Life Sciences, Vol. 33, pp. 765-768 Pergamon Press Printed in the U.S.A.

77Br-p-BROMOSPIPERONE : A LIGAND FOR IN VIVO LABELLING OF DOPAMINE RECEPTORS

Frank Owen, Mark Poulter, Robert D. Mashal, Timothy J. Crow, *Norman Veall and *Guissepe D. Zanelli

Division of Psychiatry and *Division of Radioisotopes, MRC Clinical Research Centre,

Watford Road, Harrow, Middlesex, HAl 3UJ, U.K.

(Received in final form June 6, 1983)

Summary

A high striatum : cerebellum ratio of 77Br-p-bromospiperone (77Br-BrSp) was observed in rat brain following tail vein injection of the drug. Striatal 77Br-BrSp was stereospecifically displaced by the isomers of flupenthixol. After chronic haloperidol administration striatal dopamine receptor supersensitivity was demonstrated both by increased 3H-spiperone binding to striatal

c 77 membranes in vitro and by in teased striatal Br-BrSp content. These results confirm and extend previous findings and enhance interest in the use of 77Br-BrSp for the in vivo assessment of central dopamine receptors in man.

Biochemical studies of post-mortem brains have revealed abnormal numbers of dopamine receptors in patients with schizophrenia (1,2,3), Huntington's chorea (4) and Parkinsonism (5). It has been reported (6,7,8) that the gamma emitter 77Br-p-bromospiperone (77Br-BrSp) is a ligand for the dopamine receptor and might be used to assess central dopamine receptors in living subjects by single photon emission computed tomography.

Non-radioactive BrSp has been reported to be a potent displacer of 3H-spiperone binding in vitro and to increase plasma prolactin levels (9). Following tail-vein injection of 77Br-BrSp in rats the distribution of radioactivity in the brain has been reported to be consistent with the known distribution of dopamine receptors (7). We report here additional experimental data which provide further evidence that 77Br-BrSp binds to dopamine receptors in vivo in the rat.

Materials and Methods

(a) Experimental Animals. Male Sprague Dawley rats were used in all the experiments. In studies of the displacement of 77Br-BrSp by the isomers of flupenthixol animals weighed 400-450g. Rats weighing 200-250g were used to study the effect of chronic haloperidol administration on striatal dopamine receptors.

(b) Isotopes

77Br supplied by the MRC Cyclotron Unit, Hammersmlth Hospital, London, was reacted with spiperone to produce 77Br-para-BrSp with a specific activity usually about I0 Ci/mmol, using a technique based on that described previously (6,9). 3H-spiperone (26 Ci/mmol) was obtained from Amersham International.

0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

766 I~7 Y~l~<) Dopamine Receptor Labelling Vol. 33, No. 8, 1983

(c) Druss and Chemicals

The isomers of flupenthlxol were a gift from Lundbeck Ltd, and haloperidol and spiperone were gifts from Janssen Ltd. All other chemicals were obtained commercially.

(d) Displacement of striatal 77Br-BrSp by the isomers of flupenthixol

Three groups of 5 rats each received 50 uCi (3.9 nmol) of 77Br-BrSP (13 Ci/mmol) i.v. in the tail veins. In addition to the 77Br-BrSp, one group also received ~-flupenthixol (2 mg/kg) and another group a similar dose of B-flupenthixol in the injection. Four hours after the injection the rats were decapitated and striatum and cerebellum dissected out and weighed. Radioactivity in the samples was then determined in a LKB-WalIac 1280 y-counter and the results expressed as counts per minute (cpm)/g tissue.

(e) Effect of Chronic Haloperidol Treatment on 77Br-BrSp Content in Rat

Striatum and 3H-Spiperone Binding to Striatal Membranes

Forty rats were randomly allocated to two groups of 20 animals. One group received haloperidol (1.5 mg/kg/day) in their drinking water for 3 weeks. Haloperidol was then withdrawn for 5 days to allow elimination of the drug. The other group of rats, identically housed, served as controls. Ten haloperidol treated and lO control rats then received 15 ~Ci (3 nmol) of 77Br-BrSp (5 Ci/mmol) into the tail veins. Four hours later the rats were decapitated and 77Br-BrSp concentration determined in striatum and cerebellum. The remaining I0 haloperidol treated and I0 control rats were decapitated, striatum dissected out and 3H-spiperone binding determined at 1.0 nM as described elsewhere (I).

