Liza Gantert

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 [11C]MK-4232 (2-[(8R)-8-(3,5-difluorophenyl)-6,8-[6-11C]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, [11C]MK-4232 displayed rapid brain uptake and a regional brain distribution consistent with the known distribution of CGRP-R. Monkey PET studies with [11C]MK-4232 after intravenous dosing with CGRP-R antagonists validated the ability of [11C]MK-4232 to detect changes in CGRP-R occupancy in proportion to drug plasma concentration. Application of [11C]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.

Activation of melanocortin MC4 receptors increases erectile activity in rats ex copula

Melanocortin peptide agonists, alpha-melanocyte stimulating hormone (alpha-MSH) and melanotan-II, stimulate erectile activity in a variety of species, including man. Since neither peptide discriminates amongst melanocortin receptors, it is not clear which subtype mediates these pro-erectile effects. Here, we present data that melanocortin-induced erectogenesis is mediated by melanocortin MC(4) receptors. Systemic administration of a melanocortin MC(4) receptor agonist (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1ylmethyl)piperidin-1-yl]-2-oxoethylamine; THIQ) with high selectivity over other melanocortin receptors enhanced intracavernosal pressure and stimulated erectile activity in rats ex copula. THIQ dose-dependently (1-5 mg/kg, i.v.) increased the total number of erections, to an extent comparable or greater than that produced by apomorphine (0.025 mg/kg, s.c.). Central administration of THIQ (20 microg, intracerebroventricular (i.c.v.)) increased the number of reflexive penile erections; whereas administration of both a nonselective endogenous melanocortin MC(4) receptor antagonist (agouti-related protein (AgRP), 5.5. microg, i.c.v.) and a melanocortin MC(4) receptor preferring antagonist (MPB10, 1 mg/kg, i.v.) blocked THIQ-induced erectogenesis. These pro-erectile effects were also attenuated by systemic or central administration of an oxytocin antagonist (L-368899, 1 mg/kg, i.v.). Thus, melanocortin MC(4) receptor activation is sufficient for erectogenesis and these effects may involve oxytocinergic pathways.

Preclinical Characterization of 18F-MK-6240, a Promising PET Tracer for In Vivo Quantification of Human Neurofibrillary Tangles

A PET tracer is desired to help guide the discovery and development of disease-modifying therapeutics for neurodegenerative diseases characterized by neurofibrillary tangles (NFTs), the predominant tau pathology in Alzheimer disease (AD). We describe the preclinical characterization of the NFT PET tracer 18F-MK-6240. Methods: In vitro binding studies were conducted with 3H-MK-6240 in tissue slices and homogenates from cognitively normal and AD human brain donors to evaluate tracer affinity and selectivity for NFTs. Immunohistochemistry for phosphorylated tau was performed on human brain slices for comparison with 3H-MK-6240 binding patterns on adjacent brain slices. PET studies were performed with 18F-MK-6240 in monkeys to evaluate tracer kinetics and distribution in the brain. 18F-MK-6240 monkey PET studies were conducted after dosing with unlabeled MK-6240 to evaluate tracer binding selectivity in vivo. Results: The 3H-MK-6240 binding pattern was consistent with the distribution of phosphorylated tau in human AD brain slices. 3H-MK-6240 bound with high affinity to human AD brain cortex homogenates containing abundant NFTs but bound poorly to amyloid plaque–rich, NFT-poor AD brain homogenates. 3H-MK-6240 showed no displaceable binding in the subcortical regions of human AD brain slices and in the hippocampus/entorhinal cortex of non-AD human brain homogenates. In monkey PET studies, 18F-MK-6240 displayed rapid and homogeneous distribution in the brain. The 18F-MK-6240 volume of distribution stabilized rapidly, indicating favorable tracer kinetics. No displaceable binding was observed in self-block studies in rhesus monkeys, which do not natively express NFTs. Moderate defluorination was observed as skull uptake. Conclusion: 18F-MK-6240 is a promising PET tracer for the in vivo quantification of NFTs in AD patients.

Discovery of 6-(Fluoro-18F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([18F]-MK-6240): A Positron Emission Tomography (PET) Imaging Agent for Quantification of Neurofibrillary Tangles (NFTs)

Neurofibrillary tangles (NFTs) made up of aggregated tau protein have been identified as the pathologic hallmark of several neurodegenerative diseases including Alzheimers disease. In vivo detection of NFTs using PET imaging represents a unique opportunity to develop a pharmacodynamic tool to accelerate the discovery of new disease modifying therapeutics targeting tau pathology. Herein, we present the discovery of 6-(fluoro-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine, 6 ([(18)F]-MK-6240), as a novel PET tracer for detecting NFTs. 6 exhibits high specificity and selectivity for binding to NFTs, with suitable physicochemical properties and in vivo pharmacokinetics.

Synthesis, characterization, and monkey positron emission tomography (PET) studies of [18F]Y1-973, a PET tracer for the neuropeptide Y Y1 receptor.

