In Vitro Receptor Binding and in Vivo Receptor Occupancy in Rat and Guinea Pig Brain: Risperidone Compared with Antipsychotics Hitherto Used

Home , Bromperidol, Nemonapride, Ocaperidone, Pipamperone, Ritanserin

In Vitro Receptor Binding and In Vivo Receptor Occupancy in Rat and Guinea Pig Brain: Risperidone Compared with Antipsychotics Hitherto Used

Alain Schotte, Pascal Bonaventure, Paul F.M. Janssen and Josee E. Leysen

Janssen Research Foundation, Department of Biochemical Pharmacology, Turnhoutseweg 30, B-2340 Beerse, Belgium

Received August 7, 1995 Accepted October 2, 1995

ABSTRACT-Risperidone was compared with antipsychotics hitherto used for in vitro receptor binding using animal brain or cloned (human) receptors and in vivo receptor occupancy in rat and guinea pig brain following acute treatment. Both in vitro and in vivo, risperidone, 9-OH-risperidone, SM-9018, clozapine and clocapramine showed higher affinity for 5-HT2A- than for D2-receptors, whereas mosapramine, haloperidol, bromperidol and nemonapride had a slight to strong preference for D2- compared to 5-HT2A- receptors. In vivo, risperidone showed the highest potency for 5-HT2A-receptor occupancy; To obtain the same extent of D2-receptor occupancy, a 19-times higher dosage was required. 9-OH-Risperidone, the principal active metabolite of risperidone, showed a receptor occupancy profile comparable to that of risperidone. No regional selectivity for D2-receptor occupancy in mesolimbic vs nigrostriatal areas was detected for any of the compounds. Risperidone differed from the other compounds by the remarkably shallow slope of its D2-receptor dose-occupancy curve. A greater predominance of 5-HT2A-receptor vs D2- receptor occupancy and a more gradual occupancy of D2-receptors differentiate risperidone from the other compounds. Both properties probably assist in preventing an extensive blockade of D2-receptors, the cause for extrapyramidal symptoms (EPS). The predominant 5-HT2A-receptor occupancy most likely underlies risperidone's beneficial effects on the negative symptoms of schizophrenia and an adequately low D2-receptor occupancy adds to the treatment of positive symptoms with a low liability of EPS.

Keywords: Receptor binding, Autoradiography, Risperidone, Antipsychotic, Schizophrenia

