Shil Patel

[18F]MK-9470, a positron emission tomography (PET) tracer for in vivo human PET brain imaging of the cannabinoid-1 receptor

[18F]MK-9470 is a selective, high-affinity, inverse agonist (human IC50, 0.7 nM) for the cannabinoid CB1 receptor (CB1R) that has been developed for use in human brain imaging. Autoradiographic studies in rhesus monkey brain showed that [18F]MK-9470 binding is aligned with the reported distribution of CB1 receptors with high specific binding in the cerebral cortex, cerebellum, caudate/putamen, globus pallidus, substantia nigra, and hippocampus. Positron emission tomography (PET) imaging studies in rhesus monkeys showed high brain uptake and a distribution pattern generally consistent with that seen in the autoradiographic studies. Uptake was blocked by pretreatment with a potent CB1 inverse agonist, MK-0364. The ratio of total to nonspecific binding in putamen was 4–5:1, indicative of a strong specific signal that was confirmed to be reversible via displacement studies with MK-0364. Baseline PET imaging studies in human research subject demonstrated behavior of [18F]MK-9470 very similar to that seen in monkeys, with very good test–retest variability (7%). Proof of concept studies in healthy young male human subjects showed that MK-0364, given orally, produced a dose-related reduction in [18F]MK-9470 binding reflecting CB1R receptor occupancy by the drug. Thus, [18F]MK-9470 has the potential to be a valuable, noninvasive research tool for the in vivo study of CB1R biology and pharmacology in a variety of neuropsychiatric disorders in humans. In addition, it allows demonstration of target engagement and noninvasive dose-occupancy studies to aid in dose selection for clinical trials of CB1R inverse agonists.

Synthesis, characterization, and monkey PET studies of [18F]MK-1312, a PET tracer for quantification of mGluR1 receptor occupancy by MK-5435

Two moderately lipophilic, high affinity ligands for metabotropic glutamate receptor subtype 1 (mGluR1) were radiolabeled with a positron‐emitting radioisotope and evaluated in rhesus monkey as potential PET tracers. Both ligands were radiolabeled with fluorine‐18 via nucleophilic displacement of the corresponding 2‐chloropyridine precursor with [18F]potassium fluoride. [18F]MK‐1312 was found to have a suitable signal for quantification of mGluR1 receptors in nonhuman primates and was more thoroughly characterized. In vitro autoradiographic studies with [18F]MK‐1312 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the known distribution of mGluR1, with the highest uptake in the cerebellum, moderate uptake in the hippocampus, thalamus, and cortical regions, and lowest uptake in the caudate and putamen. In vitro saturation binding studies in rhesus monkey and human cerebellum homogenates confirmed that [18F]MK‐1312 binds to a single site with a Bmax/Kd ratio of 132 and 98, respectively. PET studies in rhesus monkey with [18F]MK‐1312 showed high brain uptake and a regional distribution consistent with in vitro autoradiography results. Blockade of [18F]MK‐1312 uptake with mGluR1 allosteric antagonist MK‐5435 dose‐dependently reduced tracer uptake in all regions of gray matter to a similarly low level of tracer uptake. This revealed a large specific signal useful for determination of mGluR1 receptor occupancy in rhesus monkey. Taken together, these results are promising for clinical PET studies with [18F]MK‐1312 to determine mGluR1 occupancy of MK‐5435. Synapse 2011.

Inverse agonist histamine H3 receptor PET tracers labelled with carbon-11 or fluorine-18

Two histamine H3 receptor (H3R) inverse agonist PET tracers have been synthesized and characterized in preclinical studies. Each tracer has high affinity for the histamine H3 receptor, has suitable lipophilicity, and neither is a substrate for the P‐glycoprotein efflux pump. A common phenolic precursor was used to synthesize each tracer with high specific activity and radiochemical purity by an alkylation reaction using either [11C]MeI or [18F]FCD2Br. Autoradiographic studies in rhesus monkey and human brain slices showed that each tracer had a widespread distribution with high binding densities in frontal cortex, globus pallidus and striatum, and lower uptake in cerebellum. The specificity of this expression pattern was demonstrated by the blockade of the autoradiographic signal by either the H3R agonist R‐α‐methylhistamine or a histamine H3R inverse agonist. In vivo PET imaging studies in rhesus monkey showed rapid uptake of each tracer into the brain with the same distribution seen in the autoradiographic studies. Each tracer could be blocked by pretreatment with a histamine H3R inverse agonist giving a good specific signal. Comparison of the in vitro metabolism of each compound showed slower metabolism in human liver microsomes than in rhesus monkey liver microsomes, with each compound having a similar clearance rate in humans. The in vivo metabolism of 1b in rhesus monkey showed that at 60 min, ∼35% of the circulating counts were due to the parent. These tracers are very promising candidates as clinical PET tracers to both study the histamine H3R system and measure receptor occupancy of H3R therapeutic compounds. Synapse 63:1122–1132, 2009.

