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The generation of lipoxygenase products on the contraction elicited by prostaglandin (PG) F2 alpha was investigated in the guinea-pig isolated trachea. Indomethacin (5 x 10(-6) M) inhibited the response at low concentrations of PGF2 alpha while enhanced the response at higher concentrations of PGF2 alpha. Phenidone (10(-4) M) and nordihydroguaiaretic acid (NDGA, 3 x 10(-5) M) appeared to inhibit the PGF2 alpha response. The PGF2 alpha response augmented by indomethacin was dose-dependently inhibited by NDGA and a leukotriene (LT) antagonist, FPL55712. NDGA had no effect on the contraction elicited by histamine but markedly inhibited the contraction elicited by LTD4. The inhibition by NDGA of the LTD4-induced contraction was abolished in the presence of indomethacin (5 x 10(-6) M). FPL55712 inhibited the LTD4-induced contraction but the extent of the antagonism was not changed by indomethacin. In the presence of indomethacin PGF2 alpha (10(-8) M) did not affect the LTD4 (3 x 10(-9) M) response but significantly enhanced the arachidonic acid (AA, 6.6 x 10(-5) M)-induced contraction. FPL55712 (3 x 10(-6) M) completely inhibited the AA response augmented by PGF2 alpha. These results suggest that lipoxygenase-mediated LT-like substances are released in the response at higher concentrations of PGF2 alpha on the guinea-pig isolated trachea, and the mode of action of PGF2 alpha is different from those of histamine and LTD4.

Drugs may alter prostaglandin production by acting on the various pathways of arachidonic acid metabolism. The liberation of arachidonic acid from membrane-bound phospholipids, induced by the enzyme phospholipase A2, may be inhibited by mepacrine and the steroidal anti-inflammatory agents. The bio-transformation of the free arachidonic acid, by the enzyme cyclooxygenase, to the unstable endoperoxide intermediates is inhibited by non-steroidal anti-inflammatory agents. Thus, the generation of all the prostaglandin products is prevented. This action can explain the anti-inflammatory, analgesic, antipyretic actions as well as the ulcerogenic properties of these aspirin-like compounds. An alternative metabolic pathway of arachidonic acid, via the lipoxygenase system, can be inhibited by an acetylenic analogue and a newer compound, phenidone. The unstable endoperoxide intermediates can be transformed by blood platelets into the pro-aggregating products, thromboxanes. This pathway can be selectively inhibited by a variety of experimental compounds. Prostacyclin, a potent vasodilator and inhibitor of platelet aggregation is the major product of endoperoxide transformation in blood vessels. Its formation can be inhibited by lipid peroxides. Selective actions on one or more steps in arachidonic acid metabolism can lead to a different profile of the products subsequently generated. Such a diversion of biosynthetic pathways may be an underlying mechanism in certain pathological conditions, perhaps even in dysmenorrhea.

Topical application of TPA to mouse skin causes oedema (2–6 h) neutrophil influx (3–24 h) and epidermal cell proliferation (24–48 h). Topical application of a cyclooxygenase inhibitor (indomethacin) dual cyclooxygenase and lipoxygenase inhibitors (phenidone and BW 755C) a selective lipoxygenase inhibitor (AA 861), protein synthesis inhibitors (cycloheximide and actinomycin D) or a glucocorticosteroid (prednisolone) inhibited oedema and neutrophil influx. Systemic administration of an inhibitor of microtubule assembly (colchicine) also prevented neutrophil influx and oedema. These results suggest that the inflammatory response to TPA depends on an interaction between a protein and products of arachidonic acid metabolism to produce a neutrophil dependent oedema. Epidermal cell proliferation was inhibited by topical administration of prednisolone, indomethacin, BW 755C and cycloheximide but not systemically administered methotrexate. This suggests that inhibition of the early inflammatory response to TPA prevents the subsequent epidermal proliferation.

The actions of a number of prostaglandins (PGs) were studied in unanesthetized rabbits using an intraocular pressure (IOP) recovery-rate method. In topical doses of 0.1 to 10 micrograms, these compounds accelerated the rate at which IOP returned to control levels after an infusion of hypertonic saline. In general, PGE1 appeared more potent than the other PGs at these doses. Arachidonic acid also increased the IOP recovery rate. The effect of arachidonic acid was completely blocked by the cyclooxygenase inhibitor indomethacin. Recovery rate responses to arachidonic acid were increased further after pretreatment with the lipoxygenase inhibitor phenidone. When administered alone, phenidone itself accelerated IOP recovery; this action was also blocked by indomethacin. The IOP recovery rate method appears to be a useful tool for studying ocular effects of PGs and other products or inhibitors of arachidonic acid metabolism.

Phenidone is not a substrate for dioxygenation by soybean lipoxygenase-1 (L1) but reduces L1 Fe(III) into L1 Fe(II), as shown by EPR spectroscopy. L1 catalyzes the oxidation of phenidone by 13-HPOD, the hydroperoxide formed by dioxygenation of linoleic acid by L1, with formation of 4,5-dehydrophenidone. Two moles of 13-HPOD are used per mole of phenidone dehydrogenated. Other pyrazoline derivatives such as BW 755C, but also, in a more general manner, different compounds containing phenol, aniline, hydrazine, hydroxylamine or hydrazide functions act as reducing substrates for decomposition of 13-HPOD by L1.

