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IN VNO CHARACTERIZATION OF R/S- AND R-["cIsKF' 82957

AS A Dl AGONIST RADIOLIGAND FOR PET:

EFFECT OF DRUGS ACTING ON THE DOPAMINE SYSTEM

Eric Greenwdd

A thesis subrnitted in confomüty with the requirements for the degree of Master of Science

in the Graduate Department of Pharmacology, University of Toronto

Q Copyright by Eric Greenwald (1998)

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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

In vivo Characterization of RB- and R-["CISKF 82957 as a Dr Agonist Radioligand for

PET: Effect of Drugs Acting on the Dopamine System

Master of Science 1998

Eric R. Greenwald

Graduate Department of Pharmacology, University of Toronto

ABSTRACT

Rationale: Di receptors exist in two interconvertable states, the high- and low-affînity

state. Dl antagonists do not differentiate between these states and bind to the total density of

DI recepton in in vitro assays. In theory, only Dl agonists can bind selectively to the high

affinity state of Di receptoa. As the high-affinity state is believed to be the functional state

of the receptor, changes in the proportion of D~~~~~ may play a role in a number of human

disordea where Di receptors have been implicated. Using the DI antagonist SCH 23390,

labeled with carbon-11, no differemce in striatal Dl receptor levels were shown with positron

emission tomography (PET) in dnig naïve schizophrenics and Parkinsonians as compared to

controls. All pnvious Dl radioligands developed for PET have been antagonists and thus

cannot be used to determine changes in the proportion of D ~ ~ ~ ~ . Development of the Dl

agonist radioligand RB- and R-["CISKF 82957 may potentially allow in vivo measurement

of the high-affinity state of Di receptoa. Objectives: The objectives of this project include

deteminhg the in vivo binding of the pure :nantiorner R-["CISKF 82957 in rats and

comparing this binding profile to that of the racemic R/S-["C]SKF 82957. The effect of

endogenous dopamine on NI vivo R/S-[L'~]~~~ 82957 binding in Di rich regions was

examined In addition, the effect of chronic treatments with the DI antagonist SCH 23390

and the indirect dopamine agonist cocaine on binding of WS- and R-["CISKF 82957 was

determined and compared to that of ["C]SCH 23390. Merhodr: Time course of the regional

brin distribution and effect of saturating doses of ritanserin, sulpiride, SCH 23390 or SKF

82957 was deterrnined for R-["C]SKF 82957. R/s-["C]SKF 82957 ngional brain uptake

was evaluated following pretreatment with dmgs known to alter concentrations of synaptic

dopamine including: amphetamine, cocaine, GBR 12909, clorgyline and reserpine. In

addition, regional brain uptake of ["C]SCH 23390, R/S- and R-["CISKF 82957 was

established following chronic treatment with SCH 23390 (0.5 mgkg) for 21 days or various

dosing protocols of repeated cocaine (20 mgkg). Results: R-[' 'CISICF 82957 binds

selectively to Di receptors in vivo and has higher signal-to-noise ratios than WS-["C]SKF

82957. Binding of RIS-["C]SKF 82957 was unaltered by acute changes in endogenous

dopamine. Cerebellar binding ratios of RIS- and R-["C]SKF 82957 diffend from that of

["CISCH 23390 following chronic treatment with SCH 23390 but not with cocaine.

Conclusions: RfS- and R-["CISKF 82957 is a suitable ligand for studying Di recepton Ni

vivo as it is selective for Di recepton and is unaffected by changes in endogenous dopamine.

In addition, this study indicates that RIS- and R-["C]SKF 82957 bind to a different

population of Di receptors Ni vivo than ["CISCH 23390, and these populations are

di fferen tiall y ngulated in response to c hronic SCH 23390 treatments. Di supersensitivi ty

induced by chronic cocaine treatments was not detected by ["CISCH 23390, RB- or R-

[ll~]~KF 82957.

iii

ACKNOVVILEDGEMENTS

1 would like to thank first and foremost my supervisor, Dr. Jean N DaSilva 1 have

had the pleasun of working with Dr. DaSilva for over two years and throughout this pend

of time he has shown unwavering patience, guidance and an uncornmon devotion to his

students.

1 would also like to thank my fnends and colleagues Celia Lourenco, Alan Wilson,

Doug Hussey, Kevin Cheung, and Robert Schwartz.

This work was supportcd by scholarships from the University of Toronto and

University College.

Finally, 1 would like to thank my farnily. 1 owe a debt of gratitude to my sister Iris

and my brother Allen for keeping me sane during stressful times. Most importantly, 1 thank

my parents for their unending support, commitrnent and love. I dedicate this thesis to them.

TABLE OF CONTENTS

TITL.E

ABSTRACT

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

LIST OF THESIS PUBLICATIONS

ABBREVIATIONS

CHAF+TER 1.0: INTRODUCTION

General introduction The dopamine neuronal system Dopamine receptor subtypes Di receptor function

1.4.1 High- and low-affinity state 1.4.2 G proteins 1.4.3 Second messenger pathways Dl receptors in neuropsychiatrie disorden

1 .S. 1 Parkinson's disease 1 S.2 Schizophrenia 1.5.3 Tardive Dyskinesia 1.5.4 Huntington's disease 1.5.5 Drug dependence

Regulation of Di receptor function 1.6.1 Desensitization 1.6.2 Supersensi tization

1.6.2.1 Chronic D antagonisu 1.6.2.2 Chronic reserpine 1.6.23 Dopamine neuron lesioning with

MPTP and 6-OHDA 1.6.2.4 Chronic Di agonists 1.6.2.5 Psychostimulant dnigs

Positron emission tomography and Dl receptors

i

ii

iv

v

viii

ix

xiii

1.8 Research objectives 1.8.1 Mainobjective 1.8.2 Hypotheses

1.8.2.1 General h ypotheses 1.8.2.2 Working hypotheses

1.8.3 Specific aims 1.8.4 Description of Chapter 2 1.8.5 Descriptionof Chapter3 1.8.6 Description of Chapter 4

CHAPTER 2.0: DOPAMINE Di AGONIST R-["cIsKF 82957: SYNTHESIS AND IN VIVO CHARACTERIZATION IN RATS

2.1 Absmt 2.2 Introduction 2.3 Materiais and methods 2.4 Results 2.5 Discussion 2.6 Statement of significance

CHAPTER 3.0: IN VIVO BINDING OF THE Dl AûûNIST PET LIGAND ["CISKF 82957 IN RATS : EFFECT OF ENWGENOUS DOPAMINE AND CHRONC TREATMENT WITH SCH 23390

3.1 Abstract 3.2 Introduction 3.3 Materials and methods 3.4 Results 3.5 Discussion 3 -6 S tatement of significance

CHAPTER 4.0: IN VIVO BIM>ING OF THE Di AGONIST PET RADIOLIGAND R- AND R/S-("C]SKF 82957 FOLLOWING CHRONIC COCALNE

4.1 Abstract 4.2 Introduction 4.3 Materials and methods 4.4 Results 4.5 Discussion 4.6 Statement of significance

CHAPTER 5.0: SUMMARY AND GENERAL DISCUSSION

CHAPTER 6.0: CONCLUSIONS

8.0 APPENDIX: OTHER PUBLICATIONS

8.1 Statistical power analysis of in vivo studies in rat brain using PET radiotracers

8.2 Chronic reserpine differentially dten Ni vivo binding of Dl a onist WS- and R-["CISICF 82957 as compared to R [ CJSCH 23390 in rat brain

vii

LIST OF TABLES

Chapter 2 Page

Table 1 Biodistribution of R-["CISKF 82957 in rats, 45 min after administration 37

Table 2 Biodisiribution of RIS-["C]SKF 82957 in rats 38

Table 3 Cdculated absorbed dose to humans for RIS-["c]sKF 82957 based on biodistribution in rats 39

Chapter 3

Table 1 Striatum-to-cenbellum ratios of radioactivity in rats following various dmg pntreatments, 45 min after R/s-[~ 'CISKF 82957 administration 54

Table 2 Effect of chronic SCH 23390 treatment on region-to- cerebellum ratios of ["CISCH 23390, R- and RIS-["C]SKF 82957 in rat train, as percent change frorn conmls 57

viii

LIST OF FIGURES

Chapter 1

Figure 1 Schematic illustration of a disintegrating positron-emitting isotope

Figure 2 Schematic illustration of a positron carnera

Chapter 2

Figure l Structure of R-SKF 82957

Figure 2 Re 'onai brain uptake and cerebellar ratios of Ti R-[ CISKF 82957 in rats Figure 3 Effect of matment with various dmgs on the

regional brain uptake of R-["CISKF 82957

Chapter 3

Figure 1 Effect of pretreatment with amphetamine, cocaine, GBR 12909, clorgyline, or reserpine on regional distribution of R/S-["C]SKF 82957 in rat brain

Figure 2 Effect of chronic treatment with SCH 23390 on regionai distribution of ["C]SCH 23390, R- and RIS-["CISKF 82957 in rat brain

Figure 3 Effect of chronic treatment with SCH 23390 on cerebellar ratios of ["CJSCH 23390, R- and RB-["cIsKF 82957 in rat brain

Chapter 4

Figure 1 Effect of chronic treatment with cocaine (14 days, last injection 20 min pnor to tracer injection) on regionai distribution of ["C]SCH 23390, R- and RIS-["C]SKF 82957 in rat brain 71

Figure 2 Effect of ctuonic treatment with cocaine (14 days, 1st injection 20 min prior to tracer injection) on cerebellar ratios of ["CISCH 23390, R- and RIS-["C]SE;F 82957 72

Figure 3 Effet of chronic treatment with cocaine (14 days + 14 days withârawal) on regional distribution of ["CISCH 23390, R- and WS-["C]SKF 82957 in rat brain 73

Figure 4 Effect of chronic treatment with cocaine (14 days + 14 days withdrawal) on cenbellar ratios of ["CISCH 23390, R- and R/S-["C]SKF 82957 74

Figure 5 Effect of chronic treatment with cocaine (5 days + 2 days withdrawal) on re 'onal distribution of R ["CISCH 23390 and R-[ CISKF 82957 in rat brain 75

Figtue 6 Effect of chronic treatrnent with cocaine (5 days + 2 days withdrawal) on cenbellar ratios of ["CISCH 23390 and R-["C]SKF 82957 76

LIST OF THESIS PUBLICATIONS

ARTICLES:

To Be Submitted:

Jean N. DaSilva, Robert A. Schwartz, Eric R. Greenwaid, Celia M. Lourenco, Alan A. Wilson & Sylvain Houle. Dopamine DI Agonist R-["C]SKF 82957: Synthesis and In Vivo Chmcterization in Rats. Nuclear Medicine and Biology (to be su bmitted).

