No abstract

Voltage-gated Naϩ channels may play important roles in establishing pathological neuronal hyperexcitability associated with chronic pain in humans. Na ϩ channel blockers, such as carbamazepine (CBZ) and lamotrigine (LTG), are efficacious in treating neuropathic pain; however, their therapeutic utility is compromised by central nervous system side effects. We reasoned that it may be possible to gain superior control over pain states and, in particular, a better therapeutic index, by designing broad-spectrum Na ϩ channel blockers with higher potency, faster onset kinetics, and greater levels of state dependence than existing drugs. 2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide (PPPA) is a novel structural analog of the state-dependent Na ϩ channel blocker V102862 [4-(4-fluorophenoxy)benzaldehyde semicarbazone]. Tested on recombinant rat Na v 1.2 channels and native Na ϩ currents in cultured rat dorsal root ganglion neurons, PPPA was approximately 1000 times more potent, had 2000-fold faster binding kinetics, and Ն10-fold higher levels of state dependence than CBZ and LTG. Tested in rat pain models against mechanical endpoints, PPPA had minimal effective doses of 1 to 3 mg/kg p.o. in partial sciatic nerve ligation, Freund's complete adjuvant, and postincisional pain. In all cases, efficacy was similar to clinically relevant comparators. Importantly, PPPA did not produce motor deficits in the accelerating Rotarod assay of ataxia at doses up to 30 mg/kg p.o., indicating a therapeutic index Ͼ10, which was superior to CBZ and LTG. Our experiments suggest that high-potency, broad-spectrum, statedependent Na ϩ channel blockers will have clinical utility for treating neuropathic, inflammatory, and postsurgical pain. Optimizing the biophysical parameters of broad-spectrum voltage-gated Na ϩ channel blockers may lead to improved pain therapeutics.

A fluorescent imaging plate reader (FLIPR) membrane potential (V m ) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT 2 ) in a stable rGlyT 2 -HEK cell line. Data show that glycine activation of rGlyT 2 consistently results in a concentration-dependent V m response on the FLIPR that is blocked by the potent and selective GlyT 2 antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylaminocyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [ 3 H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT 2 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 GlyT 2 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 V m assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT 2 . (Journal of Biomolecular Screening 2005:365-373)

Voltage-gated sodium channels (NaChs) are relevant targets for pain, epilepsy, and a variety of neurological and cardiac disorders. Traditionally, it has been difficult to develop structure-activity relationships for NaCh inhibitors due to rapid channel kinetics and state-dependent compound interactions. Membrane potential (V m ) dyes in conjunction with a high-throughput fluorescence imaging plate reader (FLIPR) offer a satisfactory 1st-tier solution. Thus, the authors have developed a FLIPR V m assay of rat Na v 1.2 NaCh. Channels were opened by addition of veratridine, and V m dye responses were measured. The IC 50 values from various structural classes of compounds were compared to the resting state binding constant (K r ) and inactivated state binding constant (K i ) obtained using patch-clamp electrophysiology (EP). The FLIPR values correlated with K i but not K r . FLIPR IC 50 values fell within 0.1-to 1.5-fold of EP K i values, indicating that the assay generally reports use-dependent inhibition rather than resting state block. The Library of Pharmacologically Active Compounds (LOPAC, Sigma) was screened. Confirmed hits arose from diverse classes such as dopamine receptor antagonists, serotonin transport inhibitors, and kinase inhibitors. These data suggest that NaCh inhibition is inherent in a diverse set of biologically active molecules and may warrant counterscreening NaChs to avoid unwanted secondary pharmacology. (Journal of Biomolecular Screening 2006: 29-39)