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Nodulation is the temporally and quantitatively most important cellular defense response to bacterial, fungal and some viral infections in insects. We tested the hypothesis that prostaglandins and other eicosanoids are responsible for mediating nodulation reactions to bacterial infection in larvae of the blowfly Chrysomya megacephala. Third-instar larvae treated with Ureaplasma urealyticum formed nodules in a challenge dose-dependent manner. Nodulation was evoked shortly after injection and reached a maximum of approximately 25 nodules/larva within 8 h. Larvae treated with the glucocorticoid, dexamethasone and the cyclooxygenase inhibitors, indomethacin and piroxicam were impaired in their ability to form nodules following U. urealyticum infection. The number of nodules decreased with increasing doses of piroxicam. Contrarily, treating larvae with the lipooxygenase inhibitor, esculetin, and the dual cyclooxygenase/lipooxygenase inhibitor, phenidone did not influence nodulation reactions to infection. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, reversed the inhibitory effect of dexamethasone on nodulation. We infer from these results that eicosanoids mediate nodulation reactions to infection of a bacterial species that lacks cell walls in larvae of the blowfly, C. megacephala.

The effects of developing agents and activators in the reversal bleach upon the diffraction efficiencies of a Slavich PFG-01 plate to be used for recording transmission holograms were studied. The plates were developed using four different developers: MSC, AAC, PSC, and HSC. The four developers were derived from different developing agents of metol, l-ascorbic acid, phenidone, and hydroquinone, respectively. Sodium carbonate was added to the developer as an activator that accelerates the development by keeping the developer alkaline. A maximum diffraction efficiency of 89.0% was obtained when the AAC developer was used.

Antioxidant activities of pure chemicals and food additives were compared with that of herb extracts using a method based on the Fenton reaction for screening natural antioxidants. This method detects antioxidants classified as free radical terminators, that may compete with linoleic acid for scavenging OH radicals. Of the 39 products investigated, 19 were commercial products and 20 were aqueous and ethylic herb infusions. Commercial phenolic and flavonoid products, both hydrophilic and lipophilic, showed strong antioxidant activities, while indole, alkaloid and fs-cyclic products showed no antioxidant activity, as determined by this method. Aqueous infusions of all herbs showed antioxidant properties. Mild oregano, strong oregano, rosemary, sage and mint showed as strong antioxidant properties as pure commercial chemical and food additives, such as hydroquinone, rutin, phenidone, catechol, epicatechin, morin and BHA, in both aqueous and ethylic infusions. Also, the results suggest that, under the experimental conditions, the aqueous extractions of short duration yielded larger quantities of active compounds by reducing the degradation of their antioxidant properties.

This paper shows that the diffraction efficiency is influenced by superadditivity for the combination of the different developing agent and concentrations in the reversal bleach applied to the Slavich PFG-01 plate to be used for recording transmission holograms. The four developers based on ascorbic acid in combination with metol, phenidone, hydroquinone respectively, were studied. In diffraction efficiency aspect, the superadditivity effect of mixture of two developing agents is lower than one developing agent developer, AAC in the reversal bleach.

The role of lipoxygenase (LOX) in the elicitor-induced taxol production of Taxus chinensis cell cultures was studied. Fungal elicitor F 5 treatment effectively increased LOX activity and taxol production. The LOX activity inhibitor phenidone could inhibit induced LOX activity and taxol production, suggesting that LOX pathway might be involved in elicitor-induced taxol production. Methyl jasmonate (MJ) also activated LOX activity and taxol production. Phenidone could inhibit LOX activity and taxol production challenged by MJ, suggesting that exogenous MJ may induce taxol production via activating the intracellular LOX pathway. For better understanding the role of LOX in T. chinensis , some biochemical features of LOX were also investigated. Results showed that at least 49.18% LOX was associated with membrane and the molecular weight of LOX was 93 kDa.

This study aimed to investigate the anti-inflammatory activity of (E)-1-(3,4-dimethoxyphenyl) butadiene (DMPBD), isolated from Zingiber cassumunar Roxb., using in vivo and in vitro models. The results show that DMPBD dose-dependently inhibited the rat ear edema induced by ethyl phenylpropiolate (EPP), arachidonic acid (AA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) and it was more potent than any other standard drugs being used. In EPP-induced edema IC(50) of DMPBD and oxyphenbutazone were 21 and 136nmol per ear, respectively. The IC(50) of DMPBD and phenidone were 60 and 2520nmol per ear, respectively, in AA-induced edema whereas DMPBD was 11 times more potent than diclofenac in TPA-induced edema (IC(50)=660 and 7200pmol per ear, respectively). DMPBD and diclofenac inhibited the rat paw edema induced by carrageenan but not by platelet activating factor (PAF). In in vitro study DMPBD, aspirin and phenidone inhibited collagen-induced platelet aggregation with IC(50) of 0.35, 0.43 and 0.03mM, respectively. Whereas IC(50) of these agents in ADP, AA and PAF inductions were 4.85, 3.98 and 1.30mM; 0.94, 0.13 and 0.04mM; and 1.14, 6.96 and 2.40mM, respectively. These results indicate that DMPBD possesses a potent anti-inflammatory activity through the inhibition of CO and LO pathways and seems to have more prominent effects on the LO pathway.

Metarhizium anisopliae conidia (spores) reduced weight gain and caused death when injected into Manduca sexta larvae. When the fungus was co-injected with the eicosanoid biosynthesis inhibitor dexamethasone, larval weight gain was further reduced and mortality increased. These effects were reversed when dexamethasone was given together with the eicosanoid precursor arachidonic acid (AA). Similarly, treatment with other eicosanoid biosynthesis inhibitors (esculetin, phenidone, ibuprofen, and indomethacin) with differing modes of action enhanced the reduction in weight gain caused by mycosis. Injection of M. anisopliae conidia induced nodule formation in vivo; nodule numbers were reduced by dexamethasone, and restored by AA. Incubation of hemocytes with conidia caused microaggregation of hemocytes (indicative of nodule formation) in vitro and this was inhibited by dexamethasone, suggesting that dexamethasone acts directly on hemocytes, although inhibition was only partially reversed by AA. We suggest that the M. sexta immune response to fungal pathogens is normally modulated by physiological systems that include eicosanoid biosynthesis. This is the first demonstration that the virulence of a fungal entomopathogen can be enhanced by compromising the insect host's immune system.