Results

The effect of ~ and B-flupenth[xol on 77Br content of striatum and cerebellum is presented in Table I which shows that (i) rats receiving 77Br-BrSp alone had a high striatum to cerebellum ratio in 77Br-BrSp content; (ii) B-flupenthixol only partially displaced striatal 77Br-BrSp whereas the displacement with ~-flupenthixol reduced striatal 77Br-BrSp to cerebellum levels; (iii) neither ~ no~ B-flupenthixol had any effect on the 77Br-BrSp content of the cerebellum.

TABLE I

Effect of = and B-Flupenthixol on 77Br-BrSp in Rat Brain (values as mean ~ SD)

Treatment Striatum Cerebellum Striatum : Cerebellum cpm/g tissue cpm/g tissue

77Br-BrSp (n = 5) 35,015 + 7150 4,466 + 670 7.8 + I.I

77Br-BrSp + B-flupenthixol 21,440 + 3561 4,584 + 750 4.7 + 0.8 (n = 5)

77Br-BrSp + a-flupenthixol 4,459 + 772 4,520 + 597 0.99 + 0.2 (n = 5)

Vol. 33, No. 8, 1983 In Viuo Dopamine Receptor Labelling 767

The effect of chronic haloperidol treatment on striatal 77Br-BrSp and 3H-spiperone binding to striatal membranes is illustrated in Fig.l.

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Effect of chronic haloperidol treatment on (A) 77Br-BrSp concentration in control (C; n = I0) and haloperidol (H, n = I0) treated rats and (B) 3H-spiperone binding to striatal membranes from control (C; n = I0) and haloperidol treated (H; n = i0) rats. (Bars represent S.D.)

Specific 77Br-BrSp content was determined by subtracting the cpm/g tissue of 77Br-BrSp in cerebellum from the corresponding cpm/g tissue of 77Br-BrSp in strlatum. It has been shown previously (6) that the cere- bellum contained the lowest levels of 77Br-BrSp in the brain after tail vein injectlon and that an excess of unlabelled spiperone reduced strlatal 77Br-BrSp levels to that of the cerebellum. 77Br-BrSp content in strlat~n of haloperidol treated rats was increased by 29.3% (p <.01). 3H-spiperone binding in membrane preparations of striatum from haloperidol treated rats was increased by 24.0% (p <.025) compared with controls. The content of 77Br-Sp in cerebellum was similar in controls and haloperldol treated rats.

Discussion

The high striatum to cerebellum ratio of 77Br-BrSp content shown in Table I for rats receiving neither isomer of flupenthlxol is consistent with previous reports (6,8) and also consistent with the known distribution of dopamine receptors i.e. dense in the strlatum and absent from the cerebellum.

768 In F~O Dopamine Receptor Labelling Vol. 33, No. 8, 1983

The ~-isomer of flupenthixol is known to be many times more potent than the B-isomer in displacing ligands from the dopamine receptor (i0). Also, clinical trials have demonstrated that the anti-psychotic efficacy of flupenthixol is attributable only to the ~-isomer (II). The stereospecific displacement of 77Br-BrSp from rat striatum by the isomers of flupenthixol therefore provides firmer evidence that 77Br-BrSp is binding to dopamine receptors than simply the displacement by spiperone reported by other workers (6,7,8).

It is now well established that chronic neuroleptic administration to rats results in dopamine D2 receptor supersensitivity characterised biochemically by an increase in striatal spiperone binding sites (for review see 12). The results of the present study (Fig.l) illustrate that after chronic haloperidol administration to rats an increase in dopamine receptors can be demonstrated by 3H-spiperone binding in vitro and a corresponding increase in 77Br-BrSp binding in vivo. Thi~ str--~-~ngly suggests that 77Br-BrSp binds to striatal dopamine receptors.

So far no binding of 77Br-BrSp has been reported using membrane preparations. In preliminary studies with either standard filtration or centrifugation techniques, to separate free from bound ligand, we have observed minimal in vitro specific 77Br-BrSp binding to membrane preparations. It may be that 77Br-BrSp will not prove a useful ligand for in vitro binding studies. Nevertheless, the results of the present study, and of previous work (6,7,8) indicate that 17Br-BrSp binds to dopamine receptors in vivo and it seems worthwhile to develop suitable in vivo techniques for studies with 77Br-BrSp in man.

Acknowledsement

R.D.M. is the recipient of a Commonwealth Scholarship.

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