Eric R. Greenwaid, Jean N. DaSilva, Celia M. Lourenco, Alan A. Wilson, and Sylvain Houle. In Vivo Binding of the Di Agonist PET Ligand ["CISKI? 82957 in Rats: Effect of Endogenous Dopamine and Chronic Treatment With SCH 23390. Synapse (to be submitted).

Eric R. Greenwald, Jean N. DaSilva, Celia M. Lourenco, Alan A. Wilson, and Sylvain Houle. In Vivo Binding of the Dl Agonist PET Radioligand R- and RIS- ["CISKF 82957 Following Chronic Coeaine. Psychophnnacology (to be submitted).

BOOK CHAPTERS:

D. Hussey. J.N. DaSilva, E. Greenwald, K. Cheung, S. Kapur, A.A. Wilson & S. Houle. Statistical Power Analysis of In Vivo Studies in Rat Brain Using PET Radiotracers. In Ouantitative Functional Brain Irnagin~: with Positron Emission Tornoma~hv. eds. Carson RE, Daube-Withenpoon ME & Herscovitch P. Academic Press (1998).

ORAL PRESENTATIONS:

E. Greenwald, J.N. DaSilva, A.A. Wilson and S. Houle. In Vivo Evaluation of R-["CISKF 82957 in Rats as a Potenaal Dopamine Dr A g o ~ s t Tracer for PET. Abstract. nie F@h Annual Psychophamco logy Duy, Sunnybrook Health Science Centre, University of Toronto (October 16,1997).

ABSRACTS:

J.N. DaSilva, E. Gnenwald, R.A. Schwartz, A.A. Wilson and S. Houle. Chronic Reserpine DifferenWy A ~ R In Vivo Binding of Dl Agonist RIS- and R-["C]SKF 82957 as Compared to [ " ~ S C H 23390 in Rat Brain. Abs~ract, S o c i e ~ of Neuroscience Meeting (November, 1998).

DaSilva J.N., Wilson A.A., Greénwald E. and Houle S. R(+)-[C-11]SKF 82957: Synthesis and Evaluation in Rats as a Dopamine D-1 Agoiiist Tracer for PET. Abstract, 4 4 1 h Annual Meeting ofthe Society of Nuctear Medicine, JNM Abstract Book Supplernent, 38: 76, 1997.

Greenwaid E, DaSilva J.N., Vaiente C, Wilson A.A and Houle S. In Vivo Evaluation of WS-[C-11lSKF 82957 in Rats as a D-1 Agonist Tracer for PET: Effeet of Endogenous Dopamine and Chronic D-1 and D-2 Antagonists Abstract, 451h Annual Meeting of Socieq of Nuclear Medicine, JNM Abstract Book Supplement, 39: 236, 1 998.

Greenwald E, DaSilva LN., Wilson A.A. and Houle S. In Vivo Evaluation of R(+)-[C-111SKF 82957 In Rats as a Potenth1 Dopamine D-1 Agoaist Tracer For PET. Abstract, The Fifth Annual Psychopharmacology Day , Sunnybrook Health Science Centre, Universiry of Toronto (October 16, 1997).

Greenwald E, DaSilva J.N., Valente C, Wilson A.A and Houle S. In Vivo Evaluation of RB-["cIsKF 82957 in Rats as a Di Agonist Tracer for PET: Effect of Endogenous Dopamine and Cbroaic Dl and D2 Antagonists. Abstruct, Visions in Phnnnacology, University of Toronto (May 15. 1998). p. 59.

6-OHDA

AC

ANOVA

ATP

CAMP

CNS

DA

DAG

DAT

DMF

EPS

G protein

GDP

Gi

GS

GTP

HD

HPLC

I p 3

Kd

MVrP

NAc

ABBREVIATIONS

6-h ydroxydopamine

adenylyl cyclase

analysis of variance

adenosine triphosphate

3', 5' cyclic adenosine monophophate

central nervous system

dopamine

diac y lgl y cerol

dopamine transporter

dimethy lfomamide

extrapyramidal si& effects

guanine nucleotide binding protein

guanosine diphosphate

inhibitory G protein

stimulatory G protein

guanosine triphosphate

Hun tington' s discase

high-performance liquid chromatograph y

inositol triphosphate

dissociation constant

1 -methyl+phenyl- l,2,3,6-tetrahydropyridine

nucleus accumbens

PD

PET

PKA

PKC

PLC

TLC

Parkinson's disease

positron emission tomogniphy

protein kinase A

protein kinase C

phospholipase C

schizophrenia

hdf-li fe

tardive dyskinesia

thin-layer chrornatography

1 .O Introduction

1.0 INTRODUCTION

1.1 GENERAL INTRODUCTION

The dopamine @A) neuronal system has k e n snidied extensively over the last 30

yean due to its importance in the control of many physiological processes such as prolactin

secretion, cognition. motor function, and the development of several human brain disorden.

Included in these are dnig dependence, schizophrenia (SZ), tardive dyskinesia (TD),

Huntington's 0) and Parkinson's Disease (PD) (reviewed in (Homykiewicz, 1966; Clark

and White, 1987; Seernan et al., 1987; Joyce et al., 1988; Davis et ai., 1991; Woolverton and

Johnson, 1992; Miller and Chouinard, 1993). Although the DA system has k e n irnplicated in

al1 of these disorden. the precise mechanisms at work are still, for the most part, unclear.

In general, ceils are able to respnd to extracellular signais through receptor proteins

expressed on the ce11 surface membrane. Upon receiving the extracellular signal, recepton

may function through the opening of specific ion channels or by the formation of second

messengers. Many neurotransmitter recepton, including the DA Dl receptor, function

through interaction with a guanine nucleotide binding protein (G protein). Activation of the

G protein cm lead to the formation of intracellular second messengers, which can induce a

host of actions including ngulation of protein kinases. gene expression and other cellular

processes. Recent dmg development, molecular biological and nuclear imaging techniques

have extended our knowledge of the complexities of the DA receptor system, which now

includes several receptor isoforms. These recent advances have allowed for further study of

receptor function and their role in normal and diseased States.

1.2 l''HE DOPAMINE NEURONAL SYSTEM

DA acts as a hormonelneurotransmitter in the central nervous system (CNS) as well

as in the periphery. Wiihin the CNS, there are two major dopaminergic systems, the

mesocorticolimbic and nigrostriatal. The mesocorticolimbic system originates in the A10 DA

cells of the ventral tegmentd area and projects to the nucleus accumbens (NAc) and olfactory

tubercles. The nigrostriatal tract originates in the A9 DA cells of the substantia nigra and

projects to the striatum. These systems are not completely exclusive and contain some

overlapping anatomy (reviewed in Volkow et al., 1996; White, 1996). Despite their close

proximity, these systems control very diffennt behavioural functions. The nigrostriatal

system is mainly concemed with the initiation and execution of movements while the

mesocorticolimbic system is involved with the reward and motivational properties of stimuli.

13 DOPAMINE RECEPTOR SUBTYPES

DA receptoa belong to the superfamily of G protein coupled receptors and several . subtypes have k e n identified that differ in their structural and pharmacological

charactenstics. Multiple receptors for DA were fint proposed by Kebabian and Caine in

1979 (Kebabian and Calne, 1979). Two distinct receptor subtypes were established based on

their interaction with the effector enzyme adenylyl cyclase (AC). The DI receptor subtype

stimulates AC while the D2 receptor subtype either inhibits or is independent of AC

(Kebabian and Calne, 1979; Stwf and Kebabian, 1984; Seeman and Grigoriadis, 1987).

Severai isoforms were later discovered and these are now divided into DI-like (Dl and DS)

and D2-Like (D2, D3 and D4) (Niznik and Van Tol, 1992). Stnicturally, the Di-like receptors

have short third intracellula. loops and long carboxy terminal tails and the D2-iike receptors

have long third intracellular loops and short carboxy terminal tails (O'Dowd, 1993). DI-like

receptor genes are similar to those of other seven trammembrane receptor genes in that they

contain vimially no introns, whereas the D2-like receptor coding sequences are intempted by

introns (O'Dowd, 1993).

Recently, dmgs able to discriminate between the D2-like DA receptor subtypes have

been developed, although no such drugs are available as yet for the DI-like DA receptors. DI

and DI receptors are structurally similar however important differences exist. Despite the

finding that the Ds receptor binds dmgs with a sirnilar pharmacological profile to Di, it

displays a IO-fold higher affinity for DA (Sunahara et al., 1991). A cornparison of DI and D5

arnino acid structures reveals an overall homology of 50%, while much higher hornology is

observed within the membrane spanning regions (ODowd, 1993). The distribution of Di and

D5 mRNA also appear to be distinct from each other. Low amounts of Ds mRNA are

expressed in the brain areas where Di mRNA is abundant, such as stnatum, olfactory

tubercles and NAc (Tiberi et al., 199 1).

1.4 Dl RECEPTOR FUNCTION

1.41 KIGH- AND LOW-AIFFINITY STATE

As with other G protein coupled receptors, Di receptors exist in iwo interconvertible

states, the high-affinity and the low-affinity states. The state of DI receptors

may have as much as a 10'-fold increased affinity for DA as compared to the Iow-affinity

state (Seeman and Grigoriadis, 1987). According to the temary complex model, receptor

function is dependant on interaction between ligand, receptor and G protein (Mitchell and

Seeman, 1998). The high-affmity state in this model is described as the state in which the

receptor is functionally coupled to the G protein. The low-affinity state occurs when the

receptor is dissociated from the G protein. Other models of receptor state conformation have

been developed to address promiscuous receptors, which couple to several different types of

G proteins o f f et al., 1985), and constitutively active receptors (Bond, 19971, however, the

definition of the high- and low-affînity states remains similar. Di antagonists, such as SCH

23390, do not distinguish between the two states and bind to the total nurnber of Di recepton

in nceptor binding assays (Leff et al., 1985; Seeman and Grigoriadis, 1987; Rubinstein et al.,

1990). Only Di agonists and partiai agonists have the potentiai to bind selectively to the

functional highwaffinity state of the receptor.

