From the Department of Clinical Neuroscience

Karolinska Institutet, Stockholm, Sweden

PET IMAGING OF TWO MONOAMINERGIC NEUROTRANSMITTER SYSTEMS IN BRAIN:

STUDIES OF THE NOREPINEPHRINE TRANSPORTER AND DOPAMINE D2 RECEPTOR

Nicholas Seneca

Stockholm 2006

All previously published papers were reproduced with permission from the publisher. Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden © Nicholas Seneca, 2006 ISBN 91-7140-923-8

ABSTRACT Positron emission tomography (PET) has been widely used to study non-

invasively function of the brain, pathophysiology of disease and aid in the development of new drugs. PET and selective radiolabeled molecules allow imaging of certain critical components of neurotransmission, such as pre-synaptic transporters and post-synaptic receptors in living brain. The general aim of the present thesis was (i) to measure neuropharmacological interventions using PET (e.g., competition between synaptic neurotransmitters and radiolabeled tracers provide a useful tool to estimate changes in synaptic levels of neurotransmitters), and (ii) PET determination of drug induced receptor occupancy, examined by the curvilinear relationship between drug plasma concentration and receptor occupancy over a wide dose-range in nonhuman primate brain.

The aim of Papers I and II was to evaluate (S,S)-[18F]FMeNER-D2 as a radiotracer for the norepinephrine transporter (NET). Paper I examined the whole-body biodistribution of (S,S)-[18F]FMeNER-D2 and estimated the resulting radiation exposure to organs of the body in nonhuman primates. The estimated radiation burden of (S,S)-[18F]FMeNER-D2 was found to be comparable to that of other 18F radioligands. In Paper II, the aim was to determine if atomoxetine occupies NET in a dose-dependent fashion using (S,S)-[18F]FMeNER-D2 in nonhuman primate brain. Previous PET studies have failed to demonstrate the feasibility of measuring a dose-dependent NET occupancy. After administration of increasing doses of atomoxetine, a dose-dependent occupancy from 38% to 82% was observed in brain regions known to contain high densities of NET.

The aim of Papers III and IV was to combine PET imaging of the D2 receptor with pharmacological challenges that either increase or decrease concentrations of dopamine. In Paper III, the aim was to assess and compare in nonhuman primate brain the sensitivity of the agonist radioligand [11C]MNPA and antagonist [11C]raclopride to stimulant-induced dopamine release. [11C]MNPA binding potential was ~50% more sensitive than [11C]raclopride to pharmacological induced increases in dopamine. The aim of Paper IV was to estimate the occupancy of D2 receptors in rat brain by endogenous dopamine using PET and [11C]MNPA. The dopamine depletion paradigm increased [11C]MNPA binding potential significantly and the results indicate ~27% of D2 high affinity state receptors are occupied by endogenous dopamine during basal conditions.

The aim of Paper V was to determine D2 and 5-HT1A receptor occupancy in brain after administration of RGH-188. In addition, the intrinsic activity of RGH-188 was estimated in vivo using a dopamine D2 agonist and antagonist radioligand. RGH-188 occupied D2 receptors in a dose dependent and saturable manner, with lowest dose occupying ~5% of receptors, and highest dose more than 90%. RGH-188 was equally potent to displace both antagonist and agonist radioligand. 5-HT1A receptor occupancy was much lower compared to D2 occupancy at the same doses, with a maximal value of ~30%. These results suggest that RGH-188 binds to D2 rather than 5-HT1A receptors, and its equal potency to displace agonist and antagonist radiotracers suggests that RGH-188 is an antagonist.

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“Our life is what our thoughts make of it.”

— Marcus Aurelius, Roman Emperor, A.D. 161-180

LIST OF PUBLICATIONS The thesis is based on the following papers and manuscripts:

I. Seneca N, Andree B, Sjoholm N, Schou M, Pauli S, Mozley PD, Stubbs JB, Liow JS, Sovago J, Gulyas B, Innis R, Halldin C. Whole-body biodistribution, radiation dosimetry estimates for the PET norepinephrine transporter probe (S,S)-[18F]FMeNER-D2 in non-human primates. Nucl Med Commun 2005; 26(8):695-700.

II. Seneca N, Gulyás B, Varrone A, Schou M, Tauscher J, Vandenhende F, Kielbasa W, Farde L, Innis RB, Halldin C. Atomoxetine occupies the norepinephrine transporter in a dose-dependent fashion: A PET study in nonhuman primate brain using (S,S)-[18F]FMeNER-D2. Psychopharmacology 2006; 188(1):119-27.

III. Seneca N, Finnema SJ, Farde L, Gulyas B, Wikstrom HV, Halldin C, Innis RB. Effect of amphetamine on dopamine D2 receptor binding in nonhuman primate brain: A comparison of the agonist radioligand [11C]MNPA and antagonist [11C]raclopride. Synapse 2006; 59(5):260-269.

IV. Seneca N, Zoghbi SS, Liow JS, Hong J, Pike VW, Halldin C, and Innis RB. PET imaging in rat brain of endogenous dopamine occupancy of D2/3 receptors using the agonist radioligand [11C]MNPA. Manuscript.

V. Seneca N, Finnema S, Innis RB, Halldin C, Laszlovszky I, Kiss B, Horváth A, Pásztor G, Kapás M, Gyertyán I, Farkas S, and Gulyás B. Occupancy of dopamine D3/D2 and serotonin 5-HT1A receptors by a novel D3/D2 antipsychotic drug in nonhuman primate brain: A PET study. Manuscript.

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CONTENTS 1 INTRODUCTION ....................................................................................... 6

1.1 POSITRON EMISSION TOMOGRAPHY (PET) ........................... 6 1.1.1 Principles of PET ................................................................... 6 1.1.2 Applications of PET neuroreceptor imaging......................... 7

1.2 NORADRENERGIC SYSTEM ........................................................ 8 1.2.1 Noradrenergic projections...................................................... 8 1.2.2 Role of norepinephrine and the norepinephrine transporter . 9 1.2.3 PET imaging of the norepinephrine transporter.................... 9

1.3 DOPAMINERGIC SYSTEM.......................................................... 10 1.3.1 Dopaminergic projections.................................................... 10 1.3.2 Dopaminergic receptors ....................................................... 11 1.3.3 Functions of dopamine......................................................... 12 1.3.4 PET imaging of dopamine receptors ................................... 13

1.4 INTERACTION BETWEEN DOPAMINE AND GLUTAMATE15 1.4.1 Glutamate receptors ............................................................. 15 1.4.2 Interactions between dopamine and glutamate ................... 16 1.4.3 PET studies of dopamine glutamate interactions ................ 18

2 AIMS .......................................................................................................... 20 3 MATERIALS AND METHODS .............................................................. 21

3.1 SUBJECTS....................................................................................... 21 3.2 MATERIALS AND RADIOLIGANDS ......................................... 21 3.3 POSITRON EMISSION TOMOGRAPHY .................................... 21 3.4 PET EXPERIMENTAL PROCEDURES ....................................... 22 3.5 EXPERIMENTAL DRUGS AND DOSE SELECTION ............... 23 3.6 IMAGE ANLAYSIS........................................................................ 25 3.7 CALCULATIONS ........................................................................... 25 3.8 NEUROPHARMACOLOGICAL INTERVENTIONS ................. 26 3.9 RECEPTOR OCCUPANCY ........................................................... 27 3.10 STATISTICAL ANALYSIS........................................................ 28

4 RESULTS AND DISCUSSION................................................................ 29 5 SUMMARY OF FINDINGS..................................................................... 39 6 FUTURE PROSPECTS............................................................................. 40 7 ACKNOWLEDGEMENTS....................................................................... 41 8 REFERENCES........................................................................................... 43

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LIST OF ABBREVIATIONS ADHD Bmax BP CNS DA ED50 EPS fMRI i.v. i.p. Ki L-dopa MAP MeNER MIRD mGluR MNPA MPEP MRI mRNA MRTM2 NET NMDA %ID PET PMOD R1 ROI SA SPECT SPM2 SUV

Attention deficit hyperactivity disorder Receptor density Binding potential Central nervous system Dopamine Effective dose to occupy 50% of receptors Extrapyramidal side effects Functional magnetic resonance imaging Intravenous Intraperitoneal Equilibrium dissociation constant Levodopa Methamphetamine Methyl norethyl reboxetine Medical internal radiation dose Metabotropic glutamate receptors (R)-2-CH3O-N-n-propylnorapomorphine [2-methyl-6-(phenylethynyl)pyridine] Magnetic resonance imaging Messenger ribonucleic acid Multilinear reference tissue model Norepinephrine transporter N-methyl-D-aspartate receptors Percent injected dose Positron emission tomography Image quantification and kinetic modeling software Relative blood flow image Region of interest Specific radioactivity Single photon emission computerized tomography Statistical parametric mapping Standard uptake value

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1 INTRODUCTION 1.1 POSITRON EMISSION TOMOGRAPHY (PET)

PET has been widely used to study noninvasively function of the brain,

pathophysiology and patient treatment management. Using neuroimaging, we are able

to track biochemical changes in the brain by imaging specific neurotransmitter

systems. Changes detected in certain neurotransmitter systems can then be used as a

lead to reveal the pathophysiology of patients suffering from psychiatric, neurological

and neurodegenerative disorders.

The PET technique utilizes radiotracers labeled with relatively short-lived

positron-emitting radionuclides such as 18F, 11C, or 15O. A low amount of mass can be

used due to the relatively high specific radioactivity (SA). Radioligands allow us to

study several biological variables such as: anatomical distribution, binding in the

brain, drug-related receptor occupancy and metabolism of the tracer. Several criteria

need to be fulfilled for PET radioligands: including high affinity and selectivity for

the target site, minimal metabolism in tissues, permeability across the blood-brain

barrier, and low non-specific binding (Halldin et al., 2001a; Lee and Farde, 2006).

The radioligands typically are evaluated in several steps, from the early evaluation in

rodents, including knockout mice, nonhuman primate PET imaging, and human post

mortem autoradiography, to the study of human physiology initially in healthy

subjects and then as applied to patients with psychiatric and neurological disorders.

1.1.1 Principles of PET PET is an in vivo molecular imaging

technique that uses positron labeled

molecules to image molecular

interactions of biological processes. A

positron-emitting radionuclide ejects a

positron from the nucleus as it decays.

The positron travels a short distance in

tissue and loses energy through

interactions with atoms. The positron

will combine with an electron in tissue

and annihilate, simultaneously

producing two 511 keV gamma rays that are emitted 180º apart (Figure 1). PET

Figure 1. The physics underlying positron emission tomography (PET). (Adapted from Cherry 2001).