Diverse particulate and soluble stimuli trigger two metabolic bursts in mouse peritoneal macrophages important in the inflammatory and/or cytotoxic actions of the cells: release, oxygenation, and further metabolism of arachidonic acid from endogenous phospholipids and reduction of molecular oxygen to reactive intermediates. We tested the hypothesis that the release of arachidonic acid or formation of its metabolites are obligatory intermediate steps in triggering the NADPH oxidase that reduces O2 to O-2. With phorbol diesters as stimuli, the following inhibitors of phospholipase A2 and lipoxygenase suppressed release of H2O2 at nontoxic concentrations (microM range): p-bromophenacyl bromide, quinacrine, eicosatetraenoic acid, nordihydroguaiaretic acid, and phenidone. Indomethacin and acetylsalicylic acid were ineffective. However, the suppressive effect of the first five agents on H2O2 release could be attributed to their suppression of macrophage glucose uptake at the same concentrations, a previously unrecognized effect of these compounds. Further, concanavalin A, wheat germ agglutinin, and thrombin each stimulated abundant arachidonate release without H2O2 release. Finally, noncytolytic concentrations of cycloheximide and/or emetine suppressed arachidonate release without affecting H2O2 secretion triggered either by phorbol esters or zymosan. Release and metabolism of arachidonic acid and secretion of reactive oxygen intermediates appear to be two frequently coincident but mutually independent metabolic pathways in the mouse peritoneal macrophage.

Compound 48/80 and formyl-methionyl-leucyl-phenylalanine (fMLP), but not concanavalin A and dextran, contract isolated guinea pig tracheal spirals and lung parenchymal strips. fMLP induced a brief histamine-independent contraction of parenchymal strips. Tracheal contraction by fMLP was only observed in the presence of indomethacin, suggesting the involvement of a bronchoconstrictor lipoxygenase product of arachidonic acid (AA). Contractions induced by compound 48/80 were prolonged, were not dependent on extracellular calcium and were partly inhibited in the first 10 min by mepyramine, and after this by phenidone. Indomethacin enhanced contractions of trachea but inhibited contractions of parenchyma. Thus, histamine only contributes to the early part of compound 48/80-induced airway contractions, and lipoxygenase-derived AA products contribute after that. Bronchoconstrictor cyclooxygenase products may also contribute to parenchymal contractions. Compound 48/80 contracts the airways in a similar manner to antigen and thus may provide a useful model to examine mediator release and airway smooth muscle contraction.

A colorimetric AULO Analyser method for the estimation of hydroquinone was found to give an over-estimate when applied to developers also containing Phenidone. The apparent increase is proportional to the concentration of Phenidone, but independent of the hydroquinone concentration at normal working concentrations. Thus, after estimating the Phenidone content of a sample, it is possible to correct for the apparent increase and to determine the true hydroquinone concentration.

The addition of Phenidone to a hydroquinone developer does not increase the rate of autoxidation of the hydroquinone when the solution is exposed to air or to oxygen. The rate of autoxidation does not appear to be limited by the rate of solution of the gas because it can be varied over a wide range by changing the pH. The Phenidone has no effect at any pH, but when present in high concentration its rate of loss can become appreciable through dismutation of its semiquinone and it can compete with the hydroquinone. When the hydroquinone is present in concentrations above 2 g per litre its rate of autoxidation is insensitive to change in its concentration and is proportional to the area of the gas-solution interface. The evidence suggests that the critical stage of the autoxidation takes place at the gas-solution interface.

The activation of colour developing solutions by certain black-and-white developing agents is described. From results on measurements of the silver images produced by various combinations of developing agents and colour coupler, it is concluded that Phenidone and metol each exhibit superadditivity with the usual types of colour developing agents in the presence of a colour coupler.

Although it is now recognized that in the presence of hydroquinone and sulphite, Phenidone does not oxidize further than the l electron stage to a free radical, oxidation in alkaline solution in the absence of hydroquinone. results in a series of highly coloured products. The separation of these products by chromatography and electrophoresis is described, derivatives of Phenidone being investigated as well as Phenidone itself.

The data obtained in the investigation of the chemistry of quinone were used in conjunction with sensitometric data to relate fog, accelerated, contagious, infectious and superadditive development. Latensification and desensitization were studied. The photographic experiments have been summarized and discussed. It was concluded that hydroxyhydroquinone or its precursors were the prime cause of development fog and aerial fog. It was also suggested that hydroxyhydroquinone is the cause of accelerated, contagious and infectious development. It was further shown that the best anti-foggants against hydroxyhydroquinone were the superadditive components of developers, namely Phenidone and Elon. It was suggested that Phenidone and Elon react with hydroxyhydroquinone to form a powerful non-fogging developer. Furthermore, it appeared from the data that benzotriazole also reacted with hydroxyhydroquinone to form a non-fogging compound, whereas 6-nitrobenzimidazole was merely strongly adsorbed to fogged areas...

