Background and purpose: The p38 kinase regulates the release of proinflammatory cytokines including tumour-necrosis factor-a (TNFa) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762-0, we investigated the therapeutic potential of p38 inhibition and compared this to anti-mouse (m)TNFa antibody treatment in murine collagen-induced arthritis (CIA). Experimental approach: Pharmacological profiles of Org 48762-0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)-induced inflammation in mice. The effects of Org 48762-0 and of mTNFa-neutralization on established arthritis were examined in murine CIA. Key results: Org 48762-0 potently inhibited p38a kinase with a high degree of kinase selectivity. In cellular assays, Org 48762-0 reduced LPS-induced TNFa release. Oral administration of Org 48762-0 in mice showed drug-like pharmacokinetic properties and inhibited LPS-induced cytokine production. These pharmacological characteristics of Org 48762-0 prompted a comparison of therapeutic efficacy with mTNFa-neutralization in CIA. Org 48762-0 and anti-mTNFa antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762-0 treatment only. Further, micro-computed tomographical and histopathological analyses confirmed the protective effects of Org 48762-0 on joint damage. Conclusions and implications: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFa produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.
Previously, it was shown that beta-elimination of selenocysteine Se-conjugates by rat renal cytosol leading to pyruvate formation was not solely catalyzed by pyridoxal phosphate-dependent enzymes. It was hypothesized that selenoxidation of the selenocysteine Se-conjugates, followed by syn-elimination, may be an alternative mechanism for pyruvate formation. In this study, selenoxidation of selenocysteine Se-conjugates was studied using rat liver microsomes and recombinant human oxidative enzymes. For all six selenocysteine Se-conjugates that were tested, it was found that rat liver microsomal incubations led to the formation of pyruvate, whereas the corresponding selenoxides were not observed. Microsomal pyruvate formation from Se-benzyl-L-selenocysteine (SeBC) was NADPH-dependent, but only marginally inhibited by several P450 inhibitors. Inhibition by methimazole and by heat pretreatment and stimulation by n-octylamine indicated that flavin-containing monooxygenases are mainly responsible for pyruvate formation from the selenocysteine Se-conjugates in rat liver microsomes. In the case of S-benzyl-L-cysteine, the sulfur analogue of SeBC, pyruvate formation was not observed. For this substrate, a chemically stable sulfoxide could be observed, as previously described. By using recombinantly expressed human flavin-containing monooxygenases and P450 enzymes, it was delineated that SeBC is selenoxidized by human FMOs, but not by human P450s. The k(cat)/K(m) of selenoxidation was 3.8-fold higher for FMO-1 than for FMO-3. In conclusion, selenoxidation of selenocysteine Se-conjugates catalyzed by FMOs and subsequently syn-elimination has taken place as an alternative route for the formation of pyruvate from selenocysteine Se-conjugates. Although selenoxides are known to be easily reduced by thiol compounds, microsomal pyruvate formation from SeBC was only 75% inhibited in the presence of an excess of glutathione. This indicates that even in the presence of physiological concentrations of reducing thiol compounds, selenoxides of selenocysteine Se-conjugates may undergo syn-elimination to some extent. Whether selenoxides and/or selenenic acids that are formed are involved in the activity of chemopreventive selenocysteine Se-conjugates remains to be established.
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