H. Donald Burns

[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.

The acyclic CB1R inverse agonist taranabant mediates weight loss by increasing energy expenditure and decreasing caloric intake

Cannabinoid 1 receptor (CB1R) inverse agonists are emerging as a potential obesity therapy. However, the physiological mechanisms by which these agents modulate human energy balance are incompletely elucidated. Here, we describe a comprehensive clinical research study of taranabant, a structurally novel acyclic CB1R inverse agonist. Positron emission tomography imaging using the selective CB1R tracer [(18)F]MK-9470 confirmed central nervous system receptor occupancy levels ( approximately 10%-40%) associated with energy balance/weight-loss effects in animals. In a 12-week weight-loss study, taranabant induced statistically significant weight loss compared to placebo in obese subjects over the entire range of evaluated doses (0.5, 2, 4, and 6 mg once per day) (p < 0.001). Taranabant treatment was associated with dose-related increased incidence of clinical adverse events, including mild to moderate gastrointestinal and psychiatric effects. Mechanism-of-action studies suggest that engagement of the CB1R by taranabant leads to weight loss by reducing food intake and increasing energy expenditure and fat oxidation.

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.

Reducing Abuse Liability of GABAA/Benzodiazepine Ligands via Selective Partial-Agonist Efficacy at α1 and α2/3 Subtypes

Abuse-liability-related effects of subtype-selective GABAA modulators were explored relative to the prototypic benzodiazepine lorazepam. 7-Cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine (TPA123) has weak partial agonist efficacy at α1-, α2-, α3-, and α5-containing GABAA receptors, whereas 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) has weaker partial agonist efficacy at α2 and α3 and none at α1 and α5 subtypes. For both compounds, preclinical data suggested efficacy as nonsedating anxiolytics. Self-injection of TPA123 (0.0032–0.1 mg/kg) and TPA023 (0.0032–0.32 mg/kg) was compared with lorazepam (0.01–0.32 mg/kg) in baboons. TPA123 and lorazepam maintained self-injection higher than vehicle at two or more doses in each baboon; peak rate of self-injection of lorazepam was higher than TPA123. Self-injected lorazepam and TPA123 also increased rates of concurrently occurring food-maintained behavior. After the availability of self-administered TPA123 doses ended, an effect consistent with a mild benzodiazepine-like withdrawal syndrome occurred. In contrast with lorazepam and TPA123, TPA023 did not maintain self-administration. Positron emission tomography studies showed that TPA023 produced a dose-dependent inhibition in the binding of [11C]flumazenil to the benzodiazepine binding site in the baboon, which was essentially complete (i.e., 100% occupancy) at the highest TPA023 dose (0.32 mg/kg). In a physical dependence study, TPA023 (32 mg/kg/24 h) was delivered as a continuous intragastric drip. Neither flumazenil at 14 days nor stopping TPA023 after 30 to 31 days resulted in the marked withdrawal syndrome characteristic of benzodiazepines in baboons. In the context of other data, elimination of efficacy at the α1 subtype of the GABA/benzodiazepine receptor is not sufficient to eliminate abuse liability but may do so when coupled with reduced α2/3 subtype efficacy.

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.

A remote-controlled high pressure reactor for radiotracer synthesis with [11C]carbon monoxide.

[11C]Carbon monoxide is a versatile building block for the synthesis of PET radiotracers. However, the difficulty of trapping [11C]CO in a small solvent volume has limited its utility. We wish to report the details of a simple, remotely operated High Pressure Reactor (HiPR) system for trapping and reacting practical quantities of [11C]CO. All parts used in the HiPR are commercially available, providing an inexpensive and easily assembled system. A number of compounds have been synthesized using the HiPR via palladium mediated reactions with [11C]CO, an aryl halide, and a nucleophile dissolved in dioxane. For example, AMPA receptor modulator [11C]CX546 was synthesized from its respective precursors in 37% isolated yield, uncorrected from trapped [11C]CO.

Nuclear imaging in drug discovery, development, and approval

Explores the application of nuclear imaging as a research tool for use by pharmaceutical and biotechnology companies to aid in drug discovery, development and the approval of new pharmaceuticals.

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.

In vitro and in vivo properties of 3-tert-butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d]-[1,2,4]triazine (MRK-016), a GABAA receptor alpha5 subtype-selective inverse agonist

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human α1-, α2-, α3-, and α5-containing GABAA receptors. It has inverse agonist efficacy selective for the α5 subtype, and this α5 inverse agonism is greater than that of the prototypic α5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (α5IA). Consistent with its greater α5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than α5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC50 value of 15 ng/ml that was similar to the rhesus monkey plasma EC50 value of 21 ng/ml obtained using [11C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3–0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species (∼3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.