The relationship between chemical structure and superadditive effectiveness in photographic development was systematically investigated for different classes of substances. The existence of a stable radical is a pre-requisite for a strong superadditivity. The formation of a stable semiquinone demands a chemical structure which allows a high resonance stabilization. In phenidone, the resonance stabilization (as well as the superadditive effect) disappears for example through displacement of the phenyl ring by alkyl substitution or by the introduction of strongly electrophilic groups. Negative charge or non-coplanar structures also reduce the stability of the semiquinone and the superadditivity. Beyond that, a definite redox potential difference is necessary for superadditive effectiveness on photographic development. The results equally well support the regeneration theory and the charge barrier model as the cause of super-additivity.

Analysis of polarographic data in the range pH 2-12 shows that Phenidone is oxidized to the neutral radical obtained by abstraction of one hydrogen atom. Phenidone is a monobasic weak acid of pK 9.35 (K=4.5x10-10). The oxidation-reduction potential is given byE1/2=E∘+RTFin(K+[H+])where E˚-0.688 on the normal hydrogen scale at 20° C. Electron Spin Resonance spectra confirm that the oxidation product is a free radical.

The advantages of Phenidone/hydroquinone over metol/hydroquinone are analysed and their application is discussed in relation to liquid concentrates, developers for rapid processing, monobaths, fine-grain developers, developer replenishment, diffusion-transfer and the activation of colour developers.

A study has been made of the photographic behaviour of Phenidone and selected series of Phenidone-hydroquinone (PQ) developers, and their properties compared with those of analogous MQ developers. The results demonstrate the greater super-additivity of the PQ developing system, and have some bearing on current theories of development. Other experimental work on certain aspects of the chemistry of Phenidone shows that no sulphonation of the Phenidone takes place during development in the presence of sulphite. A mechanism for the PQ developing system is postulated which involves a coloured reversible mesomeric free-radical formed by the loss of an electron from the pyrazolidone ion.

A colorimetric method for the estimation of Phenidone, in Phenidone/hydroquinone developers, is described, which relies upon its reaction with 3-methyl-2-benzothiazolone hydrazone hydrochloride in alkaline solution and subsequent oxidation to form a meganta dye. A modification of the method is also described, for the estimation of small concentrations of Phenidone in the presence of colour developing agents and colour couplers.

The decay kinetics of the free radical produced electrochemically by monoelectronic oxidation of Phenidone has been studied by polarography, spectrophotometry and ESR spectrometry at different pH's, temperatures and reagent concentrations. The results obtained with the three techniques, together with the information of flash photolytic experiments, prove that the red-orange decaying species responsible for the cathodic polarographic wave is a primary free radical product of oxidation. The kinetic data indicate that a dismutation of the radical is the rate determining step. The preliminary results on some derivatives of Phenidone are also reported.

A colorimetric procedure using 3-methyl-benzothiazolone hydrazone hydrochloride (MBTH) has been adapted to theTechnicon Auto Analyser for the determination of Phenidone in black-and-white developers. It appears to be unaffected by developer oxidation products and various emulsion and developer addenda usually encountered in developer solutions. Reproducibility of the method is ± 1.5 per cent at 20 samples per hour. The method has been found suitable for routine estimation of Phenidone in a wide range of developers of conventional formulae.

A survey of literature on superadditivity in colour development is given, which is compared with superadditivity in black-and-white development. Generally superadditivity in colour is explained by the same regeneration mechanism as that in black-and-white. There are however some fundamental differences that throw some doubt on the correctness of this assumption. The main points are:1. the oxidation of colour developers by air in the absence of silver halide is increased by Phenidone, the oxidation of hydroquinone on the contary is not increased by Phenidone;2. the p-phenylenediamines develop with a short induction period, hydroquinone with a large one;3. the p-phenylenediamines are far better adsorbed on to silver than hydroquinone;4. the redox potential of the p-phenylenediamines is much more positive than the potential of phenidone or Metol, the potential of hydroquinone is of the same order as that of Phenidone or Metol and5. the activation of colour development is coupler-dependent. These poin...

Developer solutions containing Phenidone and hydroquinone, both carbonate and borate based, have been subjected to accelerated aeration. It has been established that although the Phenidone concentration in such solutions is usually low compared with that of the hydroquinone, its stabilization by the latter is such that the loss of Phenidone by oxidation is only slight even when the aeration is continued well beyond the point where the hydroquinone has been converted entirely to the sulphonate. The effect of EDTA on the course of such aerations has also been determined. The marked super-additivity of Phenidone with hydroquinone monosulphonate has been demonstrated experimentally, and the practical importance of this effect is discussed.

Some new analogues of Phenidone have been tested as developing agents in combination with Hydroquinone. The results are discussed in the fight of current views on the mechanism of development and superadditivity. and in relation to the structure of the compounds. The activity of some l-alkyl-3-pyrazolidinones shows that the phenyl group of Phenidone is not essential for superadditivity. although necessary for strong superadditivity.

Phenidone C9H10N2O has found wide application in chemical and food industry, It has been investigated by thermogravimetric method. The thermal stability of phenidone, its melting point and the kinetic parameters of the first partial process of the decomposition at different heating rates were determined.

