Research Collection

Doctoral Thesis

Development of novel ligands for PET imaging of the metabotropic subtype 5 (mGluR5)

Author(s): Kessler, Lea Janine

Publication Date: 2004

Permanent Link: https://doi.org/10.3929/ethz-a-004842638

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ETH Library Diss. ETH No.: 15633

Development of Novel Ligands for PET Imaging of

the Metabotropic Glutamate Receptor Subtype 5

(mGluR5)

A thesis submitted to the Swiss Federal Institute of Technology Zurich for the degree of Doctor ofNatural Sciences

presented by

Lea Janine Kessler

Eidg. Dipl. Apothekerin

born August 14th, 1974 citizen of Zurich, Switzerland

accepted on the recommendation of

Prof. Dr. P.A. Schubiger, examiner Prof. Dr. G. Folkers, co-examiner PD Dr. S.M. Ametamey, co-examiner

2004

Diss. ETH No.: 15633

Development of Novel Ligands for PET Imaging of

the Metabotropic Glutamate Receptor Subtype 5

(mGluR5)

A thesis submitted to the Swiss Federal Institute of Technology Zurich for the degree of Doctor ofNatural Sciences

presented by

Lea Janine Kessler

Eidg. Dipl. Apothekerin

born August 14th, 1974 citizen of Zurich, Switzerland

accepted on the recommendation of

Prof. Dr. P.A. Schubiger, examiner Prof. Dr. G. Folkers, co-examiner PD Dr. S.M. Ametamey, co-examiner

2004

Table of Contents I

TABLE OF CONTENTS

TABLE OF CONTENTS I

LIST OF ABBREVIATIONS III

SUMMARY V

ZUSAMMENFASSUNG IX

CHAPTER 1 1

Introduction

1.1 Glutamate Receptors 3 1.2 Positron Emission Tomography 11 1.3. Aim of the Thesis 18 1.4. References 19

CHAPTER 2 25

Synthesis, Radiolabelling, in vitro and in vivo Evaluation of [11C]-2-Methyl- 6-(3-Fluoro-Phenylethynyl)-Pyridine as Radioligand for the Metabotropic Glutamate Receptor Subtype 5 (mGluR5)

2.1 Abstract 27 2.2 Introduction 28 2.3 Chemistry 29 2.4 Pharmacology 33 2.5 Conclusion 37 2.6 References and Notes 38

CHAPTER 3 41

Synthesis, Radiolabelling, in vitro and in vivo Evaluation of [11C]-ABP688 as Radioligand for the Metabotropic Glutamate Receptor Subtype 5 (mGluR5)

3.1 Abstract 43 3.2 Introduction 44 3.3 Materials and Methods 46 3.4 Results 52 3.5 Discussion 61 3.6 References 64 II Table of Contents

CHAPTER 4 67

Synthesis and Pharmacological Evaluation of [18 F]-Fluoroethyl-ABP688 and [18F]-Fluoromethyl-ABP688 as Radioligands for the Metabotropic Glutamate Receptor Subtype 5 (mGluR5)

4.1 Abstract 69 4.2 Introduction 70 4.3 Materials and Methods 72 4.4 Results and Discussion 77

4.5 Conclusion 83 4.6 References 84

CHAPTER 5 87

Conclusion and Future Directions

PUBLICATIONS AND PRESENTATIONS 91

CURRICULUM VITAE 93

HERZLICHEN DANK 95 of Abbreviations

OF ABBREVIATIONS

ABP688 3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2- enone <>methyl-oxime AM PA 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)- propionic acid BBB blood brain barrier Bq Becquerel BSA bovine serum albumin BuLi butyl lithium CAMP cyclic adenosine monophosphate Ci curie CNS central nervous system d deuteron DAG diacylglycerol DMF dimethylformamide DMSO dimethylsulfoxide EOB end of bombardment EOS end of synthesis Et3N triethylamine G PCR G-protein coupled receptor h hour(s) HEPES 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid

H PLC high pressure liquid chromatography i.v. intravenous IC50 inhibition constant required for displacement of 50% of radioligand binding ID injected dose KD dissociation constant LTP long term potentiation Mel methyl iodide M-FPEP 2-methyl-6-(3-fluoro-phenylethynyl)-pyridine mGluR metabotropic glutamate receptor mGluR5 metabotropic glutamate receptor subtype 5 min minute(s) M-MPEP 2-methyl-6-((methoxyphenyl)ethynyl)-pyridine MPEP 6-methyl-2-(phenylethynyl)-pyridine MS mass spectrometry n neutron NMDA N-methyl-D-aspartate NM DAR N-methyl-D-aspartate receptor NMR nuclear magnetic resonance P proton p.i. post injection PET positron emission tomography PI phosphoinositol IV List of Abbreviations

PLC phospholipase C ROI region of interest SUV standard uptake value TAC time activity curve THF tetrahydrofurane Summary V

SUMMARY

Positron emission tomography (PET) is the only non-invasive imaging technique offering the possibility to visualise and quantify brain receptors in vivo. PET is therefore a promising tool for studying neuroreceptors under both physiological and

pathophysiological conditions. Although the PET technology is well advanced, the

imaging of many neurotransmitter receptors including glutamate receptors is limited

by the lack of suitable radiotracers. The metabotropic glutamate receptor subtype 5

(mGluR5) is of special interest since it has been implicated in a variety of diseases in the central nervous system including mood disorders, schizophrenia, Parkinson's disease but also chronic and inflammatory pain. Prompted by the need to develop

PET radiotracers for the neurotransmission system, four high-affinity and selective compounds were synthesised, radiolabelled and pharmacologically characterised as prospective agents for imaging mGluR5 by PET.

Recently, M PEP and its methyl analogue M-MPEP were identified as potent and highly selective non-competitive antagonists for mGluR5 and their structural modification

led to 2-methyl-6-(3-fluoro-phenylethynyl)-pyridine (M-FPEP). The synthesis,

radiolabelling and in vitro ana in vivo evaluation of [nC]-M-FPEP are described in the first part of this thesis. The syntheses of M-FPEP and its desmethyl-precursor were accomplished in a three-step reaction sequence. The radiolabelling of M-FPEP with carbon-11 was successfully achieved by reacting the lithium salt of the desmethyl-

precursor with [nC]-MeI in appropriate radiochemical yields (10%) and high specific activities (90-120 GBq/umol). [nC]-M-FPEP exhibited high in vitro affinity for mGluR5

(KD = 1.4 ± 0.1 nM) determined by Scatchard analysis, adequate lipophilicity (logD =

2.7) for good blood-brain barrier penetration and high in vitro plasma stability.

Metabolite studies of rat brain homogenates also revealed high stability of the

radioligand in vivo. Despite these promising properties, classical biodistribution studies as well as PET experiments using the quad-HIDAC small animal tomograph demonstrated a low and homogeneous accumulation of [nC]-M-FPEP in rat brain.

Blockade studies indicated that [nC]-M-FPEP binding in rat brain was non-specific.

[nC]-M-FPEP thus represents a compound in a series of MPEP derivatives, which failed to exhibit good in vivo characteristics for PET imaging of the mGluR5. We VI Summary

therefore reasoned that for our development work on mGluR5 PET ligands of a different class of compounds might be more suitable.

The second part of this thesis describes one such compound, 3-(6-methyl-pyridin-2- ylethynyl)-cyclohex-2-enone <>methyl-oxime (ABP688), obtained in a four-step

reaction sequence. ABP688 was radiolabelled with carbon-11 by O-methylation of desmethyl-ABP688 and gave [nC]-ABP688 in good radiochemical yields (30-40%) and high specific activities (100-200 GBq/umol). In vitro saturation assays revealed a

high in vitro binding affinity of [nC]-ABP688 for mGluR5 (KD = 1.7 ± 0.2 nM). The

lipophilicity (logD = 2.4) and high in vitro plasma stability of [nC]-ABP688 were

promising properties for a prospective PET ligand. Moreover, in vivo radioactive

metabolites that might enter the brain and confound the radioactivity signals were

not identified in rat brain extraction experiments. PET imaging and classical

biodistribution studies in rats demonstrated significant radioactivity accumulation in

mGluR5-rich brain regions such as hippocampus and striatum. In the cerebellum, a

region known for negligible mGluR5 expression, very little radioactivity uptake was

noted. In the receptor-rich regions the co-injection of [nC]-ABP688 and M-MPEP

resulted in a blocking effect of up to 80%, whereas in the cerebellum no change in

radioactivity uptake was observed. The comparison of mGluR5 wt- and ko-mice elegantly confirmed the specificity of [nC]-ABP688 binding in vivo. Furthermore, ex

vivo autoradiography exhibited high resolution images which clearly revealed high activity uptake in receptor-rich brain regions and subregions such as dentate gyrus and stratum radiatum. As expected, autoradiographic sections of a mGluR5 ko-

mouse showed a homogeneous distribution of the tracer throughout the brain. [nC]-

ABP688 thus represents the first PET radioligand for imaging mGluR5 in vivo.

The excellent in vivo results obtained with [nC]-ABP688 encouraged us to prepare fluoro-derivatives of ABP688 as fluorine-18 possesses better imaging characteristics and a longer half-life. Since ABP688 is not directly amenable to fluorination, fluoroethyl-ABP688 and fluoromethyl-ABP688 were generated. These two fluoro- derivatives were prepared from desmethyl-ABP688. The radiolabelling of fluoroethyl-

ABP688 with fluorine-18 was accomplished in a two-step reaction sequence whereas for fluoromethyl-ABP688 a three-step reaction sequence was used. Both derivatives were obtained in 5-10% radiochemical yield and specific activities ranged from 35 to Summary VII

55 GBq/umol. Both derivatives revealed high in vitro binding affinity, adequate lipophilicity and high plasma stability. Despite these promising in vitro characteristics both radioligand failed in vivo, partly due to low brain uptake and partly due to high non-specific binding.

In conclusion, of all four compounds synthesised and pharmacologically characterised, only [nC]-ABP688 exhibited excellent in vivo properties suitable for further evaluation in humans. This new ligand represents the first ever selective PET ligand that has been developed for the imaging of mGluR5 and will soon be tested in healthy volunteers. If [nC]-ABP688 shows in vivo specificity in humans, it will represent an invaluable tool to elucidate the function of mGluR5 in various physiological, pathophysiological or pharmacological conditions.

Zusammenfassung IX

ZUSAMMENFASSUNG

Die Positronen-Emissions-Tomographie (PET) erlaubt als einziges nicht-invasives bildgebendes Verfahren die Möglichkeit, Hirnrezeptoren in vivo darzustellen, zu quantifizieren und ihre Physiologie und Pathophysiologie zu analysieren. Obwohl die

PET-Technologie schon weit fortgeschritten ist, existieren noch keine PET-Liganden für Neurotransmitter-Rezeptoren wie zum Beispiel die glutamatergen Rezeptoren.

Der Subtyp 5 der metabotropen Glutamatrezeptoren (mGluR5) ist von besonderem

Interesse, da er an der Pathophysiologie verschiedener Krankheiten des Zentralen

Nervensystems beteiligt ist. Dazu gehören depressive Erkrankungen, Schizophrenie,

Morbus Parkinson, aber auch chronische und entzündliche Schmerzen. Diese Arbeit umfasst die Synthese und pharmakologische Charakterisierung von hochaffinen und selektiven PET-Liganden für eine Visualisierung und Analyse des mGluR5 in vivo.

Mit MPEP und seinem Methyl-Ana logon M-MPEP wurden unlängst die ersten selektiven, nicht-kompetitiven Antagonisten für den mGluR5 entwickelt. Strukturelle

Derivatisierung führte zu 2-Methyl-6-(3-Fluoro-Phenylethynyl)-Pyridin (M-FPEP). Die

Synthese, Radiomarkierung und Charakterisierung in vitro und in vivo von M-FPEP sind im ersten Teil dieser Arbeit beschrieben. M-FPEP und der entsprechende

Desmethyl-Vorläufer wurden mittels einer dreistufigen Synthese erhalten. Die

Radiomarkierung von M-FPEP erfolgte durch Reaktion von [nC]-MeI mit dem

Lithiumsalz des Desmethyl-Vorläufers. Dabei wurden eine radiochemische Ausbeute von 10% und eine hohe spezifische Aktivität (90-120 GBq/umol) erreicht.

Sättigungsstudien ergaben eine hohe in vitro Affinität von [nC]-M-FPEP für mGluR5

(KD = 1.4 ± 0.1 nM). Ebenso konnte eine ausreichende Lipophilie (logD = 2.7) für ein Passieren der Bluthirnschranke sowie eine hohe in vitro Plasmastabilität von

[nC]-M-FPEP nachgewiesen werden. Metaboliten-Analysen von Ratten h irnextrakten ergaben eine hohe Stabilität des Radioliganden in vivo. Entgegen dieser vielverspechenden Resultate zeigten klassische Biodistributions-Studien und PET-

Experimente mit dem Kleintier-Tomographen quad-HIDAC nur eine geringe

Aufnahme und homogene Verteilung des Radioliganden im Hirn. Zudem zeigte [nC]-

M-FPEP in Blockade-Studien mit co-injiziertem M-MPEP keine spezifische Bindung. M-

FPEP ist somit ein weiteres MPEP-Derivat, welches keine geeigneten in vivo X Zusammenfassung

Eigenschaften für einen PET-Liganden aufweist. Daher haben wir uns entschieden, die Entwicklung neuer mGluR5 PET-Liganden mit einer neuen Gruppe von MPEP-

Derivaten fortzusetzen.

Im zweiten Teil dieser Arbeit wurde ein solches Derivat untersucht. 3-(6-Methyl-

Pyridin-2-ylethynyl)-Cyclohex-2-enon <>Methyl-Oxim (ABP688) wie auch sein

Desmethyl-Vorläufer wurden in einer vierstufigen Synthese hergestellt. Ausgehend von Desmethyl-APB688 erfolgte die Radiomarkierung von ABP688 mit Kohlenstoff-11 via O-Methylierung, was eine sehr gute radiochemische Ausbeute (30-40%) sowie eine hohe spezifische Aktivität (100-200 GBq/umol) lieferte. In vitro

Sättigungsstudien zeigten eine hohe Affinität von [nC]-ABP688 für mGluR5 (KD = 1.7

± 0.2 nM). Die Bestimmung der Lipophilie und Plasmastabilität von [nC]-ABP688 ergaben günstige Voraussetzungen für eine Eignung dieser Substanz als PET-Tracer.

Durch Metabolitenanalysen von Rattenhirnextrakten konnte ausgeschlossen werden, dass die Bestimmung der Radioaktivitätskonzentration im Hirn durch die Bildung von

radioaktiven Metaboliten verfälscht wird. PET-Aufnahmen und Biodistributions-

Studien in Ratten zeigten eine deutliche Radioaktivitätsaufnahme in Hirnregionen mit einer hohen mGluR5-Dichte, z.B. Hippocampus und Striatum. Co-Injektion des

Radioliganden mit M-MPEP reduzierte die Radioaktivitätsaufnahme in diesen

Regionen um bis zu 80%, während für das Cerebellum, einer Region mit kleiner

mGluR5-Dichte, keine Blockade nachgewiesen werden konnte. Ein Vergleich von

mGluR5-Wildtyp und -Knockout Mäusen lieferte einen weiteren Nachweis, dass [nC]-

APB688 in vivo spezifisch an die mGluR5 bindet. Ebenso konnte in ex vivo

Autoradiographien eine Radioaktivitätsanreicherung in den mGluR5-reichen Regionen

und Subregionen, wie z.B. Gyrus dentatus und Stratum radiatum, visualisiert werden.

Wie erwartet, zeigte [nC]-ABP688 in der Autoradiographie einer mGluR5 Knockout

Maus eine homogene Verteilung. [nC]-ABP688 bietet somit als erster Radioligand geeignete Eigenschaften, um mGluR5 mittels PET darzustellen.

Der erfolgreiche Einsatz von [nC]-ABP688 gab Anlass, Fluor-18-Derivate von ABP688 zu entwickeln, da Fluor-18 bessere bildgebende Eigenschaften und eine deutlich

längere Halbwertszeit besitzt. Da ABP688 jedoch keine direkte Fluorierung erlaubt, wurden zunächst die beiden Derivate Fluoroethyl-ABP688 und Fluoromethyl-ABP688 entwickelt. Diese konnten ausgehend von Desmethyl-ABP688 hergestellt werden. Bei Zusammenfassung XI der Radiomarkierung mit Fluor-18 wurde in einer zweistufige Synthesesequenz [ F]-

Fluoroethyl-ABP688, in einer dreistufigen Synthesesequenz [18F]-Fluoromethyl-

ABP688 erhalten. Beide Radiomarkierungen lieferten angemessene radioaktive

Ausbeuten (5-10%) und spezifische Aktivitäten (35-55 GBq/umol). In vitro zeigten diese beiden Derivate viel versprechende Eigenschaften wie nanomlare Affinität für mGluR5, angemessene Lipophilie, sowie eine gute in vitro Plasmastabilität. Hingegen zeigten beide Radioliganden in vivo keine geeigneten Eigenschaften, teils aufgrund zu geringer Aufnahme im Hirn, teils aufgrund hoher unspezifischer Bindung.