The synthesis and preclinical evaluation in rhesus monkey of [18F]MK-6577 and [11C]CMPyPB glycine transporter 1 positron emission tomography radiotracers

Two positron emission tomography radiotracers for the glycine transporter 1 (GlyT1) are reported here. Each radiotracer is a propylsulfonamide‐containing benzamide and was labeled with either carbon‐11 or fluorine‐18. [11C]CMPyPB was synthesized by the alkylation of a 3‐hydroxypyridine precursor using [11C]MeI, and [18F]MK‐6577 was synthesized by a nucleophilic aromatic substitution reaction using a 2‐chloropyridine precursor. Each tracer shows good uptake into rhesus monkey brain with the expected distribution of highest uptake in the pons, thalamus, and cerebellum and lower uptake in the striatum and gray matter of the frontal cortex. In vivo blockade and chase studies of [18F]MK‐6577 showed a large specific signal and reversible binding. In vitro autoradiographic studies with [18F]MK‐6577 showed a large specific signal in both rhesus monkey and human brain slices and a distribution consistent with the in vivo results and those reported in the literature. In vivo metabolism studies in rhesus monkeys demonstrated that only more‐polar metabolites are formed for each tracer. Of these two tracers, [18F]MK‐6577 was more extensively characterized and is a promising clinical positron emission tomography tracer for imaging GlyT1 and for measuring GlyT1 occupancy of therapeutic compounds. Synapse, 2011.

In vitro characterization of [3H]MethoxyPyEP, an mGluR5 selective radioligand

We have characterized the in vitro properties of 3-[3H]methoxy-5-(pyridin-2-ylethynyl)pyridine ([3H]MethoxyPyEP), an analogue of the mGluR(5) receptor subtype antagonist MPEP [2-methyl-6-(phenylethynyl)-pyridine], in rat tissue preparations using tissue homogenates and autoradiography. Binding of [3H]MethoxyPyEP to rat cortex, hippocampus, thalamus and cerebellum membrane preparations revealed saturable, high affinity binding (3.4 +/- 0.4 nM, n = 4 in rat cortex) to a single population of receptors in all regions studied except for cerebellum. Binding was found to be relatively insensitive to pH and insensitive to DTT. High concentrations of NEM both reduce receptor concentration and binding affinity for the radioligand. In time-course studies at room temperature k(on) and k(off) were determined as 2.9 x 10(7) M(-1) min(-1) and 0.11 min(-1) respectively. The rank order of affinities, as assessed by equilibrium competition studies, of a variety of ligands suggested binding of the radioligand selectively to mGluR5 (MPEP > trans-azetidine-2,4-dicarboxylic acid congruent with (S)-4-carboxyphenylglycine congruent with (+)MK801 congruent with CP-101,606 congruent with clozapine congruent with atropine congruent with ketanserin congruent with yohimbine congruent with benoxathian). Autoradiographic studies with [3H]MethoxyPyEP showed that binding was regioselective, with high density of binding in caudate and hippocampus, intermediate binding in thalamus and very low density in the cerebellum. These data show that [3H]MethoxyPyEP is a high affinity radioligand useful for the in vitro study of mGluR5 receptor distribution and pharmacologic properties in brain.

An in vitro assay for predicting successful imaging radiotracers.

PURPOSEnTo develop an in vitro binding assay able to predict whether a radiolabel is likely to be a useful clinical tracer for positron emission tomography (PET).nnnPROCEDURESnRodent and rhesus brain sections were incubated with radioligands, most of which are tritiated or iodinated versions of known clinical PET radiotracers, and assayed for binding to brain receptors for a 20-minute period using a no-wash protocol (n=>/=3).nnnRESULTSnRadiolabeled flumazenil (RO-151788), WAY100635, N-methylscopolamine, N-methylspiperone, raclopride, citalopram, (1-)2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), paroxetine, and 4-(2-methoxyphenyl)-1-[2-[N-(2-pyridinyl)-p-flurobenzamido]ethyl]piperazine (MPPF) were assessed for binding to either rhesus caudate putamen, and/or frontal cortex, or rat whole brain sections. Specific binding for these compounds ranged from 0 to 94% by 20 minutes. Those with %-specific binding less than 10% have also been shown to not be effective as in vivo PET radiotracers. In addition, successful PET radiotracers incubated in tissue sections with target receptor either absent or present in low density behaved poorly in this assay, as expected, as did radiolabels previously shown to possess high non-specific binding.nnnCONCLUSIONSnAn in vitro binding assay using rodent and rhesus brain sections has been developed that, within the currently assayed radiotracers, is able to rapidly predict whether a radiolabeled compound is a useful clinical PET radiotracer. This method suggests significant potential for the rapid in vitro evaluation of potential in vivo PET radiotracers.