1.4.2 G PROTEINS

Over the last 20 years, knowledge of the functions and varieties of G proteins has

expanded immensely (for review see Gilman, 1987 and Bimbaumer, 1990). These membrane

bound proteins act as signal tramducen relaying information from the rcceptor to the intenor

environment of the cell. G proteins exist as heterotrimers consisting of an a-, & and y-

subunit Oessauer, 1996). The a-subunit is capable of binding guanosine diphosphate (GDP)

or guanosine triphosphate (GTP). Many types of G proteins have been purified and were

originally classified according to function, however, severai G proteins have been isolated

and cloned without their function being known. G proteins classified according to function

include the O, and Cr, proteins, which activate and inhibit AC respectively. Other G proteins

include Gt, &, Goir and Go, which have various functions, and G,, which connects to the

phosphofipase C (PLC) pathway (Dessauer, 1996).

1.43 SECOND MESSENGER PATHWAYS

Dl receptors have been known to couple to O,, which triggers the formation of CAMP

through activation of AC (Clark and White, 1987). This paihway has been well established

and Di agonists are classified according to their ability to stimulate CAMP formation. CAMP

exerts its effects through cAMPdependent protein kinases (e.g. protein kinase A or P U )

(Mitchell and Seeman, 1998). Once activated by CAMP, these protein kinases then go on to

phosphorylate specific target proteins, which can exert effects on metabolisrn and gene

expression (Seeman and &igoriadis, 1987; Siûhu, 1988).

More recentfy, Di receptors have bem reported to stimulate phosphoinositide

hydrolysis and couple with Gq (Undie and Friedman, 1990; Kimura et al., 1995; Wang et al., . .

1995). Gq stimulates PLC activity which breaks down phosphatidylinositol-bisphosphate in«,

the second messengers inositol triphosphate (IP3) and diacylglycerol @AG) (Mitchell and

Seeman. 1998). These two molecules an each able to act as second messengea through two

different pathways. IP3 diffuses rapidly through the cytoplasm and can bind to ca2+ channels,

increasing the +concentration of Ca2+ within the cell. This rise in ca2+ concentrations can

trigger many effects including the activation of ca2+-binding proteins (e.g. calmodulin) and

~a~~/calmodulin-depen&nt protein kinases. The other second messenger in this pathway,

DAG, stays in the plasma membrane and is capable of activahg a group of enzymes know

as protein kinase C (PKC). PKC has a number of phosphorylation targets including ion

channels, receptors and other protein kinases (Mitchell and Seeman, 1998).

Di receptor agonists are classified according to their ability to stimulate CAMP

accumulation, however, other pathways (including PLC) may play a significant d e .

Examination of several Dl nceptor agonists showed littie conelation between behavioural

effects in rats and ability to stimulate AC (Arnt et al., 1992; Gilmore et al., 1995;

Gnanalingharn et ai., 1995). These findings indicate that the extent to which a DI agonist

activates AC is not the sole indicator of its in vivo action and that there may be a substantiai

role for other pathways in rnediating the effects of Di agonists.

15 Di RECEPTORS IN NEUROPSYCMATRIC DISORDERS

1.5.1 PARKINSON'S DISEASE

PD is characterized by a loss of dopaminergic neurons projecting from the substantia

nigra to the striatum (Guttman, 1992). The effect of this DA depletion on the Di receptors

has yielded conflicting results. In vitro studies of striatal Di receptors have show increases

(Seeman et ai., 1987) or no change (Cortés et ai., 1989) in B, as measured by [ 3 ~ ~ ~ ~ '

23390. In vivo studies using positron emission tomography (PET) and ["C]SCH 23390 have

shown no change in the density of stnatal DI receptors in untreated PD patients (Rinne et al.,

1990). In theory, the lack of agonist stimulation in PD is expected to increase the proportion

of Dl receptors in the high-affhity state as measured in the reserpine-animal model of PD

(Rubinstein et ai., 1990).

Cumntly, the most common treatment of PD is with L-DOPA and aithough this

treatment is usually very effective for the fint several years, dyskinesias and Ioss of efficacy

emerges over time (Kopin, 1993). Thus, in order to avoid these si& effects. new treatments

involving drugs that target specific DA receptor subtypes are king developed (Jenner, 1995).

SZ is a disease commonly charactenzed by hallucinations, delusions and

psychomotor unrest (Deniker, 1990). The most common cumnt treatment of SZ is with the

use of antipsychotic dmgs. Most antipsychotic dmgs act as antagonists at D2 receptors, and a

very good comlation has been established between the dissociation constant (Kd) of an

antipsychotic for the D2 receptor and their clinical oral doses required to block psychotic

symptoms (Seeman, 1995). PET has been used to show that antipsychotic h g s should

occupy 65% to 85% of D2 receptors in order to be effective (Farde et al., 1988; Nordstrom et

al., 1993; Kapur et al., 1996). Unfortunately, a variety of negative side effects, laiown as

extrapyramidal side-effects (EPS) occur upon antipsychotic dnig administration which

include parkinsonism and tardive dyskinesia (Hietala et al., 1990). A new class of "atypical"

antipsychotics, including the dnig clozapine, have been developed which have a very low

incidence of EPS (Altar et al., 1988; Meltzer, 199 1; Farde et al., 1992). PET studies have a

shown that while the Dz occupancy of these dmgs are lower than that of the typical

neuroleptics, the Dl occupancy is much higher reaching 38% to 52% (Farde et al., 1989;

Farde et ai., 1992; Nordstrom et ai., 1995). Thus, Dl receptors may play an important role in

the generation of negative symptoms (Lynch, 1992).

Hess et al (1987) reported a loss of striatal Dl receptors (43%) in SZ. In vivo

measurement of Dl receptors using ["CISCH 23390 and PET showed no difference in h g

naive SZ as compared to controls (Sedvall et al., 1992). DA overactivity in SZ is expected to

stimulate Dl receptors. In fact, Mamelak et al. (1993) have reported a decrease in the

proportion of Dl receptors in the high-afhity state.

1 . 3 TARDiVE DYSKINESIA

Rolonged administration of typical antipsychotic drugs, such as haloperidol, can

cause TD in patients. Treated patients in their fint episode of psychosis experience lower

incidence of TD (-15%) (Chakos et al., 1996) as compared to those treated for longer periods

of time (over 30%) (Jeste and Caligiuri, 1993; Miller and Chouinard, 1993). This side effect

is characterized by involuntary movements of the oro-facial musculature and may include the

limbs and trunk. TD syrnptoms may also be produced in rats by administration of Di

agonists, whereas Di antagonists decrease this behaviour (Lubin, 1995; Miller, 1993).

Atypical antipsychotics do not produce TD and this unique characteristic may be due to their

significant occupation of Di receptoa (Casey, 1989).

1.5.4 HUNTINGTON'S DISEASE

Hû is a disorder characterized by degeneration of cells in the basal ganglia, in which

the predominant symptom is hyperkinesia (Buniham. 1989). Both Di and 4 nceptor

densities have k e n reported to be reduced in HD patients using a variety of radioligands and

quantification techniques (Cross and Rossor, 1983; Sceman et al., 1987; Filloux et al., 1990).

1.5 J DRUG DEPElYOENCE

DA has been implicated in the euphoric and habit-forming effects of a number of

commonly abused drugs. Several seemingly unrelated compounds have been shown to

increase DA concentrations in the NAc of rats, including cocaine, amphetamine, opiates,

ethanol and nicotine (Di Chiara and Imperato, 1988). A significant comlation between

cocaine occupancy of the DA transporter @AT) and feeling of "high" has been established

(Volkow et al., 1997). DI recepton appear to play a particularly important role in mediating

the behavioural and pharmacological effects of cocaine and amphetamine. Full Di agonists

produced comparable behavioural effccts to cocaine in squiml monkeys (Rosenzweig-

Lipson et al., 1994) and are partially substituted in rats trained to self-administer cocaine

(Witkin et al., 1991). In addition, the DI antagonist SCH 23390 increased cocaine self-

administration in rats when injected peripherally (Comgall and Coen, 1991) or micro-

injected direaly into the NAc (Caine et al., 1995). In mutant mice lacking the DI receptor,

cocaine does not increase locomotion and electrophysiological studies reveal a reduction in

the inhibitory effects of cocaine on the generation of action potentials (Xu et al., 1994).

1.6 REGULATION OF Di RECEPTOR FUNCTION

Agonist stimulation of G protein coupled recepton is usually of limited duration and

can be regulated. Regulation of receptor funciion can occur mainly through two processes:

(1) by regulating the number of receptors expressed on the ce11 surface, and (2) by altering

the ability of the receptor to interact with G proteins. These two processes are capable of

inducing desensitized or supersensitized responses of receptors to agonists.

1.6.1 DESENSITIZATION

The phenornenon of desensitization is observed as a loss of cellular responsiveness to

a dnig after repeated or prolonged exposure to the h g . Receptor desensitization can be

divided into two categories: (1) homologous desensitization, which involves a loss in

sensitivity of the specific receptors being stimulated without affecting the responsiveness of

other receptor systems. and (2) heterologous desensitization, which cm result in a loss in

sensi tivity of several different receptor groups.

The process of desensitization involves a rapid uncoupling of the receptor from its G

proteins followed by receptor sequestration and downregulation. Generally, homologous

desensitization involves uncoupling of receptor from G protein through phosphorylation of

senne and threonine residues in the cytoplasmic lwps and carboxyl terminus of the receptor

by G-protein-coupled nceptor kinases that act specifically on that receptor (Mitchell and

Seeman, 1998). Heterologous desensitization diffen in that this phosphorylation is canied

out by second messengei activated protein kinases such as CAMP-dependent protein kinase

(PKA) and protein kinase C (PKC) (Ferguson et al., 1996; Ferguson et al.. 1998). Receptor

sequestration involves intemalization of receptors from the outer membrane for processing,

and downregulation occm with changes in receptor gene expression.