Von diesen vier PET-Tracern, die synthetisiert und pharmakologisch charakterisiert wurden, bewies nur [nC]-ABP688 hervorragende in vivo Eigenschaften für eine

Weiterentwicklung im Menschen. Diese Verbindung stellt den allerersten selektiven

PET-Tracer für mGluR5 dar und wird demnächst in gesunden Probanden getestet.

Sollte [nC]-ABP688 gute Eigenschaften im Menschen zeigen, bietet sich dieser PET-

Tracer für die Analyse der Physiologie, Pathophysiologie und Pharmakologie dieses

Rezeptors an.

1

Introduction

Chapter 1 3

1.1 GLUTAMATE RECEPTORS

The amino acid glutamate is known as the main excitatory neurotransmitter in the

mammalian brain. Released from synapses, it binds to neurons, and thereby activates cell surface receptors. These receptors are characterised as either

ionotropic or metabotropic glutamate receptors.

1.1.1 IONOTROPIC GLUTAMATE RECEPTORS

Ionotropic glutamate receptors (iGluRs) are the principle mediators of excitatory

neurotransmission. These are ligand-gated cationic-selective channels permeable to

Na+, K+ and Ca2+. The ionotropic receptor family is subdivided into three major classes named according to their selective agonists which structurally resemble glutamte but do not occur naturally in the brain: a-amino-3-hydroxyl-5-methyl-

isoxazol-4-proprionate (AMPA), N-methyl-D-aspartate (NMDA) and kainate (KA) 13.

AM PA receptors are found in most excitatory synapses and mediate the majority of fast excitatory neurotransmission 1/2. The role of kainate receptors in excitatory

neurotransmission is less clear. They are thought to specifically activate

presynaptically the release of the inhibitory neurotransmitter GABA, whereas

postsynaptic I ly their activation leads to excitatory synaptic currents 4'5. NMDA

receptor activation depends on the coincidence of presynaptic activity (release of glutamate, presence of the co-antagonist glycine) and postsynaptic activity

(membrane depolarisation by AMPA/kainite or other excitatory inputs) 6.

Overstimulation of these receptors by glutamate is thought to be a major mechanism for Ca2+ overload in neurons, which mediates neuronal injury and death 7"9. These diseases range from acute neurological disorders such as stroke, trauma and epilepsy to more chronic neurodegenerative states such as Parkinson's and

Alzheimer's disease 10. 4 Chapter 1

1.1.2 METABOTROPIC GLUTAMATE RECEPTORS

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCR), which activate intracellular secondary messenger systems when bound by the

physiological ligand glutamate n. Activation of mGluRs results in modulating and fine-tuning effects on ion channel function via ionotropic glutamate receptors, synaptic plasticity and excitotoxicity.

To date, the mGluRs consist of eight receptor subtypes, which are subdivided into three groups, based on sequence homology, signal transduction pathways and

pharmacological properties (Table 1). Group I (mGluRl and mGluR5) mGluRs are

positively coupled to phospholipase C (PLC), thus stimulating diacylglycerol (DAG) formation and phosphoinositide (PI) turnover, resulting in an intracellular Ca2+ signalling. Group I mGluRs modulate glutamate excitability primarily via postsynaptic

mechanisms 12. Group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6,

mGluR7 and mGluR8) mGluRs inhibit adenylate cyclase (AC) and hence the cyclic adenosine monophosphate (cAMP) production 13,14. Both the group II and group III

mGluRs have been implicated in the modulation of glutamate transmission by

predominantly presynaptic mechanisms.

Group Subtype Transduction Agonist Antagonist

I mGluRl PLC, 3,5-DHPG CPCCOEt mGluR5 positively coupled CHPG M PEP

II mGluR2 AC, (2R, 4R)-APDC MCCG-I mGluR3 negatively coupled LY341495

III mGluR4 (R,S)-PPG CPPG mGluR6 AC, \\ \\ mGluR7 negatively coupled \\ \\ mGluR8

Table 1: Pharmacological classification of the members of the mGluR family 15.

The structure of mGluRs (Figure 1) is characterised by two distinctly separated topological domains: an exceptionally long extracellular hydrophilic N-terminal domain and a variable intracellular C-terminal domain. The extracellular domain is contributing to glutamate binding, agonist activation of the receptor and subtype specificity for group selective agonists 16~18. It is composed of two globular domains Chapter 1 5

with a hinge region, which represent the binding domain of glutamate. After an initial

binding to one domain the hinge is proposed to fold and trap the amino acid in a

pocket.

Extracellular and intracellular domains are connected by seven closely located

hydrophobic segments, predicted to form membrane-spanning segments, which are

19 termed the seven transmembrane domain (7TMD). Pin et al. have shown that the second intracellular loop (i2) and the amino portion of the carboxy-terminal tail of the intracellular loop 4 (i4) contribute to G-protein coupling. However, the first and the third intracellular loop are discussed to play an important role in G-protein activation. The role of the intracellular C-domain is not yet known. Some results suggest that it might be involved in the transduction mechanism of these receptors.

The presence of numerous phosphorylation sites (threonine, serine residues) at the

C-terminus also suggests a target for several types of kinases that regulate receptor activity. In all mGluR subtypes, 21 cysteine residues are conserved, 19 of them are

located in the extracellular region, suggesting that the rigid three dimensional conformation of the extracellular region is important for mGluR function.

glutamate binding site MPEP binding site

N-terminal domain with hinge region extracellular region

cystein rich domain

7TMD

G-protein coupling

intracellular region C-terminal domain

Figure 1: Schematic structure of the mGluRs with the conserved binding site of glutamate and the non-competitive binding site of mGluR5 antagonists within the 7TMD.

The mGluRs are widely expressed throughout the central nervous system (CNS).

Each subtype is specifically distributed in certain brain regions. The two mGluRs of 6 Chapter 1

group I have somewhat complementary regional expression patterns. While mGluRl

is highly expressed in the cerebellum, ventral pallidum and substantia nigra 20,21.

mGluR5 in contrast, shows little expression in these regions but displays significantly

higher levels of expression in striatum and cortex and is furthermore widely expressed in the hippocampus, 11-22-23 where it is located at the levels of dendrites and cell bodies of neurons. Group II mGluRs are primarily distributed in the forebrain

region and group III mGluRs are widely expressed throughout the CNS, except

mGluR6, which is essentially expressed in the retina.

1.1.3 GROUP I MGLURS

Function and Pathophysiology

Group I mGluRs stimulate phospholipase C as revealed by an increase in

phosphoinositide turnover and Ca2+ release from internal stores. In addition, agonists of group I mGluRs have been shown to inhibit Ca2+ channels and to regulate K+ channels. Regulation of ionotropic glutamate receptors by mGluRs has also been observed. These transduction mechanisms mediate a variety of pharmacological effects, which finally result in increased neuronal excitability and modulation of

14'15'24' synaptic transmission featuring the induction of long-term potentiation (LTP) which has been suggested being the basis for the formation of memory and learning.

Since the ubiquitous distribution of glutamatergic synapses, mGluRs have the

potential to participate in a wide variety of functions of the CNS and offer an

interesting target for modulation of pathological functions evoking several

neurological and neurodegenerative disease states. Thus, involvement in glutamate-

25,26 induced neuronal damages, like ischemia or epilepsy has been proven, and due to the import role these receptors play in motor circuits, they are assumed to be

involved in movement disorders such as Parkinson's disease 27.

Group I mGluR subtype 5 is highly expressed in the limbic system suggesting the

participation in behavioural and emotional processes of brain function. In fact, administration of selective mGluR5 antagonists was effective in animal models of

mood disorders, such as anxiety and depression, in a benzodiazepine-like manner28. Chapter 1 7

Further, mGluR5 shows modulating, yet activating effects on NMDA receptors

(NMDARS). Hypofunctional NMDA receptor activity, together with high mGluR5 expression was observed in schizophrenic brains 29~32. Whereas increased glutamate concentrations in the nucleus accumbens, a region with high mGluR5 expression, is observed for acute and repeated administration. MGIuR5-knock-out mice

lacked the reinforcing properties of cocaine and hence highly suggested involvement of the mGluR5 in drug addiction and drug abuse 33.

As mGluR5 is widely expressed throughout the central and peripheral nervous system, it also occurs in the spinal cord. There, it appears in the dorsal horn, an area which contains the spinal cord neurons which are responsible for the processing of

nociceptive transmission. These central and peripheral localisations of the mGluRs suggest a significant role in nociception. Systemic administration of mGluR5 antagonists was effective against acute, chronic and persistent pain, inflammatory

hyperalgesia and neurophathic pain. Common side effects like locomotor disturbances or acute gastric toxicity as known with traditional therapies for chronic

pain with either opioids or non-steroidal anti-inflammatory drugs were not observed

34-37

For mGluRl several studies have shown that overstimulation of this receptor subtype

is suggested implicating in several neurological and psychiatric disorders such as

ischemia, epilepsy, anxiety and pain. Due to the complementary distribution of

mGluR5 and mGluRl, their involvements in the above mentioned disorders are suggested at different levels of brain functions.

Pharmacology

Since the discovery of the mGluRs and their subtype specific potential in different disease states, excessive work has been undertaken in the development of competitive and subtype specific agonist and antagonist (for review see Conn, 2003

38 or Schoepp et ai, 199939). First synthetic ligands for mGluRs derived from amino acids like glutamate. For group I mGluRs novel subtype-selective compounds, structurally unrelated to amino acids emerged only recently. These compounds bind to a modulatory site of group I mGluRs and act in a non-competitive manner and therefore do not affect binding of glutamate. For mGluRl, chromen-1-carboxylethyl 8 Chapter 1 derivative CPCCOEt is the first non-amino acid-like selective antagonist (Table 1), acting with the seventh extracellular loop of the receptor. For mGluR5, 6-ethyl-2-

(phenylazo)-pyridine-3-ol (SIB-1757), (E)-2-methyl-6-styryl-pyridine-3-ol (SIB-1893) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP), based on a diaryl acetylene

backbone, were the first antagonists described (Figure 2). MPEP is a highly potent,

non-competitive, selective and systemically active antagonist (IC5u = 34 nM) 40.

Binding of MPEP to the mGluR5 occurs within the transmembrane regions III and VII

by interaction with non-conserved residues, Pro-655 and Ser-658 in TMIII and Ala-

810 in TMVII 41. Even though MPEP shows 10000 fold selectivity for mGluR5 versus

mGluRl it possesses only moderate affinity and low water solubility and hence lacks appropriate pharmacokinetic properties for in vivo studies. This led to structural

modifications of MPEP and revealed only recently some new highly affine and selective antagonists 42~45.

SIB-1757 SIB-1893 MPEP

Figure 2: Structures of of SIB-1757, SIB-1893 and MPEP.

Such antagonists, once offering appropriate pharmacokinetics in vivo, may be suitable radioligands, for non-invasive imaging of the mGlu5 receptor by positron emission tomography and may allow new insights into the involvement of the

receptor subtype and offer a better understanding of its role in brain function.

1.1.4 GROUP II MGLURS

One of the most important and earliest breakthroughs in mGlu receptor

pharmacology was the development of a highly selective agonist for the group II Chapter 1 9

mGlu receptors. This compound, termed LY354740, selectively activates mGluR2 and

mGluR3 at nanomolar concentrations that have no effect on any other known

receptor subtypes. Since that time a number of second and third generation agonist of group II mGlu receptors with higher affinities and improved pharmacokinetic

properties were developed. These compounds have provided extremely valuable tools for dissecting the physiological roles of group II mGluRs and the behavioural effects of group II mGluRs activation suggesting a number of therapeutic indications,

including anxiety disorders, schizophrenia, Parkinson's disease and addictive disorders.

1.1.5 GROUP III MGLURS

The molecular pharmacology for group III mGluRs, mGluR4, mGluR6, mGluR7 and

mGluR8, is not as well developed as that for the group I and II receptor classes. This

is partially due to the greater diversity of receptors within this class and partially to the paucity of pharmacological tools developed to study these targets.

Agonists for Group III include L-AP4 and L-SOP. In the last few years, new structurally diverse ligands, more potent and selective, have become available. In

particular, a-methylpropylphosphophenyl glycine and a-cyclopropylphosphophenyl glycine are quite potent and selective antagonists of group III receptors. The

increasing availability of group- and subtype-specific ligands, such as irhomo-AMPA, the first selective mGluR6 agonist, is expected to shed light on the physiological role and the therapeutic opportunities of group III mGluRs. A potential neuroprotective

role of group III receptors has already been postulated 46.

1.1.6 CONCLUSION

The discovery of the multiplicity of mGluRs and their crucial role in the modulation of glutamatergic neurotransmission appears therefore as great hope in the development of highly selective drugs. Such subtype specific ligands will first contribute to a 10 Chapter 1

further understanding of the respective roles of each of these mGluRs and may later

probably lead to the development of new therapeutic agents. Chapter 1 11

1.2 POSITRON EMISSION TOMOGRAPHY

Positron emission tomography is a non-invasive and quantitative imaging technique offering the external detection and recording of the radioactivity distribution of the administered compound radiolabelled with positron emitters in the body. The most commonly used short-lived positron emitting isotopes for radiolabelling of the

molecules of interest (e.g. water, sugar, amino acids, drugs) are carbon-11,

nitrogen-13, oxygen-15 and fluorine-18 47,48. C-ll, N-13 and 0-15 are isotopes which are present in organic molecules and therefore provide radiotracers without effecting changes in the biochemistry or the pharmacology of the drug molecule. F-18

normally substitutes hydrogen which exhibits very similar characteristics. Table 1 shows the formation process, half live, positron range and the maximum energy of some of the most commonly used PET isotopes. Due to the short half-life of these

isotopes, production of radiotracers has to take place on site with a dedicated cyclotron and rapid radiochemistry methods have to be established 49. The resulting

radiotracers show high specific activity ranging from 13.5 up to 200 GBq/nmol which

is 20 to 200 times higher than typical specific activities for tritiated compounds. This

property allows the application of tracer amounts in nanomolar range, thus avoiding any unwanted pharmacological effects. Further, the short half-life results in a low

radiation dose for subjects undergoing PET experiments as most of the activity given decays during experiment. PET can therefore safely be used even in repeated studies

in the same individual. 12 Chapter 1

Radionuclide Nuclear reaction Half-life Range in water Maximum energy

[min] [mm] [MeV] nC 20.4 4.1 0.96

nB(p,n)nC 13N 160(p,a)13N 10.0 5.4 1.72

12C(d,n) 13N 15q 14N(d,n)150 2.1 8.2 1.19

15N(p,n)150* IBp 180(p,n)18F* (F) 109.7 2.4 0.64

180(p,n)18F* (F2)

20Ne(d,a)18F (F2)

Table 2: Formation process, half-life, maximal linear range in water maximum energy of clinically used radionuclides for PET. The most widely used nuclear reactions are shown in bold. *Enriched nuclide as target material.

1.2.1 POSITRON DECAY AND PET PRINICPLE

A positron emitter is a nuclide with an excess of protons. Positron emission occurs when a proton is converted into a neutron according to the following reaction:

NpX - ViY + °!ß+ + V

The excess of energy shared between the positron (ß+) and the neutrino (v), is varying from one nuclide to the other (maximum energy for nC for example: 0.97

MeV, for 18F: 0.64 MeV). The positron is the antiparticle of the electron and has

identical properties, except from its positive charge. After being emitted by beta- decay, the positron loses its kinetic energy by collision in the surrounding matter.

Eventual combination with an electron results in positron annihilation and simultaneous emission of two gamma ray photons at an angle of 180° and each with an energy of 511 keV, corresponding to the resting mass of the electron. Due to their Chapter 1 13

relatively high energy, these photons undergo little interactions in biological tissues and are therefore detectable externally. The PET principle is illustrated in figure 3.

180°

detector 1 detector 2

electronic Ï coincidence

Figure 3: Positron annihilation and the positron emission tomography (PET) coincidence principle.

The use of a coincidence camera for the detection of the gamma ray emission takes advantage of the anti-parallel nature of the two photon directions and records only coincident scintillations. Thereby it identifies a positron annihilation along the line connecting the two detectors. This detection method leads to an improved resolution and sensitivity compared to single photon emission tomography (SPET). However, due to several factors like positron travel before annihilation, angulation of the

photon pair due to residual positron motion and detector size and type, the

resolution of clinical PET scanners are limited to 4 to 6 mm. PET cameras with a smaller diameter particularly well suited for small animal studies reach a resolution of

50-52 1 mm

1.2.2 RADIOLABELLING STRATEGIES

PET radionuclides are the products of nuclear reactions which are induced by the

bombardement of stable isotopes with highly energetic protons or deuterons.

