Zhaoda Zhang

Radiosynthesis of PET radiotracer as a prodrug for imaging group II metabotropic glutamate receptors in vivo

Group II metabotropic glutamate receptors (mGluRs) have been implicated in a variety of neurological and psychiatric disorders in recent studies. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers the possibility to visualize and study group II mGluRs in vivo under physiologic and pathologic conditions. We synthesized a PET tracer, (S,S,S)-2-(2-carboxycyclopropyl)-2-(3-[(11)C]methoxyphenethyl) glycine dimethyl ester ([(11)C]CMGDE), as a prodrug for group II mGluRs, and studied its preliminary biological properties in Sprague-Dawley rats to visualize group II mGluRs. The microPET studies demonstrated that [(11)C]CMGDE readily penetrated into the brain and the radiotracer generated from [(11)C]CMGDE had fast reversible binding in the group II mGluRs rich regions including striatum, hippocampus and different cortical areas. Blocking studies with LY341495 showed 20-30% decrease of binding of the radiotracer generated from [(11)C]CMGDE in all brain areas with the highest decrease in the striatum 31.5±3.2%. The results show [(11)C]CMGDE is the first PET tracer that is brain penetrating and can be used to image group II mGluRs in vivo.

Radiosynthesis of N-(4-chloro-3-[11C]methoxyphenyl)-2-picolinamide ([11C]ML128) as a PET radiotracer for metabotropic glutamate receptor subtype 4 (mGlu4)

N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu4 positive allosteric modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu4 PAM to show efficacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers a possibility to investigate mGlu4 expression in vivo under physiologic and pathological conditions. We synthesized a carbon-11 labeled ML128 ([(11)C]3) as a PET radiotracer for mGlu4, and characterized its biological properties in Sprague Dawley rats. [(11)C]3 was synthesized from N-(4-chloro-3-hydroxyphenyl)-2-picolinamide (2) using [(11)C]CH3I. Total synthesis time was 38±2.2min (n=7) from the end of bombardment to the formulation. The radioligand [(11)C]3 was obtained in 27.7±5.3% (n=5) decay corrected radiochemical yield based on the radioactivity of [(11)C]CO2. The radiochemical purity of [(11)C]3 was >99%. Specific activity was 188.7±88.8GBq/mol (n=4) at the end of synthesis (EOS). PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6 studies blocking with an mGlu4 modulator (4) to investigate specificity and 3 studies blocking with an mGlu5 modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [(11)C]3 (peak activity between 1-3min) in several brain areas including striatum, thalamus, hippocampus, cerebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu4 modulator 4 showed 22-28% decrease of [(11)C]3 accumulation while studies of selectivity showed only minor decrease supporting good selectivity over mGlu5. Biodistribution studies and blood analyses support fast metabolism. Altogether this is the first PET imaging ligand for mGlu4, in which the labeled ML128 was used for imaging its in vivo distribution and pharmacokinetics in brain.

Radiosynthesis and evaluation of an 18F-labeled positron emission tomography (PET) radioligand for metabotropic glutamate receptor subtype 4 (mGlu4).

Four 4-phthalimide derivatives of N-(3-chlorophenyl)-2-picolinamide were synthesized as potential ligands for the PET imaging of mGlu4 in the brain. Of these compounds, N-(3-chloro-4-(4-fluoro-1,3-dioxoisoindolin-2-yl)phenyl)-2-picolinamide (3, KALB001) exhibited improved binding affinity (IC50 = 5.1 nM) compared with ML128 (1) and was subsequently labeled with 18F. When finally formulated in 0.1 M citrate buffer (pH 4) with 10% ethanol, the specific activity of [18F]3 at the end of synthesis (EOS) was 233.5 ± 177.8 GBq/μmol (n = 4). The radiochemical yield of [18F]3 was 16.4 ± 4.8% (n = 4), and the purity was over 98%. In vivo imaging studies in a monkey showed that the radiotracer quickly penetrated the brain with the highest accumulation in the brain areas known to express mGlu4. Despite some unfavorable radiotracer properties like fast washout in rodent studies, [18F]3 is the first 18F-labeled mGlu4 radioligand, which can be further modified to improve pharmacokinetics and brain penetrability for future human studies.

Development of [(123)I]IPEB and [(123)I]IMPEB as SPECT Radioligands for Metabotropic Glutamate Receptor Subtype 5.

mGlu5 play an important role in physiology and pathology to various central nervous system (CNS) diseases. Several positron emission tomography (PET) radiotracers have been developed to explore the role of mGlu5 in brain disorders. However, there are no single photon emission computed tomography (SPECT) radioligands for mGlu5. Here we report development of [(123)I]IPEB ([(123)I]1) and [(123)I]IMPEB ([(123)I]2) as mGlu5 radioligands for SPECT. [(123)I]1 and [(123)I]2 were produced by copper(I) mediated aromatic halide displacement reactions. The SPECT imaging using mouse models demonstrated that [(123)I]1 readily entered the brain and accumulated specifically in mGlu5-rich regions of the brain such as striatum and hippocampus. However, in comparison to the corresponding PET tracer [(18)F]FPEB, [(123)I]1 showed faster washout from the brain. The binding ratios of the striatum and the hippocampus compared to the cerebellum for [(123)I]1 and [(18)F]FPEB were similar despite unfavorable pharmacokinetics of [(123)I]1. Further structural optimization of 1 may lead to more viable SPECT radiotracers for the imaging of mGlu5.