The Di receptor has been shown to undergo homologous desensitization under a

variety of experimental conditions. For example, prolonged stimulation of striatal slices with

DA attenuated subsequent stimulation of AC by DA (Memo et al., 1982). Acute

amphetamine Fatment, which releases endogenous DA, has been shown to desensitize

striatal Dl receptors in the rat (Bamett and Kuczenski, 1986; Roseboom and Gnegy, 1989).

Furthemore, Roseboom and Gnegy (1989) demonstrated that acute amphetamine did not

alter the number of Di receptors but that the proportion of receptoa in the high-affinity state

was reduced. Heterologous desensitization of the DI DA receptor has ken demonstrated in

opossum kidney cells (Bates et al., 1991) in which treatrnent with 8-Br-CAMP desensitized

DA stimulated AC in a'manner qualitative and quantitatively distinct h m that induced by

agonist exposure. In addition, heterologous desensitization of Di receptors was shown in Y1

adrenai ceils where matment with dopaminergic agonists diminished responsiveness of AC

to DA, ACTH and NaF (Olson and Schimmer, 1992).

1 SUPERSENSITUATION

Supersensitization of Dl receptors cm occur through two known mechanisms: (1)

upregulation, and (2) increased signal transduction efficiency of receptor and second

messenger systern. Upregulation of Dl receptors is an incnase in the total number of receptor

binding sites on the cell, leading to augmented responsiveness to dopaminergic stimuli. As

stated previously, the production of second messengea involves a number of steps, each of

which may be arnplified, leading to greater transduction efficiency. For example, increased

coupling of receptor and G protein could lead to an increased proportion of Dl receptors in

the high-amnity state. and enhanced responsiveness to agonist stimulation, in the absence of

increased total number of Dl recepton. This type of supersensitization could not be

quantified with an antagonist, such as ["CISCH 23390, as antagonists do not differentiate

between the high- and low-affinity state in in vitro binding assays. However, as stated

previously, Di agonists, such as RB- and R-["CISKF 82957, can theoretically bind

selectively to D~~~~ and thus be used to measure changes in the proportion of Dl receptors

in the high-affinity state. A number of different experimental treatments have been shown to

induce supersensitization of Di receptors, as descnbed below.

1m6.2m1 CEIRONIC Di ANTAGONISTS

Chronic treatment with the Dl selective antagonist SCH 23390 has k e n demonstrated

repeatedly to increase Di receptor binding sites (Creese and Chen, 1985; Chipkîn et al., 1987;

McGonigle et al., 1989; Lappalainen et al., 1992). Behaviouraily. rats treated with chconic

SCH 23390 showed a more intense stereotypy response to SKF 38393 (a partiai Di agonist)

and incnased spontaneous locomotor activity (Hess et al., 1986). In addition, DA-stimulated

AC activity was also increased by chronic SCH 23390 treatment (Hess et ai., 1986; Schettini

et al., 1992).

1.6.2.2 CHRONlC RESERPINE

Chronic administration of reserpine, a DA depleting agent, has also been shown to

induce striatal supersensitivity in rats ( h t , 1985). The AC response to Di receptor

stimulation was markedly enhanced following chronic reserpine treatment however, no

increases in the total density of DI receptors were observed as measured with L~H]SCH

23390 (Hess et al., 1986; Missale et al., 1989). Interestingly, the proportion of DI receptors in

the high-affinity state was increased as measured by DA competition for [ 3 ~ ~ ~ ~ 23390

binding (Rubinstein et al., 1990).

1.6.2.3 DOPAMINE NEURON LESIONING WITH MfTP GND &OHDA

Other treatments that reduce dopaminergic neurotransmission include lesioning the

nigrostriata1 tract with injections of 6-hydroxydopamine (6-OHDA) or 1-methyl4phenyl-

1,2,3,6-tetrahydropyridine (MlTP) to destroy dopaminergic neurons. 6-OHDA was shown to

increase the respnsiveness of globus pallidus neurons to Di agonists such as SKF 38393 and

the rnixed Di/D2 agonist apomorphine following 6-OHDA lesions (Carlson et ai., 1990),

indicating a Di receptor supersensitivity. Discrepant results have b e n reported concerning

changes in the total density of DI receptors following treatment with 6-OHDA in rats with

gmups observing increases (Porceddu et al.. 1987; Iwata et al.. 1996). decreases (Joyce,

1991) or no change (Morelli et al., 1990). M I T P matment in monkeys has also produced

variable results in terms of Di receptor density as no change (Pifi et al., 1992) and increases

(Gnanaiingham et al.. 1993) have been reported.

1.6.2.4 CHRONIC Di AGONISTS

Although the acute administration of DA agonists induces desensi tization, repeated

administration of a number of direct and indirect acting DA agonists has been shown to

induce supersensitization of Di associated behaviours. This seemingly paradoxical effect

occurs in repeated administration paradipu of the partial Di agonist SKF 38393 and the full

DI agonist SKF 81297 (Braun and Chase, 1988; White et ai., 1990; Hu et al.. 1992). These

studies, as well as othen. have show that animals which had received chronic Di receptor

stimulation, followed by a specified withdrawal period, will have altered behavioural and

electrophysiological test scores indicative of Di sensitization. Indeed, biochemical assays of

AC function have shown that this type of chronic matment will produce an enhancement of

the cyclase-associated Di receptor complex.

1.6.2.5 PSY CHOSTIMULANT DRUGS

The majority of studies on psychostimulant induced sensitization have used

amphetamine or cocaine (for review see Kalivas and Stewart. 1991). Acute administration in

rats of a low-to-moderate dose of cocaine or amphetamine produces an increase in

ambulatory activity, rearing behaviour and rotation while higher doses result in stereotyped

behaviour including repetitive head movements and sniffing (Robinson and Bemdge, 1993).

The npeated administration of a Iow- to-moderate dose produces a progressive1 y increased

locomotor response with each subsequent administration. This increased response is known

as behavioural sensitization or reverse tolerance. The human behaviouraI correlate of this

phenomenon is uncertain, however, a link towards dnig craving and addiction has been

postulated (Robinson and Bemdge, 1993).

The mechanisms responsible for this phenomenon have corne under intense

in;estigation. Several studies point to an increase in DA neurotransmission as a primary

mechanism for this phenomenon (Roy et al., 1978; Kalivas and Duffy, 1990), although it is

clear that increased DA neurotransmission is not the only mechanism at work (Kalivas and

Duffy, 1993; Heidebreder et al., 1996). Additionally, Di receptors have been reported to be

supersensitive in rats following chronic matment with cocaine (Henry and White, 1991;

Unterwald et al., 1994; Unterwald et al., 1996).

1.7 POSITRON EMISSION TOMOGRAPHY AND Di RECEPTORS

PET is a powerful, non-invasive irnaging technology, which is capable of measuring

body function and metabolism in vivo. Radioactive substrates, known as radiotracen or

tracers, which have the same pharmacokinetics as their non-radioactive analogues, emit

positrons as their atorns decay (Fig. 1). The positron then collides with an electron and this

event is termed an annihilation. The annihilation event produces two highenergy gamma

rays (51 1 keV) that travel in opposite directions. It is this pattern of simultaneous gamma

radiation that is measund by a PET scanner (Fig. 2). PET tracers are different from most

other radiotracers in that they have very short half-lives (ttn) ranging from 2 (O-lS), 20.4

Fig. l . Schematic illustration of a disintegrating positron-emitting isotope ("C). The positron (P3 nleased from the disintegrating "C nucleus collides with an elecuon (el in the tissue, leading to the annihilation of the two particles and the formation of two antiparallel gamma quanta (diagram taken from Sedvall, 199 1).

Ag. 2.Schematic illustration of a positron caniera w i th the ring of coincidencecoupled scintillation detectors surrounding the head of a human subject (diagram taken frorn Sedvall, 1991).

(C-11) or 110 (F-18) minutes (Fowler and Wolf, 1986). Due to this shoa time frame. PET

radiotracers are usually produced on-site by a cyclotron.

Animal studies can be performed in dedicated PET carneras, however, the current

spatial resolution (-5 mm) prevents quantitative measurements in smail brain regions.

Therefone, studies in animals with smaller brains (e.g. rats) are carried out by sacrificing the

animal following tracer injection, and manually dissecting the brain. Each individual

dissexted region is then measured with a gamma-counter to determine the arnount of tracer it

contains.

1.8 RESEARCH OBJECTIVES

1.8.1 MAIN OBJECTIVE

The ultimate objective of this research project is to measure in vivo the density of the

functional high-afinity sites of DI receptors in diseased and normal human brains using PET.

The main objective of this research project is to (1) determine the in vivo pharmacological . profile of the new DA Di agonist radioligand R-[' 'CISKF 82957 in rats, and (2) establish the

abifity of R- and RB-["C]SKF 82957 to rneasure changes in Di receptors, induced by

dopaminergic dmg treatments in rats, that are different from those rneasured with [*'C]SCH

23390.

1 HYPOTHESES

1.8.2.1 GENERAL HYPOTHESES

The general hypotheses of this research project include:

The Dl agonist WS- and R-["C]SKF 82957 bind selectively to the high-affinity state

of DI receptors in in vivo studies.

The Dl antagonist ["CISCH 23390 binds to the total density of Dl receptors in in

vivo studies.

R/S- and R-["CISKF 82957 bind to a different subpopulation of Di receptors as

compared to ["C]SCH 23390 and these populations are differentially rcgulatcd in

response to dopaminergic drug treatments.

1.8.2.2 WORKING HYPOTHESES

The working hypotheses of this nsearch project are:

1. The pure enantiomer R-["CJSKF 82957 will exhibit high in vivo binding in rat brain

regions rich in DI receptors and this binding will be selective for Dl recepton.

2. R-["C]SKF 82957 displays increased signal-to-noise ratios as compared to the

racemic WS-["CISKF 82957 in rat brain.

3. The elevation of synaptic DA by acute dmg treatments will decrease binding of WS-

["CISU 82957 in rat stnatum due to competition with endogenous DA for binding

to Dl receptors.

4. Following chronic treatment with the Di antagonist SCH 23390. binding of ["C]SCH

23390, WS- and R-["CISICF 82957 will d l be increased to the same extent in rat

btain.

