The PET tracer [11C]MK-6884 quantifies M4 muscarinic receptor in rhesus monkeys and patients with Alzheimer's disease.

Positron emission tomography (PET) ligands play an important role in the development of therapeutics by serving as target engagement or pharmacodynamic biomarkers. Here, we describe the discovery and translation of the PET tracer [11C]MK-6884 from rhesus monkeys to patients with Alzheimer's disease (AD). [3H]MK-6884/[11C]MK-6884 binds with high binding affinity and good selectivity to an allosteric site on M4 muscarinic cholinergic receptors (M4Rs) in vitro and shows a regional distribution in the brain consistent with M4R localization in vivo. The tracer demonstrates target engagement of positive allosteric modulators of the M4R (M4 PAMs) through competitive binding interactions. [11C]MK-6884 binding is enhanced in vitro by the orthosteric M4R agonist carbachol and indirectly in vivo by the acetylcholinesterase inhibitor donepezil in rhesus monkeys and healthy volunteers, consistent with its pharmacology as a highly cooperative M4 PAM. PET imaging of [11C]MK-6884 in patients with AD identified substantial regional differences quantified as nondisplaceable binding potential (BPND) of [11C]MK-6884. These results suggest that [11C]MK-6884 is a useful target engagement biomarker for M4 PAMs but may also act as a sensitive probe of neuropathological changes in the brains of patients with AD.

Preclinical evaluation of [11 C]L-235 as a radioligand for Positron Emission Tomography cathepsin K imaging in bone.

The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.

Discovery of [11C]MK-6884: A Positron Emission Tomography (PET) Imaging Agent for the Study of M4Muscarinic Receptor Positive Allosteric Modulators (PAMs) in Neurodegenerative Diseases.

The measurement of receptor occupancy (RO) using positron emission tomography (PET) has been instrumental in guiding discovery and development of CNS directed therapeutics. We and others have investigated muscarinic acetylcholine receptor 4 (M4) positive allosteric modulators (PAMs) for the treatment of symptoms associated with neuropsychiatric disorders. In this article, we describe the synthesis, in vitro, and in vivo characterization of a series of central pyridine-related M4 PAMs that can be conveniently radiolabeled with carbon-11 as PET tracers for the in vivo imaging of an allosteric binding site of the M4 receptor. We first demonstrated its feasibility by mapping the receptor distribution in mouse brain and confirming that a lead molecule 1 binds selectively to the receptor only in the presence of the orthosteric agonist carbachol. Through a competitive binding affinity assay and a number of physiochemical properties filters, several related compounds were identified as candidates for in vivo evaluation. These candidates were then radiolabeled with 11C and studied in vivo in rhesus monkeys. This research eventually led to the discovery of the clinical radiotracer candidate [11C]MK-6884.

Preclinical Safety Evaluation and Human Dosimetry of [18F]MK-6240, a Novel PET Tracer for Imaging Neurofibrillary Tangles.

PURPOSE: [18F]MK-6240 is a selective, high-affinity positron emission tomography tracer for imaging neurofibrillary tangles, a key pathological signature that correlates with cognitive decline in Alzheimer disease. This report provides safety information from preclinical toxicology studies and first-in-human whole-body biodistribution and dosimetry studies of [18F]MK-6240 for its potential application in human brain imaging studies. PROCEDURES: MK-6240 was administered intravenously (IV) in a 7-day rat toxicity study at × 50, × 100, and × 1000 dose margins relative to projected highest clinical dose of 0.333 µg/kg. The IV formulation of MK-6240 for clinical use and the formulation used in the 7-day rat toxicity study was tested for hemolysis potential in human and Wistar rat whole blood. Sequential whole-body positron emission tomography scans were performed in three healthy young subjects after IV bolus injection of 180 ± 0.3 MBq [18F]MK-6240 to characterize organ biodistribution and estimate whole-body radiation exposure (effective dose). RESULTS: MK-6240 administered IV in a 7-day rat toxicity study did not show any test article-related changes. The no-observed-adverse-effect level in rats was ≥ 333 µg/kg/day which provides a margin 1000-fold over an anticipated maximum clinical dose of 0.333 µg/kg. Additionally, the MK-6240 formulation was not hemolytic in human or Wistar rat blood. [18F]MK-6240 activity was widely distributed to the brain and the rest of the body, with organ absorbed doses largest for the gall bladder (202 µGy/MBq). The average (±SD) effective dose was 29.4 ± 0.6 µSv/MBq, which is in the typical range for F-18 radiolabeled ligands. CONCLUSIONS: Microdoses of [18F]MK-6240 are safe for clinical positron emission tomography imaging studies. Single IV administration of 185 MBq (5 mCi) [18F]MK-6240 is anticipated to result in a total human effective dose of 5.4 mSv and thus allows multiple positron emission tomography scans of the same subject per year.