Effluents from X-ray film developing processes feature high contaminant load (COD about 200 g/l). Identification of the main organics present in these wastewaters was performed by using liquid chromatography-mass spectrometry in electron impact mode, LC-(EI)MS. Both, unconsumed ingredients and sulphonated/hydroxylated derivatives and dimers yielded from the developing agents hydroquinone and phenidone were found to be the main organics contributing to the contaminant load of these effluents. Their potential degradation by oxidation with both photo- and thermal-Fenton reactions was investigated and the optimal degradation conditions were determined. Under these conditions the initial COD was reduced by about 97% within 6 h of treatment. The intermediates detected by LC-(EI)MS in the oxidation of hydroquinone and phenidone during the treatment were essentially hydroxylated derivatives and dimers and the residual organic matter was mainly made up of carboxylic acids. Reaction pathways for the oxidation of the developing agents hydroquinone and phenidone were proposed. The presence of many organic and inorganic components in the wastewaters did not affected the favoured oxidation route of these developers. The results obtained allow to assess the reduction of costs permitted in the treatment of X-ray effluents with the photo- and thermal-Fenton reaction without affecting the degradation degree and quality of the discharge.

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.

N-Aminoindoline derivatives were prepared and their 5-lipoxygenase inhibitory activities were evaluated in vitro and compared with those of phenidone and NDGA. Compound 4 presents the most effective 5-LO inhibition.

The effects of the second-generation leukotriene B 4 receptor (LTB 4 receptor) antagonist, 2-[2-propyl-3-{3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxy-phenoxy]-propoxy}phenoxy]benzoic acid sodium salt (LY293111Na), on leukotriene B 4 -induced leukocyte infiltration and interleukin-1-accelerated collagen-induced arthritis in mice were studied. Neutrophil infiltration induced into an air pouch by leukotriene B 4 was dose-dependently inhibited by LY293111Na and strongly so by another LTB 4 receptor antagonist, 4-[5-{4-(aminoiminomethyl)phenoxy}pentoxy]-3-methoxy- N , N -bis(1-methylethyl) ( Z )-2butenedioate (1:1) (CGS25019C). Both compounds significantly inhibited the increase of the arthritis index and the ankle bone destruction in interleukin-1-accelerated collagen-induced arthritis. Phenidone, a 5-lipoxygenase inhibitor, also inhibited interleukin-1-accelerated collagen-induced arthritis, while indomethacin and tenidap, cyclooxygenase inhibitors, had slight inhibitory effects. Injection of interleukin-1 elicited a marked increase of the leukotriene B 4 level in arthritic paws, while the prostaglandin E 2 level was slightly increased. These findings indicate clearly that leukotriene B 4 is an important mediator of interleukin-1-accelerated collagen-induced arthritis in mice. If this can be extrapolated to man, LTB 4 receptor antagonists might be useful for treatment of the acute progressive phase of human arthritis.

Acculmulating evidence indicates that a marked generation of oxygen free radicals derived from the metabolism of arachidonic acid causes neurodegeneration. Recently, we have demonstrated that the novel antioxidant actions mediated by phenidone, a dual inhibitor of cyclooxygenase/lipoxygenase pathways, may play a crucial role in preventing neuroexcitotoxicity in vitro [Neurosci. Lett. 272 (1999) 91], and that phenidone significantly attenuates kainic acid (KA)-induced seizures via inhibiting the synthesis of Fos-related antigen protein [Brain Res. 782 (1998) 337]. In order to extend our understanding of the pharmacological intervention of phenidone, we evaluated the antioxidant activity of this compound in vivo in the present study. In order to better understand the significance of a blockade of both the cyclooxygenase and lipoxygenase pathways, we studied the effects of aspirin (ASP; a non-selective inhibitor of cyclooxygenase), NS-398 (a selective inhibitor of cyclooxygenase-2), esculetin (an inhibitor of lipoxygenase) and phenidone on lipid peroxidation, protein oxidation, and glutathione (GSH) status in the rat hippocampus after KA administration. ASP (7.5 or 15 mg/kg), NS-398 (10 or 20 mg/kg), esculetin (5 or 10 mg/kg) or phenidone (25, 50 or 100 mg/kg) was administered orally five times every 12 h before the injection of KA (10 mg/kg, i.p.). The KA-induced toxic behavioral signs, oxidative stress (lipid peroxidation and protein oxidation), impairment of GSH status, and the loss of hippocampal neurons were dose-dependently attenuated by the phenidone, NS-398+esculetin, and ASP+esculetin. However, ASP, NS-398 and esculetin alone failed to protect against the neurotoxicities induced by KA. Therefore, the results suggest that protection by blockade of both cyclooxygenase and lipoxygenase pathways against KA-induced neuroexcitotoxicity is via antioxidant actions. However, a novel anticonvulsant/neuroprotective effect mediated by phenidone remains to be further characterized.

This study investigates the effect and some of the mechanisms involved following systemic treatment of mice with Mycobacterium bovis bacillus Calmette-Guerin (BCG) (1 dose per animal containing 6.4 x 10(4) colony-forming units (CFu) 20-60 days beforehand) on modulation of the kinin B1 receptor agonist-induced nociception and oedema formation in the formalin test. Intraplantar (i.p.l.) co-injection of des-Arg9-bradykinin (4-32 nmol/paw) or des-Arg10-kallidin (1-15 nmol/paw), together with sub-maximal concentrations of formalin (0.01 or 0.5%), potentiated (P < 0.01) both pain phases and the paw oedema caused by formalin in animals pre-treated with saline. However, when animals were pre-treated with BCG, the dose-response curves for both B1 agonists were shifted 2 to 8-fold to the left. These B1-mediated effects peaked at 30-45 days after BCG treatment and were still elevated at 60 days after BCG injection. The pain response and oedema formation caused by i.p.l. co-injection of des-Arg9-bradykinin, together with formalin in BCG-pre-treated animals, were dose-dependently antagonised by i.p.l. co-injection of the B1 antagonist des-Arg9[Leu8]bradykinin (1-15 nmol/paw), but were not affected by the B2 antagonist Hoe 140 (10 nmol/paw). The i.p.l. co-injection of tyrosine8-bradykinin (a B2 agonist, 3-15 nmol/paw) with formalin (0.01 or 0.5%) potentiated the pain response and paw oedema in BCG and saline-pre-treated animals to the same extent (P < 0.01). The actions caused by tyrosine8-bradykinin were antagonised by Hoe 140, while des- Arg9[Leu8]bradykinin (10 nmol/paw) had no effect. Dexamethasone (0.5 mg/kg, s.c.), given every 24 h, from day 0 to 30-45, inhibited significantly the potentiation of nociceptive response and oedema formation caused by i.p.l. co-injection of formalin plus des-Arg9-bradykinin, while indomethacin (2 mg/kg, i.p.) or phenidone (30 mg/kg, i.p.), given 1 h prior, caused less inhibition. These data show that the long-term systemic treatment of mice with BCG produced dose-related potentiation of B1 receptor agonist-mediated nociception and oedema formation, without affecting similar responses caused by the B2 receptor agonist tyrosine8-bradykinin. Thus, systemic treatment of mice with BCG induces upregulation of B1 receptors, without affecting B2-mediated responses, by a mechanism that seems to be secondary to cytokine release.

