Preclinical Characterization of the Phosphodiesterase 10A PET Tracer [(11)C]MK-8193.

PURPOSE: A positron emission tomography (PET) tracer for the enzyme phosphodiesterase 10A (PDE10A) is desirable to guide the discovery and development of PDE10A inhibitors as potential therapeutics. The preclinical characterization of the PDE10A PET tracer [(11)C]MK-8193 is described. PROCEDURES: In vitro binding studies with [(3)H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [(11)C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor. RESULTS: [(3)H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [(11)C]MK-8193 displays rapid kinetics, low test-retest variability, and a large specific signal that is displaced by a structurally diverse PDE10A inhibitor, enabling the determination of pharmacokinetic/enzyme occupancy relationships. CONCLUSIONS: [(11)C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [(11)C]MK-8193 in humans is warranted.

Methyl-substitution of an iminohydantoin spiropiperidine ß-secretase (BACE-1) inhibitor has a profound effect on its potency.

The IC50 of a beta-secretase (BACE-1) lead compound was improved ∼200-fold from 11 µM to 55 nM through the addition of a single methyl group. Computational chemistry, small molecule NMR, and protein crystallography capabilities were used to compare the solution conformation of the ligand under varying pH conditions to its conformation when bound in the active site. Chemical modification then explored available binding pockets adjacent to the ligand. A strategically placed methyl group not only maintained the required pKa of the piperidine nitrogen and filled a small hydrophobic pocket, but more importantly, stabilized the conformation best suited for optimized binding to the receptor.

Discovery of novel triazolobenzazepinones as γ-secretase modulators with central Aß42 lowering in rodents and rhesus monkeys.

Synthesis and SAR studies of novel triazolobenzazepinones as gamma secretase modulators (GSMs) are presented in this communication. Starting from our azepinone leads, optimization studies toward improving central lowering of Aß42 led to the discovery of novel benzo-fused azepinones. Several benzazepinones were profiled in vivo and found to lower brain Aß42 levels in Sprague Dawley rats and transgenic APP-YAC mice in a dose-dependent manner after a single oral dose. Compound 34 was further progressed into a pilot study in our cisterna-magna-ported rhesus monkey model, where we observed robust lowering of CSF Aß42 levels.

The relationship between glycine transporter 1 occupancy and the effects of the glycine transporter 1 inhibitor RG1678 or ORG25935 on object retrieval performance in scopolamine impaired rhesus monkey.

Reduced NMDA receptor functioning is hypothesized to underlie the cognitive and negative symptoms associated with schizophrenia. However, because direct activation of the NMDA receptor is accompanied by neurotoxicity, mechanisms that activate the glycine co-agonist site on the NMDA receptor could carry greater therapeutic potential. In the current study, the effects of two glycine transporter 1 (GlyT1) inhibitors, RG1678 and ORG25935, were characterized in the object-retrieval detour (ORD) task in scopolamine-impaired rhesus monkeys and, using positron emission tomography (PET), the GlyT1 occupancy to efficacy relationship of each compound was established. Scopolamine exerted a significant decrease in accuracy in the ORD task. Lower doses of RG1678 (0.3 and 1.0 mg/kg, p.o.) significantly attenuated the impact of scopolamine, whereas the highest dose tested (1.8 mg/kg) did not. The predicted GlyT1 occupancies of RG1678 at the effective doses were ~10 and 30 %. ORG25935 (0.1, 0.3, and 1 mg/kg, p.o.) also significantly attenuated the impact of scopolamine on the ORD task, whereas 3 mg/kg did not. The predicted GlyT1 occupancies of ORG25935 at the effective doses ranged from 16 to 80 %. These data suggest that GlyT1 inhibitors have the potential to improve performance on prefrontal cortex-dependent tests such as the ORD task, but that efficacy is lost when higher occupancies are achieved. Importantly, recent Ph2B data published by Roche suggests that low but not high doses of RG1678 improved negative symptoms in patients with schizophrenia, highlighting the potential translational nature of the current preclinical findings.

