Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain

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Abstract

2-((4-(1-[11C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC50 = 0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [11C]CH3I, ∼45% yield, >92% radiochemical purity, >370 GBq/μmol specific activity at end of bombardment (EOB). Evaluation in Sprague–Dawley rats revealed that [11C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [11C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood–brain-barrier may limit the clinical utility of [11C]MP-10 as a PDE10A PET tracer.

Graphical abstract

Structure and microPET image of [11C]MP-10.

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Introduction

Phosphodiesterase 10A (PDE10A) is a unique dual specificity phosphodiesterase that converts both cyclic adenosine monophosphate (cAMP) to adenosine monophosphate (AMP) and cyclic guanosine monophosphate (cGMP) to guanosine monophosphate (GMP).1, 2 Unlike other phosphodiesterase (PDE) families, PDE10A is uniquely expressed in the brain where it plays a critical role in dopaminergic neurotransmission.3, 4 The expression of PDE10A is highest in the medium spiny neurons of the striatum (caudate and putamen), nucleus accumbens, and olfactory tubercle of mice, dogs, cynomolgus and humans.5 Abnormal striatal levels of PDE10A impair striatal output that may contribute substantially to the pathophysiology of schizophrenia, Huntington’s disease (HD), Parkinson disease (PD), Tourette syndrome, and drug abuse.6, 7, 8, 9 PDE10A plays a major role in the regulation of cyclic nucleotide signaling cascades; inhibition of the enzyme causes cAMP response element-binding (CREB) phosphorylation and activation.10 For Huntington’s disease, western analysis of 1 μg protein extracts from the striatum of R6/1 and R6/2 Huntington’s disease mice prior to motor manifestation development displayed lower PDE10A proteins levels than in wild type mice, and may be an early marker of neuronal dysfunction. Post mortem analysis of human brain tissue has shown similar results: western analysis of 5 μg of total protein samples from the caudate, nucleus accumbens and putamen from three patients with grade 3 HD demonstrated reduced PDE10A protein levels compared with age-matched neurologically normal control subjects.10, 11, 12 Inhibition studies with 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline (TP-10), an analogue of 2-((4-(1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)-methyl)quinoline (MP-10, 7) in a rodent quinolinic acid model of Huntington’s disease suggest that PDE10A inhibition may be a novel neuroprotective approach to the treatment of HD. In addition to the potential application of PDE10A inhibitors in HD, the observation that PDE10A expression parallels D2-receptor distribution suggests that PDE10A inhibition might yield therapeutic effects similar to D2 antagonism for schizophrenia patients, without accompanying untoward effects. Inhibition of PDE10A in rodents significantly increased extra-cellular levels of cAMP in the striatum and also mediated certain clinical antipsychotic effects13, 14 with significant differences from D2-receptor antagonists in preclinical behavioral models of schizophrenia including the induction of catalepsy and prepulse inhibition of startle.14, 15

Over the past 10 years, tremendous efforts have been made to develop PDE10A inhibitors for treatment of schizophrenia and associated mental disorders.16, 17, 18, 19 MP-10 has been identified as a promising therapeutic inhibitor of PDE10A and is in clinical evaluation.16, 17 However, most evidence supporting the role of PDE10A in CNS disorders comes from behavioral studies and post mortem tissue analysis. Positron emission tomography (PET) is a non-invasive imaging modality that can provide functional information about molecular and cellular processes in living subjects. Therefore, a PET radioligand having high affinity and selectivity for PDE10A would provide a unique tool to study changes in PDE10A levels of living subjects and investigate the physiological function of PDE10A in the CNS. Our group reported initial results for imaging PDE10A in vivo.20, 21 Recently, an initial validation of an MP-10 analogue, 2-((4-(1-[18F]ethyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)-methyl)quinoline (18F-JNJ41510417) was reported.22 To confirm if MP-10 could serve as a PET probe for quantifying PDE10A in vivo, we radiosynthesized [11C]MP-10 and conducted in vivo validation studies in rodent and non-human primate models. In this paper, we will describe the radiosynthesis of [11C]MP-10, its biodistribution and validation in vivo in Sprague–Dawley rats, MicroPET imaging studies of [11C]MP-10 in rhesus macaque, as well as the metabolite analysis of the rat brain tissue, plasma and monkey plasma post-injection of [11C]MP-10 in animals.

Section snippets

Chemistry

The target compound MP-10 is a pyrazole derivative, that possesses a methyl group on the nitrogen, which gives easy access to 11C-labeling by N-methylation of the corresponding desmethyl precursor (6) with [11C]methyl iodide ([11C]CH3I). The synthesis of the N-desmethyl precursor 6 and the standard compound 7 was accomplished by starting with methyl 4-hydroxybenzoate (1) as shown in Scheme 1 following the reported procedure.16, 23 However, reference compound 7 and its isomer 8 were synthesized

General

All analytical grade chemicals and reagents were purchased from Sigma–Aldrich (Milwaukee, WI) and were used without further purification unless otherwise specified. Flash column chromatography was conducted using Scientific Adsorbents, Inc. silica gel, 60 Å, ‘40 Micron Flash’ (32–63 μ). Melting points were determined using MEL-TEMP 3.0 apparatus and uncorrected. 1H NMR spectra were recorded at 300 MHz on a Varian Mercury-VX spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS)

Conclusion

In summary, [11C]MP-10, a high affinity and high selectivity ligand for enzyme PDE10A was successfully radiosynthesized by methylating the corresponding desmethyl precursor with [11C]CH3I in DMF in the presence of sodium hydride as the base. Biodistribution study of [11C]MP-10 was conducted in male Sprague–Dawley rats and found that the highest uptake in the brain was in the striatum, the PDE10A enriched region. The highest target to non-target (striatum to cerebellum) ratio was found to be

Acknowledgment

Financial support for these studies was provided by the National Institute of Health under 5R33MH081281-04 (RHM), NS058714, 1R21NS061025-01A2 and Intramural grant MIR-11-009 of Mallinckrodt Institute of Radiology in Washington University School of Medicine. The authors gratefully thank Christina M. Zukas, John Hood, Darryl Craig, Ruike Wang, Aixiao Li and Junfeng Li for their excellent technical assistance for the microPET studies in non-human primate.

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