Gastrointestinal mucosal injury following repeated daily oral administration of conventional formulations of indometacin and other non-steroidal anti-inflammatory drugs to pigs: a model for human gastrointestinal disease.

Non-steroidal anti-inflammatory drugs (NSAIDs) vary in their propensity to cause damage in different regions of the gastrointestinal (GI) tract in laboratory animals and humans. This may depend on the type of drug formulation as well as the intrinsic pharmacological properties of the drugs. The purpose of this study was to determine the effects of NSAIDs, with cyclooxygenase 1 and 2 inhibitory activity but with different potency as inhibitors of prostaglandin production, when given orally as tablet/capsule formulations of NSAIDs for 10 days to pigs, a species that has close resemblance in structure and function of the tract to that in humans. Three capsule or tablet formulations of NSAIDs were given orally to pigs for 10 days. GI bleeding was measured by determination of radioactive iron in the faeces from (59)Fe-pre-labelled red blood cells. The blood loss was compared with the pathological changes in the GI mucosa observed at autopsy, mucosal myeloperoxidase (MPO) activity as an index of leucocyte infiltration, and plasma and mucosal concentrations of the drugs at termination assayed by high-performance liquid chromatography. Mucosal damage and bleeding varied according to the type of NSAID. Gastroduodenal ulcers and lesions occurred with the cyclooxygenase inhibitors indometacin (indomethacin) (Indocid capsules 10 or 5 mg kg(-1) day(-1) b.i.d.), aspirin (USP tablets 150 mg kg(-1) day(-1) b.i.d) and naproxen (Apotex tablets 50 or 75 mg kg(-1) day(-1) b.i.d.), and there was an increase in the cumulative (i.e. 10-day) blood loss at higher doses of indometacin and naproxen, and with aspirin. There was no statistically significant increase in gastric or intestinal mucosal MPO activity in the non-damaged mucosa with these drugs and this was confirmed by histological observations in non-lesioned areas of the mucosa. Indometacin produced focal ulcers in the caecum but this was not observed with the other drugs. All the NSAIDs produced significant blood loss coincident with gastric ulceration but no increase in gastric or intestinal MPO activity. Plasma concentrations of the non-aspirin NSAIDs were within the range encountered therapeutically in humans. The mucosal concentrations of indometacin in the gastric and intestinal mucosa correlated with mucosal injury. These findings show that: (i) NSAIDs vary in their propensity to produce mucosal injury in different regions of the GI tract according to their pharmacological properties and formulation; (ii) mucosal injury from some NSAIDs may not directly relate to blood loss at low doses of NSAIDs and this may depend on inhibition of platelet aggregation; and (iii) the occurrence of caecal ulcers uniquely observed with indometacin treatment may be relevant to the development of intestinal pathology (e.g. diaphragm-like structures) seen occasionally in humans. These results suggest that the pig model employed in the present studies may be useful for investigations of GI damage from NSAID tablets/capsules, especially in regions that are generally inaccessible to routine endoscopic investigations in humans (e.g. the proximal regions of the large intestine).

New cyclooxygenase-2/5-lipoxygenase inhibitors. 1. 7-tert-buty1-2,3-dihydro-3,3-dimethylbenzofuran derivatives as gastrointestinal safe antiinflammatory and analgesic agents: discovery and variation of the 5-keto substituent.

A series of 5-keto-substituted 7-tert-buty1-2,3-dihydro-3,3- dimethylbenzofurans (DHDMBFs) were prepared and evaluated as potential nonsteroidal antiinflammatory and analgesic agents. Interest in this class of compounds arose when a DHDMBF was found to be an active metabolite of the di-tert-butylphenol antiinflammatory agent tebufelone. We have now found that a variety of 5-keto-substituted DHDMBFs have good in vivo antiinflammatory and analgesic activity after oral administration. These compounds inhibit both cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) in vitro. The cyclooxygenase inhibition was found to be selective for the cyclooxygenase-2 isoform, and this combination of COX-2/5-LOX inhibition may be responsible for the gastrointestinal safety of compounds such as 30.

An antibody to a 17 amino acid synthetic peptide of the type I interleukin-1 receptor preferentially blocks interleukin-1 beta binding.