For the production of carbon-11, there are several useful nuclear reactions. The most commonly used is the 14N(p,a)nC-reaction using a nitrogen gas target with a small 14 Chapter 1

amount of oxygen 53. The resulting [nC] is then converted into a primary precursor, a compound either directly produced in the target or the product obtained by a rapid on-line synthesis. Primary precursors are for example [nC]-C02, [nC]-CO, [nC]-HCN or [nC]-CH4 which are rapidly transferred into more reactive secondary precursors in either batchwise or on-line productions. Secondary precursors are for example [nC]-

CH3I, [nC]-RCHO, [nC]-COCI2, [nC]-CH3Li or [nC]-RCH2N02. This wide range of carbon-11 labelled secondary precursors allows the synthesis and labelling of a broad

range of compounds of interest.

For the production of fluorine-18 there are different chemical forms of direct fluorine-

18-production. The commonly used chemical cyclotron reactions are 20Ne(d,a)18F offering [18F]-F2 and 180(p,n)18F resulting in [18F]-F. The major factor to be considered for producing fluorine-18 is the chemical form of the fluorine-18 required

54. Both electrophilic and nucleophilic forms of fluorine-18 are usable for present

radiotracer synthesis. However, the most common strategy for radiolabelling with fluorine-18 remains the nucleophilic approach, which results sufficiently high specific activities (>37 GBq/nmol). Therefore [18F]-F" is synthesised via the proton irradiation of enriched [180]-compounds, mainly [180]-water. [18F]-F" is commonly used in a complex with the aminopolyether kryptofix (K.2.2.2.) and K+. Electrophilic

radiolabelling approaches using the deuteron irradiation of neon gas leading to [18F]-

F2 generally result in very low specific activities (< 0.37 GBq/nmol) because of the carrier-added production. These two radiolabelling strategies, the nucleophilic as well as the electrophilic offer many possibilities for radiolabelling a broad range of compounds.

1.2.3 DEVELOPMENT OF NEW RECEPTOR-BINDING PET

RADIOLIGANDS

The development of new and highly potent ligands requires an extensive pre-clinical characterisation and safety documentation followed by a time- and money- consuming clinical part. Thereby, PET offers a great opportunity to reduce time and costs of drug developmet as it offers rapid information about biodistribution and Chapter 1 15

kinetics of new molecules in vivo in non-invasive experiments 55~58. Today, receptor

radioligands are being developed for various purposes such as functional,

physiological and biochemical information, pathology detection, diagnostic or therapeutic management and drug development.

Although tremendous efforts have been put in developing new radioligands for

neuroreceptor visualisation and neuroreceptor studies, only a handful of suitable tracers exist today. PET radiotracers have to fulfil several requirements (Table 3) and

must possess convenient binding characteristics that are described below.

Criterion Related parameter

In vitro affinity and selectivity KD, K,, IC5o, Km, EC5o

Concentration in target sites Bmax

Reversibility of binding k-i, koff, Ic,

Non-specific binding Log P

Toxicity LD50

Specific activity

Metabolism

Labelling strategy

Table 3: Some important criteria in the development of novel brain radioligands.

In vitro affinity and selectivity: The first two criteria to be considered for selection of a new radioligand are the affinity (KD) and selectivity (K,). The optimum affinity is closely related to the expected Bmax (see below). Ideally, the affinity of a radioligand should be highest for the site of interest by more than one order of magnitude. Lack of selectivity may be acceptable if non-target sites are separated anatomically from the target binding sites.

Concentration in target sites: It is preferable if the binding site concentration (Bmax) clearly exceeds the affinity (KD) of a radioligand in order to give a high binding

potential and to produce high target to non-target ratios. A high ratio gives maximal

response to changes in binding site concentration caused by disease or drug occupancy. 16 Chapter 1

Reversibility of binding: For both, reversible and irreversible radioligands, simple

plotting approaches, Logan and Patlak plot, are available. However, for diagnostic

imaging tracers using short living radionuclides very high binding affinity in combination with a slow clearance from tissue (slowly reversible tracers) may reduce the usefulness of the ligand (flow-limited conditions).

Non-specific binding: Non-specific binding and its clearance in vivo are difficult to

predict with absolute confidence, but within a class of structurally related compounds, non-specific interactions with tissue are generally enhanced with

increased lipophilicity 59. The optimal lipophilicity of a molecule to cross the blood-

brain barrier by simple diffusion is estimated at logP = 1-3 60.

Specific activity: Specific activity defines the relation of unlabelled to labelled compound in the final solution. Too low specific activities may result in

pharmacological or toxic effects of the radiotracer because of a high unlabelled

ligand concentration administered. For receptors with low binding site concentrations

(low Bmax), very high specific activity is required in order to exclude substantial occupation of target sites by unlabelled ligand and the saturation of the biological system of interest.

Toxicity: Toxic effects may occur only if high amounts of the radiotracer with a low specific activity and a low LD5o value are injected. Then, the target site occupation

may reach toxic levels.

Position of labelling: The radioligand should be resistant to rapid metabolism for the acquisition time and radioactive metabolites should not be taken up in the target area. Therefore, the position for radiolabelling within the molecule might be crucial for the usefulness of a radioligand.

When a potential radiotracer has been selected and a radiolabelling technique has

been developed, some preclinical evaluations have to be performed prior to PET in

humans. Characterisation of a new radioligand includes autoradiography,

biodistribution and metabolite studies as well as pretreatment and displacement studies, using in vitro, in vivo and ex vivo techniques. With the increasing ability to carry out PET studies in rodents, using dedicated small animal PET cameras

radioligand evaluation is simplified and forms an important bridge between Chapter 1 17

laboratory and clinical science 61. However, only if a radioligand has demonstrated to

possess suitable characteristics it can finally be applied to humans for PET imaging.

Binding Radioligand Reference Binding Radioligand Reference

system system ["C]-WAY100635 Dopamine [nC]-SCH 23390 62,63 Serotonin 64,65

(DO (5-HT1A)

[uC]-NAD-299 67 [nC]-NNC112 64,66

["C]-MDL100907 Dopamine [nC]-raclopride 68,69 Serotonin 70,71

(D2) (5-HT2A) [18F]-altanserin [nC]-FLB 457 72,73 74,75

[11C]-McN5652 78-80 Dopamine [18F]-ß-CIT-FP 76,77 Serotonin Transporter Transporter 81 [UC]-DASB 82 [18F]-ß-CFT (DAT)

83 [UC]-MADAM 84 [nC]-ß-CPPrT

Table 4: Some brain tracers in use for PET.

During the past two decades, the dopamine and a number of serotonin receptor and transporter systems have been studied extensively. Some of the most common PET

radiotracers for these targets are shown in Table 4. However, for many CNS targets,

no suitable radioligands have been available yet. 18 Chapter 1

1.3. AIM OF THE THESIS

Recent evidence points to the importance of the metabotropic glutamate receptor subtype 5 (mgluR5) in brain function and as a potential therapeutic target for a

number of central nervous system disorders. Non-invasive techniques like positron emission tomography (PET) might offer the possibility to visualise the mGluR5. The visualisation of mGluR5 by PET, however, has been limited by the lack of high-affinity subtype-selective compounds. The aim of this project was to develop novel and

highly selective radioligands that could be used as imaging agents for the glutamatergic neurotransmission by PET. More specifically, the following:

• Radiosynthesis and pharmacological evaluation of [nC]-M-FPEP as PET

imaging agent for mGluR5.

• Synthesis, radiolabelling and biological characterisation of [nC]-ABP688 for

the visualisation of the mGluR5.

• Synthesis and evaluation of the [18F]-fluoromethyl- and [18F]-fluoroethyl-

derivatives of ABP688 as prospective ligands for PET imaging of the mGluR5. Chapter 1 19

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Synthesis, Radiolabelling, in vitro and in vivo Evaluation of

[11C]-2-Methyl-6-(3-Fluoro-Phenylethynyl)-Pyridine as

Radioligand for Imaging the Metabotropic Glutamate

Receptor Subtype 5 (mGluR5)

Lea J. Kessler,1 Michael Honer,1 Mathias Rebsamen,1 Fabrizio Gasparini,2 Yves Auberson,2 P. August Schubiger1 and Simon M. Ametamey1*

1 Center for Radiopharmaceutical Science ofETH, PSI and USZ, 5253 Villigen-PSI, Switzerland 2 Novartis Institute for Biomedical Research, 4002 Basle, Switzerland

Journal of Bioorganic and Medicinal Chemistry Letters

(submitted)

Chapter 2 27

2.1 ABSTRACT

M-FPEP (2-methyl-6-(3-fluoro-phenylethynyl)-pyridine) was evaluated for its potential as PET imaging agent for the metabotropic glutamate receptor subtype 5. M-FPEP was therefore radiolabelled with carbon-11 by reacting [nC]-MeI with the lithium salt of the bromo-pyridine precursor. This uncommon radiolabelling pathway afforded

[nC]-labelled M-FPEP in good radiochemical yields and high specific activities.

Scatchard analysis of [nC]-M-FPEP saturation binding data revealed in a single high- affinity binding site with a KD of 1.4 ± 0.1 nM and a Bmax value of 563 ± 190 fmol/mg

protein. Dynamic PET scanning using the small animal PET camera quad-HIDAC

indicated a rapid uptake of [nC]-M-FPEP in rat brain, followed by a fast clearance.

Classical biodistribution studies in rats showed a homogeneous distribution of [nC]-

M-FPEP in all brain regions examined. Blockade studies by co-injection of [nC]-M-

FPEP with M-MPEP (1 mg/kg) confirmed the lack of specificity of [nC]-M-FPEP

binding for mGluR5 in vivo. 28 Chapter 2

2.2 INTRODUCTION

Metabotropic glutamate receptors (mGluRs) belong to the family of G-protein coupled receptors, which are activated by glutamate, the main excitatory

neurotransmitter in the mammalian nervous system.1 Binding of glutamate results in an activation of intracellular secondary messenger systems. In particular, group I

mGluRs (subtypes 1 and 5) are positively coupled to phosphoinositide/Ca2+ cascade.

Group II mGluRs (subtypes 2 and 3) and group III mGluRs (subtypes 4, 6, 7 and 8) are negatively coupled to adenylate cyclase. An excessive activation of mGluR5 is thought to be implicated in a variety of diseases that affect the nervous system.2 For example an involvement in mood disorders,3,4 schizophrenia5 and movement disorders6 such as Parkinson's disease7 has been reported. Further involvement has

been observed in neuroprotection8,9 as well as drug addiction and drug abuse.10

Recent experiments also revealed the participation of mGluR5 in nociceptive

processes such as chronic pain, inflammatory and neuropathic pain.11,12

The non-invasive imaging technique positron emission tomography (PET) offers the

possibility to visualise the mGluR5 and present an interesting tool for understanding

mGluR5's function under physiological and pathophysiological conditions. To this aim the carbon-11 labelled PET-tracer M-FPEP ([nC]-2-methyl-6-(3-fluoro-

phenylethynyl)-pyridine) was evaluated for its potential as a PET imaging agent. Chapter 2 29

2.3 CHEMISTRY

Recently, the identification of a series of non-competitive mGluR5 antagonists was described by Gasparini and co-workers.13 MPEP (2-methyl-6-(phenylethynyl)- pyridine) (Figure 1) was the first non-competitive and highly selective mGluR5 antagonist with moderate affinity (IC50 = 34 nM) reported. Its methoxy-derivative, 2- methyl-6-((3-methoxyphenyl)ethynyl)-pyridine (M-MPEP), showed equal selectivity but higher affinity in vitro (KD = 2 nM).14 Despite the promising in vitro properties post mortem biodistribution studies revealed a lack of specific binding of [nC]-M-

MPEP for mGluR5 in vivo.15 MPEP and M-MPEP are highly lipophilic compounds and exhibit clogP values of 3.4 and 3.0, respectively. Since high lipophilicity can cause high non-specific binding,16 derivatives with lower lipophilicity were sought. One such a derivative was M-FPEP with a clogP value of 2.8 and a high in vitro affinity (IC50 =

9nM).

Figure 1: Chemical structures of MPEP, M-MPEP and M-FPEP.

The radiolabelling of M-FPEP with carbon-11 was considered at the methyl group of the pyridine ring starting from the stannylated precursor 7. The syntheses of the precursor 7 and M-FPEP (6) were accomplished as shown in scheme 1. Initially, 3- fluorobenzaldehyde (1) and tetrabromomethane were condensed to intermediate 3 which was later reacted with butyllithium to give the acetylenic compound 4 in 45% yield. Compound 4 was finally reacted with 2,6-dibromopyridine to afford the bromo- pyridine precursor 5 in 60% yield. Reacting 4 with 2-bromo-6- methyl pyridine gave

M-FPEP (6) in 60% yield. 30 Chapter 2

Scheme 1: Reagents and conditions: a) Tetra bromometha ne, PPh3, CH2CI2, 0°C, 2 h (85%); b) BuLi (1.6 M), hexane, diethyl ether (1:1), -15°C, 2 h (45%); c) 2-bromo-6-methylpyridine, PdCI2(PPh3)2, Cul, Et3N, DMF, 50°C, 4 h (60%); d) 2,6-dibromopyridine, PdCI2(PPh3)2, Cul, Et3N, DMF, 50°C, 12 h (60%); e) Sn2(Bu)6, Pd(PPh3)4, toluene, reflux, 18 h (30%).

The stannylated precursor 7 was obtained by heating 5 in refluxing toluene using

Pd(PPh3)4 as a catalyst.17,18 Analytical data ^H-NMR, MS) for compounds 5, 6 and 7 were in agreement with the indicated structures.19

Cross-coupling reactions between aryl or vinyl halides with organostannanes, commonly referred to as the Stille reaction, offer a valuable synthetic tool for the

preparation of carbon-carbon bonds. As such, this approach was considered for the

radiosynthesis of [nC]-M-FPEP (Scheme 2). Cross coupling reactions with [nC]-MeI

have proven to be a reliable method for the synthesis of several [nC]-methylphenyl analogues.20

r-"^

11 Sn(Bu) CH

^^ ^^

Scheme 2: Reagents and conditions: a) K2C03, Cul, tri-o-tolylphosphine, Pd2dba3, DMF, room temperature; b) [nC]-MeI, 60°C, 10 min. Chapter 2 31

In our experiment, the stannylated precursor 7 in DMF was added to a DMF solution of K2CO3, Cul, tri-o-tolylphosphine and Pd2dba3 shortly before introducing [nC]-MeI at room temperature. The reaction mixture was then heated to 60°C and allowed to

react for 10 min. Under these conditions no synthetic useful radiochemical yield for

[nC]-M-FPEP was achieved. Neither varying solvents nor changing reaction

parameters such as temperature and reaction time, led to the desired product. The

reaction probably might have been quenched when all reagents were present in the trapping vial.17

A new radiosynthetic strategy was therefore developed and involved the addition of

methyl iodide to the lithium salt of the precursor 5. Even though not commonly used

in radiochemistry, a number of compounds have been successfully radiolabelled

using this approach.21 The radiosynthesis of [nC]-M-FPEP was successfully accomplished in a two-step reactions sequence as shown in scheme 3. The appropriate bromo-pyridine precursor 5 reacted in THF at -85°C with butyllithium for

5 min. [nC]-CH3l was added at -85°C to the lithium salt, then the mixture was warmed to 45°C and allowed to react for 8 min. The product was finally purified by

reversed-phase HPLC. The total synthesis time was 50 min and radiochemical purity was greater than 98%. Radiochemical yield was 10% on average and specific

radioactivity ranged from 90-120 GBq/nmol.

Scheme 3: Reagents and conditions: a) BuLi (1.6 M), THF, -85°C, 5 min; b) (radioactive): [nC]-MeI, 45°C, 8 min (10% radiochemical yield), purification on reversed-phase chromatography, radiochemical purity >98%, specific activity 90-120 GBq/(xmol.