We report differential effects of various compounds on inhibition of histamine release from washed leukocytes stimulated through IgE (using antigen or anti-IgE antibody) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) receptors. Inhibition of IgE-induced release was seen for several compounds listed in order of potency (IC50 in μM): CI-922 (3,7-dimethoxy-4-phenyl-N-(l-H-tetrazol-5-yl)-4-H-furo-[3,2-b]-indole-2-carboxamide, 1-arginate) < NDGA < BW755c < proxicromil < isamoxole < phenidone. Other compounds including FPL 55712, meclofenamate, and indomethacin were inactive or enhanced IgE-mediated release. When FMLP was used to stimulate histamine release the order of potency (IC50) changed: meclofenamate = FPL 55712 = proxicromil < CI-922 < NDGA = BW755c < indomethacin < isamoxole = phenidone. The inhibition of FMLP-induced histamine release by FPL 55712, meclofenamate and indomethacin in the absence of inhibition (or even enhancement) of histamine release by the IgE mechanism suggests that different pathways of activation and/or control are critical in the release process when different stimuli are used to activate basophils.

A new method for the determination of l-phenyl-3-pyrazolidone (Phenidone) in Phenidone-hydroquinone developers is based on the extraction of the Phenidone with chloroform, titration with a solution of iodine in chloroform in the presence of aqueous acid and back-titration with sodium thiosulphate. The iodine-Phenidone reaction is nearly stoichiometric under these conditions, provided that at least a 50 per cent excess of iodine is used. The titration in the chloroform-water mixture appears to inhibit secondary reactions between iodine and the oxidation products of the Phenidone.

Recent studies showed that soybean lipoxygenase inhibitors like phenidone and nordihydroguaiaretic acid (NDGA) reduce the catalytically active ferric lipoxygenase to its inactive ferrous form. Addition of 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) regenerated the active ferric form. In this paper, it is shown that in such a system the inhibitors are oxidized to free-radical metabolites. Incubation of soybean lipoxygenase and linoleic acid with p-aminophenol, catechol, hydroquinone, NDGA, or phenidone resulted in the formation of the one-electron oxidation products of these compounds. Free-radical formation depended upon the presence of the lipoxygenase and 13-HPOD. The free radicals were detected by ESR spectroscopy, and their structure was confirmed by analysis of the spectra, using a computer correlation technique. These data support the proposed mechanism for the inhibition of lipoxygenase by phenolic antioxidants.

Sulfasalazine, used therapeutically in the treatment of ulcerative colitis, is cleaved in vivo to form 5-aminosalicylic acid (5-ASA) and sulfapyridine. In an isolated preparation of rat peritoneal cells both sulfasalazine (IC50 = 0.15 mM) and 5-ASA (IC50 = 2.3 mM), but not sulfapyridine, inhibited calcium ionophore-stimulated formation of contractile leukotriene activity. This activity, although not identified directly, is attributable to a mixture of leukotriene C4 and leukotriene D4 (commonly referred to as SRS-A, or slow-reacting substance of anaphylaxis). Reference compounds evinced expected activities (IC50 = 0.024 mM for phenidone, IC50 = 0.3 microM for nordihydroguaiaretic acid, IC50 = 0.033 mM for BW 755C), whereas para-aminosalicylic acid and thiosalicylic acid were inactive. These properties of sulfasalazine may contribute to its therapeutic efficacy in vivo.

Arachidonic acid (AA) injected into hindpaws of Lewis rats produces a severe edematous response. Treatment with corticosteroids (dexamethasone, prednisolone), dual inhibitors of arachidonate metabolism (phenidone, SK & F 86002), anti-histamine/serotonin agents (chlorpheniramine, cyproheptadine) and a gold compound (auranofin) inhibited AA-induced edema. In contrast, administration of high doses of cyclooxygenase inhibitors (indomethacin, piroxicam, naproxen, ibuprofen, meclofenamic acid and tiflamizole) did not affect AA-induced hind paw edema. The involvement of lipoxygenase products and mast cell mediators in the edematous response to arachidonic acid render this model potentially useful for studying antiinflammatory agents with a mechanism of action different from that of cyclooxygenase inhibitors.

Experiments were designed to determine the role of the endothelium in the facilitation by anoxia of contractile responses of isolated coronary arteries. Rings and strips of canine coronary arteries, with or without endothelium, were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution. To determine the release of a vasoactive substance(s) from the endothelial cells, strips without endothelium were layered with strips containing endothelium. In rings and in layered preparations with endothelium, anoxia (95% N2-5% CO2) augmented contractile responses to prostaglandin F2 alpha. Hypoxia (10 or 5% O2) caused contractions in the presence of indomethacin. Removal of the endothelium abolished the anoxic facilitation, and the hypoxic contractions. Inhibitors of cyclo-oxygenase, lipoxygenase or phospholipase A2 or of adrenergic, serotonergic and histaminergic receptors did not prevent the response to anoxia. Likewise, inhibitors of the endothelium-derived factor(s) (quinacrine, phenidone and methylene blue) did not affect the anoxic facilitation. Hypoxia and anoxia caused contraction of coronary arteries without endothelium when layered with femoral arteries and veins with endothelium. Anoxic facilitation was observed in femoral arteries, but not in femoral veins, with endothelium. These experiments indicate that hypoxia and anoxia cause the release of a diffusible vasoconstrictor substance(s) from endothelial cells. The sensitivity of smooth muscle of different anatomical origin to the facilitatory mediator(s) varies.