Several studies have described metabolism of pesticidal chemicals in plants and animals by the NADPH-dependent cytochrome P-450 monooxygenases while relatively little is known regarding the role played by the peroxidative oxidation pathway. Here we describe, for the first time, hydrogen peroxide-supported N-demethylation of aminocarb by the highly purified preparations of soybean lipoxygenase. Formaldehyde generated in the reaction was estimated by the Nash reaction. N-demethylation of aminocarb was found to depend on the incubation time, concentration of the enzyme, aminocarb, and hydrogen peroxide. Under optimal conditions,Vmaxvalues of 18 nmol of formaldehyde/min/nmol of lipoxygenase or 170 nmol of formaldehyde/min/mg protein were observed. The reaction exhibitedKmvalues of 3.4 mM for aminocarb and 235 μM for hydrogen peroxide. A strong inhibition of the reaction by nordihydroguaiaretic acid, gossypol, and phenidone clearly implicates the lipoxygenase involvement as the protein catalyst. A significant decline in the formaldehyde accumulation in the presence of either reduced glutathione or dithiothreitol suggests generation of a free radical species as an initial oxidation intermediate during the demethylation of aminocarb by the lipoxygenase. The inhibition of formaldehyde generation by BHT and BHA further supports this contention. In addition to aminocarb, seven other pesticides were also found to undergo N-demethylation, albeit at relatively low rates. Collectively, the evidence presented suggests, for the first time, that certain pesticides may oxidatively undergo dealkylation via the lipoxygenase pathway in plants.

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K+ and Ca2+ channels in isolated rat type I carotid body cells. AA (2-20 microM) produced a concentration-dependent inhibition of both K+ currents and Ca2+ channel currents. The effects of AA on K+ currents were unaffected by indomethacin (5 microM), phenidone (5 microM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.

The contribution of several vasoactive mediators such as histamine, serotonin, bradykinin, arachidonic acid metabolites and PAF to vascular permeability changes was determined in a rat model of acute endotoxemia. Lipopolysaccharide (10–40 mg/kg, i.v.) from E. coli 0127:138 (LPS) elicited an increase in Evans blue extravasation in trachea, thymus, seminal vesicle and stomach, whereas other organs remained unaffected. LPS (25 mg/kg)-induced extravasation was not inhibited by intravenous pretreatment with histamine (H1) antagonist mepyramine (5 mg/kg) or bradykinin (B2) antagonist HOE-140 (0.1 mg/kg), whereas other standard drugs selectively inhibited leakage in particular tissues, e.g. the cyclooxygenase inhibitor indomethacin (5 mg/kg) in trachea (78%) and seminal vesicle (64%), the serotonin and H1 antagonist cyproheptadine (2 mg/kg) in trachea (88%) and stomach (56%) and the dual cyclooxygenase/lipoxygenase inhibitor phenidone (10 mg/kg) in seminal vesicle (87%). PAF antagonists lexipafant and UR-12460 (10 mg/kg), but not apafant, potently inhibited extravasation in trachea (59, 84%) and seminal vesicle (81, 78%) and in stomach only UR-12460 (52%), whereas all of them were ineffective in thymus. When extravasation was induced by PAF (4 μg/kg) a low dose (0.1 mg/kg) of the three PAF antagonists strongly reduced extravasation in thymus and seminal vesicle, whereas lexipafant and UR-12460 did so in trachea (82, 100%) and only lexipafant in stomach (100%). Mepyramine, cyproheptadine, HOE-140 and indomethacin did not inhibit the effect of PAF, whereas phenidone inhibited it by 58% in trachea. These results suggest that most of the LPS-induced increase in vascular permeability is mediated by secondary vasoactive mediators among which PAF plays a pivotal role, although their relative contribution may vary from tissue to tissue.

We have reported that bradykinin induces graded contraction in guinea-pig gallbladder in vitro through activation of bradykinin B2 receptors and prostanoid release, while des-Arg9-bradykinin, a selective bradykinin B1 receptor agonist, causes only a weak contraction, suggesting the presence of badykinin B1 receptors in this tissue. In the present study, we attempted to characterise the receptor subtype and the possible mechanism by which des-Arg9-bradykinin induces contraction in this preparation. Contractions induced by des-Arg9-bradykinin in guinea-pig gallbladder (1 pM to 1 microM) increased significantly as a function of time elapsed after setting up of the preparation, reaching the maximum after 6 h of equilibration (EC50 16.4 pM and Emax 0.6 +/- 0.08 g). Des-Arg9-bradykinin-induced contraction in guinea-pig gallbladder was totally prevented by cycloheximide (70 microM, an inhibitor of protein synthesis), indomethacin (3 microM), ibuprofen (30 microM), phenidone (30 microM) or Ca2+-free medium plus EGTA, and was partially antagonised by MK 571 ((3-(3-(2-(7-chloro-2-quinolinyl) ethenyl) phenyl ((3-dimethyl amino-3-oxo-propyl) thio) methyl) propanoic acid, 0.1 microM) or by nicardipine (1 microM), but was not affected by dazoxiben (30 microM), staurosporine (100 nM) or L 655,240 (240 (3-[1-(4-clorobenzil)-5-fluoro-3-metilhyindol-2il] 2,2-dimetilpropanoic acid, 1 microM). Unexpectedly, des-Arg9-bradykinin-induced contraction was unaffected by the selective bradykinin B1 receptor antagonists, des-Arg9-[Leu8]-bradykinin and des-Arg9-NPC 17761 (des-Arg0-D-Arg [Hip3, D-HipE (transtiofenil)7, Oic8]-des-Arg9-bradykinin). However, the selective bradykinin B2 receptor antagonists, HOE 140 (D-Arg0-[Hyp3, Thi5, D-Tic7, Oic8]-bradykinin) and NPC 17731 (D-Arg0 [Hyp3, DHypE (transpropyl)7, Oic8]-bradykinin), completely blocked des-Arg9-bradykinin-mediated contraction. Pre-treatment of the animals with Escherichia coli endotoxin (lipopolysaccharide, 30 microg/animal, i.v., 24 h) did not significantly change the response to des-Arg9-bradykinin induction. It is concluded that des-Arg9-bradykinin-induced contractions in guinea-pig gallbladder are mediated primarily by the release of proinflammatory eicosanoid(s) derived from the cyclo-oxygenase pathway. These effects are unrelated to thromboxane A2 and do not seem to be coupled to activation of a protein kinase C-dependent mechanism. Response to des-Arg9-bradykinin increases as a function of the equilibration period of the preparation by a mechanism dependent on protein synthesis and seems to be mediated by activation of bradykinin B2 (but not B1) receptors. Finally, in contrast to that observed for bradykinin, the contraction induced by des-Arg9-bradykinin in guinea-pig gallbladder is fully dependent on the influx of extracellular Ca2+, partially through L-type Ca2+ channels.

