European Journal of Pharmacology, 219 (1992) 35-44 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$(15.00

35

EJP 52569

Dopamine receptor occupancy in vivo" behavioral correlates using NNC-112, NNC-687 and NNC-756, new selective dopamine D 1 receptor antagonists E r i k B. N i e l s e n ~l a n d P e t e r H . A n d e r s e n

b

" Department of Behacioral Pharmacology, CNS Dit'is'ion, Not,o Nordisk A / S , DK-2760 Mfltlot', Denmark and ~ Department of Molecular Pharmacology, Bioscienee, Not'o Nordisk A / S , DK-2880 Bagst,aerd, Denmark Received 28 November 1991, revised MS received 17 March 1992, accepted 19 May 1992

The ability of dopamine D 2, mixed D j / D 2 and selective D 1 receptor antagonists, including NNC-112, NNC-687, NNC-756, to inhibit the in vivo binding of [3H]SCH 23390 or [3H]raclopride to dopamine receptors was studied in mice and rats. Furthermore, the dopamine-antagonistic effects of these drugs were also studied in various behavioral models. Significant levels of in vivo receptor blockade were required for antagonism of typical dopamine agonist-mediated behaviors. However, fewer D~ than D. receptors had to be blocked to produce similar antagonistic effects. Thus, there may be a greater receptor reserve for D 2 receptors than for D t receptors. D o p a m i n e D 1 receptors; Dopamine D 2 receptors; Binding (in vivo); Behaviour; (Rat)

1. Introduction

In recent years, P E T studies have been very informative in elucidating the in vivo level of receptor occupancy by neuroleptics necessary to maintain psychotic patients in a relatively symptom-free state (Farde et al., 1988, 1989). Thus, during effective antipsychotic drug therapy, classical neuroleptics, such as haloperidol and triflouperazine, block some 80% of the D 2 receptors in the striatum (ibid). Clozapine, however, occupies only 4 0 - 6 5 % of D 2 receptors but in addition occupies 42% of D~ receptors. O t h e r neuroleptics (flupenthixol, thioridazine) occupy fewer D 1 receptors (0-36%). These data provided the impetus to study the functional consequences of dopamine receptor blockade in vivo in various animal behavior models of dopamine function. Since data on this issue are scarce in the literature, we designed a series of experiments in which it was possible to determine the dopamine receptor occupancy necessary to obtain antagonism of amphetamine-induced behaviors (model of psychosis) or induction of catalepsy in rats (a model of neuroleptic-induced motor side-effects). Further, antagonism of

Correspondence to: E.B. Nielsen, CNS Division, Novo Nordisk A / S , Novo Nordisk Park, DK-2760 Mfilcv, Denmark.

methylphenidate-induced stereotyped gnawing and novelty-induced hypermotility in the mouse was also studied. [3H]Raclopride was used to label D 2 receptors in vivo and [3H]SCH 23390 was used to label D l receptors. Classical dopamine D 2 receptor antagonists and mixed dopamine D I / D 2 receptor antagonists were selected. As dopamine D I receptor antagonists we used SCH 23390 and the new benzazepine dopamine DI receptor antagonists NNC-112, NNC-687 and NNC-756 (see Andersen et al., this issue, for structures). These data have been presented in a preliminary form elsewhere (Andersen, 1988a; Andersen and Nielsen, 1988; Fink-Jensen et al. 1989; Nielsen and Andersen, 1991).

2. Materials and m e t h o d s

2.1. A n i m a l s

Male Wistar rats (M¢llegaards Breeding Labs., LI. Skensved, Denmark) weighing 80-150 g were used. The rats were housed in group cages with four to five r a t s / c a g e (except in the drug discrimination experiments, when individual housing was used). Male N M R I mice (bred at Novo Nordisk A / S ) weighing 20 4-2 g were used. The mice were housed in group cages with

3~ 20-30 mice/cage. The cages wcrc placed in rooms with constant temperature 1 9 - 2 2 ° C and relative humidity of 40-6(t%. The animal rooms were on a day night cycle with lights on from 06:00 to 18:(1(I h.

following scale from (I to 3: (0) latency < 15 s: (1) 15-29 s; (2) 30-59 s: (3) > 60 s.

2.2. In cico dopamine receptor binding in rats

Rats were deprived to 80-85% of their initial freefeeding weight by restricting water intake to that received in 25-min daily experimental sessions (below). These sessions were conducted in eight chambers (Skinner boxes) which were each cquippcd with two response levers and a valve-operated spigot that provided 0.1 ml deionized water reinforcements. After i.p. injection of 1 m g / k g of d-amphetamine sulphate (the 'training drug'), the animals were placed 15 rain later in individual chambers in which responding (prcssing a lever) was reinforced only on a designated ('drug') lever. After i.p. saline (control) injections, responding was reinforced only on the lever opposite to the drug lever. In order to control for olfactory cues. the assignment of drug and saline levers relative to the position (e.g. left/right) of the levers was counterbalanced within and between "running' groups of eight animals. Further, responding on the incorrect lever (relative to the injection of amphetamine or saline) never had any programmed consequences. Responding on the correct lever was reinforced after the completion of 32 responses (fixed ratio (FR) 32 schedule). Amphetamine and saline training sessions were conducted in an irregular order but there were never more than three consecutive saline or amphetamine training sessions. Stable, high (90-95%) discrimination performance was acquired rapidly, within 10-15 training sessions, as evidenced by the distribution of responses on the two levers before the delivery of the first reinforcement (i.e. in the absence of any cue other than that provided by the presence or absence of the internally discriminable effect of the respective training drugs). Accordingly, discrimination accuracy ((~4) was calculated as 100 × the number of correct responses divided by the number of correct + incorrect responses before delivery of the first reinforcer. Training was continued until the discrimination performance was > 9(1(>; ( > 75q4 for individual animals) for 5 days. Test sessions (below) were then interspersed every. 2-7 days in between regular training sessions. The ability of test drugs to antagonize the amphetamine cue was determined in the test sessions. These sessions were terminated once an animal had completed 32 responses on either lever (no reinforcement was delivered) or when 20 min had elapsed. The time required to complete the first 32 responses (i.e. 1 FR) was designated 'reaction time' and was used as an index of non-specific behavioral disruption by the test drug. If an animal did not complete 32 responses, the reaction time was assigned a value of 1200 s, the maximum session time.

