GSK256066, an exceptionally high-affinity and selective inhibitor of phosphodiesterase 4 suitable for administration by inhalation: in vitro, kinetic, and in vivo characterization.

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-({3-[(dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy) phenyl]amino}-3-quinolinecarboxamide (GSK256066), 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) <0.5 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 α 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 (>380,000-fold versus PDE1, PDE2, PDE3, PDE5, and PDE6 and >2500-fold against PDE7), inhibited PDE4 isoforms A-D with equal affinity, and had a substantial high-affinity rolipram binding site ratio (>17). When administered intratracheally to rats, GSK256066 inhibited LPS-induced pulmonary neutrophilia with ED(50) values of 1.1 µg/kg (aqueous suspension) and 2.9 µg/kg (dry powder formulation) and was more potent than an aqueous suspension of the corticosteroid fluticasone propionate (ED(50) 9.3 µg/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 discovery and initial optimisation of pyrrole-2-carboxamides as inhibitors of p38alpha MAP kinase.

A novel pyrrole-2-carboxamide series of p38alpha inhibitors, discovered through the application of virtual screening, is presented. Following evaluation of activity, selectivity and developability properties of commercially available analogues, a synthesis program enabled rapid assessment of the series' suitability for further lead optimisation studies.

Pyrazolopyridines as potent PDE4B inhibitors: 5-heterocycle SAR.

Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al. Bioorg. Med. Chem. Lett.2008, 18, 4237] the SAR of the 5-position was investigated further. A range of substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-α production from isolated human peripheral blood mononuclear cells.

The identification of a novel phosphodiesterase 4 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), with improved therapeutic index using pica feeding in rats as a measure of emetogenicity.

Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as anti-inflammatory 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-alpha production by human peripheral blood mononuclear cells was roflumilast (IC(50) = 5 nM) > EPPA-1 (38) > rolipram (269) > cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D(50) = 0.042 mg/kg) > roflumilast (0.24) > rolipram (3.34) > 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) = 0.495 mg/kg) > roflumilast (1.6) > cilomilast (6.4) > 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 alpha(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) > roflumilast (6.4) > cilomilast (1.4) > 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.

Quinolines as a novel structural class of potent and selective PDE4 inhibitors: optimisation for oral administration.

Crystallography-driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of a series of quinoline derivatives that were highly potent and selective inhibitors of PDE4 with a good pharmacokinetic profile in the rat. Quinolines 43 and 48 have potential as oral medicines for the treatment of COPD.

Quinolines as a novel structural class of potent and selective PDE4 inhibitors. Optimisation for inhaled administration.

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.

Pyrazolopyridines as a novel structural class of potent and selective PDE4 inhibitors.

Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase 4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred substituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4 active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhibits LPS induced TNF-alpha production from isolated human peripheral blood mononuclear cells and has an encouraging rat PK profile suitable for oral dosing.

Narrow Spectrum Kinase Inhibitors Demonstrate Promise for the Treatment of Dry Eye Disease and Other Ocular Inflammatory Disorders.

Purpose: The purpose of this study is to determine the potential of narrow spectrum kinase inhibitors (NSKIs) to treat inflammatory eye disorders. Methods: Human conjunctival epithelial (HCE) cells were retrieved from subjects via impression cytology. Real-time quantitative PCR (qPCR) was performed on HCE cells to determine gene expression of NSKI kinase targets and proinflammatory cytokines in dry eye disease (DED) patients versus healthy controls. qPCR also assessed p38α expression in hyperosmolar-treated Chang conjunctival epithelial cells. Interaction of NSKI TOP1362 with the kinases was evaluated in ATP-dependent Z-LYTE and competition binding assays. Anti-inflammatory activity was assessed in human peripheral blood mononuclear cells and primary macrophages. In an endotoxin-induced uveitis (EIU) study, lipopolysaccharide (LPS) was administered intravitreally to Lewis rats. TOP1362, dexamethasone, or vehicle was administered topically, and inflammatory cytokine levels were measured 6 hours after LPS injection. Results: HCE cells from DED patients showed significantly increased expression of p38α, spleen tyrosine kinase (Syk), Src, lymphocyte-specific protein tyrosine kinase (Lck), interleukin one beta (IL-1ß), interleukin eight (IL-8), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9). TOP1362 strongly inhibited the kinase targets p38α, Syk, Src, and Lck, blocked the rise in p38α expression in hyperosmolar Chang cells, and potently reduced inflammatory cytokine release in cellular models of innate and adaptive immunities. In the EIU model, TOP1362 dose-dependently attenuated the LPS-induced rise in inflammatory cell infiltration and ocular cytokine levels with efficacy comparable to that of dexamethasone. Conclusions: TOP1362 is a potent inhibitor of kinases upregulated in DED and markedly attenuates proinflammatory cytokine release in vitro and in vivo, highlighting the therapeutic potential of NSKIs for treating ocular inflammation, such as that observed in DED.

Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis.

BACKGROUND: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition. METHODS: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa. RESULTS: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays. CONCLUSIONS: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease.

Discovery of 6-aryl-7-alkoxyisoquinoline inhibitors of IkappaB kinase-beta (IKK-beta).

The identification and progression of a potent and selective series of isoquinoline inhibitors of IkappaB kinase-beta (IKK-beta) are described. Hit-generation chemistry based on IKK-beta active-site knowledge yielded a weakly potent but tractable chemotype that was rapidly progressed into a series with robust enzyme and cellular activity and significant selectivity over IKK-alpha.