Nabumetone is a neutral non-steroidal anti-inflammatory drug with a low propensity to cause gastrointestinal (GI) damage. Previous studies, in vivo, have shown that the drug has weak effects on gastric mucosal cyclooxygenase activity, which may help to explain its favourable GI profile. The present study set out to determine whether the observed effects of nabumetone on cyclooxygenase, in vivo, parallel those of its active metabolite, 6-methoxy-2-naphthylacetic acid (6MNA), in vitro. We have also studied nabumetone and 6MNA on two other systems, namely 15-prostaglandin dehydrogenase (15-PGDH) and 5-lipoxy-genase (5-LO), which when inhibited may confer mucosal protection. The results showed that 6MNA had variable effects on cyclooxygenase activity, depending on the concentration and was less potent and less effective than indomethacin. Cyclooxygenase activity was not inhibited by the reversible inhibitor, aminopyrine, but at low concentrations stimulation was observed. Sulphasalazine inhibited 15-PGDH in a concentration-dependent manner whereas 6MNA inhibited it only at high concentrations. Nabumetone was devoid of activity. Basal 5-LO activity was attenuated by phenidone and unaltered by 6MNA but increased by nabumetone at the highest concentration. In the presence of arachidonic acid, to raise 5-LO activity, nabumetone, 6MNA, BW755C and phenidone apparently inhibited this activity. However, it was possible that both nabumetone and 6MNA inhibited a prostanoid-mediated stimulatory effect on 5-LO rather than effecting enzyme inhibition per se. Nabumetone's favourable GI profile may, therefore, relate to 6MNA having weak effects on mucosal cyclooxygenase and is unlikely to involve inhibition of prostanoid metabolism or 5-LO.

Data from several laboratories suggest a role for a variety of cytokines in the process of bone resorption. SK&F 86002 [5-(4-pyridyl)-6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b) thiazole], a potent cytokine-suppressive anti-inflammatory agent, has been shown to inhibit cyclooxygenase (CO) and 5-lipoxygenase (LO) activity and to inhibit the production of cytokines both in vitro and in vivo. In the present study, SK&F 86002 inhibited fetal rat long bone (FRLB) resorption induced by parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3, tumor necrosis factor alpha, and Escherichia coli lipopolysaccharide in a dose-dependent (IC50 of 0.5–1 μM) and reversible manner. Under identical conditions, selective CO inhibitors (indomethacin, ibuprofen, naproxen) and 5-LO inhibitors (phenidone, SK&F 107649) were inactive. Analogs of SK&F 86002, which are dual CO/LO inhibitors devoid of cytokine inhibitory activity (SK&F 81114 and SK&F 86055), also failed to significantly inhibit PTH-induced FRLB resorption. Analogs of SK&F 86002, which retain cytokine inhibitory activity (SK&F 104493 and SK&F 105561), inhibit bone resorption. These data indicate that the observed inhibition of bone resorption by compounds of this class correlates with their cytokine suppressive activity.

The mechanisms responsible for diabetes mellitus-induced enhancement of prostaglandin (PG) F2 alpha response were investigated in vascular smooth muscles isolated from diabetic mice and rats. Streptozocin (150 mg/kg, i.v. bolus, 6 week-elapsed)-ddY mice and (60 mg/kg, i.v. bolus)-Wistar rats and genetically diabetic GK-rats were used. The responses to PGF2 alpha were enhanced in small blood vessels such as mesenteric arteries (diabetic rats) and veins (diabetic mice) and they were reduced in large blood vessels such as the aorta and vena cava (diabetic rats). The enhanced response to PGF2 alpha in diabetic blood vessels was significantly inhibited by nordihydroguaiaretic acid (NDGA) (0.03 mM) and phenidone (0.05 mM), lipoxygenase inhibitors, cycloheximide (1 mg/kg, i.v.), a protein synthesis inhibitor and actinomycin D (2.8 mg/kg, i.v.), a RNA polymerase inhibitor, but neither inhibited by cyclooxygenase inhibitors, a thromboxane antagonist, nor Ca2+ antagonists. The PGF2 alpha response was also enhanced with aging alone, whereas the extent of enhancement was less than that with diabetes mellitus, and not significantly blocked by NDGA. These results demonstrate that diabetes mellitus-induced imbalance in the regulation of the eicosanoid metabolic pathways (suppressed cyclooxygenase and accelerated lipoxygenase) may cause the enhancement of PGF2 alpha-induced responses in small blood vessels.

This study was performed to establish a useful method for monitoring the effects of inhibitors of 5-lipoxygenase (5-LO) and/or cyclooxygenase (CO) and for differential evaluation of these inhibitors. After oral dosing, CO inhibitors such as indomethacin (20-40 mg/kg) and ketoprofen (40-80 mg/kg), zileuton (5-LO inhibitor, 20-80 mg/kg) and MK886 (5-LO-activating-protein inhibitor, 640 mg/kg) potently suppressed arachidonic acid (AA, 0.25 mg)-induced ear edema in mice. Methysergide (serotonin antagonist, 20 mg/kg) showed a slight anti-edematous effect, while mepyramine (160 mg/kg) and bromelain (320 mg/kg) had no effect. The anti-edematous effects of indomethacin and ketoprofen were reduced by concomitant topical application of prostaglandin E2 (PGE2, 1 micrograms/ear), but not by concomitant intradermal application of leukotriene C4 (LTC4, 0.1 micrograms/ear). On the contrary, the anti-edematous effects of zileuton and MK886 were reduced by LTC4, but not by PGE2. Dual (5-LO and CO) inhibitors such as phenidone (80-160 mg/kg) and BW755C (40-80 mg/kg), which inhibited the biosynthesis of LTB4 13-15 times more potently than that of PGE2 in rat peritoneal exudate cells, also showed anti-edematous effects that were reduced by LTC4, but not by PGE2. These results suggest that the AA (0.25 mg)-induced ear edema in mice is mainly mediated by LTs and PGs and is suitable for evaluating inhibitors of 5-LO and/or CO, and that an application of LTC4 or PGE2 with AA is a useful method for differential evaluation of these inhibitors.

1. Regulatory properties of lipoxygenase activity in rat brain cytosol were studied using linoleic acid (LA) as a substrate. 2. A change in the absorbance at 234 nm was biphasic when a mixture of LA and pre-formed hydroperoxide (LA-OOH) was incubated with freshly isolated native brain cytosol. Initially, a rapid depletion of LA-OOH was observed with a concomitant formation of LA-oxo compounds. This phase was followed by LA dioxygenation. 3. Both hydroperoxidase and dioxygenase activities of lipoxygenase were inhibited by micromolar concentrations of classic lipoxygenase inhibitors (phenidone, 5,8,11-eicosatriynoic acid and nordihydroguaiaretic acid). 4. The dioxygenase activity in dialysed cytsool was stimulated by nanomolar concentrations of H2O2 and micromolar concentrations of LA-OOH and it was inhibited by serotonin, dopamine and norepinephrine (IC50 25-43 microM).

