Kenneth J. Valenzano

Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy

To date, two cannabinoid receptors have been identified, CB1 and CB2. Activation of these receptors with non-selective cannabinoid receptor agonists reduces pain sensitivity in animals and humans. However, activation of CB1 receptors is also associated with central side effects, including ataxia and catalepsy. More recently, a role for selective CB2 agonists in pain modification has been demonstrated. GW405833, a selective CB2 agonist, was recently reported to partially reverse the inflammation and hyperalgesia in a rat model of acute inflammation. In the current report, we extend the characterization and therapeutic potential of this compound. For the first time, we show that GW405833 selectively binds both rat and human CB2 receptors with high affinity, where it acts as a partial agonist (approximately 50% reduction of forskolin-mediated cAMP production compared to the full cannabinoid agonist, CP55,940). We also report for the first time that intraperitoneal administration of GW405833 (0.3-100 mg/kg) to rats shows linear, dose-dependent increases in plasma levels and substantial penetration into the central nervous system. In addition, GW405833 (up to 30 mg/kg) elicits potent and efficacious antihyperalgesic effects in rodent models of neuropathic, incisional and chronic inflammatory pain, the first description of this compound in these models. In contrast, analgesia, sedation and catalepsy were not observed in this dose range, but were apparent at 100 mg/kg. Additionally, GW405833 was not antihyperalgesic against chronic inflammatory pain in CB2 knockout mice. These data support the tenet that selective CB2 receptor agonists have the potential to treat pain without eliciting the centrally-mediated side effects associated with non-selective cannabinoid agonists, and highlight the utility of GW405833 for the investigation of CB2 physiology.

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).

Development of a Fluorescent Ligand-Binding Assay Using the AcroWell Filter Plate

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.

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.

Design and parallel synthesis of piperidine libraries targeting the nociceptin (N/OFQ) receptor.

Based on literature structures, we proposed a pharmacophore for NOP receptor ligands and used it as a guide for the design of a focused piperidine library and an optimization library. Potent NOP receptor agonists and antagonists were obtained from these libraries as well as a few potent, mu selective agonists.

A Miniaturized Column Chromatography Method for Measuring Receptor-Mediated Inositol Phosphate Accumulation

Inositol phosphates (IPs), such as 1,4,5-inositol-trisphosphate (IP3), comprise a ubiquitous intracellular signaling cascade initiated in response to G protein-coupled receptor-mediated activation of phospholipase C. Classical methods for measuring intracellular accumulation of these molecules include time-consuming high-performance liquid chromatography (HPLC) separation or large-volume, gravity-fed anion-exchange column chromatography. More recent approaches, such as radio-receptor and AlphaScreen™ assays, offer higher throughput. However, these techniques rely on measurement of IP3 itself, rather than its accumulation with other downstream IPs, and often suffer from poor signal-to-noise ratios due to the transient nature of IP3. The authors have developed a miniaturized, anion-exchange chromatography method for measuring inositol phosphate accumulation in cells that takes advantage of signal amplification achieved through measuring IP3 and downstream IPs. This assay uses centrifugation of 96-well-formatted anion-exchange mini-columns for the isolation of radiolabeled inositol phosphates from cell extracts, followed by low-background dry-scintillation counting. This improved assay method measures receptor-mediated IP accumulation with signal-to-noise and pharmacological values comparable to the classical large-volume, column-based methods. Assay validation data for recombinant muscarinic receptor 1, galanin receptor 2, and rat astrocyte metabotropic glutamate receptor 5 are presented. This miniaturized protocol reduces reagent usage and assay time as compared to large-column methods and is compatible with standard 96-well scintillation counters.