The method described by Nielsen et al. (1989) was used. Briefly, rats were injected in the tail vein with 8 #Ci of [:~H]SCH 23990 (73.4-85 C i / m m o l ; synthesized at Novo Nordisk or obtained from NEN) or [3H]raclopride (60 C i / m m o l ; obtained from Astra Alab or NEN). Fifteen minutes after the administration of [3H]SCH 23990 or [3H]raclopride, the animals were decapitated and the whole striatum (approximately 50 mg) or the ventral 'limbic' striatum (approximately 10-15 rag)was rapidly dissected and homogenized in 7 ml of buffer (pH 7.1). Conventional filtering and liquid scintillation counting techniques were used to determine bound radioactivity. Non-specific binding was defined for both radioligands as the difference in cpm in rats with and without pretreatment with cis-flupenthixol (i.p. 2 h, l(i mg/kg).

2.3. In i,it'o receptor binding in mice The methods described in detail by Andersen (1988b) and Andersen and Gr0nvald (1986) were used.

2. 4. Antagonism of d-amphetamine-induced stereotyped behacior (rats) The rats were habituated to an observation room for at least 24 h. Upon injection of the test compound (below), the animals were placed individually in wiremesh cages which were coded in a random order unknown to the observer (below). A trained observer scored the behavior of the animals according to the following scale (Nielsen, 1981); (0) inactive or asleep; (1) awake, normal activity such as eating; drinking or grooming; (2)locomotion; (3)discontinuous sniffing over a large area of the cage; (4) continuous sniffing in a restricted area of the cage; (5) continuous licking or biting the cage bars ( 6 ) a w k w a r d crouched posture with continuous self-grooming.

2.5. Induction of catalepsy (rats) The method is similar to that described by Morelli and Di Chiara (1985). Briefly, rats were injected with the test compound and placed individually on an inclined (70 o) wire-mesh screen (0.8 mm steel wire, 7 mm mesh). The extremities of the animal were gently abducted. The latency to move any extremity was used to define the intensity of catalepsy according to the

2.6. Amphetamine discrimhTatio, (rats)

37

2. 7. Inhibition of exploratory behacior (mice) Mice were injected with the test compound and then placed individually in a plexiglass box (width, length, height: 29 × 29 × 38 cm) equipped with a frame of photocell detectors (spaced equidistantly) to detect locomotor behavior. The photocells were located 1 cm above the floor. The photocell chamber was housed in a sound-insulated, dimly lit, and ventilated chest. The number of photocell crossings in a 10-min period detected by a minicomputer was used as a measure of exploratory behavior.

INHIBITION OF 3H-SCH 23390 BINDING IN VlVO IN THE MOUSE 1

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2.8. Antagonism of stereotyped gnawing behacior (mice)

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Dose (mg/kg)

The methods described by Pedersen and Christensen (1972) were used. Mice were pretreated with saline or test drugs before receiving an s.c. injection of methylphenidate (60 mg/kg). The mice were then placed in plexiglass boxes (two animals/box) resting on corrugated paper. The presence or absence of methylphenidate-induced biting of the paper was evaluated after 1 h (at least 10 bite marks were required for a positive score). Five pairs of mice were used per dose; three to four doses of test drug were tested.

Fig. 1. Ability of different dopamine antagonists to block the in vivo binding of [~[-I]SCH 23390 to dopamine D I receptors in mice. Two or three doses of antagonist were administered via the routes and at the pretreatment times indicated in table 1. Some of these data have been presented previously (Andersen and Gronvald, 1986; Andersen, 1988a, b). ( A ) Chlorpromazine; (o) clozapine; ( o ) flupenthixol, cis(Z); ( • ) fluperlapine: ( • ) NNC-112: ( • ) NNC-687; ( [] ) NNC-756: ( v ) SCH 23390.

sulpiride (Sigma) in acetic acid 1 N made up to volume with water. All drug doses refer to the salt.