Ring homologation of the known lipoxygenase inhibitor, phenidone to 1-phenyl-[2H]-tetrahydropyridazin-3-one provided A-53612, which was discovered to be a selective, orally active 5-lipoxygenase inhibitor. In contrast to phenidone, A-53612 did not cause significant inhibition of platelet 12-lipoxygenase, soybean 15-lipoxygenase, or intact rat PMNL cyclo-oxygenase at concentrations up to 100 μM.

In the search for a model of leukotriene (LT) production to provide a method to determine in vivo 5-lipoxygenase (5-LO) inhibitory activity by various compounds, a passive anaphylactic reaction in the rat peritoneal cavity was examined, refined and characterized. The reaction, produced by passive sensitization with an i.p. injection of rabbit anti-bovine serum albumin (anti-BSA) followed by an i.p. injection of BSA, resulted in the biosynthesis of large amounts of sulfidopeptide LTs measurable by immunoassay or by reversed phase high performance liquid chromatography. The oral activity of several 5-LO inhibitors has been examined using this model. An example of these is zileuton (Abbott-64077), a potent 5-lipoxygenase inhibitor now under clinical evaluation. Zileuton inhibited sulfidopeptide LT biosynthesis in the rat peritoneal cavity in a dose-dependent manner (ED50 = 3 mg/kg). WY-49,232, MK-866, BW A4C and phenidone also produced good activity with ED50 values of 6, 8, 11 and 17 mg/kg, respectively. This modified rat peritoneal anaphylaxis model appears to be a valuable tool for establishing in vivo activity of 5-LO inhibitors.

Earlier studies from this laboratory demonstrated that lipoxygenase catalyzes oxidation of a number of xenobiotics. This report presents data on the desulfuration and dearylation of parathion catalyzed by purified soybean lipoxygenase in the presence of linoleic acid. The formation of paraoxon was estimated from in vitro inhibition of acetylcholinesterse (electric eel) and further confirmed by using [ 14 C]parathion. The metabolites paraoxon and p -nitrophenol were separated on thin layer chromatography and quantified by radiometry. The maximal rate of parathion metabolism occurred in the presence of 1.0 m M linoleic acid, 25.0 μ M parathion, 20.0 n M enzyme at pH 8.0. The rate of desulfuration and dearylation reactions were dependent on incubation time, pH, linoleic acid, parathion, and enzyme concentration. Lipoxygenase inhibitors, like nor-dihydroguaiaretic acid and phenidone, significantly blocked the desulfuration and dearylation reactions. The turnover numbers (nmol product/min/nmol enzyme) were found to be 3.0 and 5.5 for paraoxon and p -nitrophenol, respectively. The rate of lipoxygenase-catalyzed parathion bioactivation was 3–20 times greater as compared to purified mammalian cytochrome P-450

A quantitative method for testing antiinflammatory agents in beagles has been developed, based on measurement of paw inflammation induced by a local injection of carrageenin. Carrageenin [0.5 mL of 2% (wt/vol) in saline] was injected into the plantar region of the hindpaws of pentobarbital-anesthetized beagles. Paw pressure changes registered from a water-filled balloon held on the top of the paw by a light adhesive tape wrapping were monitored for 240 min. In control dogs given 0.5% (wt/vol) methylcellulose (10 mL/kg orally) just before carrageenin, paw pressure increased significantly (p less than 0.05) over eightfold, from 2.9 +/- 0.8 mm Hg (mean +/- SEM, n = 29 paws) at 75 min to 26.0 +/- 3.5 mmHg at 240 min. The increase in paw pressure was significantly inhibited by the cyclooxygenase inhibitors, ibuprofen, indomethacin, and orpanoxin, and partially inhibited by the lipoxygenase inhibitor, phenidone, administered orally before carrageenin injection. Thus this model, with further characterization, could provide a convenient, quantitative way of assessing the efficacy of nonsteroidal antiinflammatory agents in dogs.

SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.

Intrapleural injection of the ionophore A23187 in rats induced leukotriene (LT) production, prostaglandin E 2 , production, plasma exudation and ieukocyte accumulation in the pleural cavity. We evaluated the inhibitory effects of orally administered drugs on 5-lipoxygenase activity by their ability to reduce the content of both peptido-LTs (LTC 4 , LTD 4 and lte 4 ) and LTB 4 in this model. TZI-41127, a novel selective 5-lipoxygenase inhibitor, significantly reduced the peptido-LTs and LTB 4 content with ed 50 values of 4.2 and 6.1 mg/kg, respectively, whereas it only reduced the prostaglandin E 2 content (cyclooxygenase activity) by 31.1% even after 100 mg/kg. Phenidone inhibited 5-lipoxygenase activity more selectively than cyclooxygenase activity. BW755C inhibited cyclooxygenase activity more selectively than 5-lipoxygenase activity. Indomethacin selectively inhibited cyclooxygenase activity. These results suggest that: (1) A23187-induced pleurisy is an convenient in vivo model for studying drug effects on 5-lipoxygenase and cyclooxygenase activities and (2) TZI-41127 is an orally active and comparatively selective inhibitor of 5-lipoxygenase activity.

This study was designed to analyse the effect of crude extract (CE) and some pure compounds isolated from M. velutina on arachidonic acid (AA)-induced ear oedema in mice. The effect of these compounds on contractions in the rat stomach induced by AA and prostaglandin E2 (PGE2) was also investigated. The CE given orally to mice (50-400 mg/kg) 1h before inhibited the ear oedema in a dose-dependent manner, with a maximal inhibition (MI) of 56%. Given topically, compound MV8612 (200-600 mg/ear) isolated from this plant inhibited the oedema in a concentration-dependent manner with a MI of 66%, while compounds MV8608 and MV8610 (100-600 mg/ear) caused less inhibition, MI 29% and 39% respectively. Compound MV8612, given i.p. (3-30 mg/kg) 30 min before, also caused a dose-dependent inhibition of AA-induced ear oedema (MI of 60.5% compared with 28% and 49% for MV8608 and MV8610). Indomethacin (0.25-1.0 mg/ear) applied topically had no effect, but orally (1-10 mg/kg) gave MI of 66%. Phenidone given orally (10-100 mg/kg) gave a MI of 30% but it was very potent topically (0.5-2 mg/ear) (MI 66%). Nordihydroguaiaretic acid applied either topically (0.5-2.0 mg/ear) or orally (10-100 mg/kg) caused MI of 63% and 47%, respectively. BW 755C, orally (10-100 mg/kg), inhibited the AA-induced oedema in a dose-dependent manner (MI 48%), but was less effective when applied topically (0.25-1 mg/ear) (MI 32%). In the rat stomach preparation, compounds MV8608 and MV8612 (0.1-20 micrograms/ml) had no significant effect on contractions to AA or PGE2, while indomethacin (0.01-3 micrograms/ml) potently inhibited AA contraction, but had no effect on the PGE2 response. These results indicate that MV8608 and MV8612 exhibit both a topical and a systemic anti-inflammatory profile, presumably by a mechanism not related to inhibition of cyclooxygenase.

