Effects of tebufelone (NE-11740), a new anti-inflammatory drug, on arachidonic acid metabolism.

Tebufelone is a novel nonsteroidal anti-inflammatory drug (NSAID), of the di-tert-butylphenol (DTBP) class, which displays potent anti-inflammatory, analgesic and anti-pyretic properties in a variety of animal models. In this report, the effects of Tebufelone on arachidonic acid (AA) metabolism are reviewed. Tebufelone potently inhibits the formation of prostaglandins (PGE2) a key mediator of pain and inflammation, in isolated enzyme preparations (IC50 = 1.5 microM, KI = 0.35 microM), two in vitro cellular systems: rat peritoneal macrophages (IC50 = 0.02 microM) and human whole blood (IC50 = 0.08 microM), and ex vivo in man. In addition to PGE2 inhibition, which is common to all NSAIDs, higher concentrations of Tebufelone block the in vitro formation of products of the lipoxygenase pathway [leukotrienes (LTB4)] in rat macrophages (IC50 = 20 microM) and human whole blood (IC50 = 22 microM). Substrate incorporation studies (14C-AA) indicate that Tebufelone reversibly inhibits cyclooxygenase (CO) and 5-lipoxygenase (5-LO) enzymes rather than regulating the release of AA. Tebufelone was shown to be a more potent CO inhibitor than indomethacin and a less potent 5-LO inhibitor than RG-5901. Comparisons to structurally related compounds under development (E-5110, Esai; KME-4, Kanagafuchi), found Tebufelone to be the most potent CO inhibitor in vitro. All three DTBP compounds were equipotent 5-LO inhibitors. It is likely that Tebufelone's inhibitory effects on AA metabolism are, in part, responsible for its in vivo efficacy and enhanced safety profile.

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.

Determination of leukotriene B4 in human plasma by gas chromatography using a mass selective detector and a stable isotope labelled internal standard. Effect of NE-11740 on arachidonic acid metabolism.

A highly selective gas chromatographic method, coupled with selected ion monitoring using a mass selective detector and positive electron ionization, was developed for the determination of leukotriene B4 (LTB4) in human plasma. Plasma was separated from whole human blood via centrifugation, proteins precipitated with acetonitrile and LTB4 recovered (approximately 82.0%) by ethyl acetate extraction. The methyl ester, bis-t-butyldimethylsilyl ether derivative of LTB4 was formed prior to analysis and determined quantitatively using [18O]2-LTB4 as an internal standard. The limit of detection (S/N = 2) was 425 pg on column (m/z 335/339) using a 1-microliter injection. Standard curves were linear over two orders of magnitude with an RSD of less than 5.0% (n = 10). NE-11740, a new anti-inflammatory drug, was shown to inhibit, in a dose-dependent manner (ED50 = 22 microM) ionophore-stimulated LTB4 biosynthesis by human whole blood in vitro.

Differential effect of platelet-activating factor (PAF) receptor antagonists on peptide and PAF-stimulated prostaglandin release in unilateral ureteral obstruction.

Unilateral ureteral obstruction (UUO) results in increased renal resistance as well as in exaggerated prostaglandin (PG) release from the obstructed hydronephrotic kidney (HNK). We have reported previously that platelet-activating factor (PAF) dose-dependently stimulates the release of PGs from both the HNK and unobstructed contralateral kidney (CLK), with CLK release being 10% that of the HNK. In the present report, we studied the interaction of PAF with its receptor by examining the effects of PAF-receptor antagonists on the release of PGs from the isolated perfused rabbit HNK and CLK stimulated by PAF; angiotensin II (AII), and bradykinin (BK) were also used as agonists. In the HNK, kadsurenone (3 microM) inhibited PAF-stimulated PGE2 and thromboxane B2 (TxB2) release by 28.2 and 62.5% respectively. CV-3988 (20 microM) and triazolam (5 microM) also preferentially diminished PAF-stimulated TxB2 release. In addition, all three drugs significantly diminished BK- and AII-stimulated TxB2 release, while CV-3988 was the only antagonist to affect peptide-stimulated PGE2 release. While effective against agonist-stimulated PG synthesis, these drugs had no direct effect on arachidonic acid metabolism to PGs. Furthermore, in the CLK, CV-3988 had no effect on BK- or AII-stimulated PGE2 release, whereas it totally inhibited PAF-stimulated release of PGE2. These results show that PAF-receptor antagonists in the HNK preferentially inhibit TxB2 release whether stimulated by PAF, AII or BK; in the CLK only PAF-stimulated PG release is affected. This biochemical difference may be of physiological significance and explain some of the functional differences between the HNK and CLK. Therefore, PAF may be an important mediator of some of the biochemical and functional changes associated with UUO.

The effect of sodium intake on renal prostaglandin production.