Schizophrenia is a devastating psychiatric disorder schizophrenic disorders, conventional neuroleptics dis- often with onset in puberty and lasting throughout life. It played major shortcomings: the induction of neuro- means a dramatic burden for the patient and others in his logical side-effects (dystonia, parkinsonism, akathisia, close environment. In addition to the mental suffering, tardive dyskinesia) and a lack of efficacy for the treatment the significant cost of the disease to society is to be consi- of the negative symptoms of schizophrenia (6, 7). With dered (1, 2). Although the disease can be manifested in the recent discovery of D3- and D4-receptors, the new several ways, schizophrenia is often characterized by the subtypes of dopamine receptors (8, 9), hypotheses were appearance of positive (hallucinations, delusions, dis- raised about possible involvement of these receptors in organized speech, disorganized behavior) and negative the pathophysiology of schizophrenia (10); they have symptoms (alogia, affective blunting, avolition, an- been proposed as potential targets for antipsychotic hedonia/asociality, attention impairment); both symp- drugs. toms, although independent, may coexist in the same Recently, renewed attention was paid to the role of patient (3 - 5). The etiology of schizophrenia is still not serotonin in schizophrenia. Upon the discovery of 5-HT2 understood. Conventional neuroleptics have been widely (now termed 5-HT2A)-receptors in 1978 (11), we noted used since the early 1960s for treatment of the positive that several neuroleptics bound to both 5-HT2A- and symptoms of schizophrenia, and a correlation was estab- D2-receptors. Pipamperone (a butyrophenone derivative) lished between the average daily therapeutic dose and stood out because of its predominant 5-HT2A-receptor in- their binding affinities to dopamine D2-receptors (6). teraction. This compound was noted in the clinic for its Although representing a breakthrough in the treatment of beneficial effect in socially withdrawn and agitated psychiatric patients and for normalizing disturbed sleep erties. In addition to the treatment of the positive symp- rhythms (12). It suggested a beneficial role of 5-HT2A- toms, risperidone was also capable of treating the nega- receptor blockade for the treatment of schizophrenia. In tive symptoms of schizophrenia with a very low liability subsequent studies, the potent and long acting 5-HT2A/2c- of extrapyramidal symptoms (24-26). receptor antagonist ritanserin was found to alleviate In the present study, risperidone was compared to negative symptoms in schizophrenics (13) and to reduce antipsychotics hitherto used to estimate the benefits of extrapyramidal symptoms (EPS) induced by neuroleptic this new compound versus the available medication for treatment (14). In animal studies it was shown that 5- the treatment of schizophrenia. For this purpose, in vitro HT2A antagonists reduced neuroleptic-induced catalepsy receptor binding assays using tissue or cell membrane (15 - 17), that they were involved in the restoration of preparations were performed to detect the target neuro- burst firing of dopaminergic neurons (18, 19) and that transmitter receptors of selected antipsychotic drugs. they would attenuate the effects of D2-receptor blockade Secondly, the occupancy of these target receptors by the in the striatum (20). 5-HT2A antagonism was suggested to test-compounds was investigated in rat brain, 2 hr after play a major role in the distinction between "atypical" s.c. administration. In vivo occupancy of the receptors and conventional neuroleptics (21). With the introduction was revealed by subsequent ex vivo radioligand binding in of risperidone (Risperdal®), it appeared that potent 5- brain sections and quantified by autoradiography, ac- HT2A antagonism combined with a milder D2 antagonism cording to a technique developed in this laboratory (22, 23) resulted in remarkably improved clinical prop- (27).