2. 9. Drugs 2.10. Statistical analysis The following drugs were dissolved in deionized water and injected in a volume of 5 m l / k g (rats; except in the drug discrimination studies: 1 m l / k g ) or 10 m l / k g (mice) unless otherwise indicated: d-amphetamine sulphate (Sigma Chemical Co., St. Louis, MO); chlorpromazine hydrochloride (Sigma); clebopride acid maleate (Laboratorios Almirall, Barcelona, Spain); clozapinc (Sandoz Pharma, Basel, Switzerland) in dilute HCI; flupenthixol dihydrochloride (H. Lundbeck, Copenhagen, Denmark); fluperlapine (Sandoz) in dilute HCI; haloperidol (Jansen Pharmaceutica, Beerse, Belgium) in propyleneglycol and 1 N tartaric acid (1 : 1) and further diluted with water; methylphenidate (Ciba-Geigy, Basel); NNC-112 (previously, NO-II2; ( + )-8-chloro-7-hydroxy-3-methyl-5-(7-benzofuranyl)-2,3,4,5-tetrahydro-lH-3-benzazepine, synthesized at Novo Nordisk, Bagsvaerd, Denmark), in dilute HC1; NNC-687 ((+)-8-nitro-7-hydroxy-3-methyl-5-(7-(2,3dihydrobenzofuranyl)-2,3,4,5-tetrahydro-lH-3-benzazepine, synthesized at Novo Nordisk); NNC-756 (previously NO-756, (+)-8-chloro-7-hydroxy-3-methyl-5(7-(2,3-dihydrobenzofuranyl)-2,3,4,5-tetrahydro-lH-3benzazepine, synthesized at Novo Nordisk), in dilute HC1; raclopride tartrate (Astra Alab, S6dertalje, Sweden); SCH 23390 hydrobromide ((+)-8-chloro-7-hydroxy-3-hydroxy-3-methyl-5-phenyl-2,3,4,5-tetrahydro1H-3-benzazepine; Schering-Plougb, N J, USA); spiperone (Jansen; dissolved as haloperidol); S ( - ) -

Log-probit methods were used to calculate dose-response curves and EDs0 values. From the curves of the inhibition of in vivo dopamine receptor binding, it was possible to determine the level of receptor occupancy at the EDs0 doses of test compounds in the behavioral tests. This measure allowed comparison of the extent of receptor occupancy by the different compounds needed for the same behavioral effect, t-Tests were used to ascertain differences in receptor occupancy between dopamine D] and D 2 receptor antagonists.

3. Results

3.1. Studies in mice Figures 1 and 2 show the ability of different dopamine antagonists to block [3H]SCH 23390 and [~H]raclopride binding in vivo, respectively. These inhibition curves provided the basis for estimation of the level of D~ and D 2 receptor occupancy necessary for blocking methylphenidate-induced gnawing (table 1). Thus, the dopamine D~ receptor antagonists blocked methylphenidate-induced gnawing at a D~ receptor occupancy of 60-75% (average 65 + 5 % (S.E.M.)). Dopamine D 2 receptor antagonists also blocked methylphenidate although at a relatively higher receptor

38 INHIBITION OF 3H-RACLOPRIOE BINDING IN VlVO IN THE MOUSE 100

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60

40 k5 o i,z

.~

20 O O.O01

0.01

0.1

1

10

100

1000

Dose (rng/kg)

Fig. 2. Ability of different dopamine antagonists to block the in vivo binding of [3}t]raclopride to d o p a m i n e D 2 r e c e p t o r s in mice. See legend to fig. 1 for other details. Some of these data have been presented previously ( A n d e r s e n , 1988a,b). ( zx ) C h l o r p r o m a z i n e ; (c~. " " ) clebopride; (o) clozapine; (c!) flupenthixol, cis-(Z); ( • ) fluperlapine: (o . . . . ) h a l o p e r i d o l ; ([] . . . . ) raclopride: (+) s p i p e r o n e ; ( * ) sulpiride, (S).

occupancy: the average level, 92 + 6%, was significantly higher than that of D~ occupancy (t = 3.6, P 0.009). The benzamides showed a high receptor occupancy ( > 99%) related to the blockade of methylphenidate-induced gnawing. It was also notable that although flupenthixol and chlorpromazine both occupied D~ and D 2 receptors, flupenthixol blocked meth-

ylphenidate-induced gnawing at a lower level of receptor occupancy than chlorpromazine. Neither elozapinc nor fluperlapine blocked methylphenidate-induced gnawing at doses which occupied almost all of the D~/D, receptors. The dopamine antagonists also blocked the noveltyinduced stimulation of locomotor activity. The dopamine D i receptor antagonist occupancy levels varied between 3 0 - 7 2 % ( a v e r a g e = 56 _+ 9%), with NNC-687 occupying the least receptors. D 2 receptor antagonists occupied almost all of the D~ receptors at doses which blocked 50% of the novelty-induced locomotor response (average 94 + 3%; again significantly higher than D t occupancy, t = 4.2, P = 0.004). The mixed antagonists likewise blocked novelty-induced locomotion in doses which occupied a significant proportion of the dopamine receptors. Under these circumstances, chlorpromazine required a lower receptor occupancy than flupenthixol. The ability of methylphenidate or d-amphetamine to displace the in vivo binding of [3H]SCH 23390 and [~H]raclopride was also studied. However, ncither methylphenidate (60 m g / k g ) nor d-amphetamine (50 m g / k g ; both substances injected s.c. 30 rain) had an effect on the binding of either ligand (data nol shown). 3.2. Studies in rats

The ability of different dopamine antagonists to inhibit [3H]SCH 23390 binding and [3H]raclopride

TABLE 1

Inhibition of methylphenidate-induced gnawing and exploratory behaviour in mice. D i, D~ or mixed antagonists were injected via the indicated route before the tests of methylphenidate-induced gnawing and exploratory behavior. The pretreatment interval (rain) is indicated under the route of administration. The data are expressed as inhibitory effect (EDsc ~) of the test compounds and the corresponding D 1 / 0 2 receptor occupancy levels (at the EDs~~ doses).