In order to determine the position of label by NMR, the synthetic procedure was

repeated under same radiochemical reaction conditions but with [13C]-CH3l. 13C-NMR 32 Chapter 2

data revealed a single intensive signal at 14.4 ppm which corresponded to the

methyl group in the 2-position of the pyridine ring.22

For [nC]-M-FPEP a logD value of 2.7 ± 0.1 was determined using the shake flask

method as described by Strijckmans et al.23 In vitro plasma stability was determined

by incubating the radioligand in human plasma for 60 min at 37°C. Analytical HPLC analysis demonstrated excellent stability of the tracer under these conditions (data

not shown). Chapter 2 33

2.4 PHARMACOLOGY

[nC]-M-FPEP was characterised pharmacologically both in vitro and in vivo. For the estimation of kinetic parameters and the dissociation constant KD, [nC]-M-FPEP was employed in saturation studies using rat whole brain membranes (without cerebellum). Filtration technique was employed for the separation of bound from free

ligand. Non-specific binding was quantified by co-incubation with 100 \iM M-MPEP and amounted to a maximum of 15% of total binding at a concentration of 8 nM of

[nC]-M-FPEP. Kinetic analysis of [nC]-M-FPEP binding at a concentration of 8 nM

resulted in an association equilibrium that was reached by 12 min (Figure 2A).

Saturation binding assays were performed in a concentration range of 0.5 - 100 nM

[nC]-M-FPEP (Figure 2B) and Scatchard transformation of the binding data resulted

in a single high-affinity binding site of [nC]-M-FPEP with a dissociation constant KD of

1.4 ± 0.1 nM (n = 3). The Bmax value amounted to 563 ± 190 fmol/mg protein

(Figure 2C).

120

10O

80-

60-

40-

20-

04—# 1 2.5 5 10 15 20 30 40

incubation time [mm]

C 0.25

3.5

0.20 3.0

j= 0.15

2.5 a

S 2.0 0.10

1.5 0.05

1.0 0.01 0.0 5 10 15 20 10 20 30 40 [UC]-M-FPEP [nM] bound [pM]

Figure 2: A Association curve of [nC]-M-FPEP binding. Maximum specific binding was set to 100%. Values are means ± SD of triplicate determinations B Saturation curve of [nC]-M-FPEP specific binding, representative example. C Scatchard analysis of [nC]-M-FPEP, representative example. 34 Chapter 2

In vivo biodistribution and pharmacokinetics of [nC]-M-FPEP were determined by a dynamic PET scan using the small animal PET camera quad-HIDAC.24 A rat (Sprague-

Dawley, 317 g) was used for the study and 25 MBq of [nC]-M-FPEP (2 umol) were

injected into the tail vein (Figure 3).

A

.

0 20 40 60 80 100

time p.i. [min]

Figure 3: A Series of coronal sections (ventral to dorsal) through the body of a rat injected with [nC]-M-FPEP. The images were obtained by reconstructing data from 0 to 40 min p.i. B Time activity curve of the rat brain for a 90 min PET scan. Data are expressed as standard uptake value (SUV).

The 90 min PET scan revealed a rather low radioactivity uptake of [nC]-M-FPEP in the rat brain. High activity accumulation was observed in the excretory organs

bladder, kidney, liver and bowel (caecum). The time activity curve showed a rapid clearance of the tracer from the brain. Chapter 2 35

Classical post mortem biodistribution studies were undertaken in order to obtain further information on the regional distribution of [nC]-M-FPEP in distinct brain

regions such as hippocampus, striatum, cortex and cerebellum under both baseline and blockade conditions. The hippocampus, striatum and cortex are regions known to contain high densities of mGluR5 whereas the cerebellum is devoid of mGluR5.25,26

[nC]-M-FPEP was administered into the tail vein of adult male rats (Sprague-Dawley,

350-470 g) and the animals were sacrificed by decapitation 30 min post injection

(p.i.). For the brain regions examined (hippocampus, striatum, cortex, cerebellum)

no significant differences in [nC]-radioactivity uptake were evident. [nC]-

radioactivity in blood was similar to that found in the brain. Furthermore, blockade studies using M-MPEP (1.0 mg/kg body weight) by co-injection with the radioligand confirmed the lack of specific binding of [nC]-M-FPEP to mGluR5 in vivo (Figure 4).

baseline

D blockade

hippocampus striatum cortex cerebellum blood

Figure 4: Biodistribution of [nC]-M-FPEP in the rat brain and in blood. The control group (n = 3) was injected each with PEG/H20 (1/1) and [nC]-M-FPEP, whereas the test group (n = 3) received M-MPEP (1.0 mg/kg body weight) and [UC]-M-FPEP co-injected. Sacrifice time point was 30 min p.i. Data are expressed as percentage of normalised injected dose per gram tissue ± SD.

Radioactive metabolites of [nC]-M-FPEP could interfere with [nC]-M-FPEP binding and detection in the brain. Therefore, in order to verify whether radioactive

metabolites were present in the brain, metabolite studies were performed. After the extraction of rat brain homogenate with heptane, aqueous and organic phases were analysed by analytical HPLC (Figure 5). 36 Chapter 2

100

0s

[11C]-M-FPEP brain

time (min)

Figure 5: HPLC chromatograms of a rat brain extract (30 min p.i) and of parent [UC]-M-FPEP.

More than 95% of the extracted radioactivity was found to be parent compound. No

radioactive metabolites were detected in the brain. These results suggest that the

lack of specific binding of [nC]-M-FPEP observed in rat brain was due to intact

parent compound and not due to radioactive metabolites. Chapter 2 37

2.5 CONCLUSION

[nC]-M-FPEP was successfully synthesised employing an uncommon radiolabelling strategy that yielded good radiochemical yields and high specific activities. In vitro properties such as affinity and lipophilicity suggested [nC]-M-FPEP to be a promising tracer for imaging mGluR5 in vivo. In vivo studies, however, revealed a lack of specific binding. [nC]-M-FPEP is therefore not a suitable candidate for imaging mGluR5 in vivo. Further derivatisation of M-FPEP is ongoing and new derivatives with lower lipophilicities might offer appropriate in vivo properties. 38 Chapter 2

2.6 REFERENCES AND NOTES

1. Pin, J. P.; Duvoisin, R., The metabotropic glutamate receptors: structure and functions. Neuropharmacology 1995, 34, (1), 1-26. 2. Spooren, W. P.; Gasparini, F.; Salt, T. E.; Kuhn, R., Novel allosteric antagonists shed light on mglu(5) receptors and CNS disorders. Trends Pharmacol Sei 2001, 22, (7), 331-7. 3. Tatarczynska, E.; Klodzinska, A.; Chojnacka-Wojcik, E.; Palucha, A.; Gasparini, F.; Kuhn, R.; Pile, A., Potential anxiolytic- and - like effects of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist. Br J Pharmacol 2001, 132, (7), 1423-30. 4. Spooren, W. P.; Vassout, A.; Neijt, H. C; Kuhn, R.; Gasparini, F.; Roux, S.; Porsolt, R. D.; Gentsch, C, Anxiolytic-like effects of the prototypical metabotropic glutamate receptor 5 antagonist 2-methyl-6- (phenylethynyl)pyridine in rodents. J Pharmacol Exp Ther 2000, 295, (3), 1267-75. 5. Ohnuma, T.; Augood, S. J.; Arai, H.; McKenna, P. J.; Emson, P. C, Expression of the human excitatory amino acid transporter 2 and metabotropic glutamate receptors 3 and 5 in the prefrontal cortex from normal individuals and patients with schizophrenia. Brain Res Mol Brain Res 1998, 56, (1-2), 207-17. 6. Kinney, G. G.; Burno, M.; Campbell, U. C; Hernandez, L. M.; Rodriguez, D.; Bristow, L. J.; Conn, P. J., Metabotropic glutamate subtype 5 receptors modulate locomotor activity and sensorimotor gating in rodents. J Pharmacol Exp Ther 2003, 306, (1), 116-23. 7. Rouse, S. T.; Marino, M. J.; Bradley, S. R.; Awad, H.; Wittmann, M.; Conn, P. J., Distribution and roles of metabotropic glutamate receptors in the basal ganglia motor circuit: implications for treatment of Parkinson's disease and related disorders. Pharmacol Ther 2000, 88, (3), 427-35. 8. Spillson, A. B.; Russell, J. W., Metabotropic glutamate receptor regulation of neuronal cell death. Exp Neurol2003,184 Suppl 1, S97-105. 9. Bruno, V.; Ksiazek, L; Battaglia, G.; Lukic, S.; Leonhardt, T.; Sauer, D.; Gasparini, F.; Kuhn, R.; Nicoletti, F.; Flor, P. J., Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective. Neuropharmacology 2000, 39, (12), 2223-30. 10. Chiamulera, C; Epping-Jordan, M. P.; Zocchi, A.; Marcon, C; Cottiny, C; Tacconi, S.; Corsi, M.; Orzi, F.; Conquet, F., Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice. Nat Neurosci 2001, 4, (9), 873-4. 11. Walker, K.; Bowes, M.; Panesar, M.; Davis, A.; Gentry, C; Kesingland, A.; Gasparini, F.; Spooren, W.; Stoehr, N.; Pagano, A.; Flor, P. J.; Vranesic, I.; Lingenhoehl, K.; Johnson, E. C; Varney, M.; Urban, L.; Kuhn, R., Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain. Neuropharmacology 2001, 40, (1), 1-9. Chapter 2 39

12. Sotgiu, M. L.; Bellomi, P.; Biella, G. E., The mGluR5 selective antagonist 6- methyl-2-(phenylethynyl)-pyridine reduces the spinal neuron pain-related activity in mononeuropathic rats. Neurosci Lett 2003, 342, (1-2), 85-8. 13. Gasparini, F.; Andres, H.; Flor, P. J.; Heinrich, M.; Inderbitzin, W.; Lingenhohl, K.; Müller, H.; Munk, V. C; Omilusik, K.; Stierlin, C; Stoehr, N.; Vranesic, I.; Kuhn, R., [(3)H]-M-MPEP, a potent, subtype-selective radioligand for the metabotropic glutamate receptor subtype 5. Bioorg Med Chem Lett 2002, 12, (3), 407-9. 14. Pagano, A.; Ruegg, D.; Litschig, S.; Stoehr, N.; Stierlin, C; Heinrich, M.; Floersheim, P.; Prezeau, L.; Carroll, F.; Pin, J. P.; Cambria, A.; Vranesic, I.; Flor, P. J.; Gasparini, F.; Kuhn, R., The non-competitive antagonists 2- methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]- chromen-la-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. J Biol Chem 2000, 275, (43), 33750-8. 15. Kokic, M. Development of novel ligands for the PET imaging of the ionotropic NMDA receptor and the metabotropic glutamate receptor subtype 5 (mGluR5). ETHZ, Zurich, 2001, No 14450. 16. O'Brien, J. A.; Lemaire, W.; Chen, T. B.; Chang, R. S.; Jacobson, M. A.; Ha, S. N.; Lindsley, C. W.; Schaffhauser, H. J.; Sur, C; Pettibone, D. J.; Conn, P. J.; Williams, D. L., Jr., A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol Pharmacol2003, 64, (3), 731-40. 17. Björkman, M., Synthesis of a nC-labelled Prostaglandin F2a Analogue Using an Improved Method for Stille Reactions with [nC]-Methyl Iodide. J. Labelled Compd. Radiopharm. 2000, 43, 1327-34. 18. Ali, H. v. L., Johan E., Synthesis of Radiopharmaceuticals via Organotin Intermediates. Synthesis-Stuttgart 1996, 4, 423-45. 19. 5: ^-NMR (300 MHz, CDCI3) 5 7.35 (d, 1H), 7.45 (s, 1H), 7.52 (d, 1H), 7.58 (t, 1H), 7.7 (d, 1H), 7.75 (d, 1H), 7.85 (t, 1H); MS (m/z) 275 (M+, 60%) 277 (M++2, 30%). 6: ^-NMR (300 MHz, CDCI3) 5 2.35 (s, 3H), 7.06 (d, 1H), 7.19 (s, 1H), 7.21 (d, 1H), 7.23 (t, 1H), 7.28 (d, 1H), 7.33 (d, 1H), 7.53 (t, 1H); MS (m/z) 211 (M+, 90%). 7: ^-NMR (300 MHz, CDCI3) 5 1.01 (t, 9H), 1.31-1.50 (m, 12H), 6.85 (d, 1H), 7.00 (s, 1H), 7.15 (d, 1H), 7.20 (t, 1H), 7.45 (m, 1H), 7.78 (d, 1H), 7.85 (t, 1H); MS (m/z) 488 (M++l, 100%), 486 (75%), 487 (60%), 484 (50%), 489 (30%), 492 (25%), 490 (15%), 493 (5%). 20. Björkman, M. Palladium-Promoted Synthesis of Compounds Labelled with nC. Uppsala University, Uppsala, 2000, No 564. 21. Oberdorfer, F., Lithiated Intermediates in the Synthesis of nC Labelled Compounds: The Preparation of D,L-a-[nC]methylornithine and D,L-a- [nC]methylvaline. Int J Appl Radiât Isot 1984, 35, (6), 559-61. 22. 13C-NMR, (300 MHz, CD3CN) d 14.4 (s, 1C); MS (m/z) 213 (M++l, 100%), 214 (15%). 23. Strijckmans, V. H., D. H.; Dolle, F.; Coulon, C; Loch, C. et al., Synthesis of a Potential M(l) muscarinic agent [Br-76]bromocaramiphen. J. Labelled Compd. Radiopharm. 1996, 38, 471-81. 40 Chapter 2

24. Honer, M.; Bruhlmeier, M.; Missimer, J.; Schubiger, A. P.; Ametamey, S. M., Dynamic imaging of striatal D2 receptors in mice using quad-HIDAC PET. J Nucl Med 2004, 45, (3), 464-70. 25. Shigemoto R, Kinoshita A, Wada E, Nomura S, Ohishi H, Takada M, Flor PJ, Neki A, Abe T, Nakanishi S, Mizuno N., Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. J Neurosci 1997,17(19):7503-7522. 26. Shigemoto R, Nomura S, Ohishi H, Sugihara H, Nakanishi S, Mizuno N., Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci Lett 1993,163(l):53-57. 3

Synthesis, Radiolabelling, in vitro and in vivo Evaluation of

[11C]-ABP688 as Radioligand for the Metabotropic

Glutamate Receptor Subtype 5 (mGluR5)

Lea J. Kessler1, Michael Honer1, Matthias T. Wyss1, Yves Auberson2, Fabrizio Gasparini2, P. August Schubiger1 and Simon M. Ametamey1

1 Center for Radiopharmaceutical Science ofETH, PSI and USZ, 5253 Villigen-PSI 2 Novartis Institute for Biomedical Research, 4002 Basle, Switzerland

Synapse (prepared for publication)

Chapter 3 43

3.1 ABSTRACT

The pharmacological profile of 3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone O-

[nC]-methyl-oxime ([nC]-ABP688), a non-competitive and highly selective antagonist for mGluR5 was evaluated for its potential as a PET imaging agent for the

metabotropic glutamate receptor subtype 5 (mGluR5). ABP688 was radiolabelled with carbon-11 by reacting [nC]-MeI with the sodium salt of the desmethyl-ABP688 in

DMF. [nC]-ABP688 was obtained in good radiochemical yields (30-40% decay corrected) and high specific activity (100-200 GBq/umol) and exhibited a moderate

lipophilicity with a logD value of 2.4 ± 0.1 nM. Saturation assays of [nC]-ABP688

using rat whole brain membranes, resulted in a single high-affinity binding site with a dissociation constant KD of 1.7 ± 0.2 nM and a Bmax value of 231 ± 18 fmol/mg

protein. Dynamic PET scanning in rats and mice using the quad-HIDAC PET tomograph indicated high radioactivity uptake in hippocampus and striatum, brain

regions known to be rich in mGluR5. Radioactivity uptake in these regions remained at almost the same level for at least 30 min, while radioactivity uptake in the cerebellum, a region with negligible mGluR5 density, was significantly lower.

Biodistribution studies showed similar distribution pattern of [nC]-ABP688 binding in the brain with high radioactivity uptake in brain regions such as hippocampus, striatum and cortex. The hippocampus, striatum and cortex to cerebellum ratios were

5.4 ± 0.1, 6.6 ± 0.1 and 4.6 ± 0.1, respectively. Blocking studies by co-injection of

[nC]-ABP688 and unlabelled M-MPEP (1 mg/kg) revealed up to 80% specific binding

in rat brain. Radioactivity uptake in mGluR5-ko-mice was fairly uniform, substantiating the specificity of [nC]-ABP688 binding to mGluR5. Ex vivo autoradiography clearly revealed the same distribution pattern as determined in

biodistribution studies, while autoradiography of a ko-mouse indicated a

homogeneous and low radioactivity uptake. [nC]-ABP688 represents the first selective tracer for imaging the mGluR5 in vivo in rodents and may offer a future tool for imaging the mGluR5 in humans using PET. 44 Chapter 3

3.2 INTRODUCTION

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors that bind glutamate to exert a modulatory influence on neuronal excitability and synaptic transmission in the central nervous system (Pin and Duvoisin, 1995). To date, eight metabotropic glutamate receptor subtypes have been identified and classified into three groups on the basis of their sequence identity, pharmacology and preferred signal transduction mechanism. Group I mGlu receptors (mGluRs 1 and 5) are coupled to phospholipase C and up/down regulate neuronal excitability (Gereau and

Conn, 1995). Group II (mGluRs 2 and 3) and group III (mGluRs 4, 6, 7 and 8) inhibit adenylate cyclase and hence reduce synaptic transmission.