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scanners contain several rings of positron-sensitive scintillation detectors, with up to

25,000 individual scintillators. The pair of photons produced from a single

annihilation will register almost simultaneously on opposing pairs of scintillation

detectors as a “coincidence event.” The rings of scintillation detectors register

thousands of coincidence events emitted from the subject per second (Phelps and

Mazziotta, 1985; Cherry, 2001). The data gathered from the coincidence events are

used to determine the source of positron annihilation at a given time. These are then

converted into a tomographic image via reconstruction algorithms.

1.1.2 Applications of PET neuroreceptor imaging Several monoamine neurotransmitter systems have been studied in animals,

healthy subjects and patient populations suffering from psychiatric disorders using

PET. The dopaminergic system has been the most extensively investigated in terms of

both pre-synaptic and post-synaptic biological markers. Exploration of the

serotoninergic system has shed light on new drugs acting on this system and has

played a role in the understanding of the pathophysiology of many psychiatric

disorders. The shortage of radioligands poses a serious limiting factor to PET

researchers. Thus, development of new radioligands is crucial to enable further study

of neurotransmitter systems in vivo such as the norepinephrine system, glutamatergic

neurotransmission pathways as well as dopamine subtype receptors and reuptake

sites.

Monoamine transporter imaging for the dopamine, serotonin and to a lesser

extent the norepinephrine transporter have proven useful in assessing transporter

occupancy of well characterized and novel drugs, and as markers for

neurodegenerative diseases (i.e., evaluation of neuronal cell loss of the dopaminergic

system and the associated loss of radioligand binding to the dopamine transporter)

(Laakso and Hietala, 2000). Biomarkers for pathophysiology of various disorders are

being developed, for example, the binding of [11C]PIB reflects the amount of β-

amyloid plaques in brain (Klunk et al., 2004). Quantification of β-amyloid plaques

provides a useful tool to gain insight into the involvement of β-amyloid plaques in the

pathology of Alzheimer’s disease and monitoring therapeutics under development

(Nichols et al., 2006).

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Small animal imaging using microPET has several advantages: aid in the

development of novel radiotracers, administration of pre-clinical drugs, study animal

models of human diseases, and cost effective compared to nonhuman primates (Phelps,

2000; Cherry, 2001). Rodent, monkey and human brain weight may vary drastically

from ~2, ~65, to ~1300 g, however, PET imaging of D2/3 receptors using

[11C]raclopride across species provides a similar outcome measure (Figure 2).

Neuroreceptor PET imaging is just one example of how PET can provide a bridge from

rodent to humans, as the same imaging experiments can be translated across species

(Cherry, 2001).

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BP

Rat Brain Human Brain Monkey Brain

Figure 2. Parametric images of [11C]raclopride binding potential estimated with a reference tissue model. (Rat PET data is courtesy of Dr. Tetsuya Suhara, NIRS, Chiba, Japan).

1.2 NORADRENERGIC SYSTEM 1.2.1 Noradrenergic projections

The major noradrenergic nucleus in

the brain is the locus coeruleus, which

is located in the rostral pons (Figure

3). These neurons give rise to most of

the noradrenergic innervation of the in

the cerebral cortex, thalamus,

cerebellum, and brain stem nuclei. In

contrast to dopaminergic neurons,

which show relatively restricted

axonal projections, noradrenergic

neurons give rise to diffuse axonal

projections and innervate virtually all

Figure 3. Noradrenergic projection systems in the brain. The major noradrenergic nuclei of the brain, the locus coeruleus and the lateral tegmental nuclei. (Adapted from Hyman and Nestler, 1993).

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areas of the brain and spinal cord. In addition, the brain also contains smaller

collections of additional noradrenergic neurons that are located in discrete regions of

the pons and medulla. These norepinephrine-containing cells project to basal forebrain,

thalamus, including certain nuclei in the hypothalamus, limbic areas such as the

amygdala, hippocampus, and brain stem.

1.2.2 Role of norepinephrine and the norepinephrine transporter Norepinephrine, also known as noradrenaline, plays an important role in human

physiology and pathology, and is involved in mood regulation, sleep regulation,

expression of behavior and the general degree of alertness and arousal (McCormick et

al., 1991; Mandela and Ordway, 2006; Mazei et al., 2002). Norepinephrine also exerts

its effects over the endocrine system and autonomic nervous system. Outside the CNS

and adrenal glands, norepinephrine is located in the sympathetic nerve endings, and the

norepinephrine content of a particular tissue reflects the extent of its sympathetic

innervation.

The norepinephrine transporter is located in the plasma membrane of

noradrenergic neurons, where it functions to take up synaptically released

norepinephrine (Mandela and Ordway, 2006). The transporter serves as the primary

mechanism for the inactivation of noradrenergic signaling. Reuptake of norepinephrine

by the transporter protein is the primary mechanism by which the biological effects of

norepinephrine in the synapse are terminated. The inactivation process through the

transporter is critical in preventing an excessive increase in the norepinephrine

concentrations in the synaptic cleft, which regulate adrenergic neurotransmission in the

brain, as well as the removal of norepinephrine from the heart and other peripheral

organs. Reuptake of norepinephrine is competitive with a variety of naturally occurring

amines and drugs. Drugs of abuse (e.g., cocaine) and antidepressants (e.g., desipramine,

reboxetine, bupropion) block the transport of norepinephrine and thereby cause an

elevation in the synaptic concentrations of norepinephrine and potentiation of the

activation of postsynaptic receptors (Zhou, 2004; Berton and Nestler, 2006).

Norepinphrine plays an important role in the CNS, and is likely to play a role in various

psychiatric disorders, including major depression and ADHD.

1.2.3 PET imaging of the norepinephrine transporter Imaging agents targeting the norepinphrine transporter (NET) in brain have

lagged due to a lack of selective NET radioligands that give a high signal to noise

ratio. Nisoxetine has high affinity for NET in vitro, nevertheless, [11C]nisoxetine

exhibits high levels of non-specific binding in vivo (Haka et al., 1989). Several

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analogs of cocaine have been radiolabeled as probes for NET, but due to high affinity

to dopamine transporter and the low density of NET in brain, the radioligands

selectively labeled the dopamine transporter in vivo (Musachio et al., 2006).

Evaluation of 11C-radiolabeled desipramine, talopram, and talsupram demonstrated

low brain uptake and lack of selective NET binding in vivo, even though they exhibit

high selectivity for NET over dopamine and serotonin transporters (Schou et al.,

2006; McConathy et al., 2004).

Recently, 11C and 18F labeled analogs of reboxetine have been prepared and

evaluated as PET radioligands in rodent, monkey and human (Wilson et al., 2003;

Schou et al., 2003; Ding et al., 2003 Andree et al., 2004). In vitro autoradiography and

in vivo PET imaging have demonstrated selective binding of [11C]MeNER and

[18F]FMeNER-D2 to NET. Further evaluation of [11C]MeNER in nonhuman primate

and human brain showed that specific binding to NET did not reach maximal values

(i.e. equilibrium) during the PET measurement. This, together with a somewhat noisy

final signal at later time points, are relative deficiencies of [11C]MeNER for quantitative

studies of NET in brain. These deficiencies lead to the preparation and evaluation of

radiofluorinated analogs of (S,S)-MeNER (Schou et al., 2004). The results indicated

that (S,S)-[18F]FMeNER-D2 is a useful radioligand for imaging NET in monkey and is

superior to [11C]MeNER given that a specific binding peak equilibrium is reached

during the PET experiment at a lower noise level. The advantage of the 18F-radioligand

lead to further development with in vitro human brain autoradiography (Schou et al.,

2005), and calculated dosimetry estimates for (S,S)-[18F]FMeNER-D2 were comparable

to other 18F-labeled radioligands allowing for multiple injections in human subjects

(Seneca et al., 2005). In addition, human NET occupancy studies using [11C]MeNER

showed a lack of complete saturation and dose-dependent occupancy across various

plasma concentrations of reboxetine and atomoxetine, but using (S,S)-[18F]FMeNER-

D2 a dose dependent and complete saturation in brain was demonstrated (Andree et al.,

2004; Wong et al., 2005; Seneca et al., 2006). In total, these data encourage further

PET studies using (S,S)-[18F]FMeNER-D2 in humans.

1.3 DOPAMINERGIC SYSTEM 1.3.1 Dopaminergic projections

Dopaminergic neurons in the substania nigra pars compacta, the ventral

tegmental area, and the hypothalamus give origin to four main pathways, the

nigrostriatal, the mesolimbic, the mesocortical, and the tuberoinfundibular (Figure 4).

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The nigrostriatal system projects from the substantia nigra to the dorsal

striatum. This system is important for the control of movement and is affected in

Parkinson’s disease and other disorders of movement. The mesolimbic system projects

from the ventral tegmental

area to limbic structures

such as the ventral striatum

(i.e., nucleus accumbens),

hippocampus and amygdala.

The mesocortical system

projects from the ventral

tegmental area to cortical

regions. Both the

mesolimbic and

mesocortical projections are

important for affect,

emotion, and motivation and are affected in schizophrenia. The fourth dopaminergic

projection originates in the arcuate nucleus of the hypothalamus and projects to the

pituitary gland, where it regulates secretion of hormones.

Figure 4. Dopaminergic projection systems in the brain. (Adapted from Hyman and Nestler, 1993).

1.3.2 Dopaminergic receptors Dopamine exerts its action by binding to specific membrane receptors, which

belong to the family of seven transmembrane domain G-protein coupled receptors. Five

distinct dopamine receptors have been divided into two sub families based on their

biochemical and pharmacological properties (Vallone et al., 2000; Missale et al., 1998;

Jaber et al., 1996). The D1-like family comprises of D1 and D5, while the D2-like

includes D2, D3, and D4. Receptors of the D1-like family stimulate adenylate cyclase,

on the other hand, the D2-like family of receptors inhibit this effector.

The D1 and D2 have the most widespread and highest levels of expression of the

dopamine receptors. The D1 receptor is mainly expressed in the striatum, ventral

striatum, cerebral cortex and amygdala. The D2 receptor is expressed predominately in

the striatum and lower densities in thalamus and cortex. The D3 receptor is concentrated

in limbic areas (e.g., islands of Calleja and nucleus accumbens) that may mediate

abnormalities of memory, speech, and focused attention in schizophrenia (Sokoloff et

al., 1990; Suzuki et al., 1998). The highest density of D4 receptors are located in

cerebral cortex, hippocampus, hypothalamus and amygdala (Primus et al., 1997). The

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D5 highest density is the hippocampus and parafascicular nucleus of the thalamus

(Jackson et al., 1994).

1.3.3 Functions of dopamine Dopamine is the most abundant catecholamine in brain and involved in the

regulation of different physiological functions in the central nervous system, including

locomotor activity, and reward mechanisms (Chinta et al., 2005; Girault et al., 2004).