5. Following chronic treatment with cocaine. WS- and R-["CISICF 82957 binding is

expected to be increased in the NAc, while binding of ["C]SCH 23390 will remain

unc hanged.

1 . 8 SPECIETC AIMS

The specific aims of this investigation are:

1. To evaiuate the tirne course of the pure enantiomer R-["CISKF 82957 and compare

the results to those obtained with the racernic RB-["CISKF 82957.

2. To establish R-["C]SKF 82957 in vivo binding selectivity for Di receptors using

saturating doses of potentiai cornpetiton.

3. To examine whether acute cimg treatments known to alter the concentration of

synaptic DA can change the in vivo binding of ["C]SKF 82957 in rat brain.

4. To study the in vivo binding of [ 1 1 ~ ] ~ ~ ~ 23390, R/S- and R-["C]SKF 82957 in rat

brain following chronic treatments with the DI antagonist SCH 23390 or the indirect

DA agonist cocaine.

If our hypotheses are correct, development of the Dl agonist WS- and R-["CISICF

82957 as PET radiotracers may dlow the measurement of high-affinity binding sites of Dl

receptors in vivo in humans. This may ultimately help in the understanding, diagnosis and

treatmcnt of several human disorders including drug addiction, SZ, PD and TD.

EUS- and R-["CISKF 82957 are the first selective Dl agonist radioligands developed

for PET. Several Dl antagonist radioligands were previously developed and permittcd in vivo

measurement of the total density of Dl receptors. These tracers, however, cannot measun

changes in the functional high-affinity sites of Dl Rceptors. Only a Dl agonist or partial

agonist is capable of diffe&ntiating between the high- and low-affinity states of Dl receptors.

The af'fïnity states of recepton are dynamic and may be easily pemirbcd by in vitro

techniques. Thus, it is important to develop methods of studying in vivo the high-affinity

state of Dl receptors.

1.8.4 DESCRIPTION OF CHAPTER 2

The synthesis. autoradiographic and in vivo evaluation in rats of WS-["C]SW 82957

(RfS-3-methyl-6-chloro-7,8-dihydroxy- 1 -phenyl-2.3,4,5-terahydro- IH-3-benzazepine) has

recently been performed (DaSilva et ai., 1996a; 1996b). This benzazepine has k e n shown to

bind with high-mnity (Ki = 0.9 nM) and selectivity to the high-affinity state of Di receptors

(Neumeyer et al.,-1991), and displays agonistic activity for AC (ECw = 0.6 (Pfeiffer et

al., 1982). Many dmgs have optical isomeric forms in which only one is active. Previous

studies have show that the affinity and activity of dmgs in the family of SKF 82957

consistently resi&s in the R-enantiomer with almost none attributcd to the Senantiorner

(Kaiser et ai., 1982; Neurneyer et al.. 1992).

Development of the pure enantiomer R-["CISICF 82957 has oniy recently ken made

possible due to the availability of R-SKF 82957 and RSKF 8 1297 (the precursor used to

make R-["CISKF 82957). The time course of regional brain uptake and cornpetition with Dl,

D2 and 5-HT2 antagonists were evaluated in rats. By charafterizing the in vivo binding profile

of R-["CISKF 82957, we hope to show that it binds selectively to Dl receptors and binds

with an increased signal-to-noise ratio as compared to WS-[l 'CISKF 82957. R-[' 'C]S w

82957 will allow for more sensitive in vivo measunments of changes in DI receptors in

cornparison to WS-[' 1 ~ ] ~ ~ ~ 82957.

1 8 DESCRIPTION OF CriAPTER 3

For any PET radioligand, the possibility exists that endogenous substances may

compete with the tracer at binding sites and thus hinder accurate measurements. Several 4

radioligands (e.g. [" ~]raclopride, [ l U ~ I B ~ ~ ) have been shown to be susceptible to

competition with endogenous DA. Thus the in vivo binding of R/S-["C]SKF 82957 was

evaluated in rats following acute pretreatment with drugs known to increase or deplete

synaptic DA levels.

Chronic SCH 23390 treatment has been shown to upregulate Dl receptos. The

proportion of DI recepton in the high-affnity state was unchanged as measured by Ni vitro

competition studies using [ 3 ~ ~ ~ ~ 23390 and DA (Hess et al., 1986). Changes in WS- and

R-["CISKF 82957 binding were then observed in rats following chronic treatment with SCH

23390 and compared with changes in ["CISCH 23390 binding. The result of this expenment

could then be used to detexmine if the binding sites of ["CISCH 23390 and RIS- or R-

["c]sKF 82957 were similarly regulated following chronic SCH 23390.

1.8.6 DESCRIPTION OF CHAPTER 4

Chronic cocaine has been shown to supersensitize Dl receptor-mediated signal

transduction in rats. The mechanisrn of this supenensitization is unclear, however, it is

possible that a shift of Di receptors toward the high-affinity state is at least partly

responsible. The effect of chronic cocaine on the in vivo binding of WS- and R-['*C]SKF

82957 was comparecf to ["CISCH 23390. If the binding of these radioligands was

di fferentiall y aitered by chronic cocaine treatment, this would provide evidence that RIS- and

R-["CISKF 82957 bind to a different subpopulation of DI receptors as compared to

[l 'CISCH 23390.

2.0 Dopamine Dl Agonist R-["CISKF 82957: S ynthesis

and In Vivo Characterization in Rats

Jean N. DaSilva, Robert A. Schwartz, Eric R. Greenwdd, Celia M. Lourenco,

Aian A. Wilson and Sylvain Houle

To be submitted to: Nuclear Medicine and Biology

1 wisted in the time course and in vivo cornpetition studies. The nature of these studies

~quires the cooperation of at least five people in order to bc carried out. 1 also performed al1

the data analysis, statistics and figures that were associated with these sections of the paper. 1

assisted in the writing of the manuscnpt concerning these sections. 1 had no part whatsoever

in the chemistry and dosimetry sections. 1 assisted in the performance only of the metabolism

studies. The dosimetry, chemistry and metabolism shidies wiii therefore aot be

discussed outside of this chapter and are not included in the overall hypothesis and

objectives of this thesis.

2.1 ABSTRACT

The active enantiomer R-SKF 82957 was labeled with "C by N-["~]meth~lation of

the full Dl agonist R-SKF 81297, using ["~]meth~l iodide in the presence of N-

ethyldiisopropylamine, in high specific activity, radiochernical purity and fields. Compared

to the Dl agonist RB-["CISKF 82957, the R-["CISKF 82957 showed higher binding in the

Di rich regions, such as striamm and olfactory tubercies (-1.7 times), thereby improving the

tissue contnist. R-["CISKF 82957 exhibited high in vivo binding selectivity for Dl receptors

in rats, since only saturating doses of Dl cornpetitors, but not D2 or 5-HT2 blocken,

significantly reduced the radioactivity levels in al1 brain areas. No metaboli tes of R-1' 'CISKF

82957 were detected in rat brain. Expected dosimetry estimates calculated frorn rat

biodisuibution have demonstrated that this radioligand can be safely administered to humans.

These results indicate that R-["cIsKF 82957 will provide mon sensitive measurements of

DI recepton in in vivo studies than the racemic mixture.

2.2 INTRODUCTION

Alteration of dopamine @A) activity has been irnplicated in the pathophysiology of

several neuropsychiatric disorders, and in h g dependence. Traditionally, DA receptors

were divided into two major groups: Di and D2 receptors (Kebabian and Calne, 1979). Both

receptors are primary targets for h g s used to treat many psychomotor disorders, and are

involved in the actions of drugs of abuse (Clark and White, 1987; Waddington and OBoyle,

1989). DI receptors exist in two interconvertible states exhibiting either high- or l ~ w - ~ n i t y

for agonists or partial agonists, while antagonisu do not differentiate between these states

and bind to the total number of Dl receptors (Leff et al., 1985; Seeman and Grigonadis,

1987; Rubinstein et al., 1990). Thereforee, only D, agonist and partial agonist radiotracers

have the potential to selëctively assess the in vivo density of the functional high-affinity sites

of D, receptors using positron emission tomography (PET). Generally, the binding of DA (or

a D, agonist) to the high-affinity state of DI recepton activates the receptors, which through

functionai coupling to a stimulatory guanine nucleotide binding protein (G-protein, GJ,

stimulates adenylyl cyclase (AC) activity leading to the formation of CAMP and subsequently

to characteristic D, dopaminergic effects (Seeman and Grigoriadis, 1987; Sidhu, 1988;

Kimura et al., 1995). Di agonists have also been shown to stimulate phospholipase-C,

phosphoinositide activities, and possibly other signaling pathways (Undie and Friedman,

1990; Kimura et al., 1995). In fact, a poor correlation was reported between agonist efficacy

to stimulate AC and certain behavioral effects produced by a series of D, agonists in normal

and 6-hydroxydopamine lesioned rats, which may indicate a significant role for signaling

pathways other than AC (Gilmore et al.. 1993; Gnanalingham et al., 1995).

Inconsistent results have been reported in postrnortem in vitro s u e s with respect to

the changes in striami DA DI receptor densities in schizophrenia (SZ), Parkinson's disease

(PD), and in the striatum of cocaine treated anirnals (Hess et al., 1987; Seeman et al., 1987;

Guttman, 1992; Mayfield et al., 1992; Alburges et al., 1993). Clinical use of the Dl

antagonist ["CISCH 23390 and PET showed no difference in striatal DI receptor densities in

dnig naive SZ and in PD, as compared to nomals (Sedvall et al., 1992; Shinotoh et al., 1993;

Laihinen et al., 1994). In theory, the lack of agonist stimulation in PD is expected to increase

the proportion of Dl recepton in their high-affinity agonist state (Rubinstein et al., 1990),

while excess of DA in SZ would produce the contrary (Mamelak et al., 1993). This

underscores the importance of developing Di agonist radioligand for irnaging Dl recepton in

living human brains with PET.