The mGluR subtype 5 shows widespread distribution throughout the central nervous system (CNS), where it is located at cell bodies and dendrites of neurons. Expression studies suggested highest levels of mGluR5 in hippocampus, moderate levels in caudate-putamen, cerebral cortex, thalamus and low levels in the cerebellar cortex

(Ohnuma et al., 1998); (Blumcke et al., 1996). Excessive activation of the mGluR5 has shown to be implicated in a variety of disorders in the central nervous system

(Spooren et al., 2001) including mood disorders (Spooren et al., 2000);

(Tatarczynska et al., 2001) and schizophrenia (Ohnuma et al., 1998), movement disorders such as Parkinson's disease (Rouse et al., 2000), neuroprotection (Bruno et al., 2000) and drug addiction (Chiamulera et al., 2001). Further, the involvement of mGluR5 in modulation of various pain states including acute, persistent and chronic pain, inflammatory pain and neuropathic pain has been observed (Walker et al.,

2001a; Walker et al., 2001b; Sotgiu et al., 2003).

Recently, the identification of a series of non-competitive mGluR5 antagonists has been described by Gasparini and co-workers (Gasparini et al., 2002). MPEP (2- methyl-6-(phenylethynyl)-pyridine) was the first non-competitive and highly selective mGluR5 antagonist with moderate affinity (IC5o = 34 nM). Its methoxy-derivative, 2- methyl-6-((3-methoxyphenyl)ethynyl)-pyridine (M-MPEP), showed equal selectivity and high affinity in vitro (KD = 3.4 nM) (Pagano et al., 2000). Despite these promising in vitro properties post mortem biodistribution studies revealed a lack of specificity of [nC]-M-MPEP for mGluR5 in vivo (Kokic, 2001). MPEP and M-MPEP are Chapter 3 45 highly lipophilic compounds and exhibit clogP values of 3.4 and 3.0, respectively.

Since high lipophilicity can cause high non-specific binding (O'Brien et al., 2003), derivatives with lower lipophilicity might be more promising. Optimal clogP values have been suggested to be in the range of 2-3 (Waterhouse, 2003). ABP688, a new promising derivative, exhibits a clogP value of 2.4 and an IC5o of 3 nM. These data suggest that ABP688 may be a useful PET ligand if labelled with an appropriate PET isotope.

In this study, we report on the synthesis, radiolabelling, in vitro and in vivo evaluation of [nC]-ABP688. 46 Chapter 3

3.3 MATERIALS AND METHODS

3.3.1 COMPOUNDS

The synthetic approach to desmethyl-ABP688 and ABP688 is shown in scheme 1.

Compound 3 was obtained by reacting 2-bromo-6-methylpyridine (1) and 2-methyl-

3-butin-2-ol (2). Treatment of 3 with sodium hydroxide in refluxing benzene gave compound 4. Reaction of 1,3-cyclohexandione (9) and bromine in the presence of

PPhb and Et3N in benzene at 60°C afforded compound 5 (85%) within 2 h, which

reacted with compound 4 to deliver 6 in 60% yield. Compound 6 was reacted with

hydroxylammoniumchloride to give desmethyl-ABP688 (7) in 70% yield. ABP688 (8) was finally obtained by reacting <>methyl-hydroxyl-amine with 6.

Scheme 1: Reagents and conditions: a) PdCI2(PPh3)2, Cul, Et3N, 55°C, 2 h (80%); b) NaOH, benzene, reflux, 1 h (50%); c) PdCI2(PPh3)2, Cul, DMF/Et3N (3/1), 55°C, 1 h (60%); d) H2NOH, pyridine, room temperature, 17 h (70%); e) H2NOMexHCI, pyridine, room temperature, 24 h (50%).

Analytical data ^H-NMR, MS) for compounds 7 and 8 were in agreement with the

indicated structures. Chapter 3 47

7: ^-NMR (300 MHz, CDCI3): 5 1.87 (t, 2H), 2.49 (q, 2H),. 2.66 (t, 2H),. 2.78 (s, 3H),

6.73 (d, 1H),7.28 (d, 1H), 7.45 (d, 1H), 7.80 (t, 1H); MS (m/z) 249 (M++ Na), 100%;

8: ^-NMR (300 MHz, CDCI3): 5 1.70 (t, 2H), 2.31 (q, 2H), 2.45 (t, 2H),2.49 (s, 3H),

3.84 (s, 3H), 6.49 (s, 1H), 7.02 (d, 1H), 7.20 (d, 1H), 7.47 (t, 1H); MS (m/z) 241 (M+

+ 1), 90%.

3.3.2 RADIOCHEMISTRY

The carbon-11 labelling of ABP688 was achieved by reacting the sodium salt of desmethyl-ABP688 in anhydrous DMF with [nC]-MeI at 120°C for 10 min (Scheme

2). The product was purified by semi-preparative HPLC (u,-Bondapak, C-18, 7.8 x 300

mm, 10 u,m; mobile phase acetonitrile : phosphoric acid 0.1% = 30:70, flow rate 6

mL/min) and the retention time was 10-11 min. After removal of the HPLC solvent by

rotary evaporation, the product was formulated using 0.15 M phosphate buffer, 10%

EtOH and 2% Tween 80 and analysed by analytical HPLC (Bondclone, C-18, 3.9 x

300, 5 urn, mobile phase: acetonitrile : phosphoric acid 0.1% = 30:70, running at 2

mL/min). The identity of the product was confirmed by co-injection of the non¬

radioactive ABP688. After a total synthesis time of 45-50 min, the product had a specific activity of 100-200 GBq/u,mol containing 0.3-1.7 ug/mL of stable ABP688.

rï"^

Nk 1JCH,

Scheme 2: Reagents and conditions: a) NaH, DMF, room temperature, 30 min; b) [nC]-MeI, 120°C, 10 min (30-40% radiochemical yield), purification on reversed phase chromatography, radiochemical purity >95%, specific activity 100-200 GBq/umol.

In order to determine the position of label by NMR, the synthetic procedure was

13, 13, repeated under the same radiochemical reaction conditions but using [ C]-CH3I. C-

NMR data showed a single intensive signal at 61.5 ppm, which corresponds to the O-

methyl-C-atom (MS (m/z) 242 (M++l)). 48 Chapter 3

A logD value of 2.4 ±0.1 for [nC]-ABP688 was obtained using the shake-flask

method as described by Strijckmans and co-workers (Strijckmans, 1996). In vitro

plasma stability was determined by incubating the radioligand in human plasma at

37°C for 60 min and the samples were analysed by HPLC. No degradation products were observed suggesting high in vitro stability of [nC]-ABP688.

3.3.3 PHARMACOLOGY

Saturation Assays

Preparation of membranes: Male rats (adult Wistar, RCC Ltd., Füllinsdorf,

Switzerland) were euthanised by decapitation. Cerebellum was quickly removed and the brain homogenised in 10 volumes of ice-cold (4°C) sucrose buffer (0.32 M sucrose, 10 mM Tris/acetate-buffer, pH 7.4) with a polytron (PT-1200 C, Kinematica

AG, Littau, Switzerland) for 1 min at setting 4. The homogenate was centrifuged at

1000 g for 15 min (4°C) to yield a crude pellet (PI). This pellet was resuspended in 5 volumes of sucrose-buffer, homogenised and centrifuged again at 1000 g for 15 min

(4°C). The resulting supernatants were combined and centrifuged at 17000 g for 20

min (4°C) to yield pellet P2. P2 was washed with ice-cold incubation buffer I (5 mM

Tris/acetate buffer, pH 7.4), homogenised and centrifuged at 17000 g for 20 min

(4°C). The pellet was resuspended in incubation buffer I and stored at -70°C. On the day of the assay, the membranes were thawed and the protein concentration determined by Bio-Rad Microassay with bovine serum albumin as a standard

(Bradford, 1976).

Saturation experiments. 500 ug/mL of whole brain (without cerebellum) rat

membranes were incubated with increasing concentrations of [nC]-ABP688 (0.5-100

nM) in incubation buffer II (30 mM NaHEPES, 110 mM NaCI, 5 mM KCl, 2.5 mM

CaCI2xH20, 1.2 mM MgCI2, pH 8) to give a total volume of 200 uL. Non-specific

binding was determined in the presence of 100 uM M-MPEP. Incubations were allowed to proceed for 45 min at room temperature before being terminated by vacuum filtration over GF/C-filters (Whatman) and pre-soaked for 1 h in incubation Chapter 3 49

buffer II in order to reduce non-specific binding. The membranes retained on the filters were rinsed twice with 4 mL ice-cold incubation-buffer. The radioactivity

retained on the filters was determined using a gamma-counter (Cobra II Auto- gamma, Camberra Packard, Groningen, The Netherlands).

Data analysis. Saturation and Scatchard analysis were performed with the computer

program, Kell-Radlig (McPherson & Biosoft, Cambridge, UK, 1997). Experiments were carried out in triplicates (n=3).

Animals

Animal care and all experimental procedures were approved by the Swiss Federal

Veterinary Office. Animals (male Sprague Dawley rats, 250-450 g; female and male

C59/BL6 wild-type (wt) and mGluR5-knock-out (ko) mice, 25-35 g) were allowed free access to food and water.

Biodistribution studies

Biodistribution studies were performed with rats and mice. A formulated solution of

[nC]-ABP688 was administered into the tail vein of awake animals (rats: 50-450 MBq

injected activity, 0.4-3.5 nmol injected mass; mice: 50-350 MBq, 0.5-2.5 nmol).

Blockade studies were carried out by co-injecting M-MPEP (1.0 mg/kg body weight; 2

mg/mL PEG/H20 1:1) with the radiotracer. The animals were sacrificed by decapitation (rats 30 min post-injection (p.i.); mice 20 min p.i.). The whole brain was

rapidly removed and dissected into specific brain regions: hippocampus, striatum, cortex and cerebellum. Blood, urine, peripheral organs like liver, kidney, muscle and

bone were taken. Each brain region was weighed and tissue radioactivity was

measured in a gamma-counter (Cobra II Auto-gamma, Camberra Packard,

Groningen, The Netherlands). For all brain regions, the tissue distribution was determined as percentage of normalised injected dose per gram wet tissue (% ID

norm/g organ). 50 Chapter 3

PET Studies

PET experiments were performed with the 16-module variant of the quad-HIDAC tomograph (Oxford Positron Systems; West-on-the-Green, UK) (Honer et al., 2004).

The resolution at the centre of field of view was 1.0 mm. The animals (rats, wt- and

ko-mice) were anaesthetised with isoflurane inhalation anaesthesia before injection of the radioligand. [nC]-ABP688 (18-22 MBq, 1-3 nmol) was administered by tail vein

injection. Scan duration was 90 min for rats and 30 min for mice. PET data were acquired in list mode and reconstructed in user-defined time frames using the OPL-

EM algorithm (0.3 mm bin size 120x120x200 matrix size) incorporating resolution

recovery. Image files were evaluated by region-of-interest (ROI) analysis using the dedicated software PMOD (Mikolajczyk et al., 1998). Time activity curves were

normalised to the injected dose per gram body weight and expressed as standardised values (SUV).

Autoradiography

[nC]-ABP688 was injected into the tail vein of a rat (730 MBq, 4.0 nmol), a wt-

mouse (110 MBq, 1.7 nmol) and a ko-mouse (202 MBq, 0.7 nmol). At 8 min p.i. the animals were sacrificed by decapitation. Brains were immediately removed and frozen in isopentane, which was cooled to -70°C. The frozen samples were cut into

20 urn sagittal sections using a cryostate, and without any washing they were placed on a phosphor imager screen for 2 h. The imaging plate data was analysed by BAS

800 II system (Fuji Film).

Metabolite Studies

[nC]-ABP688 (350-600 MBq, 2.5-4 nmol) was administered into the tail vein of awake rats (n = 2, 250-400 g) and the animals were sacrificed by decapitation 30

min p.i. Brain, blood and urine were taken and analysed for metabolites. Analytical

HPLC (Bondclone, C-18, 3.9 x 300, 5 u,m, mobile phase: acetonitrile : phosphoric acid

0.1% = 65:35, flow rate 0.4 mL/min) was used for analysis.

Brain: The rat brain was homogenised with phosphate buffer (pH=7.4; 1 mL), acetonitrile (1.5 mL) was added and the resulting homogenate was centrifuged Chapter 3 51

(4000 rpm, 5 min). The supernatant was analysed by HPLC (conditions as mentioned before).

Blood. Blood samples were centrifuged (4000 rpm, 5 min). Obtained plasma was precipitated with PCA and again centrifuged. The supernatant was analysed by HPLC

(conditions as mentioned before).

Urine. Whole sample was directly analysed by HPLC without further work-up

(conditions as mentioned before). 52 Chapter 3

3.4 RESULTS

3.4.1 SATURATION ASSAYS

For the estimation of dissociation constant (KD) and maximum number of binding sites (Bmax), [nC]-ABP688 was employed in saturation studies. Receptor binding of

[nC]-M-FPEP was found to be saturable (Figure 1A). Scatchard transformation of the saturation binding data resulted in a single high-affinity binding site with a KD of 1.7

± 0.2 nM (n = 3). Bmax value amounted to 231 ± 18 fmol/mg protein (Figure IB).

A B

1.2

1 1 1.0 2: c 0.8 ?8 5 ^D 0.6 3 4 n ^l O 1 1 J2 u 0.4

0.2

i

0 10 20 30 40 50 60 0.2 0.4 0.6 0.8 1.0 1.2

[HCJ-ABP688 [nM] bound [nM]

Figure 1: A Saturation curve of [nC]-ABP688 binding to rat brain membranes. B Scatchard analysis of [nC]-ABP688, representative example.

3.4.2 BIODISTRIBUTION STUDIES

Biodistribution studies in rats

Classical post mortem biodistribution studies were undertaken in order to obtain

information on the regional distribution of [nC]-ABP688 in distinct brain regions such as hippocampus, striatum, cortex and cerebellum under both baseline and blockade conditions. Biodistribution data of [nC]-ABP688 in rats are shown in figure 2. Chapter 3 53

0.28 n

0.24 baseline

D blockade

0.2

0.16

0.12

0.08

0.04 Ü

hippocampus striatum cortex cerebellum blood

Figure 2: Biodistribution of [UC]-ABP688 in rat brain and blood. The control group (n = 3) was injected with PEG/H20 (1/1) and [UC]-ABP688, whereas the test group (n = 3) received M-MPEP (1.0 mg/kg body weight) and [nC]-ABP688. Data are expressed as percentage of normalised injected dose per gram tissue ± SD. Animals were sacrificed 30 min p.i.

Relative high activity accumulation was observed in mGluR5-rich brain regions such as hippocampus, striatum and cortex, while radioactivity uptake in the cerebellum, a

brain region poor in mGluR5, was very low. Using cerebellum as a reference region, the ratios of radioactivity uptake in hippocampus, striatum and cortex were 5.4 ±

0.1, 6.6 ± 0.1 and 4.6 ± 0.1, respectively. The specificity of [nC]-ABP688 binding was confirmed by blockade studies with M-MPEP. For the hippocampus and striatum,

up to 80% of specific binding was observed. Tracer accumulation in the cerebellum could not be blocked.

Replication of the biodistribution study in rats in two more experiments revealed

under baseline (n = 6) and blockade (n = 6) conditions a similar distribution pattern.

Radioactivity uptake in blood, urine and peripheral regions was determined and shown in figure 3. 54 Chapter 3

0.4 i

0.36 baseline

0.32 D blockade

c 0.28

21 o 0.24 -

1" 0.2 - o

- y q 0.16 i—i É^

^ 0.12 -

0.08

0.04 - 1 1 1 É* É wr

lung liver kidney blood bone muscle

Figure 3: Biodistribution of [nC]-ABP688 in peripheral regions of rats. The control group (n = 6) was injected with PEG/H20 (1/1) and [UC]-ABP688, whereas the test group (n = 6) received M-MPEP (1.0 mg/kg body weight) and [nC]-ABP688. Data are expressed as percentage of normalised injected dose per gram tissue ± SD. Animals were sacrificed 30 min p.i.

Highest activity accumulation was observed in urine, which was on average 5-10 times higher than the radioactivity accumulation observed in hippocampus (data not shown). Moderate uptake was noted in liver and kidney and low in lung, bone and

muscle. Tracer accumulation in these regions was the same under baseline and

blockade conditions

Biodistribution studies in wt- and ko-mice

Biodistribution data of [nC]-ABP688 binding in wt- and ko-mice are shown in figure

4. Wt-mice showed a distribution pattern similar to that described for rats. The ratios of radioactivity uptake using the cerebellum as a reference region were 2.7 ± 0.1,

2.6 ±0.1 and 2.0 ±0.1 for hippocampus, striatum and cortex, respectively. Chapter 3 55

\>.i.