Fig. 1. Chemical structures of the test compounds. MATERIALS AND METHODS microscope slides (Star Frost, Knittel Glaser, Germany). The sections were then kept at - 20 C until use. In vitro receptor binding Risperidone, 9-OH-risperidone, haloperidol, brom- Ex vivo radioligand binding in brain sections and quan- peridol, nemonapride, SM-9018, clozapine, clocapramine titative autoradiography and mosapramine (chemical structures are shown in Fig. The receptors for which the compounds revealed K;- 1) were screened for inhibition of radioligand binding in values below 100 nM were studied for in vivo occupancy vitro to neurotransmitter-, peptide- and lipid-derived fac- after acute treatment: where appropriate, occupancy of tor receptors, ion channels and neurotransmitter trans- 5-HT1A-, 5-HT2A-, 5-HT2C-, D1-, D2-, D3-, a1-, a2-, porters. Systems, radioligands and assay conditions are cholinergic muscarinic- or H1-receptors was measured. summarized in Table 1. The in vivo occupancy of D4-receptors could not be in- Neurotransmitter receptors that are targets for the vestigated since no reliable model was available for D4- compounds were further assayed in vitro using homog- binding in brain sections. Receptor occupancies were enates from animal brain or cloned (human) receptors quantified in brain areas showing high receptor density, stably expressed in mammalian cell lines. Compounds which were selected after regional distribution studies were incubated under the assay conditions described performed by ourselves and by others (58-62). The in Table 1, at 10 to 12 concentrations between 10-11 to selected brain regions and their precise localization are 10-5 M. Incubations were stopped by rapid filtration listed in Table 2. Occupancy of all selected receptors was of the binding mixture over glass fiber filters (manual quantified in each individual brain (6 -12 animals per filtration manifold or Tomtec semi-automatic filtration); dose, 6-8 dosages). The filters were then rapidly rinsed, and membrane- Neurotransmitter receptors were labelled in brain sec- bound radioactivity collected on the filters was deter- tions, and autoradiograms were generated according to mined by scintillation counting for tritiated ligands the protocols summarized in Table 2. The following (Packard Tricarb or Wallac scintillation counter) or by general procedure was applied: after thawing, sections using a gamma counter for iodinated ligands (Packard were dried under a cold air stream. The sections were not Cobra). Counts were directly captured in a computer. washed prior to incubations in order to avoid dissociation Data were further calculated and analyzed using auto- of the drug-receptor complex. D3-receptors, however, can mated procedures. Radioligand binding in the presence of not be labelled in unwashed brain sections, due to occlu- non-labelled compound was expressed as a percent of the sion by an endogenous substance likely to be dopamine total binding and plotted against the log of the concen- (69). A minimal preincubation (30 sec) was therefore ap- tration of the compound. Hence, sigmoidal curves were plied to render D3-receptors partially accessible for [3H]7- generated. The curve of best fit was calculated by com- OH-DPAT. Radioligand solution (200 pl) was applied on puterized curve fitting using equations as described by each section; incubation was restricted to 10 min at room Oestreicher and Pinto (56). pIC50-values (-log of the temperature in order to minimize dissociation of the drug concentration producing 50% inhibition of specific from the receptor. Brain sections of drug-treated and radioligand binding) were derived from the curves. K;- saline-treated animals were incubated in parallel, and the values were calculated according to Cheng and Prusoff incubation time was rigorously controlled. Full associa- (57). Experiments were repeated 2-5 times independent- tion of the radioligand to the receptor was, in general, not ly. achieved after 10 min of incubation. As a consequence, radioactive labelling of the receptors remained below its Drug treatment maximal level. This loss in radioactive signal was com- Male Wistar rats (200 g) and male Dunkin-Hartley pensated for by using longer exposure times to generate guinea pigs (300 g), both bred in own facilities, were the autoradiograms. Non-specific binding was measured treated with subcutaneous injections of saline or test in adjacent sections, in the presence of an excess of an compounds at 6 or 7 dose levels ranging from 0.0025 to 40 unlabelled competitor with chemical structure different mg/kg body weight. Six to twelve animals were used per from the radioligand (Table 3). After the incubation, the dose. The animals were sacrificed by decapitation 2 hr excess of radioactivity was washed-off in consecutive after drug administration. Brains were immediately re- baths of ice-cold buffer, followed by a quick rinse in ice- moved from the skull and rapidly frozen in dry-ice cooled cold water. The sections were then dried under a cold-air 2-methylbutane (-40°C). Twenty-micron-thick frontal stream, placed in a light-tight cassette and covered with a sections were cut out of these brains using a Reichert- light-sensitive film. Ektascan GRL films (Kodak) were Jung 2800E cryostat-microtome (Cambridge Instru- used for the generation of autoradiograms by iodinated ments, Cambridge, UK) and thaw-mounted on adhesive ligands. After the exposure time, these films were deve- Table 1. Assay conditions for radioligand binding and neurotransmitter uptake Table 1. (continued)

