A range of NSAIDs and reported Cox 2 selective compounds were tested in human freshly isolated platelets and LPS-stimulated mononuclear cells to determine their potency and selectivity as inhibitors of constitutive (presumably Cox 1) and inducible (presumably Cox 2) cyclooxygenase respectively. All compounds tested were either equipotent at inhibiting constitutive and inducible cyclooxygenase or were selective for the inducible form. The most selective compound was Dup697 and the least selective, ketoprofen. Several compounds only produced a partial inhibition of constitutive cyclooxygenase as the maximum inhibitor concentration achievable in the assay was limited to 1 mM. With the exception of paracetamol, all compounds were able to produce full inhibition curves against the inducible form. Potency estimates against constitutive Cox compare closely with published data but most compounds were consistently more potent against the inducible isoform than in published data for human cloned, microsomal Cox 2. These data suggest that human mononuclear cells are either exquisitely sensitive to some NSAIDs or they may contain another Cox isoform as yet indistinguishable from Cox 2.

Oral phosphodiesterase (PDE) 4 inhibitors such as roflumilast have established the potential of PDE4 inhibition for the treatment of respiratory diseases. However, PDE4 inhibitor efficacy is limited by mechanism-related side effects such as emesis and nausea. Delivering the inhibitor by the inhaled route may improve therapeutic index, and we describe 6-, an exceptionally high-affinity inhibitor of PDE4 designed for inhaled administration. GSK256066 is a slow and tight binding inhibitor of PDE4B (apparent IC 50 3.2 pM; steady-state IC 50 Ͻ0.5 pM), which is more potent than any previously documented compound, for example, roflumilast (IC 50 390 pM), tofimilast (IC 50 1.6 nM), and cilomilast (IC 50 74 nM). Consistent with this, GSK256066 inhibited tumor necrosis factor ␣ production by lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes with 0.01 nM IC 50 (compared with IC 50 values of 5, 22, and 389 nM for roflumilast, tofimilast, and cilomilast, respectively) and by LPS-stimulated whole blood with 126 pM IC 50 . GSK256066 was highly selective for PDE4 (Ͼ380,000-fold versus PDE1, PDE2, PDE3, PDE5, and PDE6 and Ͼ2500-fold against PDE7), inhibited PDE4 isoforms A-D with equal affinity, and had a substantial high-affinity rolipram binding site ratio (Ͼ17). When administered intratracheally to rats, GSK256066 inhibited LPS-induced pulmonary neutrophilia with ED 50 values of 1.1 g/kg (aqueous suspension) and 2.9 g/kg (dry powder formulation) and was more potent than an aqueous suspension of the corticosteroid fluticasone propionate (ED 50 9.3 g/kg). Thus, GSK256066 has been demonstrated to have exceptional potency in vitro and in vivo and is being clinically investigated as a treatment for chronic obstructive pulmonary disease.

The pathogenic form of the cyclooxygenase (COX) enzyme, COX-2, is also constitutively present in the spinal cord and has been implicated in chronic pain states in rat and man. A number of COX-2 inhibitors, including celecoxib and rofecoxib, are already used in man for the treatment of inflammatory pain. Preclinically, the dual-acting COX-2 inhibitor, GW406381X tion injury model and reversed thermal hyperalgesia in the mouse partial ligation model, both models of neuropathic pain. GW406381X, was also effective in a rat model of capsaicininduced central sensitization, when given intrathecally (ED 50 ϭ 0.07 g) and after chronic but not acute oral dosing. Celecoxib and rofecoxib had no effect in this model. Several hypotheses have been proposed to try to explain these differences in efficacy, including central nervous system penetration, enzyme kinetics, and potency. The novel finding of effectiveness of GW406381X in these models of neuropathic pain/central sensitization, in addition to activity in inflammatory pain models and together with its central efficacy, suggests dual activity of GW406381X compared with celecoxib and rofecoxib, which may translate into greater efficacy in a broader spectrum of pain states in the clinic.The role of prostaglandins, formed through the action of the two isoforms of the cyclooxygenase (COX)

Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as antiinflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo [3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-␣ production by human peripheral blood mononuclear cells was roflumilast (IC 50 ϭ 5 nM) Ͼ EPPA-1 (38) Ͼ rolipram (269) Ͼ cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D 50 ϭ 0.042 mg/ kg) Ͼ roflumilast (0.24) Ͼ rolipram (3.34) Ͼ cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D 50 ϭ 0.495 mg/kg) Ͼ roflumilast (1.6) Ͼ cilomilast (6.4) Ͼ EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of ␣ 2 -adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D 50 )/(neutrophilia D 50 )] was EPPA-1 (578) Ͼ roflumilast (6.4) Ͼ cilomilast (1.4) Ͼ rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D 50 )/(neutrophilia D 50 )]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.Phosphodiesterases are a superfamily of enzymes that hydrolyze cAMP and/or cGMP to their inactive nucleotides. Phosphodiesterase 4 (PDE4) is selective for cAMP, and consists of the four subtypes A, B, C, and D. PDE4 inhibitors have shown efficacy in various in vitro and in vivo inflammatory models by increasing the intracellular levels of cAMP in many immune cells (T lymphocytes, monocytes, neutrophils, and eosinophils). As such, PDE4 inhibitors have been pursued as therapeutics for pulmonary diseases with an inflammatory component, including chronic obstructive pulmo-

Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also inhibits LPS induced production of TNF-alpha from isolated human peripheral blood mononuclear cells with a pIC(50) of 11.1. GSK256066 also has a suitable profile for inhaled dosing.