0.16- wt-mice

ko-mice

° 0.12 -

q 0.08 0.04 llLJL hippocampus striatum cortex cerebellum blood

Figure 4: Biodistribution of [UC]-ABP688 in brain regions of wt-mice (n = 4), ko-mice (n = 4) and blood. The mice were injected with [nC]-ABP688. Data are expressed as percentage of normalised injected dose per gram tissue ± SD. Animals were sacrificed 20 min p.i.

The specificity of [nC]-ABP688 binding was confirmed with mGluR5-ko-mice, which showed a homogeneous distribution of [nC]-ABP688 in the brain. For the

hippocampus and striatum a reduction in the radioactivity uptake of up to 70% was observed. Tracer accumulation in the cerebellum was similar in both wt- and ko-

mice.

3.4.3 PET STUDIES

PET study of a rat

In order to obtain information on the whole body distribution and the brain uptake of

[nC]-ABP688, a dynamic PET scan using a rat was carried out. Figure 5 shows coronal sections of a 30 min PET scan in a rat. A moderate radioactivity uptake in the

brain and a high uptake in organs such as liver and bowel were evident. This is not surprising in view of the fact that these organs are excretory organs. Areas of

highest radioactivity uptake in the brain corresponded to hippocampal and striatal

regions. In the cerebellum no activity uptake was observed. 56 Chapter 3

brain (hippocampus, striatum)

Figure 5: Series of coronal sections (ventral to dorsal) through the body of a rat injected with [nC]- ABP688. The images were obtained by reconstructing data from 0.5 to 30 min p.i.

PET studies of wt- and ko-mice

Data reconstruction of the dynamic PET scans of wt- and ko-mice resulted in high

resolution images of the mice brains, allowing a good delineation of the striatum and

hippocampus (Figure 6A), regions known to contain high densities of mGluR5. The time-activity curves of [nC]-AP688 in the hippocampus and cerebellum are shown in figure 6C. [nC]-ABP688 rapidly entered the hippocampus and remained at almost the same uptake level during the scan period of 30 min (Figure 6C). For the striatum, a similar time course and level of radioactivity uptake was observed (data not shown). Chapter 3 57

glands

um

hippocampus

glands

15 20 25 30

time p i [mn]

Figure 6: Coronal PET slices of [UC]-ABP688 in a mouse head showing summation images from 0.5- 30 min of A wild type mouse and B knock-out mouse. C Time-activity curves of [nC]-ABP688 in hippocampus and cerebellum of wt-mouse and D in the forebrain and cerebellum of ko-mouse. Data are expressed as standard uptake value (SUV).

The SUVs of tracer uptake measured for the hippocampus were more than twice the

SUVs determined for the cerebellum, a reference region known to have a low expression of mGluR5. To confirm the specificity of [nC]-ABP688 binding in vivo, a dynamic PET scan using a mGluR5-ko-mouse was performed. The brain images of the ko-mouse showed low radioactivity uptake in the forebrain and a homogeneous distribution throughout the brain (Figure 6B). The SUVs of tracer uptake measured in forebrain and cerebellum were both on the same level (Figure 6D). High radioactivity

uptake was observed in extra-cerebral regions (i.e. nasal and lachrymal glands) in wt- as well as in ko-mice. These regions are known to non-specifically accumulate

CNS PET ligands (Myers et al., 1999). 58 Chapter 3

3.4.4 EX VIVO AUTORADIOGRAPHY

Ex vivo autoradiography of a rat brain

Biodistribution of [nC]-ABP688 in rat brain was further analysed by ex vivo autoradiography. As with classical dissection experiments rat brain cryostat sections

revealed heterogeneous binding of the tracer with high uptake in receptor-rich

regions (hippocampus, striatum and cortex) while negligible uptake was observed in the cerebellum (Figure 7). The ultra high resolution of the system allowed a good delineation of different regions of the hippocampus. High radioactivity accumulation was observed in the dentate gyrus, CA1 region and the stratum radiatum. Using cerebellum as a reference region, the ratios of radioactivity uptake in hippocampus, striatum and cortex were 3.2, 3.3 and 2.8, respectively.

cingulate cortex

striatum

stratum radiatum hippocampus

hippocampus

cerebellum

Figure 7: Ex vivo autoradiography of [ C]-ABP688 (unwashed, 20 |j.m coronal section of a rat brain, 8 min p.i.). Chapter 3 59

Ex vivo autoradiography using wt- and ko-mice

Ex vivo autoradiography was performed using wt- and ko-mice in order to determine the specificity of [nC]-ABP688 binding in autoradiographic sections. The brain of the wt-mouse (Figure 8A) showed a comparable distribution pattern as observed in the

rat mentioned above. Autoradiographic sections of the brain of a mGluR5-ko-mouse confirmed the specificity of [nC]-ABP688 binding, since a uniform and low

radioactivity uptake was detected throughout the brain (Figure 8B).

Figure 8: Ex vivo autoradiograms of [ C]-ABP688 uptake in mice brains, unwashed 20 urn coronal sections of A wt-mouse and B ko-mouse.

3.4.5 METABOLITES

Figure 9 shows the results of ex vivo [nC]-ABP688 metabolite analysis in the brain,

blood and urine of rats. HPLC analysis of brain homogenate indicated that more than

95% of radioactivity in rat brain was parent compound. 60 Chapter 3

100

80-

%

[11C]-ABP688

urine

blood

time (min)

Figure 9: HPLC chromatograms of rat brain, blood and urine extracts at 30 min p.i. as well as of parent [UC]-ABP688.

Greater than 75% and 95% of the radioactivity found in blood and urine,

respectively, was attributed to metabolites. Chapter 3 61

3.5 DISCUSSION

Recent evidence points to the importance of mGlu5 receptors in brain function and as a potential therapeutic target for a number of central nervous system disorders.

Non-invasive techniques like positron emission tomography (PET) offer the possibility to visualise the mGluR5 and present an interesting tool for studying the receptor subtype under physiological and pathophysiological conditions. To date, the visualisation of mGluR5 by PET has been limited by the lack of high-affinity and subtype-selective compounds. In this work ABP688, a novel high-affinity (IC50 = 3

nM) and selective antagonist for mGluR5, was radiolabelled with carbon-11 and its

potential as an in vivo PET imaging agent was evaluated.

An efficient chemical synthesis was developed for desmethyl-ABP688 (7) and parent compound ABP688 (8) (Scheme 1). The syntheses of both compounds were accomplished in high chemical yields and the spectroscopic data confirmed the structures of both compounds (NMR, MS). The radiolabelling of ABP688 with carbon-

11 was accomplished by O-methylation of des methyl-AB P688 using [nC]-MeI as a

methylation agent and sodium hydride as a base. Reversed phase HPLC was used for the purification of the final product, which was obtained in good radiochemical yields and high specific activity. 13C-NMR data of ABP688 obtained from the reaction of

[13C]-CH3I and desmethyl-ABP confirmed the position of label at the O-methyl-group.

Saturation studies and Scatchard analysis confirmed the high in vitro affinity (KD of

1.7 ± 0.2 nM) of [nC]-ABP688 for mGluR5. These results show that the determination of in vitro receptor binding parameters such as KD for carbon-11

labelled compounds is feasible despite the physical limitations of the short half-life of carbon-11 (ti/2 = 20.3 min). The nanomolar affinity of [nC]-ABP688 for the mGluR5 combined with optimal lipophilicity and the high in vitro plasma stability encouraged a further evaluation of [nC]-ABP688 using ex vivo autoradiography, classical

biodistribution studies and PET imaging.

The lipophilicity of the tracer was determined experimentally using the shake flask

method and the logD value obtained was 2.4 ± 0.1. This value is similiar to the calculated value of 2.4 and confirmed the robustness of the method. For CNS PET 62 Chapter 3

ligands, Waterhouse (Waterhouse, 2003) postulated a logP value between 2 and 3 as an optimal range for good blood-brain barrier (BBB) penetration. As such for [nC]-

ABP688, an unhindered passage through the BBB was expected. As expected from the lipophilicity, [nC]-ABP688 penetrated rat brain. The highest uptake of

radioactivity in the rat brain was in the striatum and hippocampus, whereas a

moderate uptake of radioactivity was observed in the cortex. Radioactivity accumulation in the hippocampus, a region with high density of mGluR5, was 0.19%

ID norm/g organ at 30 min p.i. A similar value was observed for the striatum (0.22%

ID g/organ). The observed heterogeneity of tracer uptake corresponded to the

reported distribution pattern of mGluR5 (Shigemoto et al., 1993; Shigemoto et al.,

1997). The specificity of [nC]-ABP688 binding was further substantiated in post

mortem blockade studies (Figure 2). Up to 80% reduction in radioactivity uptake in

mGluR5-rich regions (hippocampus, striatum) was observed, whereas in the cerebellum no changes in radioactivity uptake were noted, suggesting also that tracer uptake in the cerebellum was non-specific binding. A more elegant way to demonstrate specificity or non-specificity of tracer binding is to utilise wt- and ko- animals provided these are available. In our studies we used also wt- and mGluR5-

ko-mice to determine whether [nC]-ABP688 binding was specific or not. While

biodistribution in wt-mice revealed the same distribution pattern as observed in rats,

mGluR5-ko-mice showed a uniform distribution of [nC]-ABP688 throughout the whole brain (Figure 4C) again indicating that binding in ko-mice was non-specific.

PET imaging using the quad HIDAC small animal PET camera resulted in high

resolution images of the mouse brain, allowing a good delineation of the striatum and hippocampus (Figure 6A). PET whole body images showed moderate

radioactivity uptake in the brain and high radioactivity accumulation in excretory organs, such as liver, bowel and bladder (Figure 5), which is in correspondence with the radioactivity accumulation observed for these regions in the dissection experiments. The high activity accumulation in these organs has to be considered when administering this tracer to humans, because in this case the excretory organs

mentioned above will be the critical organs.

From point of view of experimental procedure, ex vivo autoradiography remains to

be a challenge when using carbon-11 labelled compounds (Wang et al., 2002). Chapter 3 63

Nevertheless, unwashed ex vivo autoradiographic sections of rat brain showed a

heterogeneous distribution of [nC]-ABP688 uptake in the different brain regions. The

ultra high resolution of the method allowed differentiating between regions such as dentate gyrus, CA1 region and stratum radiatum within the hippocampus (Figure 7).

The distribution pattern observed in rat brain sections corresponded also to the

reported distribution of mGluR5 (Blumcke et al., 1996; Daggett et al., 1995).

The question whether radioactive metabolites are present in the brain is of great

importance, since radioactive metabolites could falsify the detection and hence the obtained data. Metabolite studies of [nC]-ABP688 in rat brain were therefore

undertaken. The results indicated that more than 95% of the radioactivity found in the brain was parent compound. However, high rates of metabolism were observed

in urine and blood (Figure 9). The extraction method used proved suitable, since the

recovery of radioactivity was greater than 90% in both brain and blood. HPLC analysis indicated the metabolites to be more hydrophilic than the parent compound and suggested that metabolites would be too polar to enter the brain.

In conclusion, [nC]-ABP688 showed high affinity and specificity for mGluR5 in vitro as well as in vivo. Specific and heterogeneous uptake were confirmed by ex vivo autoradiography, post mortem biodistribution studies and PET imaging. [nC]-ABP688 therefore represents the first selective tracer for imaging the mGluR5 in vivo in

rodents and may offer a future tool for imaging the mGluR5 in humans using PET. 64 Chapter 3

3.6 REFERENCES

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R. 2000. The non-competitive antagonists 2-methyl-6-(phenylethynyl)pyridine and 7-hydroxyiminocyclopropan[b]chromen-la-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. J Biol Chem 275(43):33750-33758. Pin JP, Duvoisin R. 1995. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 34(1): 1-26. Rouse ST, Marino M J, Bradley SR, Awad H, Wittmann M, Conn PJ. 2000. Distribution and roles of metabotropic glutamate receptors in the basal ganglia motor circuit: implications for treatment of Parkinson's disease and related disorders. Pharmacol Ther 88(3):427-435. Shigemoto R, Kinoshita A, Wada E, Nomura S, Ohishi H, Takada M, Flor PJ, Neki A, Abe T, Nakanishi S, Mizuno N. 1997. Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. J Neurosci 17(19):7503-7522. Shigemoto R, Nomura S, Ohishi H, Sugihara H, Nakanishi S, Mizuno N. 1993. Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci Lett 163(l):53-57. Sotgiu ML, Bellomi P, Biella GE. 2003. The mGluR5 selective antagonist 6-methyl-2- (phenylethynyl)-pyridine reduces the spinal neuron pain-related activity in mononeuropathic rats. Neurosci Lett 342(l-2):85-88. Spooren WP, Gasparini F, Salt TE, Kuhn R. 2001. Novel allosteric antagonists shed light on mglu(5) receptors and CNS disorders. Trends Pharmacol Sei 22(7):331-337. Spooren WP, Vassout A, Neijt HC, Kuhn R, Gasparini F, Roux S, Porsolt RD, Gentsch C. 2000. Anxiolytic-like effects of the prototypical metabotropic glutamate receptor 5 antagonist 2-methyl-6-(phenylethynyl)pyridine in rodents. J Pharmacol Exp Ther 295(3):1267-1275. Strijckmans VH, D. H.; Dolle, F.; Coulon, C; Loch, C. et al. 1996. Synthesis of a Potential M(l) muscarinic agent [Br-76]bromocaramiphen. J Labelled Compd Radiopharm 38:471-481. Tatarczynska E, Klodzinska A, Chojnacka-Wojcik E, Palucha A, Gasparini F, Kuhn R, Pile A. 2001. Potential anxiolytic- and antidepressant-like effects of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist. Br J Pharmacol 132(7): 1423-1430. Walker K, Bowes M, Panesar M, Davis A, Gentry C, Kesingland A, Gasparini F, Spooren W, Stoehr N, Pagano A, Flor PJ, Vranesic I, Lingenhoehl K, Johnson EC, Varney M, Urban L, Kuhn R. 2001a. Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain. Neuropharmacology 40(l):l-9. Walker K, Reeve A, Bowes M, Winter J, Wotherspoon G, Davis A, Schmid P, Gasparini F, Kuhn R, Urban L. 2001b. mGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia. Neuropharmacology 40(1): 10-19. Wang WF, Ishiwata K, Kiyosawa M, Kawamura K, Oda K, Kobayashi T, Matsuno K, Mochizuki M. 2002. Visualization of sigmal receptors in eyes by ex vivo autoradiography and in vivo positron emission tomography. Exp Eye Res 75(6):723-730. 66 Chapter 3

Waterhouse RN. 2003. Determination of lipophilicity and its use as a predictor of blood-brain barrier penetration of molecular imaging agents. Mol Imaging Biol 5(6):376-389. 4

Synthesis and Pharmacological Evaluation of [18F]-

Fluoroethyl-ABP688 and [18F]-Fluoromethyl-ABP688 as

Radioligands for the Metabotropic Glutamate Receptor

Subtype 5 (mGluR5)

Lea J. Kessler1, Michael Honer1, Yves Auberson2, Samuel Hintermann2, P. August Schubiger1 and Simon M. Ametamey1

1 Center for Radiopharmaceutical Science ofETH, PSI and USZ, 5253 Villigen-PSI, 2 Novartis Institute for Biomedical Research, 4002 Basle, Switzerland

Nuclear Medicine & Biology

(prepared for publication)

Chapter 4 69

4.1 ABSTRACT

Fluoroethyl-ABP688 (3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone <>(2-[18F]- fluoro-ethyl)-oxime) and fluoromethyl-ABP688 (3-(6-methyl-pyridin-2-ylethynyl)- cyclohex-2-enone <>(2-[18F]-fluoro-methyl)-oxime) were evaluated for their potential as PET imaging agents for the metabotropic glutamate receptor subtype 5 (mGluR5).