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and extract was washed with dichloromethane then and adjusted to pH 3.5 or 6.0 respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantified by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025–1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 ± 0.036 ng/ml) and LTD4 (1.399 ± 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)-4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.

The objective of this work was to investigate the role of leukotrienes in the production of IL-1 by activated human peripheral blood monocytes and mouse peritoneal macrophages. Using overnight adherent macrophages, stimulation with lipopolysaccharide or zymosan caused a time-dependent increase in IL-1 production. LTC4 was detected and preceded IL-1 production only in zymosan-treated macrophages. Lipopolysaccharide did not stimulate macrophages to produce LTC4. Zymosan-stimulated LTC4 production was inhibited by the lipoxygenase inhibitors, ICI207968 (3.20 microM), nordihydroguaiaretic acid (0.22 microM), phenidone (4.60 microM), REV5901 (0.20 microM), and the Merck 5-lipoxygenase "translocation inhibitor" MK886 (0.02 microM) with IC50 values as shown in parenthesis. However, none of these inhibitors reduced IL-1 production at concentrations which completely inhibited leukotriene synthesis. Taken together, these results do not support a role for leukotrienes in the production of IL-1 by zymosan-activated macrophages.

The purpose of these experiments was to assess the role of the lipoxygenation products of arachidonic acid in hamster periodontitis. Phenidone and ketoconazole were used as inhibitors of leukotriene synthesis. In an established periodontitis, both drugs administered for 30 days induced a statistically significant decrease in PMNLs in the infiltrated connective tissue and around bacterial plaque within periodontal pockets. These changes were associated with a significant decrease in osteoclastic bone resorption. The results suggest that leukotrienes, and particularly leukotriene B4, are involved during hamster periodontitis and are responsible for PMNL infiltration of the periodontal pocket. The effects on bone are probably the consequence of the reduced inflammation resulting from the decrease in PMNL chemotaxis.

Injection of zymosan in rat pleural cavity provokes an exudate which is already detectable at 15 min and which is maximum at 24 h. The leucocyte count (mostly neutrophils) increases at 2-4 h and is maximum at 48 h. In this paper the reaction has been studied up to 6 h. Evidence of histamine release, of mast cell degranulation and of reduction of the exudate by anti-H1 compounds, as well as by sodium cromoglycate, proves the active role played by histamine in the early stage of pleurisy. Serotonin (whose role was studied exclusively using antagonists) seems to have only a minor part in the early phase of the reaction. Some metabolites of arachidonic acid were determined in the pleural exudate at 1 h and 6 h. The concentration of leukotriene B4 was high at 1 h and decreased at 6 h. The thromboxane B2 level was already high at 1 h and was neatly augmented at 6 h while the amount of prostaglandin F1 alpha was high at both times. The non-steroidal anti-inflammatory substances studied all reduced the pleural exudate at 1 h but their activity then varied from each other at 6 h. Cyclooxygenase and lipoxygenase inhibitors (phenidone, BW755C) induced a reduction of the exudate at both times. Zymosan-induced pleurisy seemed thus to be an excellent model for the investigation of antiallergic and anti-inflammatory compounds active on histamine and cyclooxygenase and lipoxygenase pathways.

Intraperitoneal injection of washed, preformed ovalbumin/anti-ovalbumin immune complexes caused the production of slow-reacting substance (SRS), prostaglandin E2, 6 keto prostaglandin F1a, vascular permeability (measured as extravazation of pontamine sky blue injected previously into the bloodstream), and polymorphonuclear leukocyte (PMN) infiltration in the mouse peritoneal cavity. Arachidonic acid metabolites appeared early in the response at 7.5 min; dye extravazation and PMN infiltration peaked at 30 min and 4 hr, respectively, after immune complex injection. BW755C and phenidone given orally 30 min before immune complex injection inhibited dye extravazation and SRS formation at 7.5 and 30 min. Indomethacin by the same regimen inhibited dye extravazation at 7.5 min but not at 30 min, while enhancing SRS formation at both times. Dexamethasone inhibited dye extravazation and to some extent SRS formation at both times. The data suggests that the mouse model may be useful for demonstrating an antiinflammatory activity of dual inhibitors that is clearly distinguishable from the activities shown by classical nonsteroidal antiinflammatory drugs and steroids.

We have characterized the effects of eight different drugs on the IgE-mediated histamine release (HR) and leukotriene C4 (LTC4R) from human basophils. Arachidonic acid analogues 5,8,11 eicosatriynoic acid and 5,8,11,15 eicosatetraynoic acid inhibit the release of both mediators in the range 10(-6) to 10(-4) mol/L with almost total (80% to 100%) inhibition of release at 10(4) mol/L. The inhibition of LTC4R was significantly (p less than 0.05) greater than the inhibition of HR only at intermediate (10(-5) to 3 X 10(-5) mol/L) doses of the drugs. Two other inhibitors of phospholipase A2 (bromophenacyl bromide and phenidone) affected the release of both mediators equally. Two drugs that activate adenylate cyclase (prostaglandin E1 and dimaprit) inhibited release in a dose-dependent fashion but failed to preferentially affect either HR or LTC4R. Isoproterenol (10(-6) to 10(-4) mol/L), a third activator of adenylate cyclase, caused only moderate (30%) inhibition of HR, even when the reaction was staged, but was slightly (0.1 less than p less than 0.05) more potent against leukotriene release. The final drug tested was the phosphodiesterase inhibitor, isobutylmethylxanthine, which proved to be an effective (50% to 100%) inhibitor of both mediators in the range 10(-5) to 10(-3) mol/L.

The 5-lipoxygenase inhibitor REV-5901 [alpha-pentyl-3-(2-quinolinylmethoxy)benzene-methanol] was evaluated for effects on mediator release in vitro from fragmented guinea-pig and human lung. In guinea-pig lung, REV-5901 inhibited the antigen-induced release of immunoreactive leukotriene D4 (iLTD4) with an IC50 of 9.6 +/- 2.9 microM and immunoreactive leukotriene B4 (iLTB4) with an IC50 of 13.5 +/- 2.2 microM. REV-5901 also inhibited the calcium ionophore-induced release of immunoreactive leukotrienes from human lung in vitro with IC50 values of 11.7 +/- 2.2 MicroM versus peptide leukotrienes and 10.0 +/- 1.1 microM versus iLTB4. The inhibition of release of iLTD4 and iLTB4 with similar IC50 values suggests that REV-5901 acts by inhibiting 5-lipoxygenase in this system. At concentrations as high as 50 microM, REV-5901 did not inhibit the release of thromboxane B2 (TxB2), 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), or histamine from either lung. The lack of effect on TxB2 and 6-keto-PGF1 alpha indicates that REV-5901 did not inhibit phospholipase A2, cyclooxygenase, or thromboxane synthetase. Inhibition of leukotriene release by REV-5901 could not be reversed by washing. Among various 5-lipoxygenase inhibitors, the order of potency for inhibition of iLTD4 release from guinea-pig lung was AA-861 greater than REV-5901 greater than phenidone greater than nafazatrom greater than NDGA greater than BW755C. These findings suggest that REV-5901 is a selective and relatively potent inhibitor of leukotriene release from lung tissue in vitro.

