In vitro pharmacological profiling is increasingly being used earlier in the drug discovery process to identify undesirable off-target activity profiles that could hinder or halt the development of candidate drugs or even lead to market withdrawal if discovered after a drug is approved. Here, for the first time, the rationale, strategies and methodologies for in vitro pharmacological profiling at four major pharmaceutical companies (AstraZeneca, GlaxoSmithKline, Novartis and Pfizer) are presented and illustrated with examples of their impact on the drug discovery process. We hope that this will enable other companies and academic institutions to benefit from this knowledge and consider joining us in our collaborative knowledge sharing.

The endogenous cannabinoid, anandamide, has been suggested as an endothelium-derived hyperpolarizing factor (EDHF). We found that anandamide-evoked relaxation in isolated segments of rat mesenteric artery was associated with smooth muscle hyperpolarization. However, although anandamide-evoked relaxation was inhibited by either charybdotoxin (ChTX) or iberiotoxin, inhibition of the relaxation to EDHF required a combination of ChTX and apamin. The relaxations induced by either anandamide or EDHF were not inhibited by the cannabinoid receptor (CB 1 ) antagonist SRI41716A, or mimicked by selective CB 1 agonists. Thus, anandamide appears to cause smooth muscle relaxation via a CB 1 receptorindependent mechanism and cannabinoid receptor activation apparently does not contribute to EDHFmediated relaxation in this resistance artery.

1 Acetycholine-mediated relaxations in phenylephrine-contracted aortas, femoral and mesenteric resistance arteries were studied in vessels from endothelial nitric oxide synthase knock-out (eNOS 7/7) and the corresponding wild-type strain (eNOS +/+) C57BL6/SV19 mice. 2 Aortas from eNOS (+/+) mice relaxed to acetylcholine in an endothelium-dependent N G -nitro-L-arginine (L-NOARG) sensitive manner. Aortas from eNOS (7/7) mice did not relax to acetylcholine but demonstrated enhanced sensitivity to both authentic NO and sodium nitroprusside. 3 Relaxation to acetylcholine in femoral arteries was partially inhibited by L-NOARG in vessels from eNOS (+/+) mice, but relaxation in eNOS (7/7) mice was insensitive to a combination of L-NOARG and indomethacin and the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The L-NOARG/ODQ/indomethacin-insensitive relaxation to acetylcholine in femoral arteries was inhibited in the presence of elevated (30 mM) extracellular KCl. 4 In mesenteric resistance vessels from eNOS (+/+) mice, the acetylcholine-mediated relaxation response was completely inhibited by a combination of indomethacin and L-NOARG or by 30 mM KCl alone. In contrast, in mesenteric arteries from eNOS (7/7) mice, the acetylcholine-relaxation response was insensitive to a combination of L-NOARG and indomethacin, but was inhibited in the presence of 30 mM KCl. 5 These data indicate arteries from eNOS (7/7) mice demonstrate a supersensitivity to exogenous NO, and that acetylcholine-induced vasorelaxation of femoral and mesenteric vessels from eNOS (7/7) mice is mediated by an endothelium-derived factor that has properties of an EDHF but is neither NO nor prostacyclin. Furthermore, in mesenteric vessels, there is an upregulation of the role of EDHF in the absence of NO.

No abstract

The neurotrophin family of growth factors, comprised of nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), and neurotrophin 4 (NT4), is implicated in the physiology of chronic pain. Given the clinical efficacy of anti-NGF monoclonal antibody (mAb) therapies, there is significant interest in the development of small molecule modulators of neurotrophin activity. Neurotrophins signal through the tropomyosin related kinase (Trk) family of tyrosine kinase receptors, hence Trk kinase inhibition represents a potentially "druggable" point of intervention. To deliver the safety profile required for chronic, nonlife threatening pain indications, highly kinase-selective Trk inhibitors with minimal brain availability are sought. Herein we describe how the use of SBDD, 2D QSAR models, and matched molecular pair data in compound design enabled the delivery of the highly potent, kinase-selective, and peripherally restricted clinical candidate PF-06273340.