Various diarylheptanoids including curcumin, bis (3,4-dihydroxy-cinnamoyl)methane, and yakuchinones A & B were screened and found to be potent inhibitors of 5-HETE production by intact human neutrophils, with respective IC50 values of 8.0, 4.4, 5.4, and 4.0 μM. These diarylheptanoids were found to be more potent than BW-755C, Phenidone, and AA-861. We have confirmed a previous report that several of these diarylheptanoids inhibit cyclooxygenase. Thus, curcuminoids and yakuchinones are more accurately characterized as dual inhibitors of arachidonic acid metabolism.

The synthesis and structure--activity profile of 2-substituted benzimidazol-4-ols as inhibitors of cell-free RBL-1 5-lipoxygenase are discussed, and their potency is compared with that of the standard inhibitors phenidone, AA 861, BW 755C, and nordihydroguaiaretic acid. In contrast to the standard compounds, most did not inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in vivo when administered at 200 microM ip to rats subjected to peritoneal anaphylaxis, although five compounds containing a methoxylated benzyl group (compounds 36, 39, 42, and 43) or hydroxylated benzyl group (41) showed similar activity to that of phenidone, nordihydroguaiaretic acid, and AA 861. Of the many compounds tested, two, 5-tert-butyl-7-methyl-2-(trifluoromethyl)-1H-benzimidazol-4-ol (57) and 2-(4-methoxybenzyl)-7-methyl-1H-benzimidazol-4-ol (36), like dexamethasone, inhibited monocyte accumulation in a pleural exudate model of inflammation. Standard lipoxygenase inhibitors such as phenidone, BW 755C, and AA 861 were inactive in this system.

The subcutaneous sponge implant model of acute inflammation in the rat has been evaluated as a suitable test system for evaluating the potential anti-inflammatory efficacy of 5-lipoxygenase inhibitors. The inflammatory parameters measured were exudate volume and leukocyte recruitment. Specific radioimmunoassays were used to measure (1) 5-lipoxygenase (LPO) and cyclo-oxygenase (CO) activity in exudate leukocytes stimulated ex vivo with A23187, and (2) the LTB 4 and PGE 2 content of inflammatory exudate. The NSAIDs flurbiprofen and indomethacin inhibited cell recruitment, exudate volume and CO activity with ed 50 S of approximately 1 mg per kg p.o. but failed to inhibit LPO activity at 10 mg per kg p.o. Nafazatrom (Bayer 6575), quercetin and NDGA, which inhibit LPO activity in vitro , were inactive against all parameters when dosed at 100 mg per kg p.o. The “mixed inhibitors” BW755C and phenidone were approximately equipotent inhibitors of LPO activity but BW755C was 10 times more potent than phenidone against CO activity. BW755C was also > 10 times more potent at inhibiting cell recruitment and exudate volume than phenidone suggesting that the anti-inflammatory efficacy of the mixed inhibitors reflect their potency against CO rather than LPO activity. Time course studies demonstrated that the inhibitor effects of BW755C and phenidone on leukocyte recruitment reflected a reduction in the PGE 2 but not the LTB 4 content of the inflammatory exudate. Polyester sponges soaked in high concentrations of LTB 4 caused only a modest (2-fold) increase in leukocyte recruitment whilst physiological levels were inactive. The results taken together suggest that CO products make a major contribution to leukocyte recruitment in this model whilst the LPO product LTB 4 has little role. This model therefore is of little value for evaluating the anti-inflammatory efficacy of 5-lipoxygenase inhibitors. Moreover, the rat would appear to be unsuitable for evaluating the role of LTB 4 in acute inflammation.

Indomethacin (1 microM) enhanced histamine-induced contractions in pig and guinea-pig isolated tracheae. Mepacrine (30-50 microM) abolished this effect of indomethacin suggesting that contractile metabolites of arachidonate are involved in the response to indomethacin. Mepacrine (100 microM) in the absence of indomethacin also markedly reduced histamine-induced contractions (81.2% inhibition) in the pig trachea, without affecting responses to acetylcholine. Histamine-induced responses in the guinea-pig trachea were similarly reduced, but with a higher concentration of mepacrine (300 microM). BW755c (226 microM) enhanced histamine-induced contractions in some pig tracheal preparations and caused inhibition in others. These effects of BW755c were negatively correlated to the initial reactivity of the muscle to histamine such that weak contractions were potentiated and strong contractions were inhibited. A similar effect was seen with phenidone (100 microM). The results with BW755c and phenidone suggest that muscle reactivity (to histamine) may be partly determined by the balance between the release of inhibitory and contractile arachidonate metabolites. Mepacrine exerts a different effect indicating that histamine-induced contractions are regulated by a mepacrine-sensitive process which appears to be separate from the metabolism of arachidonate.