Table 2. Location of selected receptors Table 3. Autoradiography protocols

loped in a Kodak X-Omat processor. 3H-Hyperfilms dose-effect curve of best fit was calculated by non-linear (Amersham, Little Chalfont, UK) were used for the regression analysis by a computer program using equa- generation of autoradiograms by tritiated ligands. They tions as described by Oestreicher and Pinto (56). From were developed manually in Kodak D19 developer for 2 these dose-response curves, the ED25-, ED50- and ED75- min and fixed with Kodak Readymatic for 3 min. values (the drug dose producing 25010, 50% and 75% receptor occupancy, respectively) and the slopes of the Data analyses curves for receptor occupancy were calculated. A non- Autoradiograms were quantified by an MCID image overlap of the 95010 confidence limits was considered to analyzer (Imaging Research, St-Catharines, Ontario, reflect biologically relevant differences. Canada) (70). Optical densities were transformed into levels of bound radioactivity after calibration of the im- Materials age analyzer with grey-values generated by coexposed [3H]7-OH-DPAT (4.22 TBq/mmol or 114 Ci/mmol), commercially available polymer standards ([125I]Micro- [3H]CCK8 (2.85 TBq/mmol or 77.0 Ci/mmol), [3H]mesul- scales and [3H]Micro-scales from Amersham). Specific ergine (3.15 TBq/mmol or 85.0 Ci/mmol), [3H]spiperone binding was given as the difference between total binding (3.59 TBq/mmol or 97.0 Ci/mmol), [3H]U69593 (2.29 and non-specific binding measured in adjacent sections. TBq/mmol or 62.0 Ci/mmol), [125I]7-amino-8-iodo-ket- Ex vivo receptor labelling by the radioligand in brain anserin ([1251]AMIK (74 TBq/mmol or 2000 Ci/mmol), sections of drug-treated animals was expressed as the per- [125I]iodosulpride (74 TBq/mmol or 2000 Ci/mmol) and centage of receptor labelling in corresponding brain sec- Autoradiographic Micro-scales were purchased from tions of saline-treated animals. Percent receptor oc- Amersham. cupancy by the drug administered to the animal was given [3H] 8-OH-DPAT (4.79 TBq/mmol or 129.5 Ci/mmol), by 100% minus the percent receptor labelling in the [3H]batrachotoxin-B (1.87 TBq/mmol or 50.5 Ci/mmol), treated animal. Percent receptor occupancies were plotted [3H]clonidine (2.3 TBq/mmol or 63.5 Ci/mmol), [3H]- vs dose. For each of the 6-8 doses per compound, in- DPDPE (1.22 TBq/mmol or 33.0 Ci/mmol), [3H]flu- dividual values from 6-12 animals (mostly 6) were used. nitrazepam (3.12 TBq/mmol or 84.3 Ci/mmol), [3H]GR Each individual value was the mean of measurements 65630 (2.27 TBq/mmol or 61.4 Ci/mmol), [3H]halo- performed in 3 consecutive sections. The sigmoid log peridol (0.55 TBq/mmol or 15 Ci/mmol), [3H]leukotriene D4 (4.73 TBq/mmol or 128 Ci/mmol), [3H]nisox- Belgium (C. Janssen). etine (2.74 TBq/mmol or 74.0 Ci/mmol), [3H]nitrendipine The drugs were obtained from the companies of origin. (2.72 TBq/mmol or 73.0 Ci/mmol), [3H]PAF (5.55 TBq Risperidone, 9-OH-risperidone, haloperidol, bromperidol /mmol or 150 Ci/mmol), [3H]paroxetine (0.61 TBq/mmol were from Janssen Pharmaceutica (Beerse, Belgium). or 16.6 Ci/mmol), [3H]prazosin (0.64 TBq/mmol or 17.4 Nemonapride was from Yamanouchi (Tokyo), SM- Ci/mmol), [3H]pyrilamine (0.918 TBq/mmol or 24.8 9018 was from Sumitomo (Osaka), clozapine was from Ci/mmol), [3H]QNB (1.59 TBq/mmol or 43.0 Ci/mmol), Sandoz (Basel, Switzerland), clocapramine and mosapra- [3H]SCH-23390 (2.6 TBq/mmol or 71.1 Ci/mmol), [3H]- mine were from Yoshitomi (Osaka). substance P (1.15 TBq/mmol or 31.0 Ci/mmol), [3H]- WIN35428 (3.13 TBq/mmol or 84.5 Ci/mmol), [1251]iodo- RESULTS cyanopindolol (81.4 TBq/mmol or 2000 Ci/mmol) were purchased from New England Nuclear (Du Pont, Dreieich, In vitro competition binding of risperidone, 9-OH- Germany). risperidone, haloperidol, bromperidol, clozapine, [3H]Dexetimide (0.36 TBq/mmol or 9.78 Ci/mmol), nemonapride, SM-9018, clocapramine and mosapramine [3H]ketanserin (1.18 TBq/mmol or 32 Ci/mmol) and to neurotransmitter receptors or neurotransmitter trans- [3H]sufentanil (0.85 TBq/mmol or 23.0 Ci/mmol) were porters, as listed in Table 1, was investigated using animal synthesized by the Janssen Research Foundation, Beerse, brain or cloned (human) receptors stably expressed in