The benzazepine RB-SKF 82957 (IUS-(1)-3-rnethyl-6shloro-7,8-di hydroxy- L - phenyl-2,3,4,5-tetrahydro- 1 H-3-benzazepine) displays agonistic activity for AC (ECro = 0.6

pM (Pfeiffer et ai., 1982)). and binds with high affinity and selectivity to the high-affinity

sites of Di receptors (Ki = 0.9 RM (Neumeyer et al., 1991)). The synthesis, autoradiographic

and initial in vivo evaluation in rats of the fiat selective DI agonist PET radioligand RIS-

["CISICF 82957 was recently reported (DaSilva et al., 1996a; 1996b). With the aim of

increasing the signal-to-noise ratios (specific-to-non-specific), we undertook the synthesis

and in vivo characterization of the active enantiorner R(+)-SKF 82957 (Fig. l), labeled with

"c. We ~ p o r t here its synthesis, as well as biodistribution, cornpetition and metabolism,

and dosimetry estimates for human studies.

Fig. 1. Structure of R-SKF 82957. .

2.3 MA'IZRIALS AND METHODS

General

N-Ethyldiisopropylamine was obtained from Lancaster and was diluted in

dimethylforrnamidc (Dm (100 mg/mL solution). R/S-SKF 8 1297aHCl and CUS-SKF

82957eHCl were generous gifts from SrnithKline Beecham Pham. (King of Russia, PA,

U.S.A.). R(+)-SKF 81297aHBr and R(+)-SKF 82957WBr were purchased from Research

Biochernicals Int. (RBI, U.S.). DMF was stimd ovemight with BaO, then distilled under

reduced pressure from Ba0 and stored over 4 A molecular sieves. Racemic ["CISICF 82957

was synthesized as descn bed prcviousiy (DaSilva et ai., 1996a). Semi-preparative and

analytical HPLC were performed as previously nported for RB-["C]SKF 82957 (DaSilva et

al.. 1996a). Thin-layer chromatographic (TL,C) analyses were carried out on plastic-backed

silica gel plates (60A K6F, Merck) with methanoUtriethylarnine 95/5 as the eluting solvent

mixture. This system is capable of separating the nor-methyl SKF 81297 (Rf -0.55) from

SKF 82957 (R/ -0.7). Radio-TLC was achieved on an automated Berthold Tracemaster 20

Linear analyzer.

The following dmgs were prepared for the cornpetition stuclies in isotonic sterile

solutions at pH 4.5-6.5 and injected at 1 U g . R-SCH 23390HC1 (RBI) was dissolved in

0.9% saline. (-)-Sulpiri&*HCl (Sigma Chem. Co.) was dissolved in wam 4% ethanoUsaline.

R/S-SKF 829570HCl was prepared for injection by dissolution in ethanol/l,2-

propanedioUsaiine 5/12.5/82.5. Ritansenn*HCI (RBI) was dissolved in ethanoU12-

propanedioVsaline 5/2O#S.

R-["C]SKF 82957 was prepared using the same conditions as RB-["C]SKF 82957

(DaSilva et al.. 1996a). Briefly. ["~]meth~l iodide. produced h m [ 1 1 ~ ] ~ 0 2 , was trapped in

a 1 mL reacti-vial containing the desmethyl derivative R-SKF 8 1297eHBr (1 mg) in the

presence of N-ethyldiisopropylamine (1.9 equivalents, 10% vlv solution in DMF) and DMF

(185 pL) at -20 to -40°C. After 5 min at 85'C. R-["C~SKF 92057 was purified by semi-

pnparative HPLC. The resulting sterile and pyrogen free R-["C]SKF 82957 formulation (pH

6-7.5) was stable to radiolysis for at least 90 min, as analyzed by analytical HPLC. Identity

of the radioactive product as R-["CISKF 82957 was determined by co-injection of authentic

R-SKF 82957 using HPLC. In addition, radio-TLC of the radioactive formulation showed

one peak with the same Rf as co-spotted authentic R-SKF 82957.

In Vivo Binding Studies

Animal experiments were conducted in accordance with the recornrnendations of the

Canadian Council on Animal Care and with approval from the Animal Care Cornmittee at the

Clarke Institute of Psychiatry. Rats were maintained on a 12 hour light/dark cycle with food

and water available ad libitum. Except for the whole-body distribution snidy, in vivo studies

were done in a manner similar to ihat previously reported (DaSilva et al., 1999) in male

Sprague-Dawley rats (Charles River, Monmal, Canada) weighing 190-250 g.

TIME COURSE. Rats were injected with high specific activity of R-["C]SKF 82957

(S00 Cilmrnol, at time of fint injection). Radioactivity levels in different brain regions (see

Fig. 2 A and B for list) and blood are expressed as percent injected dose per gram (%IDlg) of

tissue.

COMPE'TITION STUDIES. Cornpetition studies were carried out by either pre-

treatment with saturating doses of sulpiride (5 mgkg, i x , 5 min prior to radioligand

injection) (Hatano et al., 1989) or ritanserin (2.5 mgkg, s.c., 100 min prior) (Leysen et ai.,

1985). followed by a tail vein injection (as above) of R-["C]SI

min postinjection), the data from the controls were pooled (n = 8) and used in statistical

calculations. P values 4 0 5 were considered significant.

Dosimetry Calcuiations

The biodistribution of R/S-["c]sKF 82957 in rats was used to caiculate the expected

human dosimetry using the MIRD methodology (Loevinger et al., 1988). The percent

injected dose per organ of the rat was converted to percent injected dose per human organ for

the brain, liver, spleen, lungs and kidneys. The conversion factor relates the ratios of organ

to body weights in the rat and human. The contents of the srnall and large intestine wen

added together and measured. Because of the short physical half-life of carbon-l 1 compared

to the mean rate of transport through the human small intestine (Eve, 1966), the total

radioactivity in the GI tract was assigned to the small intestine in Our calculations. The penis

on male rats was tied off and urine was collected directly from the bladder. The other organs

listed in Table 2 were examined to rule out unexpected high uptake by those organs but the

data were not used for the final dosimetry calculations. The estimated radiation dose was

calculated with the MiRDOSE 3 software (Stabin, 1996).

Metabolhm Studies

The metabolism of R-["CISKF 82957 was examined in plasma and brain

homogenates of a male Sprague-Dawley rat (300 g), 30 min after an injection of

approximately 4 mCi via a tail vein (as above). Upon decapitation the nit brain was rapidly

removed and stored on ice, and blood from the trunk was coiiected in a heparinized tube.

This procedure was repeated in another rat with a separate R-["CISKF 82957 formulation in

orâer to verify the reproducibility of the results.

PLASMA. Blood was centrifuged (1000 g, 5 min), and the resulting plasma (1 mL) was

rnixed with acetic acid 1% in water (3 rnL) containing RB-SKF 82957 (20 pg, as internal

standard). The solution was passed through a pnactivated (ethanol (10 mL) followed by

acetic acid 1% (20 mL)) Ci8 Sep Pak Plus (Waters Co.). Acetic acid 1% (4 ml) was then

passed through the column (twice) to ensure elution of remaining polar metabolites. The

hydrophobie fraction was eluted with ethanol (95%)/glacial acetic acid 90110 (4 mL). Al1

eluted fractions, whole blood. plasma sarnples, and the Ci8 Sep Pak contents were counted

for radioactivity in the gamma-counter. Recoveries of radioactivity were better than 97%.

The organic fraction was then prepared for analysis by evaporation to dryness under vacuum,.

re-suspended in -100 pL of the elution solvent, spotted ont0 the TLC plates, developed, and

then analyzed both by radio-TLC and ultraviolet absorption (254 nm). Control experiments

were carried out with rat blood and -200 pCi of authentic R-["CISKF 82957 to validate the

procedure.

Rotein binding of R-["CISKF 82957 to plasma was assessed via ultrafiltration

centrifugation utilizing the Centrifree (Amicon) kit. Plasma containing R-["CISKF 82957

was centrifuged (1000 g, 45 min) at ambient temperature, and the resulting filtrate (plasma

free fraction) together with a sample of plasma was counted in the gamma-counter.

BRAIN. A control rat was also killed at 30 min postinjection of saline into the lateral tail

vein, and its brain removed Both brains, the one from the rat injected with R-["C]SKF

82957 and the control rat, were homogenized (Polytron) in icecold ethanol954blwater 80120

(10 mL) containing R/S-SKF 82957 (40 pg, as an internal standard), and -200 pCi of R-

["cIsKF 82957 aûded to the control only. then centrifuged (82,000 g, 15 min). Glacial

acetic acid (1 ml) was added to the resulting supernatant and evaporated to dryness. Then i t

was analyzed by radio-TU: and UV (as above).

2.4 RESULTS

Radiochemistry

R-["CJSKF 82957 was synthesized by ~-["~]meth~lat ion of the full Di agonist R-

SKF 812974Br with ['l~]methyl iodide in the presence of N-ethyldiisopropylamine. Using

the same conditions as for RIS-["CJSKF 82957, R-["CISKF 82957 was prepared in high

radiochernical yields 45.75% (decaycorrected, based on ["CJCHS), purity (>99%) and

specific activity (>1000 Ci/mmol (>37 GBq/pol), at end-of-synthesis), in a synthesis time

of 35 min (including quality control assays).

Regional Brah Distribution Studies

The time-activity curves of regional rat brain distribution of radioactivity following

R-["CISKF 82957 injection are presented in Figure 2 (A and B). High retention of

radioactivity was found in the DI receptor-rich strianim and olfactory tubercles, while the

lowest levels were obtained in the cerebellum. a region relatively devoid of Di receptors

(Boyson et al., 1986; Savasta et al., 1986). Al1 other brain regions exhibited similar uprake

of radioactivity vs. time and similar washout rates. High stria- and olfactory tublercles-

to-cerebellum (signal-to-noise) ratios vs. time were observed (Fig. 2 C), and reached 11.3 *

1.9 and 9.7 1.5, respectively, at 45 min postinjection. Other region-to-cerebellum ratios

(e.g. frontai cortex) were approximately 2 throughout the study (Table 1). Cornparrd to WS-

["C]SKF 82957, R-["CISKF 82957 showed higher accumulation of radioactivity in the

striatum and the olfactory tubercles (-1.7 tirnes), thereby improving the tissue contrast

(Table 1).

Since the Dl-rich striatum also contains high levels of D, and 5-m2 receptors, the

effects of pre-treatment or CO-injection of Di, D2 and 5-Hï2 blockers on the regional brain

distribution of R-["CISKF 82957 in rats was detemined, and the results are depicted in Fig.