In vivo quantification of calcitonin gene-related peptide receptor occupancy by telcagepant in rhesus monkey and human brain using the positron emission tomography tracer [11C]MK-4232.

Calcitonin gene-related peptide (CGRP) is a potent neuropeptide whose agonist interaction with the CGRP receptor (CGRP-R) in the periphery promotes vasodilation, neurogenic inflammation and trigeminovascular sensory activation. This process is implicated in the cause of migraine headaches, and CGRP-R antagonists in clinical development have proven effective in treating migraine-related pain in humans. CGRP-R is expressed on blood vessel smooth muscle and sensory trigeminal neurons and fibers in the periphery as well as in the central nervous system. However, it is not clear what role the inhibition of central CGRP-R plays in migraine pain relief. To this end, the CGRP-R positron emission tomography (PET) tracer [(11)C]MK-4232 (2-[(8R)-8-(3,5-difluorophenyl)-6,8-[6-(11)C]dimethyl-10-oxo-6,9-diazaspiro[4.5]decan-9-yl]-N-[(2R)-2'-oxospiro[1,3-dihydroindene-2,3'-1H-pyrrolo[2,3-b]pyridine]-5-yl]acetamide) was discovered and developed for use in clinical PET studies. In rhesus monkeys and humans, [(11)C]MK-4232 displayed rapid brain uptake and a regional brain distribution consistent with the known distribution of CGRP-R. Monkey PET studies with [(11)C]MK-4232 after intravenous dosing with CGRP-R antagonists validated the ability of [(11)C]MK-4232 to detect changes in CGRP-R occupancy in proportion to drug plasma concentration. Application of [(11)C]MK-4232 in human PET studies revealed that telcagepant achieved only low receptor occupancy at an efficacious dose (140 mg PO). Therefore, it is unlikely that antagonism of central CGRP-R is required for migraine efficacy. However, it is not known whether high central CGRP-R antagonism may provide additional therapeutic benefit.

Discovery of MK-3168: A PET Tracer for Imaging Brain Fatty Acid Amide Hydrolase.

We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.

[(11)C]MK-4232: The First Positron Emission Tomography Tracer for the Calcitonin Gene-Related Peptide Receptor.

Rational modification of the potent calcitonin gene-related peptide (CGRP) receptor antagonist MK-3207 led to a series of analogues with enhanced CNS penetrance and a convenient chemical handle for introduction of a radiolabel. A number of (11)C-tracers were synthesized and evaluated in vivo, leading to the identification of [(11)C]8 ([(11)C]MK-4232), the first positron emission tomography tracer for the CGRP receptor.

Evaluation of [¹8F]MK-0911, a positron emission tomography (PET) tracer for opioid receptor-like 1 (ORL1), in rhesus monkey and human.

Antagonism of the central opioid receptor like-1 receptor (ORL1) has been implicated in cognition, and has been a focus of drug discovery efforts to ameliorate the cognitive deficits that remain during the stable treatment of schizophrenia with current antipsychotics. In order to facilitate dose selection for phase II clinical testing an ORL1-specific PET tracer was developed to determine drug plasma concentration versus occupancy relationships in order to ensure that the doses selected and the degree of target engagement were sufficient to ensure adequate proof of concept testing. MK-0911 is a selective, high affinity antagonist for the ORL1 receptor radiolabeled with high specific activity (18)F for positron emission tomography (PET) studies. Evaluation of [(18)F]MK-0911 in rhesus monkey PET studies showed a pattern of brain uptake which was consistent with the known distribution of ORL1. In vitro autoradiography with [(18)F]MK-0911 in rhesus monkey and human brain tissue slices showed a regional distribution that was consistent with in vivo imaging results in monkey. Pre-treatment of rhesus monkeys with high doses of structurally diverse ORL1 antagonists MK-0584, MK-0337, or MK-5757 achieved blockade of [(18)F]MK-0911 in all gray matter regions. Baseline PET studies with [(18)F]MK-0911 in healthy human subjects showed tracer distribution and kinetics similar to that observed in rhesus monkey. Quantification of [(18)F]MK-0911 uptake in repeat human baseline PET studies showed a test-retest variability in volume of distribution (V(T)) averaging 3% across brain regions. Humans dosed orally with MK-5757 showed reduced [(18)F]MK-0911 tracer concentration in brain proportional with MK-5757 dose and plasma level. [(18)F]MK-0911 was useful for determining MK-5757-induced receptor occupancy of ORL1 to guide MK-5757 dose-selection for clinical proof-of-concept studies. Additionally, [(18)F]MK-0911 may be a useful tool for studying the pharmacology of ORL1 in various human populations and disease states.

