Jane V. Peppard

Preclinical pharmacology of lumiracoxib: a novel selective inhibitor of cyclooxygenase-2

1 This manuscript presents the preclinical profile of lumiracoxib, a novel cyclooxygenase‐2 (COX‐2) selective inhibitor. 2 Lumiracoxib inhibited purified COX‐1 and COX‐2 with Ki values of 3 and 0.06 μM, respectively. In cellular assays, lumiracoxib had an IC50 of 0.14 μM in COX‐2‐expressing dermal fibroblasts, but caused no inhibition of COX‐1 at concentrations up to 30 μM (HEK 293 cells transfected with human COX‐1). 3 In a human whole blood assay, IC50 values for lumiracoxib were 0.13 μM for COX‐2 and 67 μM for COX‐1 (COX‐1/COX‐2 selectivity ratio 515). 4 Lumiracoxib was rapidly absorbed following oral administration in rats with peak plasma levels being reached between 0.5 and 1 h. 5 Ex vivo, lumiracoxib inhibited COX‐1‐derived thromboxane B2 (TxB2) generation with an ID50 of 33 mg kg−1, whereas COX‐2‐derived production of prostaglandin E2 (PGE2) in the lipopolysaccharide‐stimulated rat air pouch was inhibited with an ID50 value of 0.24 mg kg−1. 6 Efficacy of lumiracoxib in rat models of hyperalgesia, oedema, pyresis and arthritis was dose‐dependent and similar to diclofenac. However, consistent with its low COX‐1 inhibitory activity, lumiracoxib at a dose of 100 mg kg−1 orally caused no ulcers and was significantly less ulcerogenic than diclofenac (P<0.05). 7 Lumiracoxib is a highly selective COX‐2 inhibitor with anti‐inflammatory, analgesic and antipyretic activities comparable with diclofenac, the reference NSAID, but with much improved gastrointestinal safety.

Inhibition of interleukin‐1α‐induced cartilage oligomeric matrix protein degradation in bovine articular cartilage by matrix metalloproteinase inhibitors: Potential role for matrix metalloproteinases in the generation of cartilage oligomeric matrix protein fragments in arthritic synovial fluid

OBJECTIVE To determine whether matrix metalloproteinases (MMPs) degrade cartilage oligomeric matrix protein (COMP) to produce fragments similar to those found in synovial fluid (SF) from patients with arthritis. METHODS COMP fragments were generated in vitro by treating (a) bovine articular cartilage with interleukin-1alpha (IL-1alpha), (b) purified bovine COMP with MMPs, and (c) articular cartilage with MMPs. The fragments generated in each case were analyzed by Western blot, using an antibody to the C-terminal heptadecapeptide of COMP. RESULTS IL-1alpha stimulation of cartilage resulted in a fragmentation of COMP, which was inhibited by MMP inhibitors CGS 27023A and BB-94. Isolated, recombinant MMPs rapidly degraded purified COMP, as well as COMP residing in cartilage. Several COMP fragments produced in vitro had similar electrophoretic mobility to those in SF of patients with arthritis. CONCLUSION MMPs may contribute to the COMP fragments found in vivo. Quantitation of MMP-specific fragments may be useful in the evaluation of MMP inhibitors in patients with arthritis.

Transforming growth factor-β enhances secretory component and major histocompatibility complex class I antigen expression on rat IEC-6 intestinal epithelial cells

Abstract Transforming growth factor-β (TGF-β) has been implicated as having a role in inflammatory responses by inducing cellular infiltration and the release of inflammatory cytokines. In this study, the IEC-6 rat intestinal epithelial cell line was used as a model to assess the effect of TGF-β 1 on the expression of various plasma membrane determinants. TGF-β 1 induced a dose-dependent increase in the percentage of cells expressing surface secretory component (SC) and class I major histocompatibility (MHC) antigens. However, the expression of class II MHC was unaffected. In contrast, epidermal growth factor had no effect on any of the surface proteins studied. The TGF-β 1 -enhanced expression of SC was accompanied by an enhanced binding of polymeric, but not monomeric, immunoglobulin A (IgA). Preincubation of the TGF-β 1 -treated cells with an anti-human β-galactosyltransferase (β-GT) antiserum did not block the binding of the anti-SC antibody, indicating that the TGF-β-induced increase in SC staining was due to SC expression and not the polymeric immunoglobulin-binding enzyme, β-GT. These results indicate that TGF-β 1 may be important in immune functions involving intestinal epithelial cells by enhancing the expression of surface class I MHC antigens and SC, a protein responsible for the transport of polymeric IgA into the intestinal lumen.