3. Co-administration of udabeled RLSSKF 82957 and SCH 23390 significantly nduced the

retention of radioactivity in al1 brain regions to cerebellar levels. In contrast, no effect was

observed in the radioactivity levels in any brain areas following ûtatrnent with saturating

doses of the Dt antagonist sulpinde or 5 - W 2 antagonist ritanserin. Whole brain uptake was

significantly decreased only in blocking studies using Di cornpetiton. These results suggest

that R-[~~C]SKF 82957 binds selectively to Di receptors, over DI and 5-mt recepton.

Whole-Body Distribution and Dosimetry Studies

The biodistnbution of R/S-["CISKF 82957 is given in Table 2. The main pathway of

excretion is through the hepatobiliary route (60 %ID alter one hour) with significantly less

ihrough the urînary route (4 %ID at one hour). Gradua1 washout of activity from the brain is

observed with an initial value of 1.04 0.34 %ID at 5 min pst-injection and 0.28 0.08

%ID at one hour pst-injection. These data were used to estimate the human dosimetry of

WS-["C]SKF 82957 which is summarized in Table 3. Because of the high rate of

+ Rest CTX -cl- Thahmus -a- Hypothalamus + Hippocampus -rt PonslMidbrain

20 - +Olt TublCerebellum .c -C- Frontal WCerebellum

12 -

O 15 30 45 60 75 90

Time (mfn)

Fi g. 2. (A and 0) Regional brain uptake and blood distriiution, and (C) region-t~~~erebellurn ratios (for striatum, oîfactory tubetcles and frontal cortex) of radioactivity in rats, following injection of R-[l ICISKF 82957 in rats. Data (A and B) are expressed as meam of % injectecl dose per gram of tissue I S.D. (n = 4). CTX: cortex; Ob.: olfactory; Tub.: tubercles.

TABLE 1. Biodlstribution of R-["cIsKF 82957 in Rats, 45 inin after Administrationt

Brain RegionIOrgan RIS-[~'C]SKF 82957" R-["CISKF 82957

Stnatum 01 factory Tubercles Frontal Cortex Rest of Cortex Thalamus Hypothalamus Hippocampus Pons/Midbrain Cerebellum Brain Lung Heart Liver Blood

Mean of 8mIg f S.D. of control rats (n=24 for RIS-["C]SKF 82957, n = 8 for R-[' 'CISKF 82957).

* Values from Dasilva et al., 1996b. * p < 0.05 compared to cerebellum.

TABLE 2. Biodistribution of WS-["CISICF 82957 in rats.'

Organ or Tissue

Pituiîary

E Y ~

Ascending/transverse LI'

Descendhg LI'

Small in tes tine

Testicles

Ovaries

Heart wall

Lungs

Spleen

Adrends

S tomach

Urinary bladder wall

Brain

Urine

GI contents'

Liver

Kidney s

Tirne (min)

5 15 30 60

0.01 f 0.00

0.03 f 0.00

0.30 f 0.04

0.10 f 0.02

9.72 f 2.91

0.64 f 0.00

0.08 k 0.0 1

0.23 f 0.03

1.15 f 0.15

0.33 f 0.07

0.04 f 0.0 1

0.73 f 0.52

0.03 f 0.0 1

0.92 f 0.14

0.13 i NIA

20.84 f 4.08

8.83 i 1.21

2.18 i 0.42

Data are expressed as % injected dose per organ f SD. ' LI = Large intestine; GI = Gastru-intestinal.

TABLE 3. Calculateci Absorbed Dose to Human for R/S-["cIsKF 82957 B a d on Biodistribution in Rats

Whole Body Red Marrow Ovaries Testes Smail Intestine Brain Heart Wall Kidneys Liver Lungs S deen

I Control 0 Sulpiride E Ritanserin P SCH23390 I SUF82957

Fig. 3. Effect of treatment with various drugs on the regional. blood and mole brain retention of radioactivity in rats, 45 min R-[II CISKF 82957 postinjection. Data are expresPed as means of % inlected dose per gram of tissue I S.D. in = 8 for controls and n = 5 for drug treatment groups). OLF: olfactory; TUB: tubercles; CTX: cortex; THAL: thabmus: HYPO: hypothalamus; HIPPO: hippocampus: CEREB: cerebellum. ' p < 0.05 (ANOVA with bonferronnî's post-hoc test).

hepatobiliary excretion, the small intestine is the criticai organ with 0.16 rad/mCi. Since the

biodistribution of R-["CISKF 82957 is similar to that of the racemic compound, its

dosimetry is expected to be sirnilar.

Meta bolism Studies

At 30 min postinjection of R-["CISKF 82957, -12% of the total radioactivity in

plasma was present as polar metabolites in the aqueous fractions and -86% in the ethanol

eluant. following solid-phase extraction. Radio-TLC analysis of the organic fraction

revealed only the presence of unmetabolized R-["CISKF 82957. This single peak had the

same Rf as that of the co-eluted RIS-SKF 82957 (as detected by U V ) and that obtained in the

control expriment using authentic R-["CISKF 82957. Both TLC analyses of the

homogenized brain extracts from the injected and control rats indicated only the presence of

unchanged R-["CISKF 82957. The plasma protein binding fraction was found to be

approximatel y 93%.

2.5 DISCUSSION

In vitro assays involve homogenization of postmortem tissue samples, or preparation

of an autoradiographic slice in the presence of a radioligand, usually labeled with a long half-

life radioisotope such as tritium, and incubation in a physiological buffer. These in vitro

experiments may be unable to reproduce reliably in vivo conditions, especially those

involving the complex mechanisms involved in the high- and low-affînity States of Dl

receptors coupled to G-proteins. It is thus important to determine the in vivo pharmacological

profile of a Di agonist PET radioligand.

The pure enantiomer R-("CJSKF 82957 was recently developed due to the

availability of the precursor R-SKF 81297. Using the same conditions as for RI'S-["C]SKF

82957, we have prepared R-["C]SKF 82957 in higher yields (45-7596, cornpared to 2 0 4 %

for WS-["CISKF 82957 (DaSilva et al., 1996a)). In vivo evaluation of R-["CISKF 82957 in

rats revealed a regional brain distribution consistent with those pnviously reported for

selective Di receptor radioligands (Boyson et al., 1986; Dubois et al., 1986; Savasta et al.,

1986). Compared to R/S-["C]SKF 82957 (DaSilva et al., 1996b). R-["CISKF 82957 showed

higher retention of radioactivity in the striatum and olfactory tubercles (-1.7 times), thereby

improving the specific-to-non-specific binding ratios. Consequently, higher signal-to-noise

ratios are obtained with the R-enantiomer of ["C]SKF 82957 as compared to the racemic

mixture. These results are in agreement with previous studies which demonstrated that the

RD(+) enantiomer of substituted 1-phenyl-3-benzazepines bind with higher afhity and

selectivity to Di receptors in cornparison to the S-(-) enantiomer, and that agonistic activity

for AC resided almost exclusively in the R- antipode (Kaiser et al.. 1982; Neumeyer et al.,

1992). R-["C]SKF 82957 uptake was reduced uniformiy across the brain regions to

cerebellum levels, only in studies using Dl cornpetitors (SCH 23390 and SKF 82957).

indicating the presence of specific binding to Dl receptors. In contrast, pretreatment with

sarurating doses of the & antagonist sulpiride or 5-m2 antagonist ritanserin showed no

effect on R-["CISKF 82957 binding as compared to controls, suggesting high binding

selectivi ty for Di receptors.

Whole-body distribution studies of WS-["CISKF 82957 in rats revealed that most of

the radioactivity was exmted via the hepatobiliary route. Dosimetry calculations indicated

that the small intestine is the limiting organ (0.16 rad/rnCi). Similar dosimetry is expected

for R-["CISE 82957, since it displays comparable biodistribution. One major limitation of

extmpolating the rat data to humans is the potential difference in the hepatic clearance rate of

SKF 82597 in the two species. Hurnan hepatic clearance rate of ["CISKF 82957 will be

required to refine the dosimetry estimates. Brain and plasma radioactivity was examined at

30 min postinjection. Slow metabolism was observed as -86 96 of R-["CISKF 82957 was

found unchanged in the plasma, and only unchanged R-[' *C]SKF 82957 was present in the

rat brain extracts.

In conclusion, the results of this study demonstrate that R-["CISKF 82957 has a high

binding selectivity for Dl receptors, acceptable radiation dosimetry, and no metabolites in rat

brain extracts. As expected, the active R-["CISKF 82957 increased the signal-to-noise ratios

as compared to the Di agonist R/S-["C]SKF 82957, allowing more sensitive Ni vivo

measurements of Dl recepton.

2.6 STATEMENT OF SIGNIFICANCE

This work npresents an initial in vivo pharmacologicai assessrnent of the pure

enantiomer R-[I1c]sIC~ 82957 in rats. It shows that this PET radioligand is selective for Di

recepton and it improvesthe signal-to-noise ratio (striatum-to-cerebellum) compared to RIS-

[ ' 1 ~ ] ~ ~ ~ 82957.

3.0 In Vivo Binding of the Dl Agonist PET Ligand ["CISKF

82957 in Rats: Effect of Endogenous Dopamine and Chronic

Treatment With SCH 23390

Eric R, Greenw son, 'ald, Jean N. DaSilva, Celia M. Lowenco, Alan A. Wil

and Sylvain Houle

To be submitted to: Synapse

1 assisted in carrying out al1 of the biodistribution studies. The nature of these studies requires

the cooperation of at least five people in order to be carried out. 1 perfonned al1 the data

analysis, statistical analysis, figures and writing of the manuscript.

