991). Thus the 5-HT1A agonist action of PD 158771 may contribute to the antipsychotic potency of the compounds in rodents and monkeys. This may explain why the compound has a lower propensity than classical antipsychotics, such as haloperidol, to induce catalepsy in rats and EPS effects in monkeys (Corbin et al., accompanying paper). Moreover,

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behavioral studies also indicated that PD 158771 consistent with other 5-HT1A agonists or partial agonists, exhibits anxiolytic activity in a rodent model of anxiety (Corbin et al., accompanying paper). Anxiolytic activity combined with antipsychotic activity may provide a superior treatment for schizophrenics showing anxiety in addition to typical schizophrenic symptoms. As with many other antipsychotic drugs, PD 158771 showed affinities for other receptors such as histamineH1, 5-HT2A, s and alpha-adrenergic receptors. Brain s binding sites, have long been implicated in the actions of antipsychotics probably through modulation of DA system. However, Meltzer et al. (1992) have reported that there was no relationship between affinity for s binding sites and the ability to either alter DA neuronal activity or to induce extrapyramidal motor dysfunction. Thus, the significance of affinity for s binding sites is unclear since no clear function has been associated with these sites. The affinity of PD 158771 for alpha 1-and alpha 2-adrenergic sites is relatively weak as compared to the reference agents such as prazosin (alpha 1-adrenergic antagonist) and idazoxan (alpha 2-adrenergic antagonist), respectively. This indeed appears to be the case in vivo as PD 158771 did not alter NE biosynthesis in NE-enriched rat brain regions such as brainstem. This suggests that it lacks significant in vivo adrenergic receptor antagonist or agonist action and thus the liability for significant cardiovascular side effects. A preliminary cardiovascular study (PD 158771, at 10, 30 and 100 mg/kg orally) supports the latter hypothesis as only the 30 mg/kg dose of PD 158771 produces a very modest and transient (first h) hypotensive effect in rats. This effect was not seen at 10 and 100 mg/kg (Keiser, personal communication). Furthermore, PD 158771 binds with moderate affinity to 5-HT2A receptors. An antagonist to this receptor, ritanserin, was reported to improve the negative symptoms of schizophrenia, to alleviate psychosis in previously refractory schizophrenics and to show reduced incidence of EPS (Megens et al., 1994). Thus, PD 158771 binding to 5-HT2A receptors may be of benefit in explaining the antipsychotic actions of PD 158771. PD 158771 shows high affinity for both DA D2-like and 5-HT1A receptors with moderate affinities for other receptors such as 5-HT2A and histamine H1 receptors. The affinity for histamine-H1 receptors may contribute to explain any observed sedative actions (Hey et al., 1995). This relatively broad receptor binding profile particularly with regard to histamine H1 receptors may explain why in locomotor activity (Corbin et al., accompanying manuscript), PD 158771 was at least 3fold more potent than in the GBL neurochemical test. In the CNS, both DA and 5-HT systems innervate the limbic forebrain structures. This interaction can be important physiologically. Dopamine D2L/D3 agonists such as CI-1007 (Pugsley et al., 1995a) and apomorphine

(Walters and Roth, 1976) have been shown to decrease DA synthesis and release in ascending dopaminergically-innervated brain areas such as the striatum, mesolimbic regions and frontal cortex. The frontal cortex is a region thought to play a key role in the pathogenesis of schizophrenia, depression, anxiety and other psychiatric disorders. Studies in schizophrenic patients have shown evidence of dorsolateral prefrontal cortical dysfunction as measured by neuropsychological tests (Goldberg et al., 1993) and in vivo assessment of regional cerebral blood flow and glucose metabolism (Berman and Weinberger, 1991). Data from patients with schizophrenia have suggested “a diminished dopaminergic afferent activity” as a basis of the prefrontal cortical hypofunction. Results from our study show that PD 158771 did not alter DA synthesis in frontal cortex, even though 5HT synthesis was decreased. Thus, PD 158771 would not exacerbate the already diminished dopaminergic activity in prefrontal cortex. The reason for the lack of effect of PD 158771 on DA synthesis is not clear but one can postulate that it may be due to a complex interaction between serotoninergic/dopaminergic and other neurotransmitters. Thus, unlike classical antipsychotics like haloperidol, PD 158771 is a combined partial DA D2 agonist and agonist at 5-HT1A receptors that exhibits a neurochemical and behavioral profile (Corbin et al., accompanying paper) believed to improve symptoms of schizophrenia and to reduce EPS liability.

Acknowledgements We thank Dr. Roy Schwarz, Ms. Carol Spencer and Ms. Carrie Nelson for providing us with affinity data on muscarinic receptor binding.

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