Route

Methylphenidate-induced gnawing

Exploratory b e h a v i o r

Receptor occupation

Receptor occupation

EDso (mg/kg)

Dopamine D~ receptor antagonists NNC-112 s.c. 30 NNC-687 p.o. 120 NNC-756 s.c. 30 SCH 23390 s.c. 30 Dopamine D 2 receptor antagonists Clebopride i.p. 120 Haloperidol i.p. 120 Raclopride s.c. 30 Spiperone i.p. 120 Sulpiride, Si.p. 120

0.037 3.3 0.032 0.055

Dl ( ¢~;~) 70 60 55 75

7.5 0.17 5.8 0.2 > 128

Mixed dopamine D 1/ D 2 receptor antagonists Chlorpromazine i.p. 120 40 Clozapine s.c. 60 > 6(1 Flupenthixol, (Z) i.p. 120 0.14 Fluperlapine s.c. 60 > 100

D, C~)

80 > 82 3O > 99

ED5 /

(mg/kg) 0.09 0.99 0.03 0.05

> 99 71) > 99 92 > 99

6.6 0.32 6.8 0.32 58

90 > 90 20 > 85

1.6 2.9 0.28 15

DI (c5)

D, (%)

67 30 57 72

> 99 82 > 99 95 96

22 22 42 95

40 75 50 52

39 INHIBITION OF 3H-RACLOPRIDE BINDING IN VlVO IN RAT N. ACCUMBENS 100-

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INHIBITION OF 3H-SCH 23390 BINDING IN VIVO IN RAT N. ACCUMBENS

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binding in the accumbens and in the striatum, respectively, is shown in figs. 3, 4, 5 and 6. It can be seen that the compounds were generally less potent in inhibiting binding in the accumbens than in the striatum (table 2). However, the differences were not statistically significant. D 1 binding differences between these two areas yielded t = 1.8, P = 0.12, corresponding D 2 binding differences yielded t = 1.1, P = 0.3. The lowest doses of NNC-687 had a somewhat variable inhibitory effect on [3H]SCH 23390 binding.

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Fig. 5. I n h i b i t i o n of [3H]SCH 23390 b i n d i n g in vivo to D 1 r e c e p t o r s in the rat v e n t r a l s t r i a t u m ( ' n u c l e u s a c c u m b e n s ' ) by different d o p a m i n e antagonists. See l e g e n d to fig. 3 for o t h e r details. (zx) C h l o r p r o m a z i n e : (o) clopazine; (©) flupenthixol, cis-(Z); ( • ) fluperlapine; ( & ) N N C - I 1 2 ; ( v ) NNC-687; ( D ) NNC-756; ( v ) SCH 23390.

3.3. Amphetamine cue antagonism To block the cueing effects of amphetamine, the dopamine D 1 receptor antagonists had to occupy some 40-80% (average 54 + 5%) of the DI receptors in the striatum and some 22-75% (average 37 _+ 9%) of the D I receptors in the accumbens (table 3). In contrast, the dopamine D 2 receptor antagonists needed to occupy nearly all D 2 receptors in order to block 50% of the amphetamine cue (average of 86 _+ 4 and 79 _+ 5% in the striatum and accumbens, respectively). These values were significantly higher than the corresponding Dt occupancy levels in these areas (t = 4.9, P = 0.0017

INHIBITION OF 3H-RACLOPRIDE BINDING IN VlVO IN RAT STRIATUM •

.

Dole

Fig. 3. Ability of different dopamine antagonists to block (percentage of control) the in vivo binding of [3H]raclopride to dopamine D 2 receptors in the rat ventral striatum ('nucleus accumbens'). The drugs were injected via the routes and at the pretreatment times indicated in table 2. Results are expressed as the average of data from two to three animals per dose. (zx) Chlorpromazine; (© . . . . ) clebopride: (o) clozapine; (©) flupenthixol, cis-(Z): ( • ) fluperlapine; (o . . . . ) haloperidol: ( [] . . . . ) raclopride: () spiperone: ( e ) sulpiride, (S).

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INHIBITION OF 3H-SCH 23390 BINDING IN VlVO IN RAT STRIATUM

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Dose (mg/kg) Fig. 4. Ability of different d o p a m i n e a n t a g o n i s t s to inhibit in vivo b i n d i n g of [ 3 H l r a c l o p r i d e to d o p a m i n e D 2 r e c e p t o r s in the s t r i a t u m (excluding the v e n t r a l part; see Methods). See l e g e n d to fig. 3 for o t h e r details.( zx ) C h l o r p r o m a z i n e ; (© . . . . ) c l e b o p r i d e ; (o) clozapine: (©) flupenthixol, cis-(Z); ( • ) f l u p e r l a p i n e ; (o . . . . ) h a l o p e r i d o l : ([] . . . . ) r a c l o p r i d e ; () s p i p e r o n e ; ( , ) sulpiride, (S).

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Fig. 6. Inhibition of [3H]SCH 23390 binding in vivo to dopamine D t receptors in the rat stria•urn (excluding ventral part: see Methods) by different dopamine antagonists. See legend to fig. 2 for other details. ( A ) Chlorpromazine; (o) clozapine; (©) flupenthixol, cis-(Z); ( • ) fluperlapine; ( A ) NNC-] 12; ( v ) NNC-687; ([]) NNC-756; ( v ) SCH 23390.