Catechin dimers induce a large long-lasting oedema when injected in the paw of the rat. This oedema is not inhibited by methysergide, promethazine, indomethacin, phenidone, bromophenacyl bromide and colchicine. It is not modified in rats made leukopenic by methotrexate. It is slightly delayed in Brown Norway rats which were kallikrein-kininogen deficient. Similarly catechin dimers induce the formation of a large peritoneal exudate in the rat. The exudate contains insignificant levels of leucocytes and 5-hydroxytryptamine. It contains kinins but its PG content is very low. The exudate does not activate (14C)-arachidonic acid into PG. Catechin dimers induce kinin formation in rat plasma "in vitro". They inhibit the formation of PG and HETE-like compounds from (14C)-arachidonic acid by rat peritoneal cells "in vitro". Catechin dimers administered at sub-irritant doses reduced carrageenan-induced oedema. Catechin dimers at low doses have an anti-inflammatory effect which may depend on PG synthesis inhibition. At larger doses, they induce inflammatory responses which occur with almost complete lack of participation of PG.

The enzymic conversion of 5,8,11,14-eicosatetraenoic acid to the corresponding hydroperoxy fatty acids by soybean lipoxygenase (lineolate: oxygen oxidoreductase E.C. 1.13.11.12.) was investigated and a simple selective extraction method was introduced. The known inhibition of the lipoxygenase pathway by phenidone, mercuric chloride, methylmercuric chloride, methylmercuric iodide, 1,5-dihydroxynaphthalene and acetone phenylhydrazone was influenced by thiol compounds in different ways. (1) A total reactivation of lipoxygenase activity was achieved when several thiol compounds, especially gluthatione, were preincubated with the inhibitor mercuric chloride and the enzyme. (2) A remarkable reduction of the inhibitory potency of phenidone against soybean lipoxygenase was seen when thiol compounds were preincubated with the enzyme before the addition of the inhibitor. When phenidone was preincubated with lipoxygenase first, sulfhydryl agents did not restore the enzyme activity. (3) No interaction was seen, when glutathione or other thiol compounds and the lipoxygenase inhibitors 1,5-dihydroxynaphthalene, nordihydroguaiaretic acid and acetone phenylhydrazone were tested against the enzyme. Therefore, we suggest that soybean lipoxygenase inhibitors may act via different modes of action. It is important to study the mechanisms of lipoxygenase inhibitors, since mammalian lipoxygenase and their products are known to be involved in the inflammatory response.

Compounds which inhibit cellular production of ATP either by uncoupling of oxidative phosphorylation (valinomycin, carbonylcyanide-4-trifluoromethoxphenylhydrazone, and 2,4-dinitrophenol), glycolytic phosphorylation (2-deoxy-D-glucose) or by inhibiting respiratory-chain reactions (antimycin A) were all shown to inhibit calcium-ionophore A23187-induced leukotriene synthesis in rat peritoneal leukocytes at concentrations closely correlating with those needed to block ATP synthesis. In contrast, none of the compounds interfered with cyclo-oxygenase or other enzymes involved in arachidonate metabolism in these cells. Two well-known inhibitors of 5-lipoxygenase, nordihydroguaiaretic acid and phenidone, blocked LTB4 synthesis without affecting ATP production. In conclusion, rat peritoneal leukocyte leukotriene synthesis depends on intact energy metabolism.

Intrapleural injection of sodium arachidonate to rats produced both a cellular response and the generation of hydroxy fatty acid metabolites of arachidonic acid without edema. The cellular influx was inhibited by mixed 5-lipoxygenase/cyclooxygenase inhibitors and by a glucocorticoid but not by cyclooxygenase inhibitors. These results suggest that a model using intrapleural injection of sodium arachidonate may have utility for detecting systemically active 5-lipoxygenase inhibitors

The metabolism of synthetic leukotrienes by guinea-pig lung homogenates was investigated. Leukotriene (LT) A 4 , B 4 , C 4 , D 4 , and E 4 were incubated for various periods of time (0–180 min) at 370° with guinea-pig lung homogenates (20,000 X g; 15 min) and the remaining biological activity was assayed either on guinea-pig ileum longitudinal smooth muscles (for LTA 4 , LTC 4 , LTD 4 and LTE 4 ) or on guinea-pig lung parenchymal strips (for LTB 4 and on some occasions LTA 4 ) in a cascade superfusion system. The half-life of LTD 4 in our conditions was about 50 minutes but the inactivation plateaued after 120 minutes with approximately 25% of residual activity. When LTA 4 and LTC 4 were added to guinea-pig lung homogenates, increases of biological activity were measured during the first few min. (10 to 30 min) and were followed by decreases. The first activation period was probably due to rapid conversion to the more potent LTC 4 and LTD 4 . LTB 4 and LTE 4 were not inactivated by guinea-pig lung homogenates (60–120 min). Inhibitors of lipoxygenases (eicosatetraynoic acid, ETYA; BW 755C; nordihydroguaiaretic acid, NDGA; phenidone) had no effect on LTD 4 inactivation and on LTA 4 transformation. γ-glutamyl-0-carboxy-phenylhydrazide did not affect LTC 4 activation. Our results confirmed the presence of the various enzymes responsible for both the formation and inactivation of leukotrienes in the guinea-pig lungs.

The mechanism of aggregation induced by arginine vasopressin (AVP) was studied in human platelet rich plasma. AVP--over the range of 1.8-113.6 mU/ml--caused a dose-dependent aggregation with a concomitant stimulation of thromboxane B2 (TXB2) formation. d(CH2)5Tyr (Me)AVP did not by itself affect platelet aggregation or TXB2 release, but completely inhibited the action of AVP. DDAVP up to the concentration of 280 pM/ml had no effect on aggregation. Pretreatment of platelets with verapamil, trifluoroperazine or methylimidazole, a thromboxane synthetase blocker, prevented AVP-induced aggregation and TXB2 release. Neither phenidone in lower concentration nor nordihydroguaiaretic acid inhibited the ability of AVP to induce aggregation and TXB2 release. These results are consistent with the hypothesis that human platelets possess AVP receptor of the calcium-dependent vasopressor (V1) subtype and suggest that AVP-induced platelet aggregation is mediated via thromboxane release.