Table 4. In vitro receptor binding profile in animal brain (K;-values (nM), n=2-5) mammalian cell lines. Table 4 contains the in vitro recep- binding sites), were similar to those published previously tor binding profiles (Ki-values) of the compounds tested (27). in this study. Receptors for which a compound displayed Figure 2 shows the dose-occupancy curves for the test- a Ki-value below 100 nM were considered as potential compounds at the D2 receptor in the caudate-putamen targets for the compound and were selected for the inves- and 5-HT2A receptors in the frontal cortex. For each tigation of in vivo receptor occupancy. dose-level median values are plotted (n=6-12). The Autoradiograms representing the various binding ED50-values (derived from the dose-occupancy curves) for models used, obtained after labelling of brain sections of the occupancy of the various target receptors by the test- saline-treated animals (maximal amount of available compounds are listed in Table 5. The slopes of the D2-, 5-

Fig. 2. Occupancy-curves for 5-HT2A-receptors in the fourth layer of the frontal cortex (dotted line) and for D2-receptors in rat caudate-putamen (full line) by the various test compounds represented by their median occupancy value (5-HT2A-receptors: open symbols; D2-receptors: closed symbols) at each dose level (n=6-12). ED25-, ED50- and ED75-values and slopes of the D2- receptor occupancy curves calculated from all data points are listed in Tables 5 and 6. Table 5. Receptor occupancy (ED50, mg/kg) measured ex vivo by quantitative autoradiography, 2 hr after s.c. administration of the compounds

Table 6. Slopesa of D2-, 5-HT2- and al-receptor occupancy curves in rat brain areas and corresponding ED25- and ED75-values for D2 receptor occupancy with ED75/ED25 ratio's, measured ex vivo by quantitative autoradiography, 2 hr after s.c. administration of the compounds

HT2A- and adrenergic al-receptor occupancy curves are DISCUSSION listed in Table 6; also included are the ED25- and ED75- values for the occupancy of D2-receptors in the caudate- Potency of interactions with dopamine receptors putamen (striatum). Data on D2-receptor occupancy in In accordance with their antipsychotic properties, all the caudate-putamen (striatum), substantia nigra and in compounds displayed affinity in vitro D2-receptors, but the mesolimbic areas (nucleus accumbens and olfactory with a wide range of potency (Table 4): nemonapride tubercle) are listed in Table 7. showed the highest affinity (subnanomolar, 5 times higher Table 7. D2-receptor occupancy (ED50, mg/kga) in rat dopaminergic areas measured ex vivo by quantitative autoradiography, 2 hr after s.c. administration of the compounds