40

TABLE 2 Inhibition of in vivo dopamine receptor binding in the rat. Ability of D I, D, and mixed dopamine antagonists to displace the in viw) binding of [~HJSCH 23391/ binding to D~ receptors or [~H]rach)pride binding to D, receptors in the striatum (excluding the ventral part) or in the ventral part ('nucleus accumbens'). The results are expressed as ED,~, values in mg/kg. The route of administration and pretreatment interval (min) are given; ~ represents an approximate value (only one data point). Route

EDs0 ( m g / k g ) Dt receptor binding [3H]SCH 23390

D e receptor binding [3 H]raclopride

Striatum

Striatum

Accumbens

Accumbens

Dopamine D/ receptor antagonists NNC-112 NNC-687 NNC-756 SCI{ 23390

s.c. 30 p.o. 120 s.c. 30 s.c. 30

0.013 17 0.018 0.013

0.037 18 - 0.033 11.027

Doparnine D 2 receptor antagonists Clebopride Haloperidol

i.p. i.p. s.c. i.p.

1211 120 30 12(1 i.p. 120

Raclopride Spiperone Sulpiride, S

-

0.06 11.05 0.1 O.Ol 7

O. I 0.07 0. I I 0.05

4.8

1.5

3.2 48

3.0 15 0.41 12

Mixed dopamine D: / D 2 receptor antagonist.~ Chlorpromazine Clozapine Flupenthixol, (Z) Fluperlapine

i.p. s.c. i.p. s.c.

120 60 120 60

12 6 0.25 1(1

42 -17 1.3 60

0.74

1I

3.4. Antagonism of stereotyped behavior

and t = 4.3, P = 0.0037, respectively). With the mixed antagonists, chlorpromazine again showed a very high occupancy of D I / D 2 receptors while clozapine exerted cue-antagonism at the lowest level of receptor occupancy.

The dopamine D 1 receptor antagonists blocked stereotyped behavior at occupancies ranging from 50 to 80% in the striatum (average = 70 _+ 7%) and 35-75%

TABLE 3 Antagonism of the amphetamine cue in the rat. lnhibitoD' effects (EDsII; m g / k g ) of dopamine antagonists on the cuing effect of d-amphetamine. Also shown is the D t, D e receptor occupancy at EDs~ values of the antagonists. See previous tables for other details. Route

ED:q~

(mg/kg)

Dopamine receptor occupancy Striatum DI (f4)

Accumbens D2 (%)

DI (%)

D, (~;~)

Dopamine D: receptor antagonists NNC- 112 NNC-687 NNC-756 SCH 23390

s.c. 30 p.o. 120 s.c. 30 s.c. 30

0.016 32 0.012 0.017

55 65 40 57

1.6 I).28 0.33 0.083 24

-

40 3.6 0.18 10

83 35 45 13

22 60 25 40

Dopamine D 2 receptor antagonists Clebopride Haloperidol Raclopride Spiperone Sulpiride, S-

i.p. i.p. s.c. i.p. i.p.

120 120 60 1211 120

> 99 92 80 83 77

91 77 77 60 88

Mixed dopamine D : / D 2 receptor antagonists Chlorpromazine Clozapine Flupenthixol, ( Z ) Fluperlapine

i.p. 12(1 s.c. 60 i.p. 120 s.c. 60

98 1

22 48

47 8 17 57

96 20 30 48

41 TABLE 4 Antagonism of amphetamine-induced stereotypy in the rat. Inhibitory potencies (EDs0) against amphetamine stereotypy and corresponding D I, D 2 receptor occupancy in vivo for different dopamine antagonists. See previous tables for other details. Route

EDso (mg/kg)

Dopamine D I receptor antagonists NNC-112 s.c. 30 NNC-687 p.o. 120 NNC-756 s.c. 30 SCH 2339(I s.c. 30

0.026 64 0.049 0.013

Dopamine D 2 receptor antagonists Clebopride i.p. 120 Haloperidol i.p. 120 Raclopride s.c. 60 Spiperone i.p. 120 Sulpiride. Si.p. 120

1.5 0.11 0.17 0.09 67

Mixed dopamine D l / D e receptor antagonists Chlorpromazine i.p. 120 Clozapine s.c. 60 Flupenthixol, (Z) i.p. 120 Fluperlapine s.c. 60

4.3 > 30 0.14 > 30

in the accumbens (average = 59 _+ 10%) (table 4). There was a tendency for dopamine D e receptor antagonists to occupy relatively more receptors at doses which blocked amphetamine-induced stereotyped behavior (average of 80 _+ 6 and 72 _+ 7%). However, the differ-

Dopamine receptor occupancy Striatum

Accumbens

D~

D~

D I

D~

(%)

(%)

(%)

(%)