[18F]-labelled fluoroethyl-ABP688 and fluoromethyl-ABP688 were prepared in good radiochemical yields and high specific activities from desmethyl-ABP688. Saturation experiments of [18F]-fluoroethyl-ABP688 binding resulted in a single high-affinity binding site with a KD of 8.5 nM. For [18F]-fluoromethyl-ABP688 an IC50 value of 3.4 nM was determined. Dynamic PET scanning using the small animal quad-HIDAC PET camera indicated for [18F]-fluoroethyl-ABP688 a slightly higher radioactivity uptake in the forebrain, an mGluR5-rich region, than the cerebellum, a region with low mGluR5 density. After an initial radioactivity uptake followed a gradual decrease. Classical biodistribution studies of [18F]-fluoroethyl-ABP688 in rats showed no significant accumulation in the receptor-rich brain regions as well as no significant blockade effect could be observed. For [18F]-fluoromethyl-ABP688 PET scanning revealed a rapid uptake followed by a fast clearance and no radioactivity accumulation in the brain. But high radioactivity uptake was observed in the skull suggesting high in vivo defluorination. [18F]-fuoroethyl-ABP688 and [18F]-fluoromethyl-ABP688 lack in vivo specificity. 70 Chapter 4

4.2 INTRODUCTION

Glutamate is the main excitatory neurotransmitter in the mammalian brain, which acts at either ionotropic or metabotropic glutamate receptors 1. Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G-protein coupled

receptors that activate intracellular secondary messenger systems when bound by glutamate. Activation of mGluRs results in a variety of cellular responses. In

particular, group I mGluRs (subtypes 1 and 5) are positively coupled to

phosphoinositide/Ca2+ cascade, while group II (subtypes 2 and 3) and group III

(subtypes 4, 6, 7 and 8) are negatively coupled to adenylate cyclase. Excessive activation of group I mGluRs is thought to be implicated in a variety of diseases affecting the nervous system 2. For example, an involvement of mGluR5 in mood disorders and schizophrenia 3, movement disorders such as Morbus Parkinson 4,

5 neuroprotection as well as drug addiction and drug abuse has been reported 6.

Further, the participation of mGluR5 in nociceptive processes such as chronic pain,

inflammatory and neuropathic pain has recently been observed 7'8.

ABP688 (3-(6-methyl-pyridine-2-ylethynyl)-cyclohex-2-enone <>methyl-oxime) is a

non-competitive and highly selective antagonist (KD = 1.7 nM) for mGluR5 with

promising in vivo properties (Figure 1).

Fluoroethyl-ABP688 Fluoromethyl-ABP688

Figure 1: Chemical structures of ABP688 and its fluoroethyl-ABP688 and fluoromethyl-ABP688 derivatives. Chapter 4 71

Radiolabelling with fluorine-18 is of particular interest since this radionuclide shows

better imaging characteristics than carbon-11 due to its low positron energy. Its half-

life of 110 min allows for more complex synthesis and longer in vivo investigation, attractive for tracers with slow kinetic properties. Furthermore, the half-life of fluorine-18 allows satellite distribution of [18F]-labelled compounds to PET centres with no radiochemistry labs. The structure of ABP688 does not allow direct

radiolabelling with fluorine-18, therefore we prepared the fluoroethyl and fluoromethyl derivatives. Here we report on the radiosynthesis and pharmacological evaluation of [18F]-fluoroethyl-ABP688 and [18F]-fluoromethyl-ABP688 as perspective

radioligands for imaging mGluR5. 72 Chapter 4

4.3 MATERIALS AND METHODS

4.3.1 GENERAL

All reagents and solvents used were of analytical quality or high performance liquid chromatography (HPLC) grade and were purchased from Merck (Dietikon,

Switzerland) or Fluka Chemie (Buchs, Switzerland), if not otherwise stated. NMR- spectra were recorded on a Bruker AC-250 (1H:300 MHz) using tetramethylsilane

(TMS) as internal standard. The signals are reported in ppm (5) downfield. Mass spectra were recorded on a Trio 2000 Spectrometer (VC Organic, UK) using positive ion mode with electrospray as interface (ES+).

4.3.2 COMPOUNDS AND RADIOCHEMISTRY

Fluoroethyl-ABP688

Fluoroethylation of ABP688 was accomplished by reacting desmethyl-ABP688 with 1- bromo-2-fluoroethane (Fluorochem, Old Gossip, UK.) in DMSO at room temperature for 1 h. Analytical data (1H-NMR, MS) were in agreement with the indicated structure.

^-NMR (300 MHz, CDCI3): S 1.40 (t, 2H), 2.00 (q, 2H), 2.58 (t, 2H), 2.77 (s, 3H)

5.85 (dd, 2H), 6.76 (s, 1H), 7.29 (d, 1H), 7.48 (d, 1H), 7.79 (t, 1H); MS (m/z) 273

(M+), 100%.

[18F]-radiolabelling was achieved in a two-step reaction sequence. First, [18F]-2- fluoroethyltosylate was obtained by heating [18F]-KF-Kryptofix complex with ethyleneditosylate in refluxing acetonitrile for 10 min. In the second step, the [18F]- intermediate reacted with the sodium salt of desmethyl-ABP688 in DMF at 100°C for

10 min to afford [18F]-fluoroethyl-ABP688. After purification by reversed-phase HPLC

(Bondclone, C-18, 300 x 7.8 mm; mobile phase acetonitrile : phosphoric acid 0.1% =

30:70; flow rate 4 mL/min) the product was collected on a Sep-Pak filter, eluted with ethanol and diluted with 0.15 M phosphate buffer. Chapter 4 73

Fluoromethyl-ABP688

The synthesis of fluoromethyl-ABP688 was achieved by gently bubbling bromofluoromethane (ABCR, Karlsruhe, Germany) through a DMF solution containing the sodium salt of desmethyl-ABP688. The mixture was allowed to react at temperature for 10 min. Analytical data (1H-NMR, MS) were in agreement with the indicated structure.

^-NMR (300 MHz, CDCI3): S 1.21 (t, 2H), 1.77 (q, 2H), 2.39 (t, 2H), 2.55 (t, 2H),

2.60 (s, 3H), 4.30 (dd, 2H), 4.67 (dd, 2H), 6.56 (s, 1H), 7.13 (d, 1H), 7.28 (d, 1H),

7.60 (t, 1H); MS (m/z) 258 (M+); 100 %.

For the radiolabelling with fluorine-18, [18F]-fluoride was trapped on a QMA Sep-Pak cartridge and eluted with acetonitrile/water (1/1) containing Kryptofix and K2C03.

After the azeotropic evaporation of the acetonitrile/water mixture, dibromomethane was added to the [18F]-KF-Kryptofix complex and the reaction mixture heated to reflux for 5 min. The resulting radioactive volatile products were blown through four

Sep-Pak Plus silica cartridges, which were connected in series, by nitrogen flow. The product was separated and allowed to pass through a heated (190°C) AgOTf column to afford [18F]-FCH2-OTf. The radioactive yield was on average 10% (decay corrected). This is lower than the reported literature value 9.

The reaction of [18F]-FCH2-OTf with the sodium salt of desmethyl-ABP688 proceeded in acetone at room temperature for 5 min to give [18F]-fluoromethyl-ABP688 in greater than 50% radioactive yield (decay corrected). The product was finally purified by reversed-phase HPLC (u-Bondapak, C-18, 7,8 x 300 mm, 10 urn; mobile phase acetonitrile : phosphoric acid 0.1% = 30:70, flow rat 6 mL/min) and after the

HPLC eluent was evaporated, the product was formulated in 0.15 M phosphate buffer, 10% ethanol and 2% Tween 80. 74 Chapter 4

4.3.3 PHARMACOLOGY

Inhibition assays

Membranes from transfected COS1 cells were collected 2 days after transfection.

Cells were washed with phosphate-buffered saline and mechanically detached in ice- cold phosphate-buffered saline containing 10 mM EDTA. Cells were centrifuged at

4000 rpm at 4°C for 20 min and resuspended in binding buffer (30 mM NaHEPES,

110 mM NaCI, 5 mM KCl, 2.5 mM CaCI2xH20 and 1.2 mM MgCI2, pH 8). Cells were then disrupted on ice with a Polytron homogeniser for 20 s, and membranes were collected by centrifugation at 18000 rpm at 4°C for 20 min. The pellet was resuspended in binding buffer, homogenised with a Teflon homogeniser, and used immediately for binding. Ligand binding assays were performed using [3H]-M-MPEP

(2 nM) and increasing concentrations of the competing compound fluoromethyl-

ABP688. Non specific binding was determined by application of cold M-MPEP (10~6

M). The reaction was terminated after 30 min incubation at 25°C by dilution and rapid filtration through Whatman GF/B filters. The filters were washed three times with cold binding buffer, and the bound radioactivity was counted using a ß-counter in 5 mL of Ultima Gold NV Packard (Canberra Packard, Zurich, Switzerland). Specific fluoromethyl-ABP688 binding was defined as total binding minus non-specific binding in the presence of 1 x 10"6 M cold M-MPEP.

Saturation assays

Preparation of membranes: Male rats (adult Wistar, RCC Ltd., Füllinsdorf,

Switzerland) were euthanised by decapitation. Cerebellum was quickly removed and the brain homogenised in 10 volumes of ice-cold (4°C) sucrose buffer (0.32 M sucrose, 10 mM Tris/acetate-buffer, pH 7.4) with a polytron (PT-1200 C, Kinematica

AG, Littau, Switzerland) for 1 min at setting 4. The homogenate was centrifuged at

1000 g for 15 min (4°C) to yield a crude pellet (PI). This pellet was resuspended in 5 volumes of sucrose-buffer, homogenised and centrifuged again at 1000 g for 15 min

(4°C). The resulting supernatants were combined and centrifuged at 17000 g for 20 min (4°C) to yield a pellet P2. The pellet was washed with ice-cold incubation buffer

I (5 mM Tris/acetate buffer, pH 7.4), homogenised and centrifuged at 17000 g for 20 Chapter 4 75

min (4°C). The pellet was resuspended in incubation buffer I and stored at -70°C. On the day of the assay, the membranes were thawed and the protein concentration determined by Bio-Rad Microassay with bovine serum albumin as a standard 10.

Saturation experiments: 500 ng/mL of whole brain (without cerebellum) rat

membranes were incubated with increasing concentrations of [18F]-fluoroethyl-

ABP688 (0.5-250 nM) in binding buffer to give a total volume of 200 uL. Non-specific

binding was determined in the presence of 100 uM M-MPEP. Incubations were allowed to proceed for 45 min at room temperature before being terminated by vacuum filtration over GF/C-filters (Whatman) pre-soaked for 1 h in incubation buffer

II in order to reduce non-specific binding. The membranes retained on the filters were rinsed twice with 4 mL ice-cold binding-buffer. The radioactivity retained on the filters was determined using a gamma-counter (Cobra II Auto-gamma, Camberra

Packard, Groningen, The Netherlands).

Data analysis. Scatchard analysis was performed with the computer program, Kell-

Radlig (McPherson & Biosoft, Cambridge, UK, 1997).

Animals

Animal care and all experimental procedures were approved by the Swiss Federal

Veterinary Office. Animals (male Sprague Dawley rats, 350-450 g) were allowed free access to food and water.

PET studies

PET experiments were performed with the 16-module variant of the quad-HIDAC PET scanner (Oxford Positron Systems; West-on-the-Green, UK) n. The resolution at the centre of field of view was 1.0 mm. Prior to the PET studies the animals were anaesthetised with isoflurane inhalation anaesthesia. The radiotracers ([18F]- fluoromethyl-ABP688: 20 MBq, 0.5 nmol; [18F]-fluoromethyl-ABP688: 10 MBq, 0.5

nmol) were administered into the tail vein of rats. Scan duration was 90 min. PET data were acquired in list mode and reconstructed in user-defined time frames using the OPL-EM algorithm (0.3 mm bin size 120x120x200 matrix size) incorporating

resolution recovery. Image files were evaluated by region-of-interest (ROI) analysis 76 Chapter 4

using the dedicated software PMOD . Time activity curves were normalised to the

injected dose per gram body weight and expressed as standardised values (SUV).

Biodistribution studies

For biodistribution studies [18F]-fluoroethyl-ABP688 was administered into the tail vein of awake rats (20-22 MBq injected activity, 0.5-0.6 nmol injected mass; n = 3).

Blockade studies were carried out by co-injecting M-MPEP (1.0 mg/kg body weight; 2

mg/mL PEG/H2O 1:1) with the radiotracer. The animals were sacrificed by decapitation (30 min post-injection). The whole brain was rapidly removed and dissected into specific brain regions: hippocampus, striatum, cortex and cerebellum.

Each brain region was weighed and tissue radioactivity was measured in a gamma- counter (Cobra II Auto-gamma, Camberra Packard, Groningen, The Netherlands).

For all brain regions the tissue distribution was determined as percentage of

normalised injected dose per gram wet tissue (% ID norm/g organ). Chapter 4 77

4.4 RESULTS AND DISCUSSION

4.4.1 COMPOUNDS AND RADIOCHEMISTRY

Fluoroethyl-ABP688

Fluoroethylation of ABP688 was accomplished by reacting desmethyl-ABP688 with 1-

bromo-2-fluoroethane in DMSO at room temperature for 1 h. After column chromatography fluoroethyl-ABP688 was obtained in 50% yield. Scheme 1 shows the

reaction sequence leading to fluoroethyl-ABP688.

^^

Desmethyl-ABP688 Fluoroethyl-ABP688

Scheme 1: Reagents and conditions: a) NaH, DMF, room temperature, 30 min; b) l-bromo-2- fluoroethane, room temperature, 1 h, (50%).

For the radiolabelling with fluorine-18, a two-step reaction sequence was employed.

The first step involved the nucleophilic radiofluorination of ethyleneditosylate to afford [18F]-2-fluoroethyltosylate, which in the second step reacted with the sodium salt of desmethyl-ABP688 to give [18F]-fluoroethyl-ABP688 in 5% radiochemical yield

(decay corrected) (Scheme 2). The total synthesis time was 150 min and the product showed a radiochemical purity greater than 95%. The specific activity of the product was 35-55 GBq/umol. 78 Chapter 4

"Tos "Tos

r<^,

Desmethyl-ABP688 [18F]-Fluroethyl-ABP688

Scheme 2: Reagents and conditions: a) [18F]-F, K.222, K+, acetonitrile, reflux, 10 min; b) NaH, DMF, room temperature, 1 h; c) [18F]-fluorethyltosylate, 100°C, 10 min; radiochemical purity >95%, specific activity 35-55 GBq/umol.

The lipophilicity of [18F]-fluoroethyl-ABP688 was determined by the shake flask

13 method as described by Strijckmans and yielded a logD value of 2.7 ±0.1.

Fluoromethyl-ABP688

The synthesis of fluoromethyl-ABP688 was accomplished by reacting desmethyl-

ABP688 with bromofluoromethane (Scheme 3). After column chromatography pure fluoromethyl-ABP688 was obtained in 40% yield.

r^*,

/CH7F l\k 2 0

Desmethyl-ABP688 Fluoromethyl-ABP688

Scheme 3: Reagents and conditions: a) NaH, DMF, room temperature, 30 min; b) bromofluoromethane, room temperature, 10 min (40%).

For the radiolabelling of fluoromethyl-ABP688 with fluorine-18, a three-step reaction sequence was employed. The first step involved the preparation of [18F]-

bromofluoromethane, which in a second step reacted with silver triflate to afford

[18F]-fluoromethyltriflate as fluorination agent. The reaction of the [18F]- fluoromethyltriflate with the desmethyl-ABP688 proceeded well to afford [18F]- Chapter 4 79

fluoromethyl-ABP688 in a radiochemical yield of 5-10% (decay corrected) (Scheme

4). The total synthesis time was 75 min. [18F]-fluoroethyl-ABP688 was obtained in

95% radioachemical purity and a specific activity of 35-45 GBq/umol.

CH2Br2 -^ [18F]-FCH2Br [18F]-FCH2-OTf

Desmethyl-ABP688 [18F]-Fluoromethyl-ABP688

Scheme 4: Reagents and conditions: a) [18F]-F, K.222, K+, acetonitrile, reflux; b) AgOTf, 190°C; c) NaH, acetone, room temperature, 30 min; d) [18F]-FCH2-OTf, room temperature, 5 min; radiochemical purity >95%, specific activity 35-45 GBq/|amol.

For [18F]-fluoromethyl-ABP688 a logD value of 2.8 ±0.1 was obtained. Further, in

vitro plasma stability was determined by incubating [18F]-fluoromethyl-ABP688 in

human plasma for 60 min at 37°C. Analytical HPLC analysis demonstrated excellent stability of [18F]-fluoromethyl-ABP688 under these conditions. 80 Chapter 4

4.4.2 PHARMACOLOGY

Saturation assays

[18F]-Fluoroethyl-ABP688: For the estimation of dissociation constant KD, [18F]- fluoroethyl-ABP688 was employed in saturation studies using rat whole brain

membranes (without cerebellum). Filtration technique was employed for the separation of bound from free ligand. Non-specific binding was quantified by co-

incubation with 100 \iM M-MPEP. Receptor binding was found to be saturable (Figure

2A) and Scatchard transformation of the binding data resulted in a single binding site with a dissociation constant KD of 8.9 nM (Figure 2B) and a Bmax value of 560 fmol/mg protein.