3.1 ABSTRACT

Changes in endogenous dopamine @A) levels have been shown to affect binding of

several DA receptor radioligands (e.g. [' l~]raclopride). It is thereforee important to evaluate

the effect of endogenous DA on the binding of new ligands for the DA system. The purpose

of the present study was to further characterize the in vivo binding of the DI agonist, RIS-

SKF 82957 labeled with C-11. in rats subjected to acute pretreatments that change synaptic

DA levels including amphetamine. cocaine. GBR 12909. clorgyline and reserpine. The

racemic and the Renantiomer of ["C]SKF 82957 and the Di antagonist ["CISCH 23390

were tested in rats expected to have upregulated Di receptors, following chronic treatment

with SCH 23390 (0.5 mgkg, s.c., 21 days followed by 3 days washout). Regional brain

uptake of WS-["CISKF 82957 was not significantly aitered by acute h g treatments.

indicating that its binding is not sensitive to acute changes in DA levels. Chronic SCH 23390

treatment significantly increased the striatum- md olfactory tubercles-toterebellum ratios of

["C]SCH 23390 by 21.34 and 16.7% respectively. In contrast, RfS- and R-["C]SKF 82957

striatum- and olfactory tubercles-to-cerebellum ratios were not significantly increased by the

chronic ueatment. These results imply that the DI agonist RIS- and R-["C]SKF 82957 bind

differently in vivo than ["C]SCH 23390 and rnay thus have implications for the use of this

Dl agonist radioligand in PET imaging.

3.2 INTRODUCTION

Dopamine (DA) receptors belong to the superfarnily of guanine nucleotide binding

protein (G-protein) coupled receptors (Kebabian and Calne, 1979). As with other G-protein

coupled receptors, DI recepton exist in two interconvertible states, the highgaffinity state and

the low-affinity state. The current temary cornplex mode1 of ligand, receptor and G protein,

presumes that the high-affinity state occua when the receptor is functionally coupled to the

G protein. The low-affinity state represents a state in which the receptor is uncoupled from

the G protein (Seeman and Grigoriadis, 1987; Kimura et al., 1995; Mitchell and Seeman,

1998). DI antagonists, such as SCH 23390, do not distinguish between the two states of the

Dl receptor. Only DI agonists and partiai agonists can bind selectively to the functiond high-

affinity state. Upon binding of DA or a Dl agonist to the high-affinity state of Dl receptors,

the stimulatory G protein (G,) is activated, which leads to the stimulation of the enzyme

adenylyl cyclase (AC). Once activated, AC converts adenosine triphosphate (ATP) into the

second messenger 3', Scyclic adenosine monophosphate (CAMP), which is capable of

mediating many different effects (e.g. protein kinase A activation and transcription of several

genes) (Sidhu, 1988; Kimura et al., 1995). DI receptors have also been linked to the

phospholipase C second messenger systern and possibly other transduction systems (Sidhu,

1988; Mahan et al., 1990; Undie and Friedman, 1990; Niznik and Van Tol, 1992; Kimura et

al., 1995).

Dl receptors have k e n implicated in a number of human disorders including dnig

dependence, schizophrenia (SZ), Huntington' s (HD) and Parkinson's disease (PD). Although

total Di receptor densitics were found to be decreased in HD, no ciifference in striatal Dl

receptor levels were observed in drug naïve schizophrenics and in PD patients as cornparrd

to nomals (Sedvall et al., 1992; Shinotoh et al., 1993; Brownell et al., 1994). However,

changes in the binding mnity and number of high-affinity sites of Di receptors have been

reported in post-mortem brains of SZ patients in cornparison to controls (Marnelak et al.,

1993). These findings highlight the need for a Di agonist radioligand in order to study the DI

high afflnity state in SZ and other neuropsychiavic disorden of the DA system in living

human brains using positron emission tomography (PET).

We have recently reported the synthesis, autoradiographic and in vivo evaluation of

the first selective Di agonist PET radioligand R/S- and R-["C]SKF 82957 (R/S(k)- and

R(+)-34' 1~]methyi-6-chloro-7,8-dihydroxy- 1 -pheny l-2,3,4,5-tetrahydro- 1 H-3-benzazepine)

(DaSilva et al., 1996a; 1996b; 1998). RIS-SKF 82957 binds with high affinity and selectivity

to the high-affinity state of Dl receptors (Ki = 0.9 n M ) (Neumeyer et al., 1991) and displays

agonistic activity for AC (EC50 = 0.6 FM) (Pfeiffer et al., 1982). The pure enantiomer

precursor of R-["C]SKF 82957 (R-SKF 8 1297) has only recently ken made available, and

R-["CISKF 82957 is generally preferable to the racemic fom as its uptake in the sniatum

and olfactory tubercles is almost two-fold higher in cornparison to R/S-["CISKF 82957

(DaSilva et al., 1998).

The first part of the present study examines whether cfrug treatments known to alter the

concentration of synaptic DA cm change the in vivo binding of WS-["C]SKF 82957 in the

rat brain. For any PET neuroreceptor radioligand, in vivo cornpetition with the comsponding

endogenous neurotmnsmitter may affect the radiotracers ability to measure the true density of

binding sites. It is thus important to establish the effect of endogenous DA on RIS-["C]SKF

82957 binding. The binding of several D2 receptor radioligands (e.g. [ll~]raclopride,

[ l U ~ ] ~ ~ ~ ~ ) has been reported to be affected by drugs that increase or deplete endogenous

DA concentrations (Young et al., 199 1; Dewey et al., 1993; Lamelle et al., 1997).

The second part of this study evaluates the in vivo binding of ["CISCH 23390, WS- and

R-["CISKF 82957 in the rat brain following chronic treatment with the Di antagonist SCH

23390. In vitro studies have previousl y shown that chronic SCH 23390 selectivel y increased

the number of DI binding sites as meastued by [)H]SCH 23390 (Creese and Chen, 1985;

Hess et al., 1986; Chipkin et al., 1987; McGonigle et al., 1989; Lappalainen et al., 1992).

["CJSCH 23390, RB- and R-["C]SKF 82957 were injected in rats treated chronically with

SCH 23390 in order to c6rnpare their ability to mesure upregulation of the Di receptors.

3 3 MATERIALS & METHODS

RIS-, R-["CISKF 82957 and ["CISCH 23390 were sp th esiz

described (Ravert et al., 1987; DaSilva et al., 1996b; 1998). The radiochernical punty was

>95% and the specific activity >400 mCiImmol (A4.8 GBq/pmol) ai time of first injection.

R-SCH 23390eHC1, d-amphetamine sulphate, and ciorgyline*HCl were purchased from

Research Biochernicals International (RBI, MA) and dissolved in 0.9% saline. Cocaine-HC1

(British Dnig House) was dissolved in 0.9% saline. GBR 12909 dihydrochloride (RBI) was

dissolved in warm ethanou l,2-propanedioUO.9% saline 5/20/75 (vlvlv). Reserpine (RB0 was

dissolved in glacial acetic acid and then sterile water (Baxter) was added. Al1 drugs and

vehicle solutions for controls were prepared at pH 4.5-6.5 and injected in a volume of 1

a g -

Anitmats

The animal experiments were conducted in accordance with the recornrnendations of

the Canadian Council on Animal Care and with approval From the Animal Care Cornmittee at

the Clarke Institute of Psychiatry. Male Sprague-Dawley rats (200-225 g at start of study)

were obtained from the Charles River Breeding F m (Montreal, Canada) and wen

maintained on a 12 hour lightldark cycle with food and water ad libitum. Animals, in

restraining boxes (Harvard Apparatus, Canada), were administered 0.3- 1.1 mCi of ["CPCH

23390, WS- or R-["CISKF 82957 in 0.3 rnL of buffered solution via a lateral tail vein

p~viously vasodilated in a warm (-45°C) water bath. AI1 animals received approximateiy

the same mass dose of the radioligand. Rats were sacrificed by decapitation 45 minutes after

radiotracer injection and blood sarnples were taken irnrnediately. The experiments were done

45 min after radioligand injection since optimal specific-to-nonspecific ratios (striatum-to-

cerebellum) are obtained at this time point, and also due to the short t 10 of C- 1 1 (20.4 min)

(DaSilva et al., 1996b).

Acute In Vivo Studies

For each formulation of WS-["CISKF 82957, 2 treated groups (n=5 each) and a

control group (n=4) injccted with vehicle were used. Dmgs administered included

amphetamine (5 mgkg, i.v.. 5 min prior to radioligand injection), cocaine (20 mgkg, i.p., 20

min prior), GBR 12909 (10 mgkg, i-v., 35 min prior), clorgyline (2 mgkg, i.p. 2 hours pnor)

and reserpine (1.0 mgkg. S.C. 24 hours prior). These doses of amphetamine. cocaine, GBR

12909 and ciorgyline have al1 k n shown to increase extracellular DA ievels (Scheffel et al.,

1989; Paterson et al., 1991; Innis et al., 1992; Roseboom and Gnegy, 1989; Young et. al,

1991) while this dose of reserpine significantfy depletes DA levels (Innis et al., 1992). RIS-

["CISKF 82957 was injected and rats were sacrificed as described above. Brains were then

rapidly removed and the following regions were dissected out: stnatum, olfactory tubercles,

frontal cortex, rest of the cortex. hippocampus, hypothalamus, thalamus, brainstem and

cerebellum. Penpheral tissues included the whole heart, and samples of the lung and liver.

Al1 tissues were washed in saline, blotted, weighed and counted in a garnma-counter (back-

correctcd to time of first injection) together with aliquots of the injected solution. Tails and

used syringes were counted in a dose-calibrator (Capintec) and residual radioactivity

remaining within them was used to calculate the net injected dose. Due to variation in body

weight of the rats, radiotracer uptake is expressed as percent-injected dose multiplied by

body weight (in kg) per gram of tissue (%IDWg), in order to account for variability in rat

sizes in different groups. Al1 brain region values were added to give the rnean %IDWg for

the whole-brain following each treatment. As the cerebellum is relatively devoid of Di

receptoa, region-to-cerebellum ratios are a better indicator of the effect of an experimental

treatment than regional uptake. Cerebellar ratios can take into account effects such as global

changes in the blood-brain-bamier and blood flow.

Chronic In Vivo Studies

Rats w m administered either SCH 23390 (0.5 mgkg, s.c., n=7) or saline in the

control group (s.c. n=7) for 21 &ys and allowed a three &y washout period prior to

[' 'CISCH 23390, RIS- or R-["CISKF 82957 injection. Brain regional radioactivity

distribution was determined as described above. For ["CISCH 23390 and R-["CISKF 82957

trials, the studies were performed twice in order to increase the statistical power and assess

reproducibility of the results.

S tatistical Anal ysis

Statistical analysis was performed using one-way ANOVA followed by Bonferroni's

pst-hoc cornparisons tests.