52 75 75 35

75 80 75 50

-

93 70 63 85 88

20 > 82 40 > 75

60 > 32 20 > 75

-

-

8(1 60 63 60 95

-

3 > 65 15 > 30

ences between

dopamine

nists were

s t a t i s t i c a l l y s i g n i f i c a n t (t = 1.1, P = 0.3

not

D,

60 > 67 25 > 7(1

and D 2 receptor

a n d t = 1.1, P = 0.32, r e s p e c t i v e l y ) . C l o z a p i n e perlapine

failed to b l o c k s t e r e o t y p e d

doses which

induced

antagoa n d flu-

b e h a v i o r , e v e n in

a significant degree

of receptor

TABLE 5 Induction of catalepsy in the rat. Ability of neuroleptics to cause catalepsy (EDs0) and corresponding levels of dopamine receptor occupancy in vivo. See previous tables for other details. Route

EDs~~ (mg/kg)

Dopamine receptor occupancy Striatum D1 (%)

Dopamine D I receptor antagonists NNC-112 s.c. 30 NNC-687 p.o. 12(1 NNC-756 s.c. 30 SCH 23390 s.c. 30

0.064 100 0.09 0.16

65 87 82 95

Oopamine 0 2 receptor antagonists Clebopride i.p. 120 Haloperidol i.p. 120 Raclopride s.c. 60 Spiperone i.p. 120 Sulpiride, Si.p. 120

13 0.75 2.7 0.25 300

-

Mixed dopamine D 1 / D 2 receptor antagonists Chlorpromazine i.p. 120 Clozapine s.c. 60 Flupenthixol, (Z) i.p. 120 Fluperlapine s.c. 60

10 > 100 1.0 > 100

15 > 95 32 > 93

Accumbens D,

D I

(%)

(¢~)

D2 (%)

82 82 78 85

99 88 98 77 92

99 97 99 99 98

87 > 77 57 > 90

40 > 88 45 > 60

80 > 92 77 >87

42 blockade. Chlorpromazine and flupenthixol blocked the stereotyped behavior at an intermediate level of receptor occupancy. The ability of d-amphetamine to displace D l or D, binding in vivo was studied (rat whole striatum). Amphetamine (10 m g / k g injected s.c. 30 min) had no effect on the binding of either [~H]SCH 23390 or [3H]raclopride (data not shown).

3.5. Induction of catalep,sy The dopamine D 1 receptor antagonists induced catalepsy at D~ receptor occupancy levels of 65-87% (average 82_+ 6%) in the striatum, while the corresponding levels in the accumbens ranged from 78 to 85% (average 82 _+ 1.4%) (table 5). The corresponding occupancy for D, receptor antagonists was higher than for the D~ receptor antagonists (average of 98.4 +_ 0.4 and 91 _+ 4% in the striatum and accumbens, t values 2.9, P = 0.023 and 1.9, P - 0.095, respectively). The mixed antagonists clozapine and fluperlapine failed to induce catalepsy in doses at which they occupied a substantial number of D I / D 2 receptors. In contrast, flupenthixol induced catalepsy at 15-40% receptor occupancy, while chlorpromazine required 80-87?J D, occupancy and 15-40% D I occupancy.

4. Discussion

The present experiments provide, for the first time, information about the relative level of in vivo dopamine D~ and D~ blockade necessary to affect a variety of specific behavioral responses in rodents. First, the resuits demonstrated that across the species and models examined, generally fewer D~ receptors than D e receptors needed to be blocked in order to obtain functional dopaminergic antagonism. This was shown in mice with respect to antagonism of methylphenidate-induced gnawing and antagonism of novelty-induced locomotor stimulation, as well as in rats with respect to induction of catalepsy and inhibition of amphetamine-induced discriminative response control, and marginally with respect to stereotyped behavior. The present finding in several models that almost complete D, blockade was necessary to obtain dopaminergic antagonism suggests that, under normal physiological conditions, there is a relative surplus of functionally relevant spare D, receptors. Alternatively, it is possible that a functional dopamine-induced stimulation may be possible with a minimum occupation of receptors by endogenous dopamine. This possibility was supported by the finding that methylphenidate and amphetamine did not alter the in vivo binding of either D~ or D, receptor ligands. This may be explained by the low affinity of dopamine for D~/D z receptors. The

relative inability of increased levels of endogenous dopamine to displace D 1 or D~ binding furthermore indicates that marked agonist-induced behavioral effects may occur at a level of endogenous dopaminc occupancy that cannot be measured with the present techniques. This observation may have relevance for understanding the finding that occupancy levels > 99% were required for dopamine D, receptor antagonists to block completely the behavioral effects of, for example, methylphenidate. Thus, although > 99% of the receptors were blocked, there may still have been "room' for a small fraction of the D, receptors to promote a behavioral effect. The general lack of effect of methylphenidate or amphetamine on in vivo D~ or I), binding does not, of course, rule out that these treatments may have affected discrete subdivisions of the terminal dopaminergic field. Further, the present method (homogenates of whole striatum or accumbens) may have failed to detect changes in those rcgions that may be critically involved in the behaviors studied. The above conclusion is at variance with the results obtained by Arnt et al. (1988) and Meller ct a[. (1989), who used E E D Q (N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline) to inactive D~ and D, receptors when studying the involvement of these receptors in stereotyped behavior in the rat. These investigators found that a decline in a dopamine-mediated response (stereotyped behavior) closely followed a decline in D, receptor inactivation, and vice versa for D~ receptor inactivation. It can be speculated that under the conditions of the E E D Q studies, the surviving dopaminc receptors may have becomc "decoupled" or supersensitized, resulting in non-physiological receptor interactions. In accordance with this contention, short-tcrm (5 days) reserpine treatment causes dopamine receptors to become supersensitizcd to agonist effects (Arnt, 1985). Furthermore, Andersen (1988c) found that at least for D 1 receptor inactivation, E E I ) Q has a disproportionately weak effect in vivo. The contention that there is a relative surplus of D, spare receptors implies in turn that synaptic dopamine may act mainly on D~ receptors. This possibility is supported by results from drug discrimination experiments with low doses of quinpirole, which may act to decrease dopamine synthesis by autoreceptor stimulation (Weathersby and Appcl, 1986). Under these circumstances, blocking D I receptors with SCH 23390 induces a discrimination response similar to that of reduced endogenous dopamine. Thus, normal dopamine tone may bc maintained primarily by D~ receptor activation (see also discussion by Nielsen et al., 1989 and references herein). It is of interest to note that increased endogenous dopamine levels (as elicited by high doses of either amphetamine or methylphenidate) did not affect thc in

