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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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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.