Indomethacin was administered subcutaneously to rats, 4 mg/kg/day for 4 consecutive days in order to produce erosions of the small intestine which were scored at necropsy on day 5. Orally administered phenidone (up to 250 mg/kg/day), a mixed cycloocygenase-lipoxygenase inhibitor, failed to produce intestinal erosions, but tended to exacerbate indomethacin-induced erosions. A 5-LO inhibitor, diphenyldisulfide, provided significant protection at 10-100 mg/kg when given orally to indomethacin-treated rats. Sulfasalazine, auranofin and cyproheptadine, but not cimetidine, also protected, suggesting a role for mast cell activation and leukotriene generation in indomethacin-induced ulcerogenesis.

Injection of platelet-activating factor (PAF-acether) into the pleural cavity of rats induced the accumulation of a moderately intense exudate within 30 to 60 minutes. By comparison with animals given injections of the vehicle alone, the animals given this mediator had elevated levels of leukotriene C 4 -immunoreactive material (LTC 4 im) in the exudate and decreased quantities of thromboxane B 2 (TxB 2 ) and of 6-KetoF 1α -prostaglandin (6-Keto PGF 1α ). Nifedipine, verapamil, and diltiazem reduced the pleural exudate with no major effect on the mediators. Both salbutamol and theophylline reduced the exudate and the levels of LTC 4 im. Acetylsalicylic acid, phenylbutazone and indomethacin significantly inhibited the exudate, greatly lowered the quantities of cyclooxygenase derivatives and tended to increase LTC 4 im. Phenidone, which inhibits the cyclooxygenase and lipoxygenase pathways, decreased the exudate and the three mediators. The phospholipase A 2 inhibitor, chloroquine, decreased both the amount of exudate and moderately the concentration of LTC 4 im. The glucocorticoids studied had no effect on the exudate or on the mediators. These results suggest that the role of the increased LTC 4 im in the induction of the pleurisy is not clear

The synthesis and structure--activity profile of 2-substituted benzimidazol-4-ols as inhibitors of cell-free RBL-1 5-lipoxygenase are discussed, and their potency is compared with that of the standard inhibitors phenidone, AA 861, BW 755C, and nordihydroguaiaretic acid. In contrast to the standard compounds, most did not inhibit the release of slow-reacting substance of anaphylaxis (SRS-A) in vivo when administered at 200 microM ip to rats subjected to peritoneal anaphylaxis, although five compounds containing a methoxylated benzyl group (compounds 36, 39, 42, and 43) or hydroxylated benzyl group (41) showed similar activity to that of phenidone, nordihydroguaiaretic acid, and AA 861. Of the many compounds tested, two, 5-tert-butyl-7-methyl-2-(trifluoromethyl)-1H-benzimidazol-4-ol (57) and 2-(4-methoxybenzyl)-7-methyl-1H-benzimidazol-4-ol (36), like dexamethasone, inhibited monocyte accumulation in a pleural exudate model of inflammation. Standard lipoxygenase inhibitors such as phenidone, BW 755C, and AA 861 were inactive in this system.

Recently it has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated by local release of sulfidopeptide leukotriene products of the lipoxygenase pathway of arachidonic acid metabolism. In the present study the response to reduced oxygen supply of isolated porcine lobar pulmonary artery and pulmonary vein spiral strips has been studied. Contractions of the pulmonary artery (mean maximum tension 66.9 +/- 13.0 mg, n = 10) required an increase in baseline tone of the preparation followed by exposure to anoxia (mean bath PO2 O +/- 3 mm Hg), whereas contractions of the pulmonary vein (mean maximum tension 75.2 +/- 13.3 mg, n = 10) could be elicited in response to hypoxia alone (mean bath PO2 40 +/- 4 mm Hg). Indomethacin (5.6 microM), a cyclooxygenase inhibitor, attenuated the arterial contraction, but the mechanism may have been independent of the cyclooxygenase pathway since phenidone, an inhibitor of both cyclooxygenase and lipoxygenase pathways, had no effect. Inhibition by FPL 55712, a leukotriene end-organ antagonist, was achieved only at a high concentration (20 microM). In the case of the pulmonary vein, both indomethacin and phenidone inhibited the contractile response, whereas FPL 55712 had no effect. Contractile responses to reduced oxygen supply can be induced in isolated porcine pulmonary artery and vein strips, but probably are not mediated by leukotrienes.

This study examined the role of the lipoxygenase (LO) pathway in the maintenance of hypertension in rats with two-kidney, one clip (2K,1C) Goldblatt hypertension. A single dose of the lipoxygenase blocker phenidone was injected intraperitoneally to 2K,1C rats during the early phase (14 days) of the development of hypertension (mean intraarterial blood pressure 137 +/- 3.9 mm Hg). Phenidone (60 mg/kg) markedly decreased arterial pressure to nadir levels of 58.9% of resting blood pressure. The maximal changes were observed 15 min after injection and the hypotensive response was sustained for at least 2 h. Plasma renin concentration (PRC) increased from 74.3 +/- 18.9 to 281.0 +/- 6.5 ng/mL/h after injection (P less than .05). Thus, the hypotensive effect of phenidone was not due to suppression of renin secretion but presumably due to inhibition of its effects. It is suggested that arachidonate metabolites of the LO pathway at the vascular bed may be involved in maintenance of high arterial pressure in 2K,1C renovascular hypertension in rat.