Brain Imaging of Alzheimer Dementia Patients and Elderly Controls with 18F-MK-6240, a PET Tracer Targeting Neurofibrillary Tangles.

18F-MK-6240 (18F-labeled 6-(fluoro)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine) is a highly selective, subnanomolar-affinity PET tracer for imaging neurofibrillary tangles (NFTs). Plasma kinetics, brain uptake, and preliminary quantitative analysis of 18F-MK-6240 in healthy elderly (HE) subjects, subjects with clinically probable Alzheimer disease (AD), and subjects with amnestic mild cognitive impairment were characterized in a study that is, to our knowledge, the first to be performed on humans. Methods: Dynamic PET scans of up to 150 min were performed on 4 cognitively normal HE subjects, 4 AD subjects, and 2 amnestic mild cognitive impairment subjects after a bolus injection of 152-169 MBq of 18F-MK-6240 to evaluate tracer kinetics and distribution in brain. Regional SUV ratio (SUVR) and distribution volume ratio were determined using the cerebellar cortex as a reference region. Total distribution volume was assessed by compartmental modeling using radiometabolite-corrected input function in a subgroup of 6 subjects. Results:18F-MK-6240 had rapid brain uptake with a peak SUV of 3-5, followed by a uniformly quick washout from all brain regions in HE subjects; slower clearance was observed in regions commonly associated with NFT deposition in AD subjects. In AD subjects, SUVR between 60 and 90 min after injection was high (approximately 2-4) in regions associated with NFT deposition, whereas in HE subjects, SUVR was approximately 1 across all brain regions, suggesting high tracer selectivity for binding NFTs in vivo. 18F-MK-6240 total distribution volume was approximately 2- to 3-fold higher in neocortical and medial temporal brain regions of AD subjects than in HE subjects and stabilized by 60 min in both groups. Distribution volume ratio estimated by the Logan reference tissue model or compartmental modeling correlated well (R2 > 0.9) to SUVR from 60 to 90 min for AD subjects. Conclusion:18F-MK-6240 exhibited favorable kinetics and high binding levels to brain regions with a plausible pattern for NFT deposition in AD subjects. In comparison, negligible tracer binding was observed in HE subjects. This pilot study suggests that simplified ratio methods such as SUVR can be used to quantify NFT binding. These results support further clinical development of 18F-MK-6240 for potential application in longitudinal studies.

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-(18)F)-3-(1H-pyrrolo[2,3-c]pyridin-1-yl)isoquinolin-5-amine ([(18)F]-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 Alzheimer's 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.

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.

Discovery of [¹¹C]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.

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.

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.

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 B(max) /K(d) 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.

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.

Stabilization and determination of a PPAR agonist in human urine using automated 96-well liquid-liquid extraction and liquid chromatography/tandem mass spectrometry.

Two stability challenges were encountered during development of an urine assay for a proliferator-activated receptor (PPAR) agonist, I (2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid), indicated for the treatment of Type II diabetes. First, the analyte was lost in urine samples due to adsorption on container surface which is a common problem during clinical sample handling. Secondly, the acylglucuronide metabolite (III), a major metabolite of I, displayed limited stability and effected the quantitation of parent drug due to the release of I through hydrolysis. Therefore, a clinical collection procedure was carefully established to stabilize I and its acylglucuronide metabolite, III, in human urine. The metabolite was not quantitated with this method. The urine samples are treated with bovine serum albumin (BSA) equal to 1.75% of the urine volume and formic acid equal to 1% of urine volume. Compound (I) and internal standard (II) were extracted from urine with 1 mL ethyl acetate using a fully automated liquid-liquid extraction in 96-well plate format. The analytes are separated by reverse phase high-performance liquid chromatography (HPLC) with tandem mass spectrometry in multiple-reaction-monitoring (MRM) mode used for detection. The urine method has a lower limit of quantitation (LLOQ) of 0.05 ng/mL with a linearity range of 0.05-20 ng/mL using 0.05 mL of urine. The method was validated and used to assay urine clinical samples.

