Terence G. Hamill

[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.

Positron emission tomography neuroreceptor imaging as a tool in drug discovery, research and development.

Improved communication and cooperation between research-driven drug companies and academic positron emission tomography (PET) centers, coupled with improvements in PET camera resolution, the availability of small animal PET cameras and a growing list of neuroreceptor-specific PET tracers, have all contributed to a substantial increase in the use and value of PET as a tool in central nervous system drug discovery and development.

18F-FPEB, a PET Radiopharmaceutical for Quantifying Metabotropic Glutamate 5 Receptors: A First-in-Human Study of Radiochemical Safety, Biokinetics, and Radiation Dosimetry

Identification of safe and valid PET radioligands for metabotropic glutamate receptor, type 5 (mGluR5), is essential to measure changes in brain mGluR5 in neuropsychiatric disorders, to confirm central mGluR5 occupancy of drug candidates, and to guide dose selection for obtaining an optimum therapeutic window. Here we present the results of a first-in-human study assessing the safety and effectiveness of a novel PET radiopharmaceutical, 18F-3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile (18F-FPEB), for quantifying regional brain concentrations of mGluR5. Methods: Quantification of whole-body biokinetics was conducted in 6 healthy adults (3 men and 3 women). The radiation safety profile was estimated with OLINDA/EXM software. Subsequently, pairs of dynamic brain scans were obtained for 11 healthy men to identify optimal methods for derivation of regional distribution volume and binding potential and to determine the repeatability of measurement. Results: The whole-body effective radiation dose was approximately 17 μSv/MBq (62 mrem/mCi), with the gallbladder receiving the highest dose of 190 μSv/MBq. In brain studies, time–activity curves showed high accumulation in the insula/caudate nucleus, moderate uptake in the thalamus, and the lowest concentration in the cerebellum/pons. The plasma reference graphical analysis method appeared optimal for 18F-FPEB; it showed acceptable test–retest variability of nondisplaceable binding potential (<10%) and identified the highest nondisplaceable binding potential values (from ∼0.5 in the globus pallidus to ∼3.5 in the insula) for target regions. Safety assessments revealed no clinically meaningful changes in vital signs, electrocardiogram, or laboratory values. Conclusion: 18F-FPEB is safe and well tolerated, and its regional cerebral distribution is consistent with previous reports in the literature for metabotropic glutamate receptors. The repeatability of measurement suggests that 18F-FPEB is suitable for quantifying mGluR5 in humans.

Non-invasive radiotracer imaging as a tool for drug development.

Non-Invasive Radiotracer Imaging (NIRI) uses either short-lived positron-emitting isotopes, such as 11C and 18F, for Positron Emis ion Tomography (PET) or single photon emitting nuclides, e.g., 123I, which provide images using planar imaging or Single-Photon Emission Computed Tomography (SPECT). These high-resolution imaging modalities provide anatomical distribution and localization of radiolabeled drugs, which can be used to generate real time receptor occupancy and off-rate studies in humans. This can be accomplished by either isotopically labeling a potential new drug (usually with 11C), or indirectly by studying how the unlabelled drug inhibits specific radioligand binding in vivo. Competitive blockade studies can be accomplished using a radiolabeled analogue which binds to the site of interest, rather than a radiolabeled version of the potential drug. Imaging, particularly PET imaging, can be used to demonstrate the effect of a drug through a biochemical marker of processes such as glucose metabolism or blood flow. NIRI as a development tool in the pharmaceutical industry is gaining increased acceptance as its unique ability to provide such critical information in human subjects is recognized. This section will review recent examples that illustrate the utility of NIRI, principally PET, in drug development, and the potential of imaging advances in the development of cancer drugs and gene therapy. Finally, we provide a brief overview of the design of new radiotracers for novel targets.

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.

Efficient radiosynthesis of 3'-deoxy-3'-18F-fluorothymidine using electrowetting-on-dielectric digital microfluidic chip.

