Efficiency gains in tracer identification for nuclear imaging: can in vivo LC-MS/MS evaluation of small molecules screen for successful PET tracers?

Positron emission tomography (PET) imaging has become a useful noninvasive technique to explore molecular biology within living systems; however, the utility of this method is limited by the availability of suitable radiotracers to probe specific targets and disease biology. Methods to identify potential areas of improvement in the ability to predict small molecule performance as tracers prior to radiolabeling would speed the discovery of novel tracers. In this retrospective analysis, we characterized the brain penetration or peak SUV (standardized uptake value), binding potential (BP), and brain exposure kinetics across a series of known, nonradiolabeled PET ligands using in vivo LC-MS/MS (liquid chromatography coupled to mass spectrometry) and correlated these parameters with the reported PET ligand performance in nonhuman primates and humans available in the literature. The PET tracers studied included those reported to label G protein-coupled receptors (GPCRs), intracellular enzymes, and transporters. Additionally, data for each tracer was obtained from a mouse brain uptake assay (MBUA), previously published, where blood-brain barrier (BBB) penetration and clearance parameters were assessed and compared against similar data collected on a broad compound set of central nervous system (CNS) therapeutic compounds. The BP and SUV identified via nonradiolabeled LC-MS/MS, while different from the published values observed in the literature PET tracer data, allowed for an identification of initial criteria values we sought to facilitate increased potential for success from our early discovery screening paradigm. Our analysis showed that successful, as well as novel, clinical PET tracers exhibited BP of greater than 1.5 and peak SUVs greater than approximately 150% at 5 min post dose in rodents. The brain kinetics appeared similar between both techniques despite differences in tracer dose, suggesting linearity across these dose ranges. The assessment of tracers in a CNS exposure model, the mouse brain uptake assessment (MBUA), showed that those compound with initial brain-to-plasma ratios >2 and unbound fraction in brain homogenate >0.01 were more likely to be clinically successful PET ligands. Taken together, early incorporation of a LC/MS/MS cold tracer discovery assay and a parallel MBUA can be an useful screening paradigm to prioritize and rank order potential novel PET radioligands during early tracer discovery efforts. Compounds considered for continued in vivo PET assessments can be identified quickly by leveraging in vitro affinity and selectivity measures, coupled with data from a MBUA, primarily the 5 min brain-to-plasma ratio and unbound fraction data. Coupled utilization of these data creates a strategy to efficiently screen for the identification of appropriate chemical space to invest in for radiotracer discovery.

Discovery of a potent, dual serotonin and norepinephrine reuptake inhibitor.

The objective of the described research effort was to identify a novel serotonin and norepinephrine reuptake inhibitor (SNRI) with improved norepinephrine transporter activity and acceptable metabolic stability and exhibiting minimal drug-drug interaction. We describe herein the discovery of a series of 3-substituted pyrrolidines, exemplified by compound 1. Compound 1 is a selective SNRI in vitro and in vivo, has favorable ADME properties, and retains inhibitory activity in the formalin model of pain behavior. Compound 1 thus represents a potential new probe to explore utility of SNRIs in central nervous system disorders, including chronic pain conditions.

3-Phenyl-5-isothiazole carboxamides with potent mGluR1 antagonist activity.

The disclosed 3-phenyl-5-isothiazole carboxamides are potent allosteric antagonists of mGluR1 with generally good selectivity relative to the related group 1 receptor mGluR5. Pharmacokinetic properties of a member of this series (1R,2R)-N-(3-(4-methoxyphenyl)-4-methylisothiazol-5-yl)-2-methylcyclopropanecarboxamide (14) are good, showing acceptable plasma and brain exposure after oral dosing. Oral administration of isothiazole 14 gave robust activity in the formalin model of persistent pain which correlated with CNS receptor occupancy.

Preclinical pharmacological profile of the selective 5-HT1F receptor agonist lasmiditan.