than haloperidol) and clozapine showed the lowest affin- more potent than its D2 affinity), also showed a sixfold ity for D2-receptors (100 times lower than haloperidol). decrease in ratio of D2 vs D3 interaction potency as com- The affinities of risperidone and 9-OH-risperidone for pared to its in vitro ratio. D2-receptors were two to three times lower than that of D1-receptor occupancy could be demonstrated for haloperidol. Clozapine was the only drug to show higher clozapine, with a two times lower potency than its D2- affinity in vitro for D4-receptors than D2-receptors. Its receptor occupancy. D4 affinity, however, was not very high. Nemonapride The measurement of D4-receptor occupancy in the rat and SM-9018 had almost equivalent affinity for D4- and brain still awaits the validation of a reliable model for D2-receptors. Mosapramine had the lowest ratio, showing D4-receptor labelling, which has not been achieved to date a 25-times lower affinity for D4-receptors than D2-recep- despite numerous attempts. tors. D1 affinities were at least 100 times lower than the drug's D2 affinities, except for clocapramine (20 times Predominance of interaction with S-HT2A-receptors vs lower) and clozapine (4 times lower). In vivo, mosapra- D2-receptors mine, haloperidol, bromperidol and nemonapride were The predominance of 5-HT2A- vs D2-antagonism is confirmed to predominantly occupy D2-receptors, believed to be an important factor for obtaining the nemonapride being extremely potent, i.e., 27 times more therapeutic properties of "atypical neuroleptics"; tenfold potent than haloperidol (Table 5). The rank order of higher affinity in vitro for 5-HT2A-receptors vs D2-recep- potencies for D2-receptor occupancy was in good agree- tors being the minimal requirement (21). Among the ment with the in vitro binding affinities. Clocapramine, compounds analyzed here, only risperidone, 9-OH- however, which had still a moderate affinity for D2-recep- risperidone and clozapine fulfil this requirement. tors in vitro, showed the lowest potency for D2-occupancy However, the present study goes a step further by also in vivo. Fifty percent occupancy of D2-receptors was not analyzing the ratios of the in vivo occupancy of receptors entirely reached by clocapramine at its highest dosage at 2 hr after s.c. administration. Peak occupancy of (40 mg/kg, s.c., 2 hr), indicating probably a more rapid receptors usually occurs within 1 hr after s.c. admin- clearance/metabolization of the compound. Its ED50 dose istration. was estimated, under the present conditions, to be around Risperidone, 9-OH-risperidone, SM-9018, clozapine 65 mg/kg in the rat caudate-putamen. and clocapramine showed higher affinity for 5-HT2A- than For most compounds, the ratio of D2- vs D3-occupancy for D2-receptors in vitro. Mosapramine, haloperidol, potency in vivo was reduced as compared to the in vitro bromperidol and nemonapride showed a slight to high affinity ratio. This may be due to a competition with endog- predominance of D2-receptor compared to 5-HT2A-recep- enous dopamine (35), which shows nanomolar affinity for tor affinity. In vivo data confirmed the in vitro observa- the D3-receptor (8) and could hinder the access of the anti- tions (Tables 4 and 5). Both in vitro and in vivo, risperi- psychotics to the D3-receptors in the islands of Calleja. done displayed the highest predominance for 5-HT2A- Nemonapride, which is the only test compound with receptor vs D2-receptor interaction (17- and 19-fold, subnanomolar affinity in vitro for D3-receptors (slightly respectively). For 9-OH-risperidone predominance of 5- HT2A-receptor vs D2-receptor interaction was also obvi- can be achieved with risperidone better than with any of ous (16- and 11-fold, in vitro and in vivo, respectively). the other compounds analyzed in this study. Only risperidone and 9-OH-risperidone showed a greater than tenfold higher potency for the in vivo occupancy of Interaction with various neurotransmitter receptors 5-HT2A-receptors vs D2-receptors. For risperidone, 9-OH- Besides affinity for 5-HT2A- and D2-receptors, the com- risperidone, SM-9018 and clocapramine, 5-HT2A-receptor pounds also showed moderate to high occupancy of a1- occupancy was also the primary property in vivo. The ra- receptors, except for clocapramine and nemonapride. tios for 5-HT2A-receptor vs D2-receptor occupancy in vivo Yet, the significance of central adrenergic a1-receptor oc- were in good agreement with the ratios of in vitro binding cupancy is not quite clear. Only risperidone and clozapine affinity for all compounds, except for clozapine that had a occupied adrenergic a2-receptors. Clozapine's primary 46-fold higher affinity for 5-HT2A-receptors in vitro but affinity in vitro is for H1-receptors, which was 30 only a fivefold preference in vivo. times higher than its D4 affinity and 70 times higher than its D2 affinity. The in vivo data confirmed both its Possible significance of predominant 5-HT2A-receptor predominant occupancy of H1-receptors (47 times more antagonism vs D2-receptor antagonism potent than D2-receptor occupancy) and potent occu- A complex interaction between the serotonergic and pancy of cholinergic-receptors (equivalent to D2-receptor dopaminergic systems seems to exist. It has been shown occupancy). The occupancy of muscarinic receptors by that the 5-HT2A antagonist