Characterization of plasma ß-secretase (BACE1) activity and soluble amyloid precursor proteins as potential biomarkers for Alzheimer's disease.

Reduction in cerebrospinal fluid (CSF) amyloid ß42 (Aß42) and elevation in total tau and phospho-thr181 tau consistently differentiate between Alzheimer's disease (AD) and age-matched control subjects. In contrast, CSF ß-site APP-cleaving enzyme activity (BACE1) and soluble amyloid precursor proteins α and ß (sAPPα and sAPPß) are without consistent patterns in AD subjects. Plasma sampling is much easier, with fewer side effects, and is readily applied in primary care centers, so we have developed and validated novel plasma BACE activity, sAPPß, and sAPPα assays and investigated their ability to distinguish AD from age-matched controls. Plasma BACE activity assay was sensitive and specific, with signal being immunodepleted with a specific BACE1 antibody and inhibited with a BACE1-specific inhibitor. Plasma sAPPß and sAPPα assays were specific, with signal diluting linearly, immunodepleted with specific antibodies, and at background levels in APP knockout mice. In rhesus monkeys, BACE1 but not γ-secretase inhibitor led to significant lowering of plasma sAPPß with concurrent elevation of plasma sAPPα. AD subjects showed a significant increase in plasma BACE1 activity, sAPPß, sAPPα, and Aß42 (P < 0.001) compared with age-matched controls. In conclusion, plasma BACE activity and sAPP endpoints provide novel investigative biomarkers for AD diagnosis and potential pharmacodynamic biomarkers for secretase inhibitor studies.

Quantification of the glycine transporter 1 in rhesus monkey brain using [18F]MK-6577 and a model-based input function.

BACKGROUND: Glycine transporter 1 (GlyT1) inhibitors have emerged as potential treatments for schizophrenia due to their potentiation of NMDA receptor activity by modulating the local concentrations of the NMDA co-agonist glycine. [18F]MK-6577 is a potent and selective GlyT1 inhibitor PET tracer. Although differences in ligand kinetics can be expected between non-human primates and humans, the tracer pre-clinical evaluation can provide valuable information supporting protocol design and quantification in the clinical space. The main objective of this work was to evaluate the in vivo kinetics of [18F]MK-6577 in rhesus monkey brain. Additionally, a method for estimating the tracer input function from the tracer brain tissue kinetics and venous sampling was validated. This technique was applied for determination of the dose-occupancy relationship of a GlyT1 inhibitor in monkey brain. METHODS: Compartmental and Logan graphical analysis were utilized for quantification of the [18F]MK-6577 binding using the measured tracer arterial input function. The stability of the tracer volume of distribution relative to scan length was assessed. The proposed model-based input function method takes advantage of the agreement between the tracer concentration in arterial and venous plasma from ~5 min. The approach estimates the initial peak of the input curve by adding a gamma like function term to the measured venous curve. The parameters of the model function were estimated by simultaneously fitting several brain time activity curves to a compartmental model. RESULTS: Good agreement was found between the model-based and the measured arterial plasma curve and the corresponding distribution volumes. The Logan analysis was the preferred method of analysis providing reliable and stable volume of distribution and occupancy results using a 90 and possibly 60 min scan length. CONCLUSION: The model-based input function method and Logan analysis are well suited for quantification of [18F]MK-6577 binding and GlyT1 occupancy in monkey brain.