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5-Lipoxygenase (LOX) is an important arachidonic acid-metabolizing enzyme producing leukotrienes and other proinflammatory lipid mediators with potent pathophysiological functions in asthma and other inflammatory diseases. 4-(3-(4-(1-Methyl-1H-pyrazol-5-yl)phenylthio)phenyl)-tetrahydro-2H-pyran-4-carboxamide (PF-4191834) is a novel, selective non-redox 5-lipoxygenase inhibitor effective in inflammation and pain. In vitro and in vivo assays were developed for the evaluation of a novel 5-LOX inhibitor using conditions of maximal enzyme activity. PF-4191834 exhibits good potency in enzyme- and cell-based assays, as well as in a rat model of acute inflammation. Enzyme assay results indicate that PF-4191834 is a potent 5-LOX inhibitor, with an IC(50) = 229 +/- 20 nM. Furthermore, it demonstrated approximately 300-fold selectivity for 5-LOX over 12-LOX and 15-LOX and shows no activity toward the cyclooxygenase enzymes. In addition, PF-4191834 inhibits 5-LOX in human blood cells, with an IC(80) = 370 +/- 20 nM. This inhibitory concentration correlates well with plasma exposures needed for in vivo efficacy in inflammation in models of inflammatory pain. The combination of potency in cells and in vivo, together with a sustained in vivo effect, provides PF-4191834 with an overall pharmacodynamic improvement consistent with once a day dosing.

The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.

The cyclooxygenase isoforms, COX-1 and COX-2, are involved in the biosynthesis of prostaglandin E2, a major prostaglandin involved in epidermal homeostasis and repair. Cancer originating in the epidermis can develop when keratinocyte proliferation and apoptosis become dysregulated, resulting in sustained epidermal hyperplasia. COX-2 inhibitors, which demonstrate significant in vivo selectivity relative to COX-1, suppress both ultraviolet-induced epidermal tumor development and progression, suggesting that prostaglandin regulation of keratinocyte biology is involved in the pathogenesis of epidermal neoplasia. In this study, we characterized the expression of COX-1 and COX-2, as well as keratinocyte proliferation, differentiation, and apoptosis, following acute ultraviolet irradiation in the hairless SKH-1 mouse. Following acute ultraviolet exposure, COX-2 expression was predominantly induced in the basal keratinocyte layer coincident with an increase in keratinocyte proliferation and apoptosis. The role of COX-2 was further evaluated using a selective COX-2 inhibitor, SC-791, as well as the traditional nonsteroidal COX inhibitor, indomethacin. Following acute ultraviolet irradiation, inhibition of COX-2 with either inhibitor decreased epidermal keratinocyte proliferation. Likewise, keratinocyte apoptosis was increased with COX-2 inhibition, particularly in the proliferating basal keratinocyte layer. There was also a modest inhibition of keratinocyte differentiation. These data suggest that COX-2 expression is probably necessary for kera-tinocyte survival and proliferation occurring after acute ultraviolet irradiation. We hypothesize that selective COX-2 inhibition, as described herein, may lead to enhanced removal of ultraviolet-damaged keratinocytes, thereby decreasing malignant transformation in the epidermis.

In this study, we evaluated the ability of low molecular weight manganese-based superoxide dismutase mimetics to attenuate neutrophil-mediated oxygen radical damage to human aortic endothelial cells in vitro. Human neutrophils, when exposed to tumor necrosis factor-alpha and the complement compound C5a, induced endothelial damage assessed by the release of 51Cr into the medium. This damage correlated with the amount of superoxide generated by neutrophils. Three superoxide dismutase mimetics, with catalytic rate constants for superoxide dismutation ranging from 4 to 9 x 10(7) M-1 S-1, inhibited neutrophil- or xanthine oxidase-mediated endothelial cell injury in a concentration-dependent manner. A similar manganese-based compound with no detectable superoxide dismutase activity was ineffective in inhibiting injury. Fluorescent studies of the neutrophil respiratory burst showed that the superoxide dismutase mimetics were protective without interfering with the generation of superoxide by activated neutrophils. Catalase, elastase inhibitors, and desferrioxamine mesylate (an iron chelator and hydroxyl radical scavenger) were not protective against cell injury. This investigation demonstrates that neutrophil-mediated human aortic endothelial cell injury in vitro is mediated by the superoxide anion and that low molecular weight manganese-based superoxide dismutase mimetics are effective in abrogating this damage.