Dysregulation of dopaminergic neurotransmission has been associated with multiple

neurological and psychiatric conditions such as Parkinson’s disease, and schizophrenia.

Parkinson’s disease

Parkinson’s disease is one of the most common neurodegenerative disorders,

and affects approximately 1.5% of individuals over the age of 65, and 2.6% of

individuals over the age of 80 (Stoessl and Fernandez, 2003). Dopamine deficiency,

caused by the degeneration of nigrostriatal dopaminergic neurons, is the cause of the

major clinical motor symptoms of Parkinson’s disease. Within the substantia nigra,

neuronal loss tends to be estimated to be 60 to 70% at the onset of symptoms (Lang and

Lozano, 1998). This results in severe dopamine depletion in the striatum, responsible

for the motor symptoms associated with Parkinson’s disease. The primary symptoms of

Parkinson’s disease include muscular rigidity, resting tremor, difficulty with movement

initiation (bradykinesia), slowness of voluntary movement, difficulty with balance, and

difficulty with walking.

Levodopa (L-dopa) remains the most effective treatment for Parkinson’s

disease, this drug remains the most effective treatment of the slowness of movement,

increased muscle tone, and tremor that are typical of Parkinson’s disease (Olanow,

2002). The therapeutic response of L-dopa in Parkinson’s disease can be attributed to

several factors: 1) neuronal responses to L-dopa are mediated by its metabolic

transformation to dopamine and noradrenaline, 2) dopamine produced by L-dopa

activates D1-like and D2-like receptors, 3) facilitates noradrenaline mediated

activation of α- and β- adrenoceptors following its conversion to noradrenaline.

Although L-dopa reduces many of the motor symptoms of Parkinson’s disease, it

does not affect non-motor symptoms and does not halt the progression of the

degeneration of dopamine-containing neurons in the substantia nigra. A main

problem associated with Parkinson’s disease is the fact that dopamine-containing

neurons continue to die and ultimately there is not enough release of dopamine, even

after L-dopa treatment, to maintain essential functions (Mercuri et al., 2005).

Schizophrenia

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Schizophrenia is a debilitating mental illness that affects 1% of the population.

Symptoms of schizophrenia are often divided into positive symptoms that include

hallucinations, delusions, thought disorganization, and negative symptoms such as

blunted affect, social withdrawal, and decreased cognitive function. The “classical”

dopaminergic hypothesis of schizophrenia proposed that excessive release of dopamine

during synaptic transmission underlies at least some aspects of the pathogenesis of

schizophrenia has received its primary support from pharmacological studies (Carlsson

and Lindqvist, 1963). This was supported by two observations 1) the correlation

between antipsychotic potency of neuroleptics and their potency to block D2 receptors,

and 2) drugs that increase the level of dopamine, such as L-dopa, cocaine, and

amphetamine can induce psychotic episodes resembling schizophrenia.

Antipsychotic drugs are effective for the treatment of schizophrenia.

Antagonism at the dopamine D2 receptor is considered an essential component of the

mechanism of action (Creese et al., 1976; Seeman et al., 1975). Classical or “typical”

antipsychotics (e.g. haloperidol, chlorpromazine) are associated with high affinity for

D2 receptors and effectively treat the positive symptoms of schizophrenia. On the other

hand, “atypical” antipsychotics such as clozapine also improve negative symptoms and

cognition, and are useful in patients who have failed treatment with typical

antipsychotics (Casey et al., 1989; Meltzer et al., 1991). In addition, clozapine causes

fewer extrapyrmidal side effects (EPS) than typical antipsychotics, possibly because of

low striatal D2 receptor occupancy and its actions at other targets (e.g. antagonist at

serotonin 5-HT2A receptors).

1.3.4 PET imaging of dopamine receptors In vitro and in vivo neuroreceptor imaging studies using autoradiography and

PET imaging have contributed to a greater understanding of the dopamine system. The

dopamine receptor system is by far the most studied neurotransmitter system of the

brain. This is primarily due to the availability of a number of well-characterized

radiotracers and to great interest in this system, especially in psychiatry. PET

radioligands for the dopamine D1 and D2/3 receptors have been studied extensively. The

present thesis focuses on PET imaging of dopamine D2/3 receptor subtypes:

Dopamine D2/3 receptors

Early brain imaging studies using D2/3 receptor radioligands (N-[methyl 11C]methylspiperone (NMSP), [11C]raclopride, and [123I]IBZM) enabled imaging of

D2/3 receptors in striatum (Halldin et al., 2001a). Studies comparing parameters of D2

receptor binding in patients with schizophrenia and healthy controls have been

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extensively studied (Wong et al., 1986; Farde et al., 1990; Hietala et al., 1994; Laruelle

et al., 1996).

In vivo neuroreceptor binding techniques can be used to measure acute

fluctuations in the concentration of endogenous transmitters. Competition between

radioligands and agents that increase synaptic dopamine concentrations like

amphetamine have been shown in human and nonhuman primates to decrease D2

receptor radioligand binding (Innis et al., 1992; Farde et al., 1992; Volkow et al., 1994;

Laruelle et al., 1995; Carson et al., 1997; review: Laruelle, 2000). Several studies

reported that amphetamine induced dopamine release is increased in patients with

schizophrenia compared to matched healthy controls, and resulted in an worsening of

positive symptoms (Laruelle et al., 1996; Breier et al., 1997). These results provide

direct evidence that exaggerated activiation of dopamine transmission at D2 receptors

mediates the expression of psychotic symptoms following amphetamine challenge.

Similarly, agents that reduce synaptic dopamine levels (e.g., reserpine or alpha-methyl-

para-tyrosine) decrease competition by the endogenous transmitter and thereby

”unmask” D2 receptors and increase radioligand binding (Ginovart et al., 1997;

Laruelle et al., 1997; Verhoeff et al., 2001). Comparing D2 receptor availability at

baseline and in the dopamine depleted state provides an indirect measure of the

proporation of D2 receptors occupied by dopamine in the baseline state. The results of

pharmacological challenges, both amphetamine stimulated dopamine release and

endogenous dopamine depletion provide direct evidence of the dopamine hypothesis of

schizophrenia.

The majority of PET imaging studies of the D2 receptor have been performed

with antagonist radioligands, such [11C]raclopride. Antagonists of G-protein coupled

receptors, such as the D2 receptor, have equal affinity for receptors in the high (i.e.,

coupled) or low (i.e., uncoupled) affinity state (Creese et al., 1984; George et al., 1985;

Seeman et al., 1985; Sibley et al., 1983). Since dopamine is the endogenous agonist for

the D2 receptor, it would more effectively compete with the binding of an agonist,

compared to an antagonist radioligand. Recent development of three D2 agonist

radioligands, [11C]NPA, [11C]MNPA, and [11C](+)-PHNO make it possible to test this

hypothesis (Hwang et al., 2000; Finnema et al., 2005; Wilson et al., 2005). The use of a

D2 agonist radioligand may shed more light into the pathophysiology of the functional

state of the D2 receptor as initial evaluation studies in rodents and nonhuman primates

can now be measured in healthy control and neuropsychiatric patients.

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PET can also be used as a tool in drug development in either a direct or indirect

manner to assess the in vivo activities of a drug (Halldin et al., 2001b; Farde and Lee

2006; Farde et al., 1996; Talbot et al., 2002; Brooks et al., 2005). Several well-

characterized PET radioligands have been used to measure receptor occupancies of

antipsychotic agents at several neurotransmitter systems. The main focus has been on

the D2 receptor in striatum using [11C]raclopride and in extrastriatal regions with

[11C]FLB 457 (Halldin et al., 1995; Farde et al., 1986). PET studies using

[11C]raclopride have demonstrated a correlation between the percentage of striatal D2

receptor occupancy and clinical response in patients treated with a range of

antipsychotics (Farde et al., 1988; Farde et al., 1989). The therapeutic effect of typical

antipsychotic medications occurs at striatal D2 receptor occupancies of ∼ 65-70%, with

significantly increased risk of EPS at occupancies >80% (Farde et al., 1992; Nordstrom

et al., 1993; Kapur et al., 2000). In comparison to these standard agents, atypical

antipsychotics, such as clozapine and quetiapine, have a reduced tendency to induce

EPS and have lower occupancy of striatal D2 receptors (Farde et al., 1992; Nordstrom

et al., 1995; Kapur et al., 2000).

1.4 INTERACTION BETWEEN DOPAMINE AND GLUTAMATE 1.4.1 Glutamate receptors

The major excitatory transmitter in brain is the amino acid L-glutamate.

Glutamate is involved in many brain functions, such as differentiation, neuronal cell

survival and death, proliferation and development of neuronal and glial cells, and

Figure 5. Glutamate receptors and their effector systems. (Adapted from Spooren, 2004).

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plastic changes in efficacy of synaptic transmission (Nakanishi, 1992). Glutamate

neurotoxicity can result in neurodegeneration and neuronal cell death in disorders

such as Alzheimer and Huntington’s disease. Glutamate activates two families of

receptors: ionotropic, which are ligand-gated ion channels and metabotropic

receptors, G-protein coupled receptors linked to second messenger pathways (Conn et

al., 1997; Schoepp et al., 1999) (Figure 5).

Ionotropic and metabotropic glutamate receptors

Ionotropic glutamate receptors are glutamate-gated ion channels that when

activated increase cellular excitability. The NMDA receptor is one subgroup of

ligand-gated channel receptors, which is highly permeable to Ca2+, Na+, and K+, and

the resultant increase of intracellular Ca2+ is thought to be responsible for evoking

both neuronal plasticity and neurotoxicity (Nakanishi et al., 1994). On the other hand,

metabotropic glutamate receptors (mGluR) have been classified into three groups

based on sequence similarities, signal transduction pathways and pharmacological

characterization (Pin et al., 2003). Group I receptors (mGluR 1 and 5) are linked to

the activation of phospholipase C and generally mediate postsynaptic excitatory

effects, whereas group II (mGluR 2 and 3) and group III (mGluR 4, 6, 7 and 8)

receptors are negatively coupled to adenylyl cyclase and generally mediate

presynaptic inhibitory influences on neurotransmitter release (Hermans et al., 2001).

While glutamate binds to the large extracellular region, the action of non-competitive

antagonists such as MPEP, are thought to bind within the seven-transmembrane

domain (Pagano et al., 2000). It is speculated that the action of the non-competitive

antagonists binding within the seven-transmembrane domain of mGluR5s act to

stabilize the inactive state of the receptor and therefore inhibit the constitutive

activity.

1.4.2 Interactions between dopamine and glutamate Abnormalities of various neurotransmitter systems have been proposed in

patients suffering from schizophrenia. Research has been primarily driven by the

dopamine hypothesis of schizophrenia proposing a hyperactivity of dopamine

transmission that is responsible for the positive symptoms associated with this disorder.