Coccidiosis: characterization of antibody responses to infection with Eimeria nieschulzi.

Summary The antibody responses of rats to infection with the intestinal intracellular protozoan parasite Eimeria nieschulzi were examined by a sensitive radio‐immunoassay with a soluble preparation of sporulated oocysts as antigen. Specific antibodies of the IgM, IgGl, IgG2a and IgG2b isotypes were found in the blood circulation and IgA antibodies were detected in the bile and in intestinal washings. The IgM response was rapid, its peak was relatively brief and it was not recalled by the reinoculation of oocysts. There were some differences between the responses in the different subclasses of IgG but they all reached a peak between 20–30 days after the initiation of the primary infection and there was an anamnestic response to a challenge inoculation of oocysts. IgA antibodies to E. nieschulzi antigen in the bile and in intestinal washings increased and decreased after both primary and secondary inocula. Antibodies of all isotypes tested were virtually absent in the blood circulation of infected athymic rats. These findings are discussed with reference to antibody responses to other parasitic infections and to the role of antibodies in immunity to coccidiosis.

Identification of a splice variant of neutrophil collagenase (MMP-8)

We have identified a splice variant of human neutrophil collagenase (MMP‐8) transcript (MMP‐8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP‐8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP‐8, the translation product of MMP‐8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP‐8alt protein may lead to uncover novel roles it plays in cellular physiology.

EFFECT OF SELECTIVE AND NON-SELECTIVE CYSTEINE PROTEASE INHIBITORS ON THE INTRACELLULAR PROCESSING OF INTERLEUKIN 6 BY HEPG2 CELLS

SummaryThe effects were measured and compared of three nonselective cysteine cathepsin inhibitors (leupeptin, trans-Epoxysuccinyl-l-Leucylamido(4-guanidino)-butane (E-64), and Z-Phe-Ala-CH2F) and a selective cathepsin B inhibitor, CA074Me, on the intracellular processing of 125I-labeled human recombinant Interleukin 6 (IL-6) by HepG2 cells. The uptake and processing of 125I-IL-6 by cells treated with inhibitors was followed over a 7-h period. All inhibitors caused an increased residence time of IL-6 inside the cell and a corresponding decrease in the output of non-trichloroacetic acid-precipitable fragments of radiolabeled protein. Maximal effect was achieved with leupeptin at 200 µM, with which the rate of IL-6 digestion was reduced to 50% that of control cells. The specific inhibitor CA074Me was the least effective in slowing the intracellular processing of IL-6. The effects of all of the inhibitors on the production of haptoglobin, either stimulated by IL-6 or basal, was negligible over a similar time period, indicating continued cell viability. The data from this model suggest that cathepsin inhibitors would not interfere with lysosomal processing to an extent which would prohibit the development of selective and potent cathepsin inhibitors for the treatment of diseases in which individual cysteine cathepsins play clearly pathophysiological roles.

Inhibition of LTB4 biosynthesis in situ by CGS 23885, a potent 5-lipoxygenase inhibitor, correlates with its pleural fluid concentrations in an experimentally induced rat pleurisy model

Abstract An intrapleural injection of carrageenan in rats induced LTB4 and LTC4/D4/E4 biosynthesis, exudate formation, and cellular influx in the pleural cavity. An injection of calcium ionophore (A23187, 100nmol) 16–18h after carrageenan injection augmented leukotriene biosynthesis and exudate formation, but not cellular influx. The carrageenan-induced pleurisy model modifid by A23187 administration was used to study the oral effect of CGS 23885 (N-hydroxy-N-[(6-phenoxy-2H-1-benzopyran-3-yl)-methyl]urea), a potent 5-lipoxygenase (5-LO) inhibitor, on inflammatory parameters. CGS 23885 dose-dependently (1 to 30mg/kg) inhibited the enhanced LTB4 and LTC4/D4/E4 (1 to 10mg/kg) biosynthesis, but had no effect on enhanced exudate formation. An inhibitory effect of CGS 23885 of small magnitude on cellular influx due to carrageenan stimulation was seen at 30mg/kg. The concentrations of CGS 23885 in the pleural fluid were dose-related, and a positive correlation (r2=0.989) between pleural fluid concentration of LTB4 and CGS 23885 was observed. The results confirm that CGS 23885 is a specific, orally active 5-LO inhibitor which can achieve concentrations in the pleural cavity sufficient to inhibit production of LTB4 and LTC4/D4/E4 in an ongoing inflammatory response.