By stopped-flow kinetic analysis, we have directly evaluated the superoxide dismutase (SOD) activity of a number of organic nitroxides and iron- and manganese-based complexes that have been attributed with having SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (kcat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal inhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (FeTPEN) was eight times more active than Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine(Fe-TPAA) in the cytochrome c assay, but only FeTPAA catalyzed the first-order decay of superoxide (kcat = 2.15 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl)porphine (FeTMPP) was active at low micromolar concentrations in both the cytochrome c and stopped-flow assays. At high micromolar concentrations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) were inhibitory in the cytochrome c assay, but showed no detectable SOD activity by stopped-flow. None of the tested compounds inhibited xanthine oxidase activity as shown by the measurement of urate production. Under the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMPOL oxidized reduced cytochrome c which rationalizes the false positives for these compounds in this assay. The inhibitory activities of Mn(II) and the manganese desferal complexes in the cytochrome c assay appear to be due to a stoichiometric, not catalytic, reaction with superoxide as catalytic amounts of these agents do not induce a first-order decay of superoxide as shown by stopped-flow.

In this report, we show that apoptosis signal-regulating kinase 1(-/-) (ASK1 KO) mice were resistant to inflammatory arthritis induced in the K/BxN serum transfer model of rheumatoid arthritis (RA). The p38 inhibitor, SD-0006 was administered to wild type (WT) mice as a comparator. Both ASK1 KO and p38 inhibition resulted in marked attenuation of edema, cartilage damage, bone resorption, and general inflammatory responses. Transcriptional profiling of mRNA prepared from paw tissue demonstrated that the production of many proinflammatory genes including cytokines, chemokines, and extracellular matrix degradative enzymes were maintained at basal levels by either ASK1 KO or prophylactic p38 MAPK inhibition. In the mouse whole blood (MWB) assay, tumor necrosis factor-α (TNF-α)-induced KC and CCL2 levels and also LPS-induced interleukin-6 (IL-6), CCL2, and KC levels in MWB from ASK1 KO were significantly lower than those from WT. Furthermore, both p38 and JNK were activated by TNF-α in human synovial fibroblasts isolated from RA patients (RASF). SD-0006 or SP600125, a JNK inhibitor, partially blocked the elevation of IL-6 production in RASF following stimulation with TNF-α. In contrast, dual inhibition with both p38/JNK inhibitors almost completely abolished TNF-α-induced IL-6 production from these cells. Ablation of ASK1 expression in RASF using siRNA for ASK1 resulted in inhibition of TNF-α-induced IL-6 and PGE(2) production. This study is the first to suggest that ASK1 is critical for the development of RA and that ASK1 may be involved in the production of proinflammatory mediators in response to TNF-α stimulation in the RA joint.

The classical nuclear factor kappaB (NF-kappaB) signaling pathway is under the control of the IkappaB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-kappaB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-kappaB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of kappaB alpha (IkappaB alpha). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

Zileuton, a redox and iron chelator 5-lipoxygenase (5-LOX) inhibitor and, leukotriene receptor antagonists are presently used clinically in the long term treatment of asthma. Recent data implicate 5-LOX pathway in pain signaling. We report 5-LOX expression in the central nervous system (CNS) and analyze the pain efficacy of a new class of non redox, non iron chelating 5-LOX inhibitor. CJ-13610, 4-(3-(4-(2-methyl-1H-imidazol-1-yl) phenylthio) phenyl)-tetrahydro-2H-pyran-4-carboxamide, demonstrated antihyperalgesic activity in inflammatory pain models including the acute carrageenan model and the chronic inflammatory model using complete Freund's adjuvant. Following complete Freund's adjuvant stimulus leukotrieneB(4) concentration in the brain was elevated (9+/-1 ng/g, mean+/-S.E.M.) by about 3 times that of the control group (3+/-0.11, mean+/-S.E.M.). Hyperalgesia and leukotrieneB(4) concentration were both reversed following CJ-13610 treatment. Furthermore, we demonstrate CJ-13610 efficacy against osteoarthritis like pain using the rat medial meniscal transection model. CJ-13610 at oral doses of 0.6, 2 and 6 mg/kg/day reversed two modalities of pain in this model; tactile allodynia and weight bearing differential. Taken together, these data suggest that 5-LOX pathway and the leukotriene products are important mediators of pain.