Imaging of Metabotropic Glutamate Receptors (mGluRs)

The ubiquitous amino acid L-glutamate is thought to act as a neurotransmitter at the majority of synapses in the brain. It mediates the major excitatory pathways in the brain, and is referred to as an excitatory amino acid (EAA). The EAA plays a role in a variety of physiological processes, such as long-term potentiation (learning and memory), the development of synaptic plasticity, motor control, respiration, cardiovascular regulation, emotional states and sensory perception (Bliss & Collingridge, 1993). The excessive or inappropriate stimulation of EAA receptors leads to neural cell damage or loss by a mechanism known as excitotoxicity (Lucas & Newhouse, 1957; Oney, 1978). EAA receptors are classified in two general types (Kornhuber & Weller, 1997). Receptors that are directly coupled to the opening of cation channels in the cell membranes of the neuron are termed ‘ionotropic’, which include NMDA, AMPA, and kainate receptors. The second type of receptors are the G-protein or second messenger-linked ‘metabotropic’ EAA receptors. This second type is coupled to multiple second messenger systems that lead to enhanced phosphoinositide hydrolysis, activation of phospholipase D, increase or decrease in cAMP formation, and changes in ion channel function (Kozikowski et al., 1998). Metabotropic glutamate receptors belong to Class C of a superfamily of G-protein coupled receptors (GPCRs). Class C GPCRs possess a large extracellular domain that is responsible for endogenous ligand recognition (Pin et al., 2003), in addition to the seven strand transmembrane domain, which is characteristic of all GPCRs. The mGluRs possess a large bi-lobed extracellular N-terminus of ~560 amino acids which has been shown by mutagenesis studies to confer glutamate binding, agonist activation of the receptor, and subtype specificity for group selective agonists (Schoepp et al., 1999). Since mGluRs have neuromodulatory role in the control of both glutamatergic and GABAergic neurotransmission, there has been much interest to develop novel mGluR ligands for therapeutic purposes of a variety of neurological and psychiatric conditions. The mGluRs have been proposed to be involved in physiological and pathophysiological processes of a number of CNS disorders, including anxiety, pain, depression, neurodegenerative disorders, schizophrenia, epilepsy, and drug abuse. In order to

Synthesis and evaluation of N-(methylthiophenyl)picolinamide derivatives as PET radioligands for metabotropic glutamate receptor subtype 4.

In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinsons disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/μmol (n=6) and 463±263GBq/μmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications.

Re-exploring the N-phenylpicolinamide derivatives to develop mGlu4 ligands with improved affinity and in vitro microsomal stability.

In recent years, mGlu4 has received great attention and research effort because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinsons disease (PD). Many positive allosteric modulators of mGlu4 have been developed. To better understand the role of mGlu4 in healthy and disease conditions, we are interested in developing an mGlu4 selective radioligand for in vivo studies. Thus, we had synthesized and studied [(11)C]2 as a PET tracer for mGlu4, which demonstrated some promising features as a PET radioligand as well as the limitation need to be improved. In order to develop an mGlu4 ligand with enhanced affinity and improved metabolic stability, we have modified, synthesized and evaluated a series of new N-phenylpicolinamide derivatives. The SAR study has discovered a number of compounds with low nM affinity to mGlu4. The dideuteriumfluoromethoxy modified compound 24 is identified as a very promising mGlu4 ligand, which has demonstrated enhanced affinity, improved in vitro microsomal stability, good selectivity and good permeability.

CHAPTER 10:mGlu4 PET Ligands as Enablers of Target Biology Understanding

Identifying the biological origin of a disease and the potential targets for intervention are some of the first steps in the discovery of a medicine. The metabotropic glutamate receptor 4 (mGlu4) has lately received much attention due to its potential role in various neuronal diseases such as Parkinson’s disease (PD) and other disorders. To better understand the role of mGlu4 in health and disease conditions, we were interested in developing an mGlu4-selective radiotracer for in vivo studies. Herein we discuss our efforts to develop novel mGlu4 positron emission tomography (PET) radiotracers from mGlu4 positive allosteric modulators (PAMs). We have investigated the biology of this receptor in vitro using cells expressing mGlu4, as well as in vivo, using the 6-hydroxydopamine (6-OHDA)-lesion rat model of Parkinson’s disease. Results of cell studies showed the important role of endogenous glutamate in inducing changes in the affinity of allosteric modulators of mGlus. Results from PET imaging studies showed the interplay between presynaptic mGlu4 and postsynaptic mGlu5 expression in the lesioned side of the brain, while the results of pharmacological MRI studies of the hemodynamic response showed enhanced signal changes in cortical areas in the lesioned side after challenge with mGlu4 PAMs or mGlu5 negative allosteric monitors (NAMs).