43

vivo binding of either [3H]SCH 23390 or [3H]raclopride. This means that the levels of antagonist occupancy under conditions of increased endogenous dopamine (e.g. cue, stereotypy and gnawing tests) were probably not affected by any shift in antagonist 'efficacy' caused by competition with increased levels of dopamine. However, the finding that a differential occupancy of D ~ / D 2 receptors was not involved in the blockade of amphetamine-induced stereotypies indicates that the level of agonist stimulation may modulate the importance of either receptor with respect to antagonist occupancy. Another general observation that can be made based on the present data is that relative small differences exist between striatal and accumbens receptor binding. Although such differences have been reported previously, they are generally of small magnitude (Arnt, 1985; LeFur et al., 1980; K6hler et al., 1986; 1981). Thus, the findings support the contention that dopamine receptors in striatal and mesolimbic areas are not different entities (Leonard et al., 1987), although small differences have been found (Altar et al., 1986). It was of interest to note that in the mouse, antagonism of methylphenidate-induced gnawing and of novelty-induced motility required about the same degree of receptor occupancy although antagonism in either model required a much higher D 2 receptor occupancy than D~ receptor occupancy. With the mixed dopamine D ~ / D 2 receptor antagonists, there was no clear indication that occupancy of either receptor had an additive action on the antagonism of methylphenidate-induced gnawing whereas this may have been the case with respect to the inhibition of novelty-induced motility. In the rat models, it is of interest to note that the induction of catalepsy required almost complete D 2 blockade, i.e. similar to the blockade of novelty-induced locomotion in the mouse. Again, the D~ occupancy needed in this model was higher than the corresponding D~ occupancy. Amphetamine discrimination has been shown previously to depend critically on activation of mesolimbic dopamine systems (Nielsen and Scheel-Kriiger, 1988, 1986; Nielsen and Jepsen, 1985). Thus, the relatively low level of D~ receptor blockade required for antagonism of the amphetamine cue may suggest high antipsychotic potential for dopamine D j receptor antagonists. With respect to the mixed antagonists, the results may be taken to suggest D ~ / D 2 synergism, except for chlorpromazine, which generally required a high occupancy of D i D 2 receptors for a dopamine-antagonistic effect. The 'atypical' neuroleptics, clozapine and fluperlapine, failed to block amphetamine-induced stereotypies and did not elicit catalepsy in doses that substantially blocked both Dj and D 2 receptors. Thus,

compensatory mechanisms (e.g. cholinergic) may operate to dampen these effects of the drugs. In contrast, clozapine and fluperlapine blocked amphetamine discrimination at a relatively low combined D j / D 2 blockade, as has been reported previously (Nielsen and Jepsen, 1985; Kilbey and Ellinwood, 1979). This is in accordance with the observation that there is no cholinergic modulation of dopamine antagonist-induced inhibition of amphetamine discrimination (Nielsen, 1987). In summary, these results indicate that dopamine antagonist-induced inhibition of behaviors associated with increased dopaminergic neurotransmission requires significant occupation of dopamine receptors. The D1 receptors requires a lower level of occupation than the De receptor in order to obtain similar antidopaminergic effects in vivo. This, however, may be dependent on the level of agonist exposure. The levels of dopamine receptor blockade are comparable to those that have been found after clinically relevant doses of neuroleptics in psychotic patients. Further, the present results indicate that neuroleptic-induced catalepsy or loss of motor behavior is associated with near maximal levels of dopamine D 2 receptor occupation. However, D¿ occupation was relatively more 'effective' than corresponding D 2 blockade. In contrast, inhibition of pharmacologically increased dopaminergic neurotransmission occurs at levels of dopamine receptor blockade which correspond to those required to 'control' psychosis. The fact that relatively low D~ blockade was associated with antagonism of amphetamine-induced discrimination may suggest high antipsychotic potential for such compounds.

Acknowledgements B. Petersen, B. Lehmann, K. Jonsson, A. Hansen, H.F. Bech and D. Andersen are thanked for skillful technical assistance as is H.F. Estrup for preparing the manuscript. The various pharmaceutical companies listed in the Methods section are thanked for their generous gifts of drugs.