Nevertheless, some patients fail to respond to treatment with dopamine antagonists and

while amphetamine causes psychosis, administration of ketamine or PCP have been

found to induce both positive and negative symptoms similar to that of patients

suffering from schizophrenia. NMDA receptor hypofunction has been suggested with

17

patients suffering from schizophrenia (Olney and Farber, 1995; Tamminga et al., 1995).

NMDA receptor blockade by acute administration of ketamine has been shown to

induce both positive and negative symptoms in healthy and schizophrenic patients.

NMDA hypofunction in schizophrenic patients most likely occurs chronically rather

than acutely, sustained dysfunction may lead to alterations in dopamine transmission

(cortical dopamine deficit and subcortical dopamine hyperactivity).

A multi-factorial view has been proposed in which neurotransmitter interactions

may shed more light into the pathophysiology of schizophrenia (Carlsson et al., 1999;

Carlsson et al., 2000). The interaction between glutamate and dopamine has lead to the

theory in which a ‘feedback loop’ exists to protect the cerebral cortex against any

elevated concentrations of both neurotransmitters regulated via a brake and accelerator.

For example, if dopamine is enhanced with amphetamine, a negative feedback

regulatory circuit appears to be activated leading to counter act the excess release. It has

been proposed that the ‘brake’, which counteracts the excess of dopamine tone fails to

function properly in patients suffering from schizophrenia. The interaction between

dopamine and glutamate has not been limited to ionotropic glutamate receptors, more

recently interactions between mGluR have been shown to affect dopamine-mediated

responses.

In vitro microdialysis studies by Golembiowska et al., 2003 demonstrated that

the effects of a systematic injection of MPEP reduced pharmacological induced

dopamine release. This inhibition reduced dopamine levels by nearly 50% compared to

methamphetamine only pretreatment. These data suggest that blockade of mGluR5 may

protect striatum terminals of dopamine neurons against methamphetamine induced

toxicity. Nevertheless, local administration of MPEP in the striatum did not decrease

but even increased dopamine levels. Thus, the modulation of excessive dopamine

release may be due to the actions of mGluR5 located outside the striatum. Further data

by Pietraszek et al., 2004 supported the interactions of mGluR5 inhibition of the

amphetamine effect in a rodent model of locomoter hyper-activity induced by

amphetamine. Labeling of dopaminergic synapses in monkey striatum have found

significant post-synaptic labeling for mGluR5 in terminals of nigrostriatal axons

(Paquet et al., 2003), while anterograde labeling determined these receptors originate

from presynaptic mGluR5 located in the primary motor cortex and other cortical

regions.

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1.4.3 PET studies of dopamine glutamate interactions The modulation of amphetamine induced dopamine release by pretreatment of

glutamatergic drugs has been studied in nonhuman primate and human brain in vivo

with PET (van Berckel et al., 2006; Kegeles et al., 2000). The effects of LY354740

(mGluR group II agonist) on the modulation of amphetamine-induced dopamine

release using PET and [11C]raclopride was studied in baboons under ketamine and

gaseous anesthesia (van Berckel et al., 2006). Acute administration of LY354740

increased the effect of amphetamine on striatal [11C]raclopride binding potential.

Amphetamine (0.5 mg/kg) alone reduced the binding potential by approximately

28%, and after pretreatment with LY354740, the amphetamine-induced reduction in

binding potential was 35%. Although these results demonstrate a greater reduction in

the outcome measure, several factors should be taken into account when interpreting

these results: (1) ketamine was used to initially to immobilize the monkey and

isoflurane was used during the PET measurements, (2) the effects of LY354740 alone

on [11C]raclopride binding was only tested twice and in the same monkey, resulting in

an 13% increase in the binding potential, (3) LY354740 is a group II agonist, which

has been found to have no effect on amphetamine induced locomotor activity

(Cartmell et al., 1999).

Sustained NMDA transmission deficiency, induced by prolonged infusion of

ketamine has been studied to determine if this leads to an increase in amphetamine

induced dopamine release in humans (Kegeles et al., 2000). Amphetamine (0.25

mg/kg) alone was found to decrease [123I]IBZM binding potential by approximately

5%. Ketamine enhanced the amphetamine induced decrease in [123I]IBZM binding

potential to approximately 12%. Acute alteration of NMDA transmission by ketamine

increased amphetamine induced dopamine release in humans. This observation

supports the hypothesis that, in schizophrenia, a deficiency of glutamatergic control of

dopamine activity might underlie the increase in amphetamine induced dopamine

release previously reported in schizophrenic patients (Breier et al., 1997; Laruelle et al.,

1996). Two important limitations should be considered when interpreting these results.

First, acute NMDA transmission deficiency most likely does not simulate the chronic

NMDA transmission deficiency occurring in patients suffering from schizophrenia

(Olney and Farber, 1995). Second, amphetamine induced a rather small change (-5.5%)

in [123I]IBZM binding potential compared to previously published brain imaging

studies using similar paradigms (Laruelle et al., 1999; Kegeles et al., 1999). The small

19

reduction in binding potential after amphetamine administration is within the range of

test-retest reproducibility (5-10%) at baseline for PET radioligands (Volkow et al.,

1993; Kim et al., 2006). Nevertheless, the increase in amphetamine induced dopamine

release induced by ketamine (greater than two-fold) was comparable in magnitude to

the exaggerated response seen in patients with schizophrenia. Due to limitations in

previous brain imaging studies on modulation of amphetamine induced striatal

dopamine release by glutametergic drugs, we performed a PET study to further

understand dopamine glutamate interactions in vivo.

2 AIMS The main objectives of the work presented in this thesis were as follows:

1. Evaluation of (S,S)-[18F]FMeNER-D2 as a potential radiotracer for the norepinephrine transporter in nonhuman primate brain (Papers I and II).

2. To measure neuropharmacological interventions using PET (e.g., competition

between synaptic neurotransmitters and radiolabeled tracers provide a useful tool to estimate changes in synaptic levels of neurotransmitters) (Papers III and IV).

a. To assess and compare the sensitivity of the agonist radioligand

[11C]MNPA and antagonist [11C]raclopride to stimulant-induced dopamine release in nonhuman primate brain (Paper III).

b. To estimate the occupancy of D2 receptors by endogenous dopamine in

rat brain using PET and [11C]MNPA (Paper IV). 3. PET determination of drug induced receptor occupancy, examined by the

curvilinear relationship between drug plasma concentration and receptor occupancy over a wide dose-range in nonhuman primate brain (Papers II and V).

a. To determine if atomoxetine (a NET reuptake inhibitor) occupies

norepinephrine transporter in a dose-dependent fashion using (S,S)-[18F]FMeNER-D2 in nonhuman primate brain (Paper II).

b. To determine dopamine D2 and serotonin 5-HT1A receptor occupancy in

nonhuman primate brain after administration of RGH-188 (a novel antipsychotic drug). In addition, the intrinsic activity of RGH-188 was estimated in vivo using a dopamine D2 agonist ([11C]MNPA) and antagonist ([11C]raclopride) radioligand (Paper V).

20

3 MATERIALS AND METHODS Detailed description of the utilized methods is provided in the individual papers. More

general characterization of the methods is presented here.

3.1 SUBJECTS Nonhuman primates

In papers I, II, III and V, Astrid Fagraeus Laboratory, Swedish Institute for

Infectious Disease Control, Solna, Sweden supplied seven (5 male and 2 female)

cynomolgus monkeys (Macaca fascicularis) weighing 2.5 – 4.0 kg. The Animal

Research Ethical Committee of the Northern Stockholm Region approved the studies.

Rodents

In paper IV, male Sprague-Dawley rats (300 - 400 g) were obtained from

Taconic Farms (Germantown, NY, USA). All animal procedures were performed in

accordance with the Guide for Care and Use of Laboratory Animals and approved by

the National Institute of Mental Health Animal Care and Use Committee.

3.2 MATERIALS AND RADIOLIGANDS The precursor and standard for FMeNER-D2, and atomoxetine (brand name

Strattera®) were obtained from Eli Lilly, Indianapolis, USA (Papers I and II). Gedeon

Richter Ltd. (Budapest, Hungary) provided RGH-188 (Paper V). We obtained

chemicals and drugs of analytical grade from various commercial sources. The

solutions were prepared as an intravenous stock-solution batch according to Good

Laboratory Practice standards.

Radiosynthesis of (S,S)-[18F]FMeNER-D2, [11C]raclopride, [11C]MNPA, and

[carbonyl-11C]WAY-100635 were prepared in high specific radioactivity (SA) as

described previously (Schou et al., 2004; Langer et al., 1999; Finnema et al., 2005;

Krasikova et al., 2003).

3.3 POSITRON EMISSION TOMOGRAPHY PET system

Nonhuman primate studies

Whole-body transmission and emission scans were acquired on a Siemens

ECAT EXACT HR, which was run in 2D mode (paper I). The spatial resolution is

about 6.0 mm full width half maximum and the field of view equal to 10.8 cm. All

other nonhuman primate studies measured radioactivity in brain with the Siemens

ECAT Exact HR 47 in 3D-mode (Wienhard et al., 1994). A three ring detector block

architecture gives a 15 cm wide field of view. The transversal resolution in the

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22

reconstructed image is about 3.8 mm full width at half maximum and an axial

resolution of 3.125 mm. The attenuation correction of the data was obtained with three

rotating 68Ge line sources. Raw PET data were then reconstructed using the standard

filtered back projection consisting of the following parameters: 2-mm Hanning Filter,

scatter correction, a zoom factor of 2.17 and a 128 x 128 matrix size (Wienhard et al.,

1994).

Rodent studies

We measured radioactivity in rat brain (Paper IV) with the small animal PET

scanner (NIH Advanced Technology Laboratory Animal Scanner) with an effective

transaxial field of view of 6.0 cm and an axial field of view of 2.0 cm (Seidel et al.,

2003). PET data were reconstructed into 17 coronal slices by 3D Ordered-Subset

Expectation Maximization algorithm, achieving a 1.6 mm full width at half maximum

resolution at the center (Johnson et al., 2002; Liow et al., 2003). The reconstructed

voxel size was 0.56 x 0.56 x 1.12 mm. No attenuation or scatter corrections were

applied.

3.4 PET EXPERIMENTAL PROCEDURES Nonhuman primate studies

In papers, I, II, III and V anesthesia was induced and maintained by repeated

intramuscular injections of a mixture of ketamine hydrochloride (3.75 mg/kg-1h-1

Ketalar , Pfizer) and xylazine hydrochloride (1.5 mg/kg-1h-1 Rompun Vet., Bayer).