A dual PPAR alpha/gamma agonist increases adiponectin and improves plasma lipid profiles in healthy subjects.

BACKGROUND: The objective of these studies was to evaluate the pharmacokinetics and pharmacodynamics of MK-0767, a prototypical dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist, following administration of single and multiple oral doses in healthy male subjects. METHODS: The first study was a double-blind, randomised, placebo-controlled, alternating two-panel, rising dose protocol in which single doses of 1-80 mg of MK-0767 were administered. The second study was a double-blind, randomised, placebo-controlled, staggered incremental dose, parallel-group protocol in which multiple doses of 0.3-25 mg of MK-0767 were administered once daily for 14 days. In both studies at each dose level, six subjects received MK-0767 and two subjects received placebo. RESULTS: Plasma area under the concentration-time curve and maximum plasma concentration increased with single and multiple doses of MK-0767 over the dose ranges studied. The apparent terminal half-life of MK-0767 averaged approximately 36 hours following single and multiple doses. Steady-state plasma concentrations were achieved following approximately 8 days of multiple doses. Compared with placebo, MK-0767 produced dose-dependent reductions in triglycerides (-26 +/- 8% [p = 0.002] and -33 +/- 13% [p = 0.008]) and free fatty acids (-50 +/- 11% [p < 0.001] and -67 +/- 23% [p = 0.008]) following single and multiple doses, respectively. Significant (p < or = 0.050) dose-dependent alterations in adiponectin (332 +/- 36%), low-density lipoprotein cholesterol (-29 +/- 5%), total cholesterol (-19 +/- 3%), non-high-density lipoprotein cholesterol (-28 +/- 4%), and fasting plasma glucose (-6 +/- 2%; only in the 25 mg group) were observed after multiple doses. CONCLUSIONS: The observed effects of MK-0767 on adiponectin, free fatty acids and lipids, even after single doses, demonstrate that this prototypical dual PPAR alpha/gamma agonist has clinically meaningful activity in vivo.

Synthesis, characterization, and first successful monkey imaging studies of metabotropic glutamate receptor subtype 5 (mGluR5) PET radiotracers.

Three metabotropic glutamate receptor subtype 5 (mGluR5) PET tracers have been labeled with either carbon-11 or fluorine-18 and their in vitro and in vivo behavior in rhesus monkey has been characterized. Each of these tracers share the common features of high affinity for mGluR5 (0.08-0.23 nM vs. rat mGluR5) and moderate lipophilicity (log P 2.8-3.4). Compound 1b was synthesized using a Suzuki or Stille coupling reaction with [11C]MeI. Compounds 2b and 3b were synthesized by a SNAr reaction using a 3-chlorobenzonitrile precursor. Autoradiographic studies in rhesus monkey brain slices using 2b and 3b showed specific binding in cortex, caudate, putamen, amygdala, hippocampus, most thalamic nuclei, and lower binding in the cerebellum. PET imaging studies in monkey showed that all three tracers readily enter the brain and provide an mGluR5-specific signal in all gray matter regions, including the cerebellum. The specific signal observed in the cerebellum was confirmed by the autoradiographic studies and saturation binding experiments that showed tracer binding in the cerebellum of rhesus monkeys. In vitro metabolism studies using the unlabeled compounds showed that 1a, 2a, and 3a are metabolized slower by human liver microsomes than by monkey liver microsomes. In vivo metabolism studies showed 3b to be long-lived in rhesus plasma with only one other more polar metabolite observed.

Fully automated liquid-liquid extraction for the determination of a novel insulin sensitizer in human plasma by heated nebulizer and turbo ionspray liquid chromatography-tandem mass spectrometry.

I, 2-{[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy}-2-methyl propionic acid is an alpha peroxisome proliferator-activated receptor (PPAR) agonist with some gamma activity being investigated for potential use in the treatment of Type II diabetes mellitus and dyslipidemia. Two automated liquid-liquid extraction methods were developed and validated for the determination of I in human plasma. Concentrations of I were determined over a wide range of clinical doses. For Method A, plasma was acidified and extracted with ethyl acetate using a fully automated procedure. Analysis was performed by LC-MS/MS with a turbo ionspray source in negative ion mode. For Method B, a larger volume of plasma was extracted and a heated nebulizer source was used on the mass spectrometer. Method A was linear from 0.05 to 50 ng/mL and Method B from 0.2 to 1000 ng/mL. Validation procedures showed that both methods were robust, specific and reproducible.