INTRODUCTION: Lasmiditan (also known as COL-144 and LY573144; 2,4,6-trifluoro-N-[6-[(1-methylpiperidin-4-yl)carbonyl]pyridin-2yl]benzamide) is a high-affinity, highly selective serotonin (5-HT) 5-HT(1F) receptor agonist. RESULTS: In vitro binding studies show a K(i) value of 2.21 nM at the 5-HT(1F) receptor, compared with K(i) values of 1043 nM and 1357 nM at the 5-HT(1B) and 5-HT(1D) receptors, respectively, a selectivity ratio greater than 470-fold. Lasmiditan showed higher selectivity for the 5-HT(1F) receptor relative to other 5-HT(1) receptor subtypes than the first generation 5-HT(1F) receptor agonist LY334370. Unlike the 5-HT(1B/1D) receptor agonist sumatriptan, lasmiditan did not contract rabbit saphenous vein rings, a surrogate assay for human coronary artery constriction, at concentrations up to 100 µM. In two rodent models of migraine, oral administration of lasmiditan potently inhibited markers associated with electrical stimulation of the trigeminal ganglion (dural plasma protein extravasation, and induction of the immediate early gene c-Fos in the trigeminal nucleus caudalis). CONCLUSIONS: Lasmiditan presents a unique pyridinoyl-piperidine scaffold not found in any other antimigraine class. Its chemical structure and pharmacological profile clearly distinguish it from the triptans. The potency and selectivity of lasmiditan make it ideally suited to definitively test the involvement of 5-HT(1F) receptors in migraine headache therapy.

Biodistribution and dosimetry in humans of two inverse agonists to image cannabinoid CB1 receptors using positron emission tomography.

PURPOSE: Cannabinoid subtype 1 (CB(1)) receptors are found in nearly every organ in the body, may be involved in several neuropsychiatric and metabolic disorders, and are therefore an active target for pharmacotherapy and biomarker development. We recently reported brain imaging of CB(1) receptors with two PET radioligands: (11)C-MePPEP and (18)F-FMPEP-d (2). Here we describe the biodistribution and dosimetry estimates for these two radioligands. METHODS: Seven healthy subjects (four men and three women) underwent whole-body PET scans for 120 min after injection with (11)C-MePPEP. Another seven healthy subjects (two men and five women) underwent whole-body PET scans for 300 min after injection with (18)F-FMPEP-d (2). Residence times were acquired from regions of interest drawn on tomographic images of visually identifiable organs for both radioligands and from radioactivity excreted in urine for (18)F-FMPEP-d (2). RESULTS: The effective doses of (11)C-MePPEP and (18)F-FMPEP-d (2) are 4.6 and 19.7 microSv/MBq, respectively. Both radioligands demonstrated high uptake of radioactivity in liver, lung, and brain shortly after injection and accumulated radioactivity in bone marrow towards the end of the scan. After injection of (11)C-MePPEP, radioactivity apparently underwent hepatobiliary excretion only, while radioactivity from (18)F-FMPEP-d (2) showed both hepatobiliary and urinary excretion. CONCLUSION: (11)C-MePPEP and (18)F-FMPEP-d (2) yield an effective dose similar to other PET radioligands labeled with either (11)C or (18)F. The high uptake in brain confirms the utility of these two radioligands to image CB(1) receptors in brain, and both may also be useful to image CB(1) receptors in the periphery.

Imaging and quantitation of cannabinoid CB1 receptors in human and monkey brains using (18)F-labeled inverse agonist radioligands.

UNLABELLED: We recently demonstrated that (11)C-MePPEP, a PET ligand for CB(1) receptors, has such high uptake in the human brain that it can be imaged for 210 min and that receptor density can be quantified as distribution volume (V(T)) using the gold standard of compartmental modeling. However, (11)C-MePPEP had relatively poor retest and intersubject variabilities, which were likely caused by errors in the measurements of radioligand in plasma at low concentrations by 120 min. We sought to find an analog of (11)C-MePPEP that would provide more accurate plasma measurements. We evaluated several promising analogs in the monkey brain and chose the (18)F-di-deutero fluoromethoxy analog ((18)F-FMPEP-d(2)) to evaluate further in the human brain. METHODS: (11)C-FMePPEP, (18)F-FEPEP, (18)F-FMPEP, and (18)F-FMPEP-d(2) were studied in 5 monkeys with 10 PET scans. We calculated V(T) using compartmental modeling with serial measurements of unchanged parent radioligand in arterial plasma and radioactivity in the brain. Nonspecific binding was determined by administering a receptor-saturating dose of rimonabant, an inverse agonist at the CB(1) receptor. Nine healthy human subjects participated in 17 PET scans using (18)F-FMPEP-d(2), with 8 subjects having 2 PET scans to assess retest variability. To identify sources of error, we compared intersubject and retest variability of brain uptake, arterial plasma measurements, and V(T). RESULTS: (18)F-FMPEP-d(2) had high uptake in the monkey brain, with greater than 80% specific binding, and yielded less radioactivity uptake in bone than did (18)F-FMPEP. High brain uptake with (18)F-FMPEP-d(2) was also observed in humans, in whom V(T) was well identified within approximately 60 min. Retest variability of plasma measurements was good (16%); consequently, V(T) had a good retest variability (14%), intersubject variability (26%), and intraclass correlation coefficient (0.89). V(T) increased after 120 min, suggesting an accumulation of radiometabolites in the brain. Radioactivity accumulated in the skull throughout the entire scan but was thought to be an insignificant source of data contamination. CONCLUSION: Studies in monkeys facilitated our development and selection of (18)F-FMPEP-d(2), compared with (18)F-FMPEP, as a radioligand demonstrating high brain uptake, high percentage of specific binding, and reduced uptake in bone. Retest analysis in human subjects showed that (18)F-FMPEP-d(2) has greater precision and accuracy than (11)C-MePPEP, allowing smaller sample sizes to detect a significant difference between groups.