ritanserin increased both burst clozapine may contribute to its particular clinical profile firing and firing rate of midbrain dopaminergic neurons but may also be responsible for side effects like hypersali- (18), indicating that 5-HT exerts (probably indirectly) an vation. The latter effect may in part be explained by inhibitory control of midbrain dopaminergic neurons. In clozapine's selective agonism for muscarinic M4-receptors an animal model of hypofrontality, a phenomenon that (although being an antagonist at the other muscarinic has been associated with negative symptoms of subtypes) (71). The extensive sedation caused by cloza- schizophrenia, the activity of midbrain dopaminergic pine (72) is most likely due to the H1-receptor occupancy. neurons projecting to the frontal cortex is deadened to Risperidone, and to a lesser extend 9-OH-risperidone, pacemaker activity by local cooling of the frontal cortex. also showed an occupancy of H1-receptors in the guinea 5-HT2A antagonists can restore the dopaminergic burst pig cerebellum. However, from in vitro binding experi- firing in these conditions (19). Similarly, in animals treat- ments using cloned human H1-receptors, it appeared that ed with low doses of the dopamine antagonist raclopride, risperidone shows a 6-times lower affinity for the human co-treatment with ritanserin could restore burst firing H1-receptor than for the guinea pig one, whereas cloza- (=signal) and reduce tonic activity (=noise) of midbrain pine shows the same affinity for both receptors (K. De dopaminergic neurons. However, restoration of burst Loore, manuscript in preparation). In vitro affinity for firing by the 5-HT2A antagonist could no longer be a1-receptors was reported for haloperidol, bromperidol, achieved when higher doses of raclopride were used (19). nemonapride and SM-9018, but since a participation of This suggests the importance of the ratio between 5- this receptor in the therapeutic properties of antipsychotic HT2A- and D2-receptor occupancy for obtaining the op- compounds seems unlikely, occupancy of the latter site timal regulation of the dopaminergic neurotransmission was not investigated in vivo. by 5-HT2 antagonism. The restoration of regular burst firing of midbrain dopaminergic neurons is likely to play Course of interaction and regional interaction with do- a role in the treatment of the negative symptoms of pamine D2-receptors schizophrenia. 5-HT2A antagonism is also known to For increasing dosages, risperidone displayed a more reduce the neuroleptic-induced side effects both in rats gradual D2-receptor occupancy than the other drugs. 9- and patients (14-17). It was demonstrated in this respect OH-Risperidone showed similarly a tendency towards that blockade of 5-HT2A-receptors attenuated the effects more gradual occupancy of D2-receptors in the striatum. of D2 blockade on dopaminergic transmission in rat The drugs classified as typical neuroleptics like striatum but not on mesolimbic dopaminergic neurons haloperidol and bromperidol showed steeper curves for (20), supporting the idea that 5-HT2A antagonism has an the occupancy of D2-receptors in the rat brain; the slopes important role in the reduction of EPS. However, to of the curves were significantly different from that of benefit from these regulating properties, an extensive risperidone (Table 6). The more gradual occupancy curve blockade of 5-HT2A-receptors needs to be combined with of risperidone is in line with previous behavioral observa- a milder D2 antagonism, leaving enough D2-receptors un- tions which showed that a much wider dose-range was re- occupied by the antipsychotic drug to receive the signal of quired between the inhibition of evoked dopaminergic the restored dopaminergic transmission. This condition responses (low D2-receptor blockade) and the occurrence of catalepsy in rats (high D2-receptor blockade) with done in schizophrenic patients. risperidone than with haloperidol (22). In other experiments it was demonstrated in rats that the disinhibition by Acknowledgments The technical assistance of Walter Gommeren, Lieve Heylen, Luc risperidone of locomotor activity induced by a high dose Gijs, Ariane Vreys, Mai Cox, Nick Meeuws and Geert Van Hecke of amphetamine was maintained over a wider dose range for the in vitro binding experiments was fully appreciated. Dr. before reaching immobility (high D2-receptor blockade) Walter Luyten and co-workers are acknowledged for receptor clon- (73) than with haloperidol. The slopes of the occupancy ing and Dr. Anne Lesage and Paul Van Gompel for gene expression. curves of adrenergic al-receptors were never significantly Many thanks to Jan Voeten for the development of the curve fitting different from the slopes for D2-receptor occupancy program. Lambert Leijssen and co-workers are acknowledged for curves in the caudate-putamen. The slopes of the 5- the art-work. HT2A-receptor occupancy curves were not different from those of D2-receptor occupancy in the caudate-putamen REFERENCES except for risperidone that showed a steeper 5-HT2A- 1 Andrews G, Hall W, Goldstein G, Lapsley H, Bartels R and receptor occupancy and clozapine that displayed a more Silove D: The economic costs of schizophrenia: implication for gradual 5-HT2A-receptor occupancy than their respective public policy. 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