Summary Erythroid progenitor cells synthesize 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) when stimulated by erythropoietin (Ep). Maximal stimulation of 12-HETE production occurred at one hour, whereas 15-HETE activity remained constant in response to Ep for 24 hours. Lipoxygenase-selective inhibitors of arachidonic acid metabolism blocked HETE production and Ep-stimulated growth and differentiation of erythroid progenitor cell-derived colonies (CFU-E). On the other hand, specific inhibitors of cyclooxygenase (aspirin and meclofenamate) did not significantly inhibit Ep-induced erythroid colony formation. It is hypothesized that the stimulation of HETE production from arachidonic acid (AA) is an essential step in the mechanism of action of Ep.

Induction of epidermal ornithine decarboxylase (ODC) by a topical application of 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, was inhibited by treatment of mouse skin with phenidone (3-90 mumol/mouse), nordihydroguaiaretic acid (30 mumol/mouse) or 3-amino-1-[m-(trifluoromethyl)-phenyl]-2-pyrazoline (BW 755C, 30 mumol/mouse), which are well-known lipoxygenase inhibitors. Phenidone and BW 755C are also to be cyclooxygenase inhibitors. Inhibition of TPA-induced ODC by indomethacin (1.12 mumol/mouse), a selective cyclooxygenase inhibitor, was counteracted by prostaglandin E2 (PGE2) (140 nmol/mouse). This counteracting effect of PGE2 was reversed by the treatment of mice with nordihydroguaiaretic acid (30 mumol/mouse) or phenidone (30 mumol/mouse). ODC activity which was suppressed by nordihydroguaiaretic acid or phenidone at a dose of 180 mumol/mouse was not further inhibited by indomethacin (1.12 mumol/mouse). In addition, the counteracting action of PGE2 (140 nmol/mouse) was not observed in mice treated with nordihydroguaiaretic acid or phenidone at a dose of 180 mumol/mouse. Thus, the suppressive effect of nordihydroguaiaretic acid or phenidone on the ODC induction by TPA would be due to the inhibition of lipoxygenase. The above findings strongly suggest that not only cyclooxygenase product (i.e., PGE2) but also lipoxygenase product(s) are involved in the mechanism of ODC induction in mouse epidermis, and a lack of either cyclooxygenase product or lipoxygenase product(s) causes a failure of ODC induction by TPA.

34- 7. ÖZEI Koyun trakea düz kasında asetillcolinin kontraktil cevaplarına AA ürünlerinin etkisini incelemek amacıyla ya pılan bu çalışmada ; 1 - Indometasin ile ön uygulamadan sonra asetilko- linin kontrakxil cevapları azaldı. Bu ise koyun trakea düz kasında siklooksijenaz yolağının baskın metabolitinin PGF2 alfa olduğunu düşündürdü. 2 - Penidon ile ön uygulamadan sonra asetilkolinin kontraktil cevapları daha büyük azalma oluşturdu. Bu da adı geçen dokuda LT'lerin kısmen de olsa bronşiyal tonüse iştirak ettiğini gösterdi. 3 - îndometasin ile ön uygulamadan sonra asetilko- line verilen kontraktil cevapların artmaması koyun trakea düz kasında siklooksijenaz inhibisyonundan sonra AA meta bolizmasının lipoksijenaz yolağına kaymadığını gösterdi. -35- 8. SUMMARY In this study which has been made on sheep tracheal smooth muscle to investigate the effects of arachidonic acid products on the contractil effects of acetylcholine, these results were ooserved : 1 - Indomethacin pre treatment reduced the contra fi til effects of acetylcholine, so it has been suggested that in the sheep tracheal smooth muscle PGF2 alpha, an arachi donic acid cyclooxygenase product, is the most predominant metabolite of arachidonic acid. 2 - Pretreatment with phenidone also reduced the contractil effects of acetylcholine being more potent than indomethacin. This shows that in this tissue leukotrienes at least partially play role in the regulation of bronchi al tonus. ???-.. 3 -.After the- inhibition of cyclooxygenase the path way in the arachidonic acid metabolism does not transfer to lypoxygenase pathway because the contractil effects of acetylcholine were not increased by pretreatment with in domethacin. 45

The oxygenation of [1-14C]-arachidonic acid by a soluble soybean lipoxygenase (E.C.1.13.11.12) preparation was determined in the presence of various cyclo-oxygenase and lipoxygenase inhibitors. The results showed that several non-inhibitory compounds drastically blunted the inhibitory potency of potent lipoxygenase inhibitors. Studies on the combined effects of a variety of structurally unrelated inhibitors of lipoxygenase, cyclo-oxygenase or both oxygenation pathways provided strong evidence for the existence of a supplementary binding site on soybean lipoxygenase which reduces the effective interactions of inhibitors with the catalytic site. Thus several cyclo-oxygenase inhibitors (which do not inhibit at the lipoxygenase catalytic site), as well as low concentrations of lipoxygenase inhibitors, interact with this putative supplementary site and blunt the inhibitory efficacy of potent lipoxygenase inhibitors. Although the degree of interaction with the catalytic site determines the absolute potency of inhibitors, the additional interaction at the putative supplementary binding site is also obligatory for inhibitory potency. In this new multiple-site model the potent lipoxygenase inhibitors (e.g. acetone phenylhydrazone, phenidone) possess high affinities for both sites, whereas weak inhibitors and certain cyclo-oxygenase inhibitors (e.g. benoxaprofen, phenylbutazone, indomethacin) interact predominantly with the supplementary site on the lipoxygenase but lack affinity for the catalytic site.