The effect of chronic alterations in dietary sodium intake on renal arachidonic acid (AA) metabolism was studied in male Wistar rats who were maintained for 14 days on a diet consisting of sodium-deficient food and either deionized water (low salt intake, LSI), 1% saline (normal salt intake, NSI), or 2% saline (high salt intake, HSI). 24 h Urinary Sodium (UNaV) and plasma renin activity (PRA) measurements were shown to validate the dietary protocol. Microsomal preparations from the cortices and medullae were incubated with radiolabeled exogenous AA, and endogenous urinary prostaglandin (PG) levels were assayed by RIA to quantify renal PG synthesis. Cortical PGF2 alpha and PGE2 synthesis was found to be the greatest following LSI. In contrast, medullary PGF2 alpha was shown to be the least following LSI and to increase with increased sodium intake. Likewise, urinary PGF2 alpha levels significantly increased with increasing sodium intake. Changes in urinary PGE2 levels showed the same trend as PGF2 alpha but did not achieve statistical significance. These data show that dietary sodium differentially affects renal cortical and medullary PG synthesis and may reflect physiological differences in the regulation of cyclooxygenase in these zones. These data further suggest that the major source of urinary PGs is the renal medulla since the relationship of urinary levels to sodium intake mimics that described for the synthesis of PGs by the medullary tissue.

Indications and contraindications for the use of nonsteroidal antiinflammatory drugs in urology.

Prostaglandins are ubiquitous biologically active compounds that are involved in inflammatory reactions, hemostasis, and, under certain circumstances, the maintenance of renal function. NSAIDs, which inhibit PG synthesis, are used therapeutically most often as antiinflammatory agents in conditions of inflammation and pain, mostly of a nonurologic nature. However, since NSAIDs inhibit systemic PG synthesis, administration of NSAIDs can lead to adverse side effects. For example, the gastrointestinal irritation caused by NSAIDs probably reflects removal of a cytoprotective effect of gastrointestinal PGs. Similarly, the kidney may be especially susceptible to adverse effects of NSAIDs. In diseases such as peptic ulcers, diabetes, hypertension, congestive heart failure, liver disease with ascites, and renal insufficiency, PGs seem to play a protective role in the kidney. This protective role, which results from increased synthesis of vasodilator PGs in the face of elevated vasoconstrictors, is diminished in the presence of NSAIDs. Other side effects include the antagonism by NSAIDs of the action of diuretics, such that the dose of the diuretic must be adjusted accordingly. The diuretic triamterene should not be used in conjunction with indomethacin due to several reported cases of toxicity. Another drug interaction involves the salicylates, which have been shown to diminish the uricosuric effects of probenecid and sulfinpyrazone. Likewise, since corticosteroids increase the renal clearance of salicylates, it is important to monitor the patient carefully following termination of steroid treatment in patients receiving large doses of salicylates, since this change in elimination can precipitate toxicity. In addition, the NSAIDs bind to plasma proteins and, as such, can displace or be displaced by other drugs that bind in the same manner and can result in either decreased efficacy or toxicity. Despite the fact that the kidney may not be the target of NSAID therapy, renal function may be adversely affected by NSAID treatment. It has therefore been proposed that a renal-sparing NSAID would be a very useful therapeutic agent. Sulindac (Clinoril) has been suggested to be such an agent, eg, able to inhibit systemic PG synthesis (usually monitored by measuring serum thromboxane synthesis) without an apparent effect on renal PG synthesis (monitored by measurement of urinary PGs). However, recent data have suggested that Sulindac does inhibit renal PG synthesis and does not exhibit selectivity. The reasons for the discrepancy are not clear, but may relate to the doses or time intervals examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-activating factor is a potent stimulus for renal prostaglandin synthesis: possible significance in unilateral ureteral obstruction.

Platelet-activating factor (PAF), a lipid with potent platelet-stimulating and hypotensive properties, has been shown to stimulate the release of prostaglandins (PGs) from a number of cell types. It is produced by a variety of inflammatory cells and the renal medulla. However, no studies to date have examined the effect of PAF on the intact kidney. We, therefore, studied the effect of bolus injections of PAF on the isolated perfused rabbit kidney subjected to aseptic ureteral ligation for 72 hr. Intrarenal resistance and release of PGs by both the hydronephrotic (HNK) and contralateral kidney (CLK) were quantified. Intrarenal administration of PAF causes a dose-dependent stimulation of the release of PGE2, thromboxane B2 (TxB2) and the PGI2 metabolite, 6-keto-PGF1 alpha from the HNK and CLK. The magnitude of the release from the HNK is much greater than that for the CLK. A 100-ng bolus injection of PAF into the HNK results in the release of 1561.0 +/- 312.0 and 117.7 +/- 38.2 ng of PGE2 and TxB2, respectively, whereas administration of this dose to the CLK causes 291.0 +/- 35.0 ng of PGE2 and 19.0 +/- 4.2 ng of TxB2 to be released. Renal vascular resistance (RVR) is increased by PAF in the HNK. Product identity was confirmed using selective inhibitors and bioassay. These data show that PAF is a potent stimulus for renal PG release and that this release may have vascular consequences.