The cyclooxygenase isoforms, COX-1 and COX-2, are the rate limiting enzymes in the biosynthesis of prostaglandin E(2), a major prostaglandin involved in epidermal homeostasis and repair. Epidermal injury results in transient hyperplasia and induction of COX-2 expression. The role of COX-2 in this hyperplasia is unknown, however. In this study, we characterized the epidermal expression of COX isozymes following wounding by abrasion in SKH-1 mice using immunohistochemistry, in situ hybridization, and Western analysis. In addition, we evaluated pivotal keratinocyte functions necessary for the reparative hyperplasia, including proliferation by 5-bromo-2'deoxy-uridine labeling and differentiation by the expression of involucrin, keratin 1, and keratin 6. Although COX-1 expression in keratinocytes remained unchanged during wound healing, COX-2 expression was induced coincidentally with keratinocyte proliferation and keratin 6 expression, suggesting a role for COX-2 in epidermal repair. The role of COX-2 was also evaluated using the selective COX-2 inhibitor SC-791 and the traditional COX inhibitors indomethacin and diclofenac. Neither inhibitor altered keratinocyte proliferation or differentiation following abrasion, in contrast to dexamethasone, which delayed these responses. Our results indicated that, although COX-2 expression was coincident with transient epidermal hyperplasia and keratinocyte proliferation/differentiation during the healing of epidermal injury, it does not play a pivotal role in this repair process.

SummaryBackground The inducible cyclooxygenase-2 (COX-2) enzyme is upregulated in inflammatory diseases, as well as in epithelial cancers, and has an established role in angiogenesis and tissue repair. Objective Because of these physiological effects and the widespread use of the selective COX-2 inhibitor, celecoxib, we wanted to determine if inhibition of COX-2 would affect incisional skin wound healing. Methods Using a cutaneous full-thickness, sutured, incisional wound model in hairless SKH-1 mice, we evaluated the role of COX-2 in the wound healing process by comparing the effects of a nonselective COX inhibitor, diclofenac, with a selective COX-2 inhibitor, SC-791. Healing was monitored for up to 28 days postincision histologically and for recovery of wound strength. Results COX-2 expression was observed over the first week of healing, peaking at day 3 and was not affected by treatment with the selective COX-2 or nonselective COX inhibitors. Infiltrating macrophages, as well as keratinocytes and dermal fibroblasts at the wound site, expressed COX-2. Neither selective COX-2, nor nonselective COX inhibition had a significant effect on the macroscopic or microscopic morphology of the wounds, whereas dexamethasone treatment resulted in epidermal and granulation tissue atrophy. In addition, neither selective COX-2, nor nonselective COX inhibition altered keratinocyte proliferation and differentiation, dermal angiogenesis or the recovery of wound tensile strength, whereas dexamethasone reduced the tensile strength of the wounds by 30–38% throughout the healing period. Conclusions These data indicate that selective COX-2 inhibition does not affect the healing of surgical skin wounds.

Objective To examine cyclooxygenase-2 (COX-2) enzyme expression, its regulation by interleukin-1 beta (IL-1 beta), and the role of prostaglandin E(2) (PGE(2)) in proteoglycan degradation in human osteoarthritic (OA) cartilage. Methods Samples of human OA articular cartilage, meniscus, synovial membrane, and osteophytic fibrocartilage were obtained at knee arthroplasty and cultured ex vivo with or without IL-1 beta and COX inhibitors. COX expression was evaluated by immunohistochemistry and Western blot analysis. The enzymatic activity of COX was measured by conversion of arachidonic acid to PGE(2). Cartilage degradation was evaluated by measuring the accumulation of sulfated glycosaminoglycans in the medium. Results IL-1 beta induced robust expression of COX-2 and PGE(2) in OA meniscus, synovial membrane, and osteophytic fibrocartilage explants, whereas low levels were produced in OA articular cartilage. IL-1 beta also induced cartilage proteoglycan degradation in OA synovial membrane-cartilage cocultures. Increased proteoglycan degradation corresponded to the induction of COX-2 protein expression in, and PGE(2) production from, the synovial membrane. Dexamethasone, neutralizing IL-1 beta antibody, or the selective COX-2 inhibitor, SC-236, attenuated both the IL-1 beta-induced PGE(2) production and cartilage proteoglycan degradation in these cocultures. The addition of PGE(2) reversed the inhibition of proteoglycan degradation caused by SC-236. Conclusion IL-1 beta-induced production of COX-2 protein and PGE(2) was low in OA articular cartilage compared with that in the other OA tissues examined. IL-1 beta-mediated degradation of cartilage proteoglycans in OA synovial membrane-cartilage cocultures was blocked by the selective COX-2 inhibitor, SC-236, and the effect of SC-236 was reversed by the addition of exogenous PGE(2). Our data suggest that induction of synovial COX-2-produced PGE(2) is one mechanism by which IL-1 beta modulates cartilage proteoglycan degradation in OA.