The head was immobilized with a fixation device (Karlsson et al., 1993). Body

temperature was maintained with a forced-air heated air blanket (Bair Hugger-Model

505, Arizant Healthcare Inc, MN) and monitored by a rectal thermometer (Precision

Thermometer, Harvard Apparatus, MA). Cardiac and respiratory rates were measured

every 20 min.

In paper I, whole-body transaxial images were acquired for a total of approx.

4.5 hours from six bed-positions from head to mid-tail. The acquisition sequence for

each frame consisted of the following: starting an emission scan at the first bed position

for the head, moving the bed caudally to the next section, scanning a total of 6 sections

consecutively for the same period of time. Each of the 6 sections was imaged 21 times

with the following sequence of frame acquisitions: 4 x 0.50 min; 6 x 1 min; 5 x 2 min,

6 x 4 min. Emission data for paper II were collected continuously for 240 min

according to a pre-programmed series of 34 frames. The five initial frames were 1 min

each, followed by five scans of 3 min each, five scans of 6 min each and the remaining

23

frames of 10 min. In papers III and V emission data were collected continuously for 93

min according to a pre-programmed series of 20 frames starting immediately after

injection of radioligand. The three initial frames were 1 min, followed by four scans of

3 min, and the remaining frames of 6 min.

Rodent studies

In paper IV, anesthesia was induced and maintained using 1 - 1.5% isoflurane

and 100% O2 inhalation through a noise cone. Radioligand was administered as a bolus

followed by a continuous infusion via penile vein catheter (Intramedic PE-10

polyethylene catheter; Aster Industries, Harmony, PA, USA) using a syringe pump

(Harvard PhD 2000, Harvard Apparatus, Holliston, MA, USA). Emission data were

collected continuously for 90 min, according to a pre-programmed series of six initial

frames of 20 s, followed by five scans of 1 min, four scans of 2 min, three scans of 5

min, three scans of 10 min, and the remaining frames of 20 min. Body temperature in

the anesthetized rats was monitored with a rectal temperature probe and maintained at

36.5-37.5°C with a heating lamp and heating pad.

3.5 EXPERIMENTAL DRUGS AND DOSE SELECTION Atomoxetine is a selective NET reuptake inhibitor, which has been found to be

effective in the treatment of ADHD in both children and adults (Spencer et al., 1998;

Michelson et al., 2001). The aim of paper II was to determine if atomoxetine occupies

NET in a dose-dependent fashion using PET in nonhuman primate brain. Each monkey

participated in a total of four PET measurements: one at baseline and one each after

administration of atomoxetine at three different doses. A prolonged intravenous

infusion design was utilized rather than a bolus injection to avoid high initial

atomoxetine concentrations, to better mimic a human oral absorption profile and to

reach plasma steady state during the PET measurements. Monkey doses were based on

a therapeutic concentration range in adults. As such, an examination of NET occupancy

in monkey at clinically-relevant exposures could be examined during the PET study.

In papers III and IV, pharmacological challenges that either increase or decrease

synaptic concentrations of dopamine are combined with PET imaging of the D2

receptor. Agents that increase synaptic dopamine concentrations like amphetamine

have been shown in human and nonhuman primates to decrease D2 receptor radioligand

binding (Innis et al., 1992; Farde et al., 1992; Volkow et al., 1994; Laruelle et al., 1995;

Carson et al., 1997; review: Laruelle, 2000). While agents that reduce synaptic

dopamine levels (e.g., reserpine or α-methyl-para-tyrosine) decrease competition by the

24

endogenous transmitter and thereby ”unmask” D2 receptors and increase radioligand

binding (Ginovart et al., 1997; Laruelle et al., 1997; Verhoeff et al., 2001).

In paper III, amphetamine was administered intravenously as a 30 s bolus

approximately 20 min prior to radioligand injection at four doses (0.1, 0.2, 0.5, and 1.0

mg/kg). Previous studies utilizing in vivo microdialysis in nonhuman primates have

demonstrated that after administration of 0.1 mg/kg of amphetamine produced an

increase of 100% over basal extracellular dopamine levels, 0.2 mg/kg (increase of 459 -

549%), 0.5 mg/kg (~1300%) and the maximum dose assessed 1.0 mg/kg (1600-2000%)

(Moghaddam et al., 1993; Saunders et al., 1994; Laruelle et al., 1997; Breier et al.,

1997). The maximum increase after amphetamine enhanced extracellular dopamine

levels in striatum by more than 20-fold, which reached a peak 40 min after

administration.

Dopamine depleting agents such as α-methyl-para-tyrosine and reserpine are

typically administered at low doses because of side effects, including hypotension. α-

methyl-para-tyrosine is a competitive and reversible inhibitor of tyrosine hydroxylase

(Mignot et al., 1985; Bennett et al., 1981) and has restricted dose limits in humans due

to excretion of crystals in urine at higher doses (Engleman et al., 1968). Reserpine acts

as an inhibitor of vesicular uptake of catecholamines and causes prolonged depletion

because of its irreversible binding to the vesicular transporter (Ponzio et al., 1984;

Guldberg et al., 1971). In paper IV, dopamine depletion was achieved in rats by

administration of reserpine (5 mg/kg, i.p.) given 24 h prior to the PET measurement.

On the day of the PET experiment, the reserpine-treated rats received two injections of

α-methyl-para-tyrosine (200 mg/kg, i.p.) at 4 and 1 h before radioligand injection. This

dual dopamine depletion paradigm has been found to reduce dopamine tissue

concentrations by 95% in rat striatum (Guo et al., 2003).

In paper V, we determined receptor occupancy of RGH-188, a novel

antipsychotic drug that exhibits high affinity for the D2 and D3 receptors, and moderate

affinity for 5-HT1A receptors. Monkeys participated in three consecutive PET

measurements per experimental day. The first PET measurement was performed at

baseline conditions, the second after a low dose (1 - 5 µg/kg) of RGH-188, and the

third after administration of a high dose (30 - 300 µg/kg). RGH-188 was injected

intravenously over 30 s, 15 min prior to radioligand injection.

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3.6 IMAGE ANLAYSIS In paper I, estimates of whole-body biodistribution over time of (S,S)-

[18F]FMeNER-D2 were performed on compressed planar images as previously

described (Tipre et al., 2004). The planar images were analyzed with PMOD software

(PMOD technologies Ltd, Adliswil, Switzerland). Regions of interest (ROI) were

drawn on planar images and a single generous sized ROI was drawn over the total

body, urinary bladder, brain, kidneys, liver, vertebra, heart and lungs. The use of

planar images for the data analysis provided conservative estimates of radiation

exposure, since the large regions of interest included overlying tissues

In paper II, mean PET images of (S,S)-[18F]FMeNER-D2 were generated with

the highest radioactivity from cortical and sub-cortical brain structures and the least

contribution from skull. PET measurements of experiments with low, medium and high

doses of atomoxetine were co-registered to baseline measurement using the “co-register

and re-slice function” in SPM2 (Statistical Parametric Mapping, Wellcome Department

of Cognitive Neurology, UK). The mean image of the baseline PET measurement was

transformed into standard anatomical space using the monkey version of the Human

Brain Atlas developed at Karolinska Institute (Roland et al., 1994). A standard template

of ROIs was generated on an average monkey MRI scan and applied to each PET

study. Anatomical regions of interest were delineated for the thalamus, locus

coeruleus, whole brain, caudate, mesencephalon, cingulate gyrus, frontal, parietal,

temporal and occipital cortex.

In papers III-V, parametric images of binding potential (BP) and relative blood

flow (R1) were generated from the original reconstructed PET data using a reference

tissue model (Ichise et al., 2003). The R1 PET image was fused with the BP image

using a tool in PMOD software (PMOD Technologies Ltd, Adliswil, Switzerland).

Anatomical regions of interest were manually defined on the fused image for left and

right striatum and cerebellum for dopamine D2 radioligands; temporal and frontal

cortex (defined as forebrain), raphe nuclei, and cerebellum for the 5HT1A radioligand.

All parametric imaging was performed in PMOD (Mikolajczyk et al., 1998) installed

on a PC workstation.

3.7 CALCULATIONS Dosimetry

Activity in the source organs (not decay-corrected) were expressed as a

percentage of injected dose and plotted versus time (Paper I). The residence times

26

from the monkey were calculated by converting the corresponding human values by

multiplication with a factor to scale organ and body weights (in kg) as

(wm,b/wm,o)(wh,o/wh,b), where wm,b is the monkey body weight, wm,o, is the monkey

organ weight, wh,b, is the human body weight, and wh,o is the human organ weight.

This allometric scaling factor is identical to that using SUV (standard uptake value),

which expresses uptake as (%ID/g organ)* g body weight. Thus, the scaling used in

this paper assumes the SUV in the monkey organ is equal to that in the human organ.

Target organ absorbed radiation doses, effective dose and effective dose equivalent

were calculated by MIRDOSE 3.1 (Loevinger et al., 1991). The dynamic bladder

model, implemented in MIRDOSE 3.1 software, was applied to calculate residence

time of the urinary activity with voiding intervals of 2.4 h, 4.8 h and no urine voiding.

3.8 NEUROPHARMACOLOGICAL INTERVENTIONS In paper III, regional radioactivity was normalized to injected activity and body

weight by use of a standardized uptake value (%SUV) [%SUV = (% injection dose/cm3

brain) x body weight (g)], which normalizes for injected activity and body weight. In

paper IV, regional time-activity curves were normalized to injected activity by use of

%(IA per h) per cm3 = [% injected radioactivity per h/cm3 brain].

The radioactivity in the cerebellum was used as an approximate value for free

and non-specifically bound radioligand concentration in the brain. The time curve for

the ratio of radioactivity was calculated for each ROI to the cerebellum. Specific

binding to D2 receptors in striatum was defined as the difference between the total

radioactivity concentration in striatum and cerebellum.

In paper IV, two approaches were used to calculate the outcome measure: 1.)

An equilibrium analysis was applied to PET data obtained from 17.5 to 90 min. The

averaged values obtained during these time frames for the striatum and cerebellum

uptake were applied using a specific to non-displaceable ratio: BPequil = (striatum –

cerebellum)/cerebellum. Second, BP was also estimated by the reference tissue model

MRTM2 (Ichise et al., 2003).

The change in the binding potential after administration of amphetamine or

dopamine depleting agents was calculated according to the equation:

Change (%) = ((BPpost-drug – BPbaseline)/ BPbaseline)*100

Under depleted conditions by α-methyl-para-tyrosine plus resperine, one can

assume that the total pool (100%) of D2/3 receptors are available for radioligand

binding. The baseline estimation is confounded by competition between endogenous

27

dopamine and the radioligand. By comparing the BPdepleted to the BPbaseline, the

difference in the signal is related to the amount of endogenous dopamine occupying

D2/3 receptors during basal conditions. We can estimate the occupancy of endogenous

dopamine by the following equation: (BPdepleted – BPbaseline)/ BPdepleted)*100

3.9 RECEPTOR OCCUPANCY Paper II and V, regional radioactivity was expressed as a standardized uptake

value (%SUV), which normalizes for injected activity and body weight. To calculate

dopamine and serotonin receptor occupancy, two quantitative methods were applied.