Pharmacologic profile of CGS 24128, a potent, long-acting inhibitor of neutral endopeptidase 24.11.

We compared the pharmacologic profiles of thiorphan, a neutral endopeptidase (NEP) inhibitor which is cleared rapidly from the circulation, and CGS 24128, an inhibitor with a much longer half-life (t1/2). Thiorphan and CGS 24128 inhibited NEP in vitro with IC50 values of 5.0 +/- 0.2 and 4.3 +/- 0.2 nM, respectively. After administration at 10 mg/kg intravenously (i.v.), the concentrations of CGS 24128 in the plasma were > 500 nM for 4 h but plasma thiorphan was detectable for only 60 min. Thiorphan 3 mg/kg administered intraarterially (i.a.) increased plasma atrial natriuretic peptide immunoreactivity (ANPir) levels by 58 +/- 12% in rats administered exogenous ANP(99-126). This response lasted < 60 min, whereas the same dose of CGS 24128 produced an average increase of 191 +/- 19% in ANPir concentrations that persisted for 4 h. ANP-induced (1 microgram/kg i.v.) natriuresis was significantly potentiated in anesthetized rats pretreated (60 min) with a bolus of CGS 24128 10 mg/kg i.v. The change in urinary sodium excretion (UNaV) produced by ANP was 28.8 +/- 4.0 and 15.8 +/- 1.8 muEq/kg/min in CGS 24128- and vehicle-treated rats, respectively. ANP-induced natriuresis was also greater during continuous infusion of thiorphan (5 mg/kg bolus + 0.1 mg/kg/min i.v.; delta UNaV = 29.2 +/- 5.8 and 13.8 +/- 3.2 muEq/kg/min in drug- and vehicle-treated rats, respectively) but not when thiorphan was administered as a bolus (10 mg/kg i.v.) 60 min before the ANP challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple and rapid radioimmunoassay for human interleukin-6

Studies were undertaken to develop a sensitive radioimmunoassay for human IL-6 using commercially available reagents. The assay utilized a polyclonal rabbit anti-IL-6 binding to iodinated IL-6; the reaction was carried out in solution and the immune complexes were precipitated using anti-rabbit IgG coupled to magnetic particles. Using a format where sample IL-6 was added in advance of the radiolabelled IL-6, the working range of the assay was found to fall between 0.1 and 10 ng/ml (IC50 around 1 ng/ml) for a 50 microliters sample volume, and was run overnight. However, the assay could be completed in 2 h, using a direct competitive format when less sensitivity is required (working range 1.5-25 ng/ml, IC50 12 ng/ml). The RIA correlated directly with a standard functional assay for IL-6 (proliferation of the mouse hybridoma B9.9) for both recombinant IL-6 and natural IL-6 from human monocytes. The total assay variance was less than 12% and no reactivity with interleukins-1-5 was found. Using the RIA, IL-6 produced in culture by human monocytes in response to various stimuli (LPS, IL-1, dibutyryl cAMP) was measured.

The Discovery and Characterization of an Interleukin 6 Cytokine Family Antagonist Protein from A Marine Sponge, Callyspongia sp.

The process of drug discovery in the pharmaceutical industry often begins with the setting up of a bioassay whereby many thousands of synthetic compounds, or less characterized substances such as natural product extracts, may be screened in high throughput mode. During the early 1990s, we started a search using this approach for inhibitors of the cytokine Interleukin 6 (IL-6). One of the sources for screening was extracts of marine organisms provided to the former Ciba-Geigy organization by Harbor Branch Oceanographic Institute (HBOI). This chapter describes the finding of activity inhibiting IL-6 function from an extract of the marine sponge Callyspongia sp., and work which was carried out during the effort to characterize this activity more fully.