Leukotrienes (LT) C4, D4, and E4 are major contributors to the pathobiology of human bronchial asthma. Therefore, it is likely that compounds that antagonize the action or inhibit the formation of LTs will be useful therapeutic agents. We have studied the effects of LT antagonists, 5-lipoxygenase inhibitors and selected standards in a model of LT-mediated allergic bronchospasm in guinea pigs. Sensitized animals were pretreated with mepyramine, indomethacin and propranolol to eliminate the influence of histamine, prostaglandins, thromboxanes and circulating catecholamines. In these animals, inhalation of antigen resulted in a bronchospasm consistent with a LT-mediated response that was slow in onset, of long duration and was inhibited by the selective LTD4, antagonists FPL-55712, LY-171,883 and ICI-198,615. ICI-198,615 was approximately 50-times more potent than FPL-55712 by the intravenous and intratracheal routes. However, of thirteen compounds known to inhibit 5-lipoxygenase and LT biosynthesis in vitro only phenidone, piriprost and AA-861 were active in this in vivo model. The allergic bronchospasm was inhibited by bronchodilators (e.g. PGE2, aminophylline and forskolin) and by some mast cell stabilizers, but was otherwise insensitive to other pharmacological classes of compounds including calcium channel blockers and antagonists of serotonin, acetylcholine and platelet-activating factor. This model seems useful and reasonably selective for the evaluation of new antianaphylactic compounds that are LT antagonists. The inactivity of many 5-lipoxygenase inhibitors in this model suggests they do not inhibit LT formation in vivo.

We have studied the effects of a lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on antagonism of leukotriene (LT) C4-induced contractions of isolated guinea-pig trachea and the results were compared to that of a cyclooxygenase inhibitor indomethacin. NDGA (30 microM) as well as indomethacin (5 microM) inhibited LTC4-induced contractions. But, in the presence of indomethacin NDGA was ineffective to inhibit the LTC4 response, whereas two other lipoxygenase inhibitors, phenidone (3-30 microM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 microM), markedly inhibited it. The antagonist action of an LTD4 receptor antagonist FPL55712 against LTC4-induced contractions was significantly reduced by NDGA (10-30 microM), but indomethacin had no effect on it. NDGA possessed the same inhibitory effect on the LTC4 antagonism in the presence of indomethacin, but 0.3 microM phenidone and 1 microM ETYA which did not inhibit the LTC4 response had no effect on it. NDGA also inhibited the relaxant response of isoproterenol on the contraction elicited by 30 nM LTC4, but did not affect those of forskolin and aminophylline. The relaxant response of isoproterenol on the LTC4 response was not inhibited by indomethacin, 0.3 microM phenidone and 1 microM ETYA. In the presence of a gamma-glutamyltranspeptidase inhibitor, L-serine borate (SB, 45 mM), NDGA had no effect on the LTC4 antagonism and the relaxant response of isoproterenol. In contrast, NDGA significantly inhibited the relaxant response of isoproterenol on 30 microM histamine- and 30 microM acetylcholine-induced contractions, but it did not affect the histamine antagonism by a histamine H1-blocker pyrilamine. These results suggest that some putative non-prostanoids are involved in LTC4-induced contractions of guinea-pig trachea and which regulate the effects of LTD4 antagonism and beta-adrenoceptor activation.

1 Pharmacological modulation of antigen-induced anaphylaxis in actively sensitized guinea-pigs with intravenously administered indomethacin (10 mg/kg), pyrilamine (2.0 mg/kg) and propranolol (0.1 mg/kg) resulted in a delayed onset, slowly developing bronchoconstriction indicative of a slow-reacting substance of anaphylaxis (SRS-A) response. 2 Measurements of pulmonary mechanics on the drug-pretreated animals challenged with ovalbumin demonstrated a more prominent effect on dynamic compliance than resistance. This is consistent with the more potent effects of SRS-A on peripheral rather than central airways. 3 The slowly developing bronchoconstriction obtained after treatment with indomethacin, pyrilamine and propranolol was inhibited by the standard SRS-A antagonist, FPL 55712 and the SRS-A synthesis inhibitors, phenidone, BW 755C and nordihydroguaiaretic acid. 4 Plasma SRS-A levels were determined in guinea-pigs following antigen challenge. The appearance of SRS-A in the plasma preceded the onset of bronchoconstriction and SRS-A levels remained elevated throughout its development. Coincident with the inhibition of bronchoconstriction by the SRS-A synthesis inhibitor, phenidone, was a dose-dependent reduction in plasma SRS-A. The intravenous ED50 in each case was 4 mg/kg. 5 This model of antigen-induced SRS-A-mediated bronchoconstriction should prove useful for the in vivo evaluation and development of therapeutics which regulate the synthesis of SRS-A.