The first approach was the peak ratio method (Farde et al., 1992, Andree et al., 1998,

Nyberg et al., 1996). Briefly, the peak ratio method calculates the ratio of

(Bound)/(Free) between the total radioactivity in the region of interest (Bound) and the

total radioactivity in a reference region (Free) with negligible density of D2 and 5-HT1A

receptors. The radioactivity in the cerebellum was used as an approximate value for

free and non-specifically bound radioligand concentration, bearing in mind the

negligible density of D2 (Hall et al., 1994; Hall et al., 1996; Civelli et al., 1993) and 5-

HT1A receptors in cerebellum (Hall et al., 1997). The ratio B/F obtained at the peak of

specific binding time-activity curve is assumed to represent transient equilibrium

(Farde et al. 1989). To calculate D2 and 5-HT1A receptor occupancy, the striatum, and

forebrain and raphe nuclei were used as the region of interest, respectively. Receptor

occupancy was defined as the percentage reduction in the ratio of B/F after drug

administration as compared to B/F at baseline.

The second method to calculate receptor occupancy was the percent reduction

of BP (estimated by MRTM2) after administration of RGH-188 compared to BP at

baseline:

Receptor occupancy (% reduction in BP)

= ((BPpost-drug – BPbaseline)/ BPbaseline)*100%

A hyperbolic curve fit (one-site binding) of NET occupancy plotted as a

function of steady state N-desmethyl-atomoxetine concentration (ng/mL). Transporter

occupancy (%) was defined by change in BP (MRTM2) after drug administration

compared to at baseline. Points represent values estimated from monkey 1 and 2. The

calculation is represented as: Occupancy (%) = Occmax*[Concdrug]/Kd + [Concdrug] in

which Occmax is the maximal occupancy that can be induced by the drug (was set to

100), [Concdrug] is the Css of N-desmethyl-atomoxetine and Kd expresses the drug

concentration at which 50% transporter occupancy is induced.

28

3.10 STATISTICAL ANALYSIS In paper III, a comparison of baseline [11C]MNPA (a selective D2 agonist) and

[11C]raclopride (an antagonist at D2 and D3 receptors) binding potential were assessed

by a repeated measured analysis of variance, with the radioligand as the repeated

condition (n = 8 for each radioligand). For each radioligand the between monkey

differences at baseline were investigated by a one-way analysis of variance (ANOVA),

with monkey as group factor (n = 8 for each radioligand). In addition, the effect of

amphetamine on the change in binding potential for each radioligand was evaluated by

ANOVA. The change in binding potential for each radioligand was designated as the

dependent variable and the main effect assessed for dose, with an interaction between

the radioligands and ligand*dose. The minimum level of significance was designated as

p < 0.05.

In paper IV, a two-tailed independent t-test was applied to the PET image data

and ex vivo radiometabolite data comparing the control and dopamine depletion groups

with a minimum level of significance designated as p < 0.05. All statistical analyses

were performed using SPSS version 13.

4 RESULTS AND DISCUSSION Whole-Body Biodistribution, Radiation Dosimetry Estimates for the PET

Norepinephrine Transporter Probe (S,S)-[18F]FMeNER-D2 in Non-Human

Primates (PAPER I)

An important safety as well as limiting factor for the clinical usability of a

radioligand is set by the relationship between radiation absorbed doses in different

source organs of the body following the radioactivity dose injected. The amount of

radiation absorbed dose delivered by internal administration radiopharmaceuticals is

based on the fact that radiopharmaceuticals have a certain biological, physical and

effective half-life based on the radionuclide half-lives. Paper I measured the whole-

body biodistribution over time of (S,S)-[18F]FMeNER-D2 and estimated the resulting

radiation exposure to organs of the body. The use of planar images for the data

analysis provided conservative estimates of radiation exposure, since the large regions

of interest included overlying tissues. Absorbed doses were calculated from

cynomolgus monkey biodistribution data. The MIRD scheme was applied as an

accurate determination of the time dependent activity of the target regions of the body.

Figure 6. Whole-body images demonstrating biodistribution of (S,S)-[18F]FMeNER-D2 in monkey at 3, 8, 30 and 110 minutes after radioligand injection.

In planar images, urinary bladder, brain, kidneys, liver, lungs, entire abdomen

(GI tract) and vertebra were visually identified as organs with moderate to high

activity (Figure 6). The peak values of the percent injected dose (%ID) at time after

radioligand injection were calculated for the lungs (26.76% ID at 1.42 min), kidneys

(13.55% ID at 2.18 min), whole brain (5.65% ID at 4.48 min), liver (7.20% ID at 2

min), red bone marrow (5.02% ID at 2.06 min), heart (2.36% ID at 1.42 min) and

29

30

urinary bladder (23% ID at 250 min). The cumulative urinary excretion of (S,S)-

[18F]FMeNER-D2 was 20.8% at 150 min. Human residence times were extrapolated

from the average of two monkeys calculated from whole-body planar images.

Radiation absorbed dose estimates were calculated with MIRDOSE 3.1 computer

program, with urine voiding intervals of 2.4 h, 4.8 h and no voiding. Assuming a

urine voiding interval of 2.4 h, the four organs with highest exposures in µGy/MBq

(mrad/mCi) were: kidneys 126 (468), heart wall 108 (399), lungs 88.4 (327) and

urinary bladder 114 (422). The effective doses were estimated with and without urine

voiding at a range of 123 (33) and to 131 (35.5) mrad/mCi (µGy/MBq).

This study indicated that (S,S)-[18F]FMeNER-D2 may be a suitable

radioligand for studying NET in brain. The calculated dosimetry results seem

comparable with those for other 18F labeled brain imaging agents. These results in

primates can be used to estimate the limit of radioactivity that can be administered at

a low risk to human subjects. Nevertheless, a human biodistribution study would

provide more accurate estimation of organ radiation absorbed doses.

Atomoxetine Occupies the Norepinephrine Transporter in a Dose-Dependent

Fashion: A PET Study in Non-Human Primate Brain Using (S,S)-[18F]FMeNER-

D2 (PAPER II)

PET can be used as a tool in drug development in either a direct or indirect

manner to assess the in vivo activities of a drug (Farde et al., 1996; Talbot et al., 2002;

Brooks et al., 2005). A direct approach is to radiolabel a potential new drug with 11C or 18F and trace its uptake, anatomical distribution, and binding in brain. An indirect

approach measures the degree to which administration of the new drug competes with

the specific binding of a conventional PET radioligand. Thus, PET studies can confirm

brain distribution, and drug binding selectivity to various subtypes of receptors.

Furthermore, correlation between plasma drug concentration and receptor occupancy

may provide valuable data to establish clinical dose levels (Halldin et al., 2001b; Farde

and Lee 2006; Farde et al., 1996; Talbot et al., 2002; Brooks et al., 2005).

The aim of paper II was to determine if atomoxetine occupies NET in a dose-

dependent fashion in nonhuman primate brain. In this PET study, we used (S,S)

[18F]FMeNER-D2 to measure NET occupancy of atomoxetine in the locus coeruleus,

thalamus, mesencephalon and cingulated gyrus. PET data was analyzed to define

relationships between doses, plasma concentrations and NET occupancy for

atomoxetine. Doses were based on the therapeutic concentration range of atomoxetine

31

in humans. As such, the transporter occupancy data may be informative to guide dosing

strategies in drug development or in determining the occupancy level necessary to

induce clinical effects.

During baseline conditions, (S,S)-[18F]FMeNER-D2 uptake was highest in NET

rich regions such as the locus coeruleus, thalamus, mesencephalon and the cingulate

gyrus, whereas the radioactivity in the

caudate was low. Quantification of

cortical uptake of (S,S)-[18F]FMeNER-D2

was difficult due to defluorination

resulting in skull-bound radioactivity that

increased cortical uptake over time. After

administration of low, medium and high

doses of atomoxetine, there was a dose-

dependent reduction of the binding

potential estimated by a reference tissue

model. The relationship between N-

desmethyl-atomoxetine concentration and NET occupancy in the locus coeruleus was

described by a hyperbolic function (Figure 7), resulting in 51% at the lowest dose (0.03

mg/kg/h) to 91% at the highest dose (0.12 mg/kg/h).

0 20 40 600

20

40

60

80

100

Locus coeruleus

N-desmethylatomoxetine (ng/mL)

NET

occ

upan

cy (%

)

80

Figure 7. A hyperbolic curve fit (one-site binding) of NET occupancy plotted as a function of steady-state N- desmethylatomoxetine

This study demonstrated that steady-state infusion of atomoxetine occupies

NET in non-human primate brain in a dose-dependent fashion ranging from 38 to 82

%. NET occupancy using the longer half-life of (S,S)-[18F]FMeNER-D2 compared to

[11C]MeNER allows a more realiable determination of NET occupancy since transient

specific binding equilibrium is attained during the time frame of the PET measurement.

This is the first in vivo PET study to successfully demonstrate the ability to measure a

dose-dependent change in NET occupancy in brain using (S,S)-[18F]FMeNER-D2.

Effect of Amphetamine on Dopamine D2 Receptor Binding in Non-Human

Primate Brain: A Comparison of the Agonist Ligand [11C]MNPA and Antagonist

Radioligand [11C]Raclopride (PAPER III)

Pharmacological challenges that increase synaptic concentrations of dopamine

are combined with PET imaging of the D2 receptor using tracer doses of the

radioligand. For example, agents that increase synaptic dopamine concentrations like

amphetamine or methylphenidate have been shown in human and nonhuman primates

to decrease D2 receptor radioligand binding. These endogenous competition studies

have typically been performed with antagonist radioligands, such as [11C]raclopride.

32

Antagonists of G-protein coupled receptors, such as the D2 receptor, have equal affinity

for receptors in the high (i.e., coupled) and low (i.e., uncoupled) affinity states. In

contrast, agonists bind preferentially to the high affinity state. Since dopamine is the

endogenous agonist for the D2 receptor, it would more effectively compete with the

binding of an agonist, compared to an antagonist radioligand. The aim of paper III was

to examine whether the striatal uptake of the agonist radioligand [11C]MNPA is more

sensitive than the antagonist [11C]raclopride to stimulant-induced dopamine release.

Cynomolgus monkeys were examined before and after increasing doses of intravenous

amphetamine. Finally, the data were used in an attempt to estimate the proportion of D2

receptors in the high and low affinity states in the anesthetized nonhuman primate.