Quantitation of cannabinoid CB1 receptors in healthy human brain using positron emission tomography and an inverse agonist radioligand.

[11C]MePPEP is a high affinity, CB1 receptor-selective, inverse agonist that has been studied in rodents and monkeys. We examined the ability of [11C]MePPEP to quantify CB1 receptors in human brain as distribution volume calculated with the "gold standard" method of compartmental modeling and compared results with the simple measure of brain uptake. A total of 17 healthy subjects participated in 26 positron emission tomography (PET) scans, with 8 having two PET scans to assess retest variability. After injection of [11C]MePPEP, brain uptake of radioactivity was high (e.g., 3.6 SUV in putamen at approximately 60 min) and washed out very slowly. A two-tissue compartment model yielded values of distribution volume (which is proportional to receptor density) that were both well identified (SE 5%) and stable between 60 and 210 min. The simple measure of brain uptake (average concentration of radioactivity between 40 and 80 min) had good retest variability ( approximately 8%) and moderate intersubject variability (16%, coefficient of variation). In contrast, distribution volume had two-fold greater retest variability ( approximately 15%) and, thus, less precision. In addition, distribution volume had three-fold greater intersubject variability ( approximately 52%). The decreased precision of distribution volume compared to brain uptake was likely due to the slow washout of radioactivity from brain and to noise in measurements of the low concentrations of [11C]MePPEP in plasma. These results suggest that brain uptake can be used for within subject studies (e.g., to measure receptor occupancy by medications) but that distribution volume remains the gold standard for accurate measurements between groups.

Synthesis, ex vivo evaluation, and radiolabeling of potent 1,5-diphenylpyrrolidin-2-one cannabinoid subtype-1 receptor ligands as candidates for in vivo imaging.

We have reported that [methyl- (11)C] (3 R,5 R)-5-(3-methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one ([(11)C] 8, [(11)C]MePPEP) binds with high selectivity to cannabinoid type-1 (CB 1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely, the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy- d 2 (21, FMPEP- d 2), and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high, selective potency at CB 1 receptors in vitro (K b < 1 nM). Each ligand (30 microg/kg, iv) was injected into rats under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand uptakes were high (22.6-48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced (6.5-17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB 1 receptors, we then established and here describe methods for producing [(11)C] 8, [(11)C] 16, and [(18)F] 20- 22 in adequate activities for evaluation as candidate PET radioligands in vivo.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas.

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

Positron emission tomography imaging using an inverse agonist radioligand to assess cannabinoid CB1 receptors in rodents.

[11C]MePPEP is an inverse agonist and a radioligand developed to image cannabinoid CB1 receptors with positron emission tomography (PET). It provides reversible, high specific signal in monkey brain. We assessed [11C]MePPEP in rodent brain with regard to receptor selectivity, susceptibility to transport by P-glycoprotein (P-gp), sensitivity to displacement by agonists, and accumulation of radiometabolites. We used CB1 receptor knockout mice and P-gp knockout mice to assess receptor selectivity and sensitivity to efflux transport, respectively. Using serial measurements of PET brain activity and plasma concentrations of [11C]MePPEP, we estimated CB1 receptor density in rat brain as distribution volume. CB1 knockout mice showed only nonspecific brain uptake, and [11C]MePPEP was not a substrate for P-gp. Direct acting agonists anandamide (10 mg/kg), methanandamide (10 mg/kg), CP 55,940 (1 mg/kg), and indirect agonist URB597 (0.3 and 0.6 mg/kg) failed to displace [11C]MePPEP, while the inverse agonist rimonabant (3 and 10 mg/kg) displaced >65% of [11C]MePPEP. Radiometabolites represented ~13% of total radioactivity in brain between 30 and 120 min. [11C]MePPEP was selective for the CB1 receptor, was not a substrate for P-gp, and was more potently displaced by inverse agonists than agonists. The low potency of agonists suggests either a large receptor reserve or non-overlapping binding sites for agonists and inverse agonists. Radiometabolites of [11C]MePPEP in brain caused distribution volume to be overestimated by approximately 13%.