References Altar, C.A., A.M, Wasley, R.F. Neale and G.A. Stone, 1986, Typical and atypical antipsychotic occupancy of D2 and $2 receptors: an autoradiographic analysis in rat brain, Brain Res. Bull. 16, 517. Andersen, P.H., 1988a, The dopamine DI receptor, a potential target for antipsychotic drugs, CINP-Meeting, Mfinchen, Germany, Aug. 15-19. Andersen, P.H., 1988b, Comparison of the pharmacological characteristics of ['H]raclopride and [3H]SCH 23390 binding to dopamine receptors in vivo in mouse brain, Eur. J. Pharmacol. 146, 113. Andersen, P.H., 1988c, Differential effects of E E D Q on the dopamine D-1 receptor in viw) and in vitro, Eur. J. Pharmacol. 152, 153.

44 Andersen, P.H. and F.C. Gronvald, 1986, Specific binding of ~H-SCH 23390 to dopamine DI receptors in viw) (published erratum appears in Lifc Sci. 1986 Sept. 22; 39 (12): 1117), Life. Sci. 38, 1507. Andersen, P.H. and E.B. Nielsen, 1988, The dopamine D1 receptor: occupancy and behavioral effects, 7th ESN-Meeting, G6teborg, Sweden, June 12-17. Arnt, J., 1985, Behavioural stimulation is induced by separate dopamine D-1 and D-2 receptor sites in reserpine-prctreated but not in normal rats, Eur. J. Pharmacol. 113, 79. Arnt. J., J. Hyttel and E. Meier, 1988, Inactivation of dopamine D-1 or D-2 receptors differentially inhibits stereotypies induced by dopamine agonists in rats, Eur. J. Pharmacol. 155, 37. Farde, L., F.-A. Wiesel, H. Hall, ('. ttalldin and G. Sedvall, 1988, Central D2-dopaminc receptor occupancy in schizophrenic patients treated with antipsychotic drugs, Arch. Gem Psychiat. 145, 71. Farde, L., F.A. Wiesel, A.-L. Nordstr6m and G. Sedvall, 1989, DI and D2-dopamine receptor occupancy during treatment with conventional and atypical neuroleptics, Psychopharmacology 99, $28. Fink-Jensen, A., E.B. Nielsen and P.H. Anderscn, 1989, Dopamine receptor occupancy in viw): functional correlates, in Mesolimbic dopamine system: from motivation Io action (Abstract), Malta, Sept. 25-29. Kilbey, M.M. and E.H. Ellinwood, Jr.. 1979, Discriminative stimulus properties of psychomotor stimulants in the cat, Psychopharmacology (Berlin) 63, 151. K6hler. C.. L. Haglund. S.-O. O~ren and T. Angeby, 1981, Regional blockade by neuroleptic drugs of in vivo ~H-spiperone binding in the rat brain. Relation to blockade of apomorphine induced hyperactivity and stereotypies, J. Neural Transm. 52, 163. K6hler, C., H. Hall and L. Gawell, 1986, Regional in viw) binding of the substituted benzamide [3H]eticlopride in the rat brain: evidence for selective labelling of dopaminc receptors, Eur. J. Pharmacol, 1211, 217. LeFur, G., F. Guilloux and A. Uzan, 1980, In viw) blockade of dopaminergic receptors from different rat brain regions by classical and atypical neuroleptics, Biochem. Pharmacol. 29, 267.

Leonard. M.N., C.A. Macey and P.G. Strange, 1987, Heterogeneity of D2 dopamine receptors in different brain regions. Biochem. J. 248, 595. Meller, E., F. Bordi and K. Bohmakcr, 1989. Behavioral recovery after irreversible inactivation of D-1 and D-2 dopamine receptors, Life Sci. 44. 11119. Morelli, M. and G. Di Chiara. 1985, ('atalepsy induced by S('tt 23391t in rats, Eur. J. Pharmacol. 117, 179. Nielsen, E.B., 1981, Rapid decline of stcrcotyped behavior in rats during constant one week administration of amphetamine via implanted A L Z E T osmotic minipumps, Pharmacol. Biochcm. Behav. 15, 161. Nielsen, E.B., 1987, Lack of cholincrgic modulation of mesolimbic dopamine function (Abstract), Dopaminc '87, l lunter Valley. Australia, I U P H A R Sattelite Meeting. Nielsen E.B. and P.H. Andersen, lt~91, Drug discrimination approaches to be behavioral role of the D-I receptor (Abstract), Br. Assoc. Psychopharmacol. July 21-24. Nielsen, E.13. and S.A. Jepsen, 1985, Antagonism of the amphetamine cue by both classical and atypical antipsychotic drugs, Eur..1. Pharmacol. 111, 167. Nielsen E.B. and ,1. ScheeI-Kri~gcr, 1986. ('ueing effccls of am phelaminc and LSD: clicitation by direct microinjcction of the drugs into the nucleus accumbens, Eur. J. Pharmacol. 125, S5. Nielsen E.B. and J. SchceI-Kri~lger, 1988, ('cnlra] nervous system stimulants: ncuropharmacological mechanisms. Psychopharmacc~l. Ser. 4. 57. Nielsen, E.B., K. Randrup and P.H. Andersen, ]989, A m p h c t a m i n c discrimination: effects of dopamine reccptor agonists. Eur. J. Pharmacol. 16/), 253. Pedersen, V. and A.V. Christenscn, 1972, Antagonism of mcthylphenidate-induced stereotyped gnawing in mice, Acta Pharmacol. Toxicol. (Copenhagen) 31,488. Weathersby, R.T. and J.B. Appe], 1986, Dopaminc D2 recepto~ mediation of the discriminative stimulus properties of I.Y 171555 (quinpirole), Eur. J. Pharmacol. 132, 87.