Following injection of [11C]MNPA in the baseline condition, activity

concentrated in striatum, with lower levels in the thalamus that were still higher than

the nonspecific uptake in cerebellum. Under baseline conditions, [11C]raclopride BP

(5.76 ± 0.95, n = 8) was significantly higher than that for [11C]MNPA BP (1.31 ± 0.21,

n = 8). When comparing the difference between the four monkeys BP values for each

radioligand, no significant between subject effects were found for [11C]MNPA or

[11C]raclopride.

The effect of amphetamine on the binding of [11C]MNPA and [11C]raclopride

was investigated by administering amphetamine intravenously (0.1, 0.2, 0.5 and 1.0

mg/kg) approximately 20 min prior to injection of radioligand. Amphetamine caused a

reduction in [11C]MNPA binding potential of 4% at 0.1, 23% at 0.2, 25% at 0.5 and

46% at 1.0 mg/kg. [11C]Raclopride binding potential was reduced to a lesser extent by

2% at 0.1, 16% at 0.2, 15% at 0.5 and 23% at 1.0 mg/kg of amphetamine (Figure 8). A

statistically significant greater change in BP for [11C]MNPA compared to

[11C]raclopride was observed (P = 0.024; ANOVA).

Based on calculations proposed by Narendran et al. (2004) and the assumption

that 10% of receptors are occupied by endogenous dopamine at baseline, our results

suggest that 61% of the D2 receptors are configured in the high affinity state. This

calculation is based upon the assumption that 10% of the receptors are occupied by

dopamine at baseline and that extrasynaptic receptors cannot be displaced by stimulant-

induced dopamine release. This 61% in the high affinity state is distributed as: 10%

occupied by dopamine at baseline, 23% are synaptic and 28% are extra-synaptic. By

this calculation the remaining receptors (39%) would be in the low affinity state (ie.,

39% = 100% - 61% in the high affinity state).

33

Paper III provides evidence that the agonist radioligand [11C]MNPA is more

sensitive than the antagonist [11C]raclopride to displacement by endogenous dopamine.

At all doses examined, the change in [11C]MNPA BP was approximately 50% more

sensitive than [11C]raclopride to pharmacological induced increases in synaptic

dopamine. These results agree with those of Cumming et al. (2002) and Narendran et

al. (2004), who similarly investigated the effect of amphetamine on the binding of

agonist and antagonist radioligands in mice and non-human primates.

Figure 8. Parametric images of [11C]raclopride and [11C]MNPA BP estimated by a reference tissue model at baseline and postamphetamine conditions. Images represent the same monkey for each dose of amphetamine.

The current results can been interpreted based on the following assumptions: 1)

with the understanding that [11C]raclopride binds to a greater number of D2 receptors

than [11C]MNPA, because the antagonist binds to both the high and low affinity states

of the receptor. In fact, there may be other reasons for differences in Bmax′ (available

receptor density) between these two tracers. For example, the ligands may differ in

affinity for the monomer and dimer forms of the receptor. 2) effects of anesthesia

should be taken into account, a mixture of ketamine and xylazine was administered

every 40 min at baseline and during amphetamine challenge conditions. It cannot be

ruled out that ketamine has some effect on [11C]MNPA and [11C]raclopride binding at

both baseline and pretreatment conditions. 3) the greater reduction in BP after

amphetamine administration for [11C]MNPA compared to [11C]raclopride may also be

secondary to agonist induced receptor internalization. The decreased uptake of

radioligand may be the result of the loss of available binding sites by internalization

34

and a subsequent loss of appropriate three dimensional binding to antagonists as well as

agonists. In summary, [11C]MNPA is a promising radioligand for PET-imaging of the

high affinity state of D2 receptors in the primate brain. Stimulation of dopamine release

with amphetamine caused a marked decreases in BP of [11C]MNPA in a dose-

dependent fashion, thus demonstrating that the radioligand is sensitive to the

endogenous dopamine concentration.

Imaging Endogenous Dopamine Occupancy of the D2 Receptor in Rat Brain With

[11C]MNPA (PAPER IV)

Endogenous dopamine levels during basal conditions have been estimated using

PET and SPECT in human and nonhuman primate brain. Dopamine depletion induced

by reserpine and/or α-methyl-para-tyrosine decreases competition by endogenous

dopamine to the D2 receptor and increases radioligand binding. The increase in binding

compared to baseline measurements provides an estimate of the number of D2 receptors

occupied by dopamine under basal conditions. Occupancy of the D2 receptor by

endogenous dopamine in brain using antagonist radioligands has ranged from

approximately 10% to 30%. The aim of paper IV was to estimate the occupancy of D2

receptors by endogenous dopamine in rat brain in vivo using PET and the agonist

radioligand [11C]MNPA. In addition, ex vivo radiometabolite studies were performed to

determine if the increase in radioligand binding after dopamine depletion was due to a

change in radiometabolites entering brain.

Following injection of [11C]MNPA in the baseline condition, activity

concentrated in striatum, with lower levels in cerebellum. [11C]MNPA baseline BP

values were similar between the outcome

measures BPequil and MRTM2, 1.04 ± 0.14

and 1.08 ± 0.12, respectively.

Nonradioactive raclopride administered

during steady state, 2 and 4 mg/kg, induced

a significant displacement in striatal activity

by ~ 60% and ~ 80%, respectively. The

specific binding of [11C]MNPA was nearly

completely inhibited by this D2/3-antagonist,

consistent with the radioligand also binding

to D2/3 receptors. Depletion of endogenous

dopamine with reserpine and α-methyl-

Control Depletion0.5

1.0

1.5

2.0

[11C

]MN

PA B

P

Figure 9. [11C]MNPA BP measured at baseline and after dopamine depletion. Dopamine depletion resulted in a significant increase in striatal [11C]MNPA binding (two-tailed p = 0.0005).

35

para-tyrosine significantly increased activity in striatum but had little effect in

cerebellum. Under baseline conditions [11C]MNPA BP was 1.08 ± 0.12 (n = 5) and

after dopamine depletion resulted in a significant increase of 38% in BP 1.49 ± 0.12 ((n

= 5); p = 0.0005), Figure 9. Assuming a complete depletion of endogenous dopamine in

rat striatum following reserpine and α-methyl-para-tyrosine administration, these

results indicate a baseline D2/3 high affinity state receptor occupancy by endogenous

dopamine of ~ 27%, calculated by comparing the BP during baseline conditions and

after acute dopamine depletion. The effect of dopamine depletion was also

demonstrated by ex vivo studies where the brain to plasma parent concentration ratios

in the depleted rats was higher than that in control rats by 30%. Brain radioactivity

composition of radiometabolite in control condition was not significantly different after

dopamine depletion. Thus, the increase in radioligand binding after dopamine depletion

is not attributed to an increase in radiometabolite binding. Both NPA and MNPA had

high binding affinities for D2 receptors (Ki = 4.7 and 9.4 nM, respectively) (Paper IV,

unpublished data). PHNO binding affinity was highest to D3 receptors (Ki = 0.53),

consistent with previous reports (Parker et al., 2006; Freedman et al., 1994). Thus, NPA

and MNPA have similar binding affinities for D2/D3 receptors, while PHNO bound

preferentially to D3 receptors.

Our PET imaging in rats confirm prior studies in monkeys (Finnema et al.,

2005; Seneca et al., 2006) that [11C]MNPA is a useful agonist radioligand for D2/3

receptors. One potential reason for increased activity in striatum after dopamine

depletion can be attributed to an increase of radiometabolites entering brain.

Nevertheless, our ex vivo experiments under control and dopamine depletion conditions

found no significant difference in radiometabolite composition or parent radioactivity

in whole brain samples. Instead, the increase in striatal binding could have been caused

by at least four factors: 1) an increase in the number of D2 receptors, 2) trafficking of

the receptor to the surface membrane, assuming that the internalized receptor does not

bind MNPA, 3) an increase in the percentage of the D2 receptors in the high affinity

state, 4) removal of competition by endogenous dopamine. Several homogenate

binding studies have found no increase in the density of D2 receptors after acute

dopamine depletion (Laruelle et al., 1997; Ross et al., 1989). In agreement with the

homogenate binding studies, in vivo Scatchard analysis using [11C]raclopride binding

reflected an increase in receptor affinity but no change in D2 receptor density in

nonhuman primate brain (Ginovart et al., 1997). Thus, the first possibility (i.e., an

36

increase in the number of D2 receptors) seems unlikely. Nevertheless, the remaining

three mechanisms are plausible and cannot be excluded by our results. In conclusion,

we estimate that dopamine occupies ∼ 27% of high affinity D2/3 receptors in brains of

rats under gaseous anesthesia.

Occupancy of Dopamine D2 and Serotonin 5-HT1A Receptors by a Novel D2/D3

Antipsychotic Drug in Non-Human Primate Brain: A PET Study (PAPER V)

RGH-188 is a novel antipsychotic drug that binds with high selectivity and

affinity to D2 and D3 and moderate affinity to 5-HT1A receptors. The aim of paper V

was to determine if RGH-188 occupies dopamine D2 and serotonin 5-HT1A receptors

in nonhuman primate brain. We used both an antagonist [11C]raclopride and agonist

[11C]MNPA radioligands to measure D2 receptor occupancy in striatum and

[carbonyl-11C]WAY-100635 to measure 5HT1A receptor occupancy in raphe nuclei

and forebrain. Following the injection of [11C]raclopride or [11C]MNPA, striatum

showed the highest activity, with lower levels in cerebellum. RGH-188 occupied D2

receptors in a dose dependent and saturable manner, with lowest dose (1 µg/kg)

occupying ~5% of receptors, and highest dose (300 µg/kg) more than 90% (Figure

10A). A comparison of antagonist/agonist radioligand ratio of receptor occupancy

may provide an in vivo estimation of intrinsic activity of RGH-188. RGH-188 was

equally potent to displace both [11C]raclopride and [11C]MNPA with an ED50 of ~5

µg/kg i.v., which suggests that RGH-188 is an antagonist.

A B.

0 50 100 150 200 250 300 3500

20

40

60

80

100

Binding PotentialPeak Ratio

RGH-188 (µg/kg)

5-H

T 1A o

ccup

ancy

(%)

0 50 100 150 200 250 300 3500

20

40

60

80

100

[11C]Raclopride (Binding Potential)[11C]Raclopride (Peak Ratio)

EPS

[11C]MNPA (Binding Potential)[11C]MNPA (Peak Ratio)

RGH-188 (µg/kg)

2D

occ

upan

cy (%

)

Figure 10. A hyperbolic curve fit of dopamine D2 (A) and serotonin 5-HT1A (B) receptor occupancy plotted as a function of RGH-188 dose (µg/kg). Receptor occupancy (%) was estimated by two approaches: the percent reduction of B/F (Peak ratio method: shown as triangles with dotted line) and change in BP estimated by a reference tissue model (shown as circles with solid line).