Neuropeptide Y receptor subtype 1 (NPY Y1) has been implicated in appetite regulation, and antagonists of NPY Y1 are being explored as potential therapeutics for obesity. An NPY Y1 PET tracer is useful for determining the level of target engagement by NPY Y1 antagonists in preclinical and clinical studies. Here we report the synthesis and evaluation of [(18)F]Y1-973, a novel PET tracer for NPY Y1. [(18)F]Y1-973 was radiolabeled by reaction of a primary chloride with [(18)F]KF/K2.2.2 followed by deprotection with HCl. [(18)F]Y1-973 was produced with high radiochemical purity (>98%) and high specific activity (>1000 Ci/mmol). PET studies in rhesus monkey brain showed that the distribution of [(18)F]Y1-973 was consistent with the known NPY Y1 distribution; uptake was highest in the striatum and cortical regions and lowest in the pons, cerebellum nuclei, and brain stem. Blockade of [(18)F]Y1-973 uptake with NPY Y1 antagonist Y1-718 revealed a specific signal that was dose-dependently reduced in all regions of grey matter to a similarly low level of tracer uptake, indicative of an NPY Y1 specific signal. In vitro autoradiographic studies with [(18)F]Y1-973 in rhesus monkey and human brain tissue slices revealed an uptake distribution consistent with the in vivo PET studies. Highest binding density was observed in the dentate gyrus, caudate-putamen, and cortical regions; moderate binding density in the hypothalamus and thalamus; and lowest binding density in the globus pallidus and cerebellum. In vitro saturation binding studies in rhesus monkey and human caudate-putamen homogenates confirmed a similarly high B(max)/K(d) ratio for [(18)F]Y1-973, suggesting the tracer may provide a specific signal in human brain of similar magnitude to that observed in rhesus monkey. [(18)F]Y1-973 is a suitable PET tracer for imaging NPY Y1 in rhesus monkey with potential for translation to human PET studies.

[(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.

Discovery of [(11)C]MK-8193 as a PET tracer to measure target engagement of phosphodiesterase 10A (PDE10A) inhibitors.

Phosphodiesterase 10A (PDE10A) inhibition has recently been identified as a potential mechanism to treat multiple symptoms that manifest in schizophrenia. In order to facilitate preclinical development and support key proof-of-concept clinical trials of novel PDE10A inhibitors, it is critical to discover positron emission tomography (PET) tracers that enable plasma concentration/PDE10A occupancy relationships to be established across species with structurally diverse PDE10A inhibitors. In this Letter, we describe how a high-throughput screening hit was optimized to provide [(11)C]MK-8193 (8j), a PET tracer that supports the determination of plasma concentration/PDE10A occupancy relationships for structurally diverse series of PDE10A inhibitors in both rat and rhesus monkey.

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

PurposeA 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 [11C]MK-8193 is described.ProceduresIn vitro binding studies with [3H]MK-8193 were conducted in rat, monkey, and human brain tissue. PET studies with [11C]MK-8193 were conducted in rats and rhesus monkeys at baseline and following administration of a PDE10A inhibitor.Results[3H]MK-8193 is a high-affinity, selective PDE10A radioligand in rat, monkey, and human brain tissue. In vivo, [11C]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[11C]MK-8193 is a useful PET tracer for the preclinical characterization of PDE10A therapeutic candidates in rat and monkey. Further evaluation of [11C]MK-8193 in humans is warranted.

2-[(3aR,4R,5S,7aS)-5-{(1S)-1-[3,5-Bis(trifluoromethyl)phenyl]-2-hydroxyethoxy}-4-(2-methylphenyl)octahydro-2H-isoindol-2-yl]-1,3-oxazol-4(5H)-one: A Potent Human NK1 Receptor Antagonist with Multiple Clearance Pathways

Hydroisoindoline 2 has been previously identified as a potent, brain-penetrant NK1 receptor antagonist with a long duration of action and improved profile of CYP3A4 inhibition and induction compared to aprepitant. However, compound 2 is predicted, based on data in preclinical species, to have a human half-life longer than 40 h and likely to have drug-drug-interactions (DDI), as 2 is a victim of CYP3A4 inhibition caused by its exclusive clearance pathway via CYP3A4 oxidation in humans. We now report 2-[(3aR,4R,5S,7aS)-5-{(1S)-1-[3,5-bis(trifluoromethyl)phenyl]-2-hydroxyethoxy}-4-(2-methylphenyl)octahydro-2H-isoindol-2-yl]-1,3-oxazol-4(5H)-one (3) as a next generation NK1 antagonist that possesses an additional clearance pathway through glucuronidation in addition to that via CYP3A4 oxidation. Compound 3 has a much lower propensity for drug-drug interactions and a reduced estimated human half-life consistent with once daily dosing. In preclinical species, compound 3 has demonstrated potency, brain penetration, and a safety profile similar to 2, as well as excellent pharmacokinetics.

Discovery of pyrazolopyrimidine phosphodiesterase 10A inhibitors for the treatment of schizophrenia.

Herein, we present the identification of a novel class of pyrazolopyrimidine phosphodiesterase 10A (PDE10A) inhibitors. Beginning with a lead molecule (1) identified through a fragment-based drug discovery (FBDD) effort, lead optimization was enabled by rational design, X-ray crystallography, metabolic and off-target profiling, and fragment scaffold-hopping. We highlight the discovery of PyP-1, a potent, highly selective, and orally bioavailable pyrazolopyrimidine inhibitor of PDE10A. PyP-1 exhibits sub-nanomolar potency (PDE10A Ki=0.23nM), excellent pharmacokinetic (PK) and physicochemical properties, and a clean off-target profile. It displays dose-dependent efficacy in numerous pharmacodynamic (PD) assays that measure potential for anti-psychotic activity and cognitive improvement. PyP-1 also has a clean preclinical profile with respect to cataleptic potential in rats, prolactin secretion, and weight gain, common adverse events associated with currently marketed therapeutics. Further, PyP-1 displays in vivo preclinical target engagement as measured by PET enzyme occupancy in concert with [(11)C]MK-8193, a novel PDE10A PET tracer.