Access to diverse PET tracers for preclinical and clinical research remains a major obstacle to research in cancer and other disease research. The prohibitive cost and limited availability of tracers could be alleviated by microfluidic radiosynthesis technologies combined with a high-yield microscale radiosynthetic method. In this report, we demonstrate the multistep synthesis of 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) with high yield on an electrowetting-on-dielectric (EWOD) microfluidic radiosynthesizer, previously developed in our group. We have identified and established several parameters that are most critical in the microscale radiosynthesis, such as the reaction time, reagent concentration, and molar ratios, to successfully synthesize 18F-FLT in this compact platform. Methods: 18F-FLT was synthesized from the 3-N-Boc-1-[5-O-(4,4′-dimethoxytrityl)-3-O-nosyl-2-deoxy-β-d-lyxofuranosyl] thymine precursor on the EWOD chip starting from the first solvent exchange and 18F-fluoride ion activation step to the final deprotection step. The fluorination reaction was performed in a mixture of thexyl alcohol and dimethyl sulfoxide. The crude product after deprotection was collected from the chip and purified on a custom-made solid-phase extraction cartridge and subjected to quality control testing. The purified 18F-FLT was suitable for small-animal PET studies in multiple nude mice xenografted with the A431 carcinoma cell line. Results: 18F-FLT was successfully synthesized on the EWOD microdevice coupled with an off-chip solid-phase extraction purification with a decayed-corrected radiochemical yield of 63% ± 5% (n = 5) and passed all of the quality control tests required by the U.S. Pharmacopeia for radiotracers to be injected into humans. We have successfully demonstrated the synthesis of several batches of 18F-FLT on EWOD, starting with approximately 333 MBq of radioactivity and obtained up to 52 MBq (non–decay-corrected) of 18F-FLT on cartridge purification. The specific activity of 2 representative preparations of 18F-FLT synthesized on the EWOD chip were measured to be 1,800 and 2,400 GBq/μmol. Conclusion: The EWOD microchip and optimized synthesis method in combination represent an effective platform for synthesizing 18F-FLT with high yield and of good quality for imaging. This compact platform, with configurable synthesis steps, could potentially form the basis of a stand-alone system that decouples PET probe production from the cyclotron and specialized radiochemistry facilities and increases diversity and flexibility in probe production.

Equivalent Dynamic Human Brain NK1-Receptor Occupancy Following Single-Dose i.v. Fosaprepitant vs. Oral Aprepitant as Assessed by PET Imaging

The type 1 neurokinin receptor (NK1R) antagonist aprepitant and its i.v. prodrug fosaprepitant have been approved for prevention of acute and delayed nausea and vomiting associated with chemotherapy. This study evaluated the magnitude and duration of brain NK1R occupancy over a period of 5 days after single‐dose i.v. infusion of 150‐mg fosaprepitant and single‐dose oral administration of 165‐mg aprepitant, using serial [18F]MK‐0999 positron emission tomography (PET) in 16 healthy subjects. Each subject underwent three scans. Brain NK1R occupancy rates after i.v. fosaprepitant at time to peak concentration (Tmax; ~30 min), 24, 48, and 120 h after the dose were 100, 100, ≥97, and 41–75%, respectively. After aprepitant, NK1R occupancy rates at these time points (Tmax ~4 h) were ≥99, ≥99, ≥97, and 37–76%, respectively. Aprepitant plasma concentration profiles were comparable for the two dosage forms. The study illustrates the utility of PET imaging in determining central bioequivalence in a limited number of subjects.

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.

Potent, Brain-Penetrant, Hydroisoindoline-Based Human Neurokinin-1 Receptor Antagonists

3-[(3aR,4R,5S,7aS)-5-{(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy}-4-(4-fluorophenyl)octahydro-2H-isoindol-2-yl]cyclopent-2-en-1-one (17) is a high affinity, brain-penetrant, hydroisoindoline-based neurokinin-1 (NK(1)) receptor antagonist with a long central duration of action in preclinical species and a minimal drug-drug interaction profile. Positron emission tomography (PET) studies in rhesus showed that this compound provides 90% NK(1) receptor blockade in rhesus brain at a plasma level of 67 nM, which is about 10-fold more potent than aprepitant, an NK(1) antagonist marketed for the prevention of chemotherapy-induced and postoperative nausea and vomiting (CINV and PONV). The synthesis of this enantiomerically pure compound containing five stereocenters includes a Diels-Alder condensation, one chiral separation of the cyclohexanol intermediate, an ether formation using a trichloroacetimidate intermediate, and bis-alkylation to form the cyclic amine.