37

After administration of [11C]WAY-100635, activity concentrated in forebrain

and raphe nuclei, with lower levels in cerebellum. Administratrion of 30 µg/kg of

RGH-188 slightly reduced activity in raphe nuclei compared to baseline and had no

effect in forebrain. 5-HT1A receptor occupancy could be described by a hyperbolic

funtion and was much lower compared to D2 occupancy at the same doses, with a

maximal value of ~30% for the raphe nuclei (Figure 10B).

In conclusion, RGH-188 occupies dopamine D2 receptors in a dose-dependent

and saturable manner and has much lower occupancy of serotonin 5-HT1A receptors.

The finding of a relatively low maximal 5-HT1A receptor occupancy suggests that

RGH-188 will have minimal pharmacological actions via this target. These PET data

demonstrated an ED50 value of 5 µg/kg at the D2 receptor and a 5 times greater dose

induced only 11-18% occupancy at the 5-HT1A receptor. RGH-188 primary CNS

actions are related to the D2 and D3 receptor, with minimal 5-HT1A related effects at

therapeutic dose levels. Imaging studies using both dopamine D2 receptor agonist and

antagonist radioligands may be used to define the affinity of a drug to the low and

high affinity states of a receptor and thereby provide an in vivo estimate of intrinsic

activity. RGH-188 had similar occupancy measured with both an agonist

([11C]MNPA) and an antagonist ([11C]raclopride) radioligand, confirming in vitro

studies that RGH-188 is an antagonist.

Interactions between Dopamine and Glutamate: A PET study in rat and non-

human primate brain to elucidate the enhancement of endogenous dopamine with

amphetamine by Group I mGluR antagonist, MPEP. (Unpublished work

performed by Seneca together with the PET imaging facility at NIRS, Chiba,

Japan. Manuscript is under preparation by Seneca, Maeda, Tokunaga, Higuchi,

Innis, Halldin, and Suhara).

The aim of this study was to explore the inhibition of methamphetamine

induced dopamine release by pretreatment with an mGluR group I antagonist, MPEP,

using the D2 agonist radioligand, [11C]MNPA. In awake conditions, cynomolgus

monkeys (n = 4) and rats (n = 3) were examined with [11C]MNPA at baseline (test-

retest conditions), following MPEP pretreatment, after methamphetamine

administration and MPEP plus methamphetamine conditions. Additionally,

microdialysis was applied to rat in order to quantify extracellular dopamine content in

the drug challenge conditions identical to those in the PET study.

38

[11C]MNPA baseline binding potential values were similar between monkey

and rat, 1.00 ± 0.05 and 0.82 ± 0.06, respectively. The test–retest binding potential

variability was relatively small, ranging from 3% to 6%. MPEP alone did not

significantly change the D2 receptor availability for the radioligand. Methamphetamine

caused a reduction in [11C]MNPA binding potential by 17 ± 6% and 25 ± 8% in

monkeys and rats, respectively. Meanwhile, pretreatment with MPEP reversed

methamphetamine induced reduction of striatal [11C]MNPA binding to baseline levels

(Figure 11A). The extracellular dopamine concentration in rat striatum was increased

by approximately 10 fold at 10 min after methamphetamine administration. MPEP

pretreatment nearly halved the impact of methamphetamine administration on the

dopamine contents (Figure 11B). This observation is in line with the PET data,

substantiating the interference of MPEP with modulation of dopamine release by

pharmacological increase in extracellular dopamine release.

0 6TestRetest

MPEP MAP

MPEP+MAP60

80

100

120

RatMonkey

BP

Cha

nge

(% B

asel

ine)

-20 -10 0 10 20 30 40 50 60 70 80 90 1000

200400600800

10001200140016001800

MAPMPEP+MAP

MPEP (1.0 mg/kg i.v.)

MAP (0.5 mg/kg i.v.)

Time (min)

DA

rele

ase

(% o

f bas

elin

e)

A. B.

Figure 11. Change in striatal BP of [11C]MNPA in conscious monkey and rat during modulation of dopamine release (A). Changes in extracellular dopamine levels in rat striatum during microdialysis (B). (MAP methamphetamine; MPEP mGluR group I antagonist).

In conclusion, these results provide evidence for the modulation of striatal

dopamine release by Glutamate-Dopamine interactions across species. The mechanistic

view that MPEP inhibits mGluR group I located on dopaminergic neurons and

suppressively acts on stimulation of dopamine release from the striatal terminals

without altering the baseline state of dopamine transmission.

5 SUMMARY OF FINDINGS The present thesis focused on examining two monoaminergic neurotransmitter

systems in rodent and nonhuman primate brain.

The estimated radiation burden of (S,S)-[18F]FMeNER-D2 is comparable to

that of other 18F radioligands. These results in primates can be used to

estimate the limit of radioactivity that can be administered at a low risk to

human subjects.

Peak equilibrium measurements were achieved using (S,S)-[18F]FMeNER-D2

in contrast to the previously reported data for (S,S)-[11C]MeNER. After

administration of atomoxetine, a dose-dependent occupancy from 38 to 82%

was observed for brain regions known to contain high densities of NET.

The agonist radioligand [11C]MNPA, was found to be more sensitive than the

antagonist [11C]raclopride to displacement by endogenous dopamine. Since an

antagonist radioligand binds with equal affinity to both high- and low-affinity

states, a radiolabeled agonist would be advantageous since it only binds to

receptors in the high- affinity state, likely leading to more sensitivity to

endogenous dopamine.

PET imaging in rats confirm prior studies in monkeys that [11C]MNPA is a

useful agonist radioligand for D2/3 receptors. We estimate that dopamine

occupies ∼ 27% of high affinity D2/3 receptors in brains of rats under gaseous

anesthesia.

The administration of increasing doses of RGH-188 (a novel antipsychotic

drug) resulted in a dose-dependent and saturable reduction in the specific

binding of [11C]raclopride and [11C]MNPA. Administration of RGH-188 at 30

µg/kg induced a higher striatal D2 receptor occupancy of ∼ 87% compared to

only 21% for 5-HT1A receptor. RGH-188 had similar occupancy measured

with both an agonist and an antagonist radioligand, confirming in vitro studies

that RGH-188 is an antagonist.

39

6 FUTURE PROSPECTS Future prospects for NET radioligands: (S,S)-[18F]FMeNER-D2 has the

potential to be a useful radioligand for quantitative imaging of NET in brain.

Nevertheless, a radioligand with a higher signal to noise ratio and lower non specific

binding would be advantageous. NET radioligands have more challenges than other

transporter radioligands such as the dopamine or serotonin transporter. A high basal

occupancy of NET by norepinephrine and other endogenous monoamines such as

serotonin and dopamine may compete for the same binding site as the radioligand. A

future study using a reserpine depletion paradigm (depletes not only dopamine

concentrations, but norepinephrine) can be performed to determine the occupancy of

NET by endogenous monoamines. New radioligands with higher affinity for NET

may help improve the signal since sparse densities of NET in brain compared to other

monoamine transporters. The use of a higher resolution PET camera may help

delineate smaller brain regions such as the caudate for the reference region and

regions known to contain high densities of NET (e.g. the locus coeruleus).

Future prospects with pharmacological challenges: Based on the results from

paper III and IV that [11C]MNPA was more sensitive to changes in endogenous

dopamine than antagonist radioligands, the findings should be confirmed in healthy

subjects and patients suffering from altered dopamine neurotransmission.

Future prospects with receptor occupancy studies: PET studies employing dual

agonist and antagonist radioligands may lead to a novel approach to further understand

the in vivo mechanisms of action of antipsychotic drugs. The data provided in paper V

should be expanded in both animal and human studies to determine if one can assess

the intrinsic activity of a drug. Studies employing, antagonists, partial agonists and

agonists would help lead further support into the notion that PET imaging using dual

radioligands can determine intrinsic activity.

40

7 ACKNOWLEDGEMENTS I would like to express my sincere gratitude and appreciation to everyone who has contributed to this work. Especially I would like to thank: Prof. Christer Halldin, my KI mentor, for his optimism, endless enthusiasm and his keen eye for developing new radioligands. Dr. Robert B. Innis, my NIH mentor, for accepting and supporting me as a NIH/KI student in the graduate partnership program, and for teaching me how to be precise in scientific thinking, studies and writing. Prof. Lars Farde, for teaching me that too few words is much better than too many. So I will keep it short!! National Institute of Radiological Sciences (NIRS) in Chiba, Japan. I wanted to thank Dr. Tetsuya Suhara for accepting the proposed study and allowing me the opportunity to perform PET studies at NIRS. Drs. Tokunaga and Maeda for being gracious hosts and for making my stay fruitful and enlightening. PET imaging group at Karolinska Institute, Dr. Bengt Andree for making my transition to Sweden and KI PET group as smooth as possible, Dr. Per Karlsson for his discussions about sports and PET imaging, Kjerstin Lind for making the workplace feel like home during the holiday season, Dr. Andrea Varrone for his friendship and valuable insight into data analysis, Julio Gabriel for reconstructing the PET data as quickly as possible! Nils Sjoholm for making sure the PET camera is always working and Dr. Stefan Pauli for his volume rendering images. Jacqueline Borg, Drs. Judit Sóvágó, Zsolt Cselenyi, Johan Lundberg and Simon Cervenka for many interesting discussions about research and life in general. The members of the PET radiochemistry group at Karolinska, for what seems like a 99% success track record on radioligand synthesis, and endless commitment to producing the highest specific activity radiotracers. I appreciated their help and collaboration: Magnus Schou, Anu Airaksinen, Phong Truong, Arsalan Amir, and Jan Andersson. Assoc. Prof. Balázs Gulyás – For all of your help with the monkey PET experiments, teaching me how to handle and work with nonhuman primates, and fruitful discussions about life and science. My office-mate, Sjoerd Finnema, fellow student and friend, the many hours, no years of working together to figure out the intricacies of PET imaging, no stone should be left unturned. Ulla-Kajsa Pehrsson and Karin Zahir – for valuable Swedish translation and helping with the many administrative issues that have arisen over the years.

41

42

Thanks to all the members of the radiochemistry and PET imaging group at NIMH. PET imaging group at the molecular imaging branch (NIMH/NIH), Dr. Jeih-San Liow for his help with microPET studies and for his friendship, Garth Terry for his assistance during the microPET studies, and Dr. Sami Zoghbi for performing the radiometabolite analysis and for his attention to detail. My family and friends – For never giving up hope and always being there when I needed them! Zoya, for your love and endless support, and for the great life we have together.

The National Institutes of Health and Karonlinska Institutet for financial support.

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