Human colon fibroblasts (HCF) produce tissue-type plasminogen activator (t-PA) in culture, but after 24-48 h, t-PA ceases to accumulate in the medium. Here, we report negative feedback regulation of t-PA expression, exerted by t-PA or complexes of t-PA with its physiological inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Inhibition of t-PA expression could be induced by addition of exogenous t-PA or t-PA·PAI-1 complexes and reversed by monoclonal antibody directed against the active site of t-PA. Analysis of metabolically radiolabeled protein and cellular mRNA showed that both t-PA protein and mRNA levels declined considerably after 24 h. When 125I-labeled t-PA or t-PA·PAI-1 complexes were incubated with HCF, monensin-inhibitable endocytosis and catabolism were observed. The low density lipoprotein receptor-related protein (LRP) was found to be expressed by HCF and to mediate these events. Addition of the 39-kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, removed the block to t-PA expression and restored its accumulation in the medium. Moreover, RAP completely prevented the degradation of exogenous 125I-labeled t-PA by HCF, suggesting that LRP is the endocytic receptor for t-PA in these cells. These results demonstrate that cellular modulation of t-PA expression in HCF involves LRP receptor-mediated clearance of t-PA. This LRP receptor-mediated event results in down-regulation of t-PA expression at the mRNA level.

The events that lead to irreversible neutrophil-mediated cell damage are poorly understood. Evidence indicates that the mechanism of injury varies with regard to cell type and the means of neutrophil (PMN) stimulation. The molecular basis of neutrophil-mediated cell injury and inhibitors of this process were evaluated in two in vitro assays: a neutrophil-mediated cytotoxicity assay, which measures the release of endothehal bound radiolabel and detects both cell injury and detachment; and a cytochrome c reduction assay which measures neutrophil-derived superoxide (O2-) activity. Human aortic endothelial (HAE) and human dermal microvascular endothelial (HDME) cells were found to differ in susceptibility to neutrophil-mediated injury. The amount of HAE cell injury correlated well with the amount of Or generated by stimulated PMN. HDME cells were injured in the presence of lower concentrations O2-than HAE cells and by PMN stimulated with inflammatory mediators (LPS, PAF, and fMLP) which appeared to generate little or no O2-. Neutrophil-mediated injury to both HAE and HDME cells was found, however, to be inhibitable by superoxide dismutase (SOD) in a dose dependent manner. The injury was not abrogated by catalase of the iron chelator, deferoxamine mesylate (desferol). When the amount of nitric oxide (NO) released from endothelial cells and unstimulated PMN (measured as nitrite) was examined, HDME cells and PMN were found to release significantly more NO than HAE cells. Stimulation of PMN to generate O2-resulted in significant decreases in basal nitrite levels. Furthermore, induction of NO synthesis by bradykinin or addition of exogenous NO significantly enhanced neutrophilmediated HAE cell injury. This injury was abolished by SOD. These results indicate that the superoxide anion is directly involved in the neutrophil-mediated killing of HAE and HDME cells. These data also suggest a role for peroxynitrite (formed upon interaction of NO and O2-) in oxidative damage. Differential release of NO from cell types may explain variation in susceptibility to neutrophil-mediated injury.

The drastically reduced virus yields obtained from monkey cells abortively infected with adenovirus 2 (Ad2) have been attributed primarily to a severe decrease in the accumulation of the virion protein fiber (IV), a product of the most pomoter distal late gene family, L5. Here we report that the accumulation of virion protein IIIa, a product of the proximal late gene family, L1, is also severely depressed. In contrast, the i-leader protein LO-13.6K and L1 protein(s) 52K/55K are expressed with the same time course and in equal amounts in monkey cells abortively infected by Ad2 or productively infected by the Ad2-simian virus 40 (SV40) hybrid Ad2+ND1 or by the host range mutant Ad2+ND3 hr602. L1-52K/55K is phosphorylated in abortively infected CV-1 or CV-C monkey cells as well as in productively infected human and monkey cells. As with fiber expression, the failure to produce IIIa appears to be due partly to reduced or delayed IIIa mRNA accumulation. The small amount of IIIa protein that is synthesized in monkey cells is stable. Since the accumulation of both IIIa and fiber protein is deficient, the mechanism of abortive infection cannot be attributed solely to the absence of the auxiliary fiber leader sequences (1).