Ketoconazole inhibits in vitro (IC50:2.6 X 10(-5) M) the formation of 5-HETE and LTB4 by isolated, carrageenin-elicited rat peritoneal PMN leukocytes, challenged with the Ca2+-ionophore A23187 in the presence of [14C]-arachidonic acid ([14C]-AA). The relative potency of various compounds tested in this respect is NDGA greater than nafazatrom greater than phenidone greater than ketoconazole greater than BW 755C. In contrast to the other compounds studies, ketoconazole in vitro, up to 1 X 10(-4) M, has no effect on the fatty acid cyclo-oxygenase or the 12-lipoxygenase-mediated metabolism of [14C]-AA by isolated human platelets; however, it stimulates the 15-lipoxygenase activity in phenylhydrazine-induced rabbit reticulocytes. After oral administration (10-40 mg/kg, -2 hr), ketoconazole inhibits in a dose-dependent way, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigs. This study demonstrates that ketoconazole is a comparatively specific and orally active inhibitor of the 5-lipoxygenase activity bearing on the production of leukotrienes derived from arachidonic acid.

5,8,11,14-Eicosatetraynoic acid (ETYA), a compound which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism, antagonized the contraction of segments of guinea-pig ileal longitudinal muscle produced by SRS-A (IC50 = 2.73 microM). This activity was unaffected by pretreatment of the tissues with 10 microM indomethacin. Phenidone, another mixed cyclooxygenase-lipoxygenase inhibitor, was inactive. FPL-55712, an SRS-A antagonist, was a very potent inhibitor (IC50 = 0.011 microM). BW755C and NDGA nonselectively inhibited the contractions of the guinea-pig ileal longitudinal muscle induced by SRS-A or histamine. ETYA antagonized the contraction of the guinea-pig ileal strip produced by 6 nM synthetic LTC4 (IC50 = 9.3 microM). FPL-55712 demonstrated an IC50 of 0.3 microM in a similar series of experiments. ETYA, 1, 3 or 10 microM did not inhibit the contractions elicited by 0.5 microM of histamine. This was not a tissue-selective effect since 100 microM ETYA antagonized the LTC4-induced contraction of the guinea-pig lung parenchymal strip preparation. These data demonstrate that ETYA antagonized the contractile effect of the leukotrienes on tissues from the gastrointestinal tract and lung. Furthermore, the inability of indomethacin or phenidone to inhibit the contractile response suggests that antagonism by ETYA may occur by a mechanism independent of cyclooxygenase and lipoxygenase enzymes.

Rat renal mesangial cells possess morphological and functional features of smooth muscle cells in culture, such as intracellular myosin filaments, A II receptors and a contractile response to A II. Furthermore, they represent the main glomerular site of PGE2 synthesis. In the presence of A II (50 nM), their PGE2 production rate was significantly increased, this effect being potentiated by arachidonic acid (3 micrograms/ml). After a prior inhibition of arachidonic acid metabolism by cyclooxygenase inhibitors (indomethacin 1 microM or naproxen 0.02 microM) or by a cyclo- and lipoxygenase inhibitor (phenidone 670 microM), the percentage of cells contracting in response to various A II concentrations (10 pM to 10 nM) was significantly enhanced as compared to the basal conditions. On the contrary, the percentage of cells presenting a contractile response to A II in the presence of exogenous PGE2 (50 ng/ml) or of arachidonic acid (3 micrograms/ml) was significantly decreased. These modifications were not related to some changes in the A II receptor properties of the cells, i.e. the number of receptor sites and their affinity constant. The data demonstrate that the contractile response of glomerular mesangial cells is modulated by the PGE2 content of their incubation medium. Since their own PGE2 production rate is enhanced in the presence of A II, this cellular response may represent a local regulatory mechanism of their contractile properties.

Injections of zymosan in mouse and rat paws provoke inflammatory reactions, the kinetics of which are different. In both models, inflammation occurs at an early stage but oedema is maximal at 30 min in rat paw and 6 h in mouse paw. In this study the two reactions have been studied up to 6 h. The reduction of oedema by anti-H 1 compounds, as well as by disodium cromoglycate, proves the active role played by histamine in rat paw oedema. In mouse its role appears to be minor or non-existent. Serotonin seems to be clearly implicated in the early stages of the oedema in mouse, somewhat less in rat. In the two species, non-steroidal anti-inflammatory compounds only reduce the 4–6 h phase. BW755C and phenidone reduce the early and late phase of paw oedema in both species, with the exception of phenidone which is inactive on the 4–6 h phase in the mouse. We can hypothesize that in the two species some leukotrienes seem to be implicated principally in the early phases, while derivatives of cyclooxygenase play a more important role in the late phases. Theophylline reduces inflammation in the two models, hydrocortisone acetate, however, is only active on the late phases. These results indicate that there are important differences in the participation of the various mediators studied in the two models.

The effects of SK&F 86002 [5-(4-pyridyl)-6 (4-fluorophenyl)-2,3-dihydroimidazo (2,1-b) thiazole] on the generation of eicosanoids in vitro and on inflammatory responses in vivo are described and compared to other non-steroidal anti-inflammatory drugs. SK&F 86002 inhibited prostaglandin H2 (PGH2) synthase activity (IC50 120 microM) as well as prostanoid production by rat basophilic leukemia (RBL-1) cells (IC50 70 microM) and its sonicate (IC50 100 microM) and human monocytes (IC50 1 microM). In addition, SK&F 86002 inhibited the generation of dihydroxyeicosatetraenoic acid (diHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE) by a high speed supernatant fraction of RBL-1 cells (IC50 10 microM). Cellular production of 5-lipoxygenase products was inhibited by SK&F 86002 as measured by leukotriene B4 (LTB4) generation from human neutrophils (IC50 20 microM), leukotriene C4 (LTC4) generation by human monocytes (IC50 20 microM), and 5-HETE production by RBL-1 cells (IC50 40 microM). The in vivo profile of anti-inflammatory activity of SK&F 86002 supports the dual inhibition of arachidonate metabolism as indicated by its activity in inflammation models that are insensitive to selective cyclooxygenase inhibitors. The responses of arachidonic-acid-induced edema in the mouse ear and rat paw, as well as the cell infiltration induced by carrageenan in the mouse peritoneum and by arachidonic acid in the rat air pouch, were inhibited by SK&F 86002 and phenidone but not by the selective cyclooxygenase inhibitors naproxen and indomethacin.

Summary Cyclic voltammetry, controlled potential electrolysis and double potential pulse techniques have been used to study the oxidation of 1-phenyl-3-pyrazolidinone at high pH (in 1.0 M KOH solution). Oxidative mechanisms previously proposed for lower pH values are confirmed and shown to hold in this environment.

Acetylcholine (ACh) caused concentration-dependent relaxation of strips of rabbit thoracic aorta precontracted with noradrenaline if the endothelium was intact. More than ten-fold higher concentrations of ACh also stimulated the release of prostacyclin (PGI 2 ) and PGE 2