On the basis of their relative hydropathy and alpha-helical structure, we prepared antibodies to four synthetic peptides with amino acid sequences homolgous to four hydrophilic, extracellular regions of the murine 80 kDa type I interleukin-1 receptor (IL-1RI). Antibodies to each of the four peptides recognized their specific immunogen. Human [125I]-IL-1 alpha or -beta was crosslinked to murine EL4 and D10S cells. Antiserum to peptide 150-166 precipitated the IL-1/IL-1R complex, whereas antibodies to peptide 66-84, 190-200, or 266-285 did not. Antibody to peptide 150-166 did not precipitate the type II IL-1R. Anti-IL-1RI150-166 blocked 71% of the binding of radiolabeled human IL-1 beta to EL4 cells and 50% of the binding to D10S cells. Using affinity-purified anti-IL-1RI150-166, we compared the ability of this antibody to inhibit the binding of murine or human IL-1 alpha to that of murine or human IL-1 beta. At a concentration of 20 ng/ml, affinity-purified anti-IL-1RI150-166 blocked 50% binding of murine IL-1 beta. At 1 microgram/ml, 90% blockage was observed. In contrast, no significant blockade of IL-1 alpha binding was observed at concentrations as high as 3 micrograms/ml of anti-IL-1RI150-166. The selective blockade of IL-1 beta forms was not due to differences in the affinities of these ligands for receptors on these cells. The antibody also blocked the binding of human IL-1 beta but not human IL-1 alpha to EL4 cells. The biologic activity of murine IL-1 beta but not IL-1 alpha on EL4 cells was also inhibited by this antibody. These data suggest (1) that antibody to a specific epitope on the extracellular domain interferes with the binding of IL-1 beta but not IL-1 alpha, (2) the differential inhibition of binding of IL-1 beta but not IL-1 alpha by anti-IL-1RI150-166 also blocks biologic activity, and (3) IL-1 alpha and IL-1 beta may transduce different signals by binding to separate loci on the IL-1RI.

Transcription, translation and secretion of interleukin 1 and tumor necrosis factor: effects of tebufelone, a dual cyclooxygenase/5-lipoxygenase inhibitor.

We examined the effect of tebufelone, a dual cyclooxygenase (CO)/5-lipoxygenase (LO) inhibitor, on the synthesis, secretion and gene expression of interleukin (IL) 1 beta and tumor necrosis factor (TNF)-alpha by human peripheral blood mononuclear cells (PBMC). Basal concentrations of immunoreactive IL 1 beta and TNF-alpha after 18-24 h, in the absence or presence of tebufelone (less than or equal to 12.5 microM), were near the limit of detection (100 pg/ml). By contrast, preincubation (1 h) of cells, in amounts of tebufelone which decrease the formation of leukotriene (LT) B4, markedly enhanced (up to 500%) the synthesis of IL 1 beta and TNF-alpha following lipopolysaccharide (LPS), heat-killed Staphylococcus epidermidis or concanavalin A stimulation. Moreover, a disproportionate amount of the overall increase in IL 1 (alpha and beta) was secreted in contrast to the amount which remained cell associated, an effect unrelated to cell damage or leakage as tebufelone had no effect on either lactate dehydrogenase release by PBMC, or mitochondrial dehydrogenases of adherent monocytes as detected by enzymatic cleavage of the substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. There was no inverse correlation between the changes in prostaglandin (PG)E2 levels and TNF-alpha or IL 1 beta synthesis, and when PG formation was maximally inhibited by preincubating the cells in indomethacin, tebufelone, added 1 h before the stimulus, continued to enhance the synthesis of IL 1 beta although not that of TNF-alpha. The addition of the CO/5-LO inhibitor 2 h after LPS stimulation, however, did not interfere with IL 1 beta synthesis, suggesting that tebufelone interacts with an early event(s) in the activation of PBMC. For IL 1 beta and TNF-alpha, basal and stimulated (4 h post LPS) mRNA levels were not increased by tebufelone, despite a concomitant increase in the synthesis of IL 1 beta. In conclusion, we have demonstrated that tebufelone enhances IL 1 (alpha and beta) and TNF-alpha synthesis at concentrations which suppress leukotriene formation. These findings argue against a role of 5-LO products as necessary intermediates of IL 1 (alpha and beta) and TNF-alpha synthesis.