4 r

E. 3

T3 U> C Q_ 3 CD g <

1 .

50 100 150 200 250 12 3

[18F]-FE-ABP688 [nM] bound [nM]

Figure 2: A Saturation curve of [iaF]-fluoroethyl-ABP688 (FE-ABP688) B Scatchard analysis of [iaF] FE-ABP688, representative example.

[18F]-Fluoromethyl-ABP688: n vitro binding characteristics of fluoromethyl-ABP688 were studied in membranes of recombinant human mGluR5 expressing L(tk-) cells and using the filtration technique to separate the bound from the free ligand.

Inhibition of [3H]-M-MPEP binding revealed an IC5o value for fluoromethyl-ABP688 of

3.4 n M (data not shown). Chapter 4 81

PET studies

[18F]-Fluoroethyl-ABP688: In order to determine the in vivo distribution of [18F]- fluoroethyl-ABP688, a PET study was performed in a rat. The PET scan revealed a

rather low radioactivity uptake of [18F]-fluoroethyl-ABP688 in rat brain. The time activity curve indicated a gradual decrease of [18F]-fluoroethyl-ABP688. The forebrain containing mGluR5 receptor rich regions (hippocampus, striatum) showed a slightly

higher radioactivity uptake than the cerebellum, a region with low expression of

mGluR5 (Figure 3B). These results suggest that [18F]-fluoroethyl-ABP688 has high

non-specific binding in vivo. High radioactivity uptake was observed in the excretory organs such as bladder and bowel (Figure 3A).

0 10 20 30 40 50 60

time p.i. [mm]

Figure 3: A Series of coronal sections (ventral to dorsal) through the body of a rat injected with [18F]-fluoroethyl-ABP688. The images were obtained by reconstructing data from 0 to 30 min p.i. B Time activity curve in the rat forebrain and cerbellum for a 60 min PET scan. Data are expressed as standard uptake value (SUV).

[18F]-Fluoromethyl-ABP688: A PET study of [18F]-fluoromethyl-ABP688 was

undertaken in order to non-invasively determine its distribution in a rat. The images

in figure 4A clearly show that from 0-30 min p.i. radioactivity uptake in rat brain was

negligible. The time-activity curve in figure 4B of [18F]-fluoromethyl-ABP688 indicated an initial high uptake which was followed by a rapid clearance. A substantial bone

uptake of radioactivity was observed in the skull suggesting that defluorination was taking place in vivo. Whole body images revealed high radioactivity in bones and 82 Chapter 4

excretory organs such as the bladder and bowel. [18F]-fluoromethylated compounds

have been reported to be relatively unstable in comparison to their corresponding

[18F]-fluoroethylated and [nC]-methylated compounds 14.

'liver

bow«

^^ bladder

Ipi Ui '• § "

40 50 60

time p.i. [min]

Figure 4: A Series of coronal sections (ventral to dorsal) through the body of a rat injected with [18F]-fluoromethyl-ABP688. The images were obtained by reconstructing data from 0-30 min p.i. B Time activity curve in the rat whole brain for a 90 min PET scan. Data are expressed as standard uptake value (SUV).

Biodistribution studies

[18F]-Fluoroethyl-ABP688: Classical post mortem biodistribution studies were

undertaken in order to obtain further information on the regional distribution of [18F]- fluoroethyl-ABP688 in distinct brain regions such as hippocampus, striatum, cortex and cerebellum under both baseline and blockade conditions. Biodistribution data of

[18F]-fluoroethyl-ABP688 in rats showed a similar uptake of radioactivity in all brain

regions examined including mGluR5-rich brain regions such as hippocampus, striatum and cortex. Blockade studies by co-injection of M-MPEP showed no significant decrease in radioactivity uptake (data not shown) at 30 min p.i. A possible

reason for the lack of specificity in vivo might be the high lipophilicity (logD = 2.7) of [18F]-fluoroethyl-ABP688. Chapter 4 83

4.5 CONCLUSION

The two fluoro-derivatives, fluoroethyl-ABP688 and fluoromethyl-ABP688, were successfully synthesised and radiolabelled with fluorine-18 in reasonable

radiochemical yields. Despite the promising in vitro properties of both tracers, in vivo evaluation revealed high non-specific uptake for both compounds. For [18F]- fluoroethyl-ABP688, the lack of specificity for mGluR5 was determined by blockade studies. Furthermore, for [18F]-fluoromethyl-ABP688 PET images suggested a rapid defluorination and consequent accumulation in the bones. Both [18F]-fluoroethyl-

ABP688 and [18F]-fluoromethyl-ABP688, are therefore not suitable ligands for

imaging the mGluR5 in vivo. 84 Chapter 4

4.6 REFERENCES

(1) Pin, J. P.; Duvoisin, R. The metabotropic glutamate receptors: structure and functions. Neuropharmacology 1995, 34, 1-26. (2) Spooren, W. P.; Gasparini, F.; Bergmann, R.; Kuhn, R. Effects of the prototypical mGlu(5) receptor antagonist 2-methyl-6-(phenylethynyl)-pyridine on rotarod, locomotor activity and rotational responses in unilateral 6-OHDA- lesioned rats. Eur J Pharmacol 2000, 406, 403-410. (3) Tatarczynska, E.; Klodzinska, A.; Chojnacka-Wojcik, E.; Palucha, A.; Gasparini, F. et al. Potential anxiolytic- and antidepressant-like effects of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist. Br J Pharmacol 2001, 132, 1423-1430. (4) Kinney, G. G.; Burno, M.; Campbell, U. C; Hernandez, L. M.; Rodriguez, D. et al. Metabotropic glutamate subtype 5 receptors modulate locomotor activity and sensorimotor gating in rodents. J Pharmacol Exp Ther 2003, 306, 116- 123. (5) Bruno, V.; Ksiazek, L; Battaglia, G.; Lukic, S.; Leonhardt, T. et al. Selective blockade of metabotropic glutamate receptor subtype 5 is neuroprotective. Neuropharmacology 2000, 39, 2223-2230. (6) Chiamulera, C; Epping-Jordan, M. P.; Zocchi, A.; Marcon, C; Cottiny, C. et al. Reinforcing and locomotor stimulant effects of cocaine are absent in mGluR5 null mutant mice. Nat Neurosci 2001, 4, 873-874. (7) Walker, K.; Bowes, M.; Panesar, M.; Davis, A.; Gentry, C. et al. Metabotropic glutamate receptor subtype 5 (mGlu5) and nociceptive function. I. Selective blockade of mGlu5 receptors in models of acute, persistent and chronic pain. Neuropharmacology 2001, 40, 1-9. (8) Walker, K.; Reeve, A.; Bowes, M.; Winter, J.; Wotherspoon, G. et al. mGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia. Neuropharmacology 2001, 40, 10-19. (9) Iwata, R.; Pascali, C; Bogni, A.; Furumoto, S.; Terasaki, K. et al. [18F]fluoromethyl triflate, a novel and reactive [18F]fluoromethylating agent: preparation and application to the on-column preparation of [18F]fluorocholine. Appl Radiât Isot 2002, 57, 347-352. (10) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. AnalBiochem 1976, 72, 248-254. (11) Honer, M.; Bruhlmeier, M.; Missimer, J.; Schubiger, A. P.; Ametamey, S. M. Dynamic imaging of striatal D2 receptors in mice using quad-HIDAC PET. J Nucl Med 2004, 45, 464-470. (12) Mikolajczyk, K.; Szabatin, M.; Rudnicki, P.; Grodzki, M.; Burger, C. A JAVA environment for medical image data analysis: initial application for brain PET quantitation. Med Inform (Lond) 1998, 23, 207-214. (13) Strijckmans, V. H., D. H.; Dolle, F.; Coulon, C; Loch, C. et al. Synthesis of a Potential M(l) muscarinic agent [Br-76]bromocaramiphen. J. Labelled Compd. Radiopharm. 1996, 38, 471-481. Chapter 4 85

(14) Zhang, M. R.; Maeda, J.; Ogawa, M.; Noguchi, J.; Ito, T. et al. Development of a new radioligand, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[18F]fluoroethyl-5- methoxybenzyl)acetami de, for pet imaging of peripheral benzodiazepine receptor in primate brain. J Med Chem 2004, 47, 2228-2235.

5

Conclusion and Future Directions

Chapter 5 89

So far, the visualisation of mGluR5 by PET has been limited by the lack of

high-affinity subtype-selective compounds. This project is focused on the development of new and highly selective radioligands for the visualisation of mGluR5

by PET.

Four compounds namely, M-FPEP, ABP688 and its fluoro-derivatives fluoroethyl-

ABP688 and fluoromethyl-ABP688 were synthesised and evaluated in vivo as imaging agents for the mGluR5. [nC]-ABP688 was the only compound that exhibited good in

vivo characteristics such as high specificity for the mGluR5. [nC]-ABP688 represents the first radiotracer for imaging the mGluR5 in rodents and is therefore a promising candidate for the imaging of mGluR5 in humans by PET. Before initiating clinical trials critical issues such as toxicity studies and radiation burden estimation have to be

resolved. A full GMP validation of the synthetic procedure must also be established. It

is planned to examine this radioligand in ten healthy volunteers. If successful, PET

imaging of patients suffering from neuronal disorders or neurodegenerative diseases

like Alzheimer's or Parkinson's disease, in which mGluR5 has been implicated will be

undertaken. These future perspectives will lead to completely new insights of

mGluR5 involvement in the above mentioned diseases and the physiological role of

metabotropic receptors especially the mGluR5 in brain function would be better

understood. Further, such a highly selective radiotracer may also be used in drug development to estimate the level of receptor occupancy achieved by therapeutic doses of mGluR5 antagonists currently in clinical trials.

The disappointing results obtained with the fluoro-derivatives should not discourage future work in developing fluorine-18 labelled derivatives of ABP688. Fluoro- derivatives are of high interest since fluorine-18 possesses best imaging characteristics due to its low positron range. Furthermore, its longer half-life would allow more extended in vivo investigations, especially attractive for tracers with slow

kinetic properties, as well as satellite distributions to PET centres without PET chemistry labs. A point to consider in future derivatisation is the lipophilicity of the compound. Since high lipophilicity can cause high non specific binding, compounds with increased hydrophilicity should be considered for evaluation.

Publications and Presentations 91

PUBLICATIONS AND PRESENTATIONS

PUBLICATIONS

M. Kokic, M. Honer, L.J. Kessler, M. Grauert, P.A. Schubiger and S.M. Ametamey;

Synthesis, in vitro ana in vivo evaluation of [11C]-Methyl-BIII277CI, a potential tracer for imaging the PCP-binding site of the NMDA receptor, J. Recept. Signal Tr. R. 2002,

22, 123-139

L.J. Kessler, M. Honer, M. Rebsamen, F. Gasparini, Y. Auberson, P.A. Schubiger and

S.M. Ametamey; Synthesis, in vitro and in vivo evaluation of [nC]-2-methyl-6-(3- fluoro-phenylethynyl)-pyridine as radioligand for the metabotropic glutamate receptor subtype 5 (mGluR5), Bioorg. Med. Chem. Lett., submitted.

L.J. Kessler, M. Honer, M.T. Wyss, F. Gasparini, Y. Auberson, P.A. Schubiger and

S.M. Ametamey; Synthesis, radiolabelling, in vitro and in vivo evaluation of [nC]-

ABP688 for the metabotropic glutamate receptor subtype 5 (mGluR5), prepared for publication.

L.J. Kessler, M. Honer, S. Hintermann, Y. Auberson, P.A. Schubiger and S.M.

Ametamey; Synthesis and pharmacological evaluation of [18F]-fluoroethyl-ABP688 and [18F]-fluoromethyl-ABP688 as radioligands for the metabotropic glutamate receptor subtype 5 (mGluR5), prepared for publication.

Patent, Case4-33364 PI, Novel pyridylacetylene derivatives, their preparation, their use as radiotracers/markers and compositions containing them, filed in September

2003

PRESENTATIONS

L.J. Kessler, M. Honer, Y. Auberson, F.Gasparini, P.A. Schubiger and S.M. Ametamey;

Radiolabelling, in vitro and in vivo evaluation of [nC]-M-FPEP as PET radioligand for 92 Publications and Presentations

imaging the metabotropic glutamate receptor subtype 5 (mGluR5); 2nd Day of Clinical

Research, Universitiy Hospital Zurich, April 2003.

L.J. Kessler, M. Honer, Y. Auberson, F.Gasparini, P.A. Schubiger and S.M. Ametamey;

Radiolabelling, in vitro and in vivo evaluation of [nC]-M-FPEP as PET radioligand for imaging the metabotropic glutamate receptor subtype 5 (mGluR5); PharmaDay,

Pharmacenter Basel-Zurich, June 2003.

S.M. Ametamey, L.J. Kessler, M. Honer, Y. Auberson, F. Gasparini and P.A.

Schubiger; Synthesis and evaluation of [nC]-M-FPEP as PET ligand for imaging the metabotropic glutamate receptor subtype 5 (mGluR5); J. Labelled Cpd. Radiopharm.

2003, 46Suppl.l, S188, Sydney, August 2003

L.J. Kessler, M. Honer, Y. Auberson, F. Gasparini, P.A. Schubiger and S.M.

Ametamey; Radiolabelling, in vitro and in vivo evaluation of [nC]-M-FPEP as PET radioligand for imaging the metabotropic glutamate receptor subtype 5 (mGluR5);

ZNZ-Symposium 2003, Zurich, October 2003.

L.J. Kessler, M. Honer, M. Rebsamen, F. Gasparini, Y. Auberson, P.A. Schubiger and

S.M. Ametamey; MPEP-derivatives for imaging the metabotropic glutamate receptor subtype 5 by positron emission tomography; Aktuelle Forschung Pharmazie-ETHZ,

Zurich, March 2003 CURRICULUM VITAE

Lea Janine Kessler

Citizen of Zurich

Born August 14th, 1974

Education

1987-1993 Gymnasium, KS Freudenberg, Zurich - Matura Typus B

1994-1997 University education in basic pharmacy at the Swiss Federal Institute of Technology (ETH) Zurich

1997-1998 Practical experience in pharmacy

1998-2000 University education in specialised pharmacy at the Swiss Federal Institute of Technology (ETH) Zurich - Diploma in pharmacy

2000 Dissertation at the Center for Radiopharmaceutical Science of ETH, PSI and USZ

2001-2004 Thesis at the Center for Radiopharmaceutical Science of ETH, PSI and USZ, Villigen, Switzerland

Courses

2001-2002 Introductory course in Neurosciene at the ZNZ

2001-2002 Attendance of lectures in economy, ETH

2002 Pharma-Business und Pharma-Marketing für Einsteiger, Pharmacenter, Basel-Zurich

2003 Presentations - Publishing - Communicating, Didaktikzentrum, ETH

Additional experience

1993-1994 Au-pair in London, Certificate of Proficiency in English

1998 Trainee in molecular biology at the University of Ouro Preto, Brazil

HERZLICHEN DANK

....an alle die mich während meiner Dissertation in verschiedenster Weise unterstützt

haben.

Prof. P. August Schubiger danke ich für die Möglichkeit, dass ich meine Dissertation

in seiner Gruppe ausführen durfte und so das Gebiet der Radiopharmazie näher

kennen lernte. Prof. Gerd Folkers danke ich für die Übernahme des Korreferats dieser

Arbeit.

Herzlich bedanke ich mich auch bei PD Dr. Simon Ametamey, der dieses Projekt

betreute und mir mit seinem grossen Erfahrungsschatz im Bereich der PET-Chemie

und seinem Enthusiasmus über so manche Hürde half. Dr. Michael Honer danke ich für die fruchtbaren Diskussionen und die Unterstützung auf dem komplexen Gebiet der Neuropharmakologie. Ein spezielles Dankeschön richtet sich an Claudia Keller und

Erika Sinnig für die grosszügige Hilfsbereitschaft bei der Durchführung der

Experimente. Bei Dr. Gerrit Westera und Tibor Cervenyak bedanke ich mich herzlich für die freundschaftliche Aufnahme in die PET-Gruppe am Universitätsspital Zürich.

Ein liebes Dankeschön geht an alle Freunde und Kollegen am Zentrum für

Radiopharmazie, insbesondere Dr. Matthias Wyss, Dr. Milen Blagoev, Dr. Cécile

Dumas und Dr. Linjin Mu, sowie alle Doktoranden, insbesondere Dr. Marko Kokic, Dr.

Anass Johayem, Drs. Martina und Albert Stichelberger, Monika Heiz, Nikiaus Marti,

Dominique Rüegg, Cristina Müller, Karin Knogler und Eva Geissler für die kollegiale

Zeit am PSI.

Besonderen Dank gebührt meiner Familie, Freunden und ganz besonders Roberto für

ihre verständnisvolle Liebe und Unterstützung zu jeder Zeit.