Sam Victory

4-(2-pyridyl)piperazine-1-carboxamides: potent vanilloid receptor 1 antagonists.

A series of 4-(2-pyridyl)piperazine-1-carboxamide analogues based on the lead compound 1 was synthesized and evaluated for VR1 antagonist activity in capsaicin-induced (CAP) and pH (5.5)-induced (pH) FLIPR assays in a rat VR1-expressing HEK293 cell line. Potent VR1 antagonists were identified through SAR studies. From these studies, 18 was found to be very potent in the in vitro assay [IC(50)=4.8 nM (pH) and 35 nM (CAP)] and orally available in rat (F%=15.1).

DiPOA ([8-(3,3-Diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]dec-3-yl]-acetic Acid), a Novel, Systemically Available, and Peripherally Restricted Mu Opioid Agonist with Antihyperalgesic Activity: I. In Vitro Pharmacological Characterization and Pharmacokinetic Properties

Mu opioid receptors are present throughout the central and peripheral nervous systems. Peripheral inflammation causes an increase in mu receptor levels on peripheral terminals of primary afferent neurons. Recent studies indicate that activation of peripheral mu receptors produces antihyperalgesic effects in animals and humans. Here, we describe the in vitro pharmacological and in vivo pharmacokinetic properties of a novel, highly potent, and peripherally restricted mu opioid agonist, [8-(3,3-diphenyl-propyl)-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl]-acetic acid (DiPOA). In a radioligand binding assay, DiPOA inhibited [3H]-diprenorphine binding to recombinant human mu receptors with a Ki value of ∼0.8 nM. The rank order of affinity for DiPOA binding to recombinant human opioid receptors was mu > kappa ≈ ORL-1 >> delta. DiPOA showed potent agonist effects in a human mu receptor guanosine 5′-O-(3-[35S]thio)triphosphate functional assay, with an EC50 value of ∼33 nM and efficacy of ∼85% {normalized to the mu agonist, [d-Ala2,MePhe4,Gly(ol)5]enkephalin}. Low potency agonist activity was also seen at ORL-1 and kappa receptors. DiPOA bound competitively to the opioid binding site of human mu receptors as demonstrated by a parallel rightward shift in its concentration-response curve in the presence of increasing concentrations of naltrexone. High and sustained (≥5 h) plasma levels for DiPOA were achieved following intraperitoneal administration at 3 and 10 mg/kg; central nervous system penetration, however, was ≤4% of the plasma concentration, even at levels exceeding 1500 ng/ml. As such, DiPOA represents a systemically available, peripherally restricted small molecule mu opioid agonist that will aid in understanding the role played by mu opioid receptors in the periphery.

Validation of a fluorescent imaging plate reader membrane potential assay for high-throughput screening of glycine transporter modulators.

A fluorescent imaging plate reader (FLIPR) membrane potential (Vm) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT2) in a stable rGlyT2-HEK cell line. Data show that glycine activation of rGlyT2 consistently results in a concentration-dependent Vm response on the FLIPR that is blocked by the potent and selective GlyT2 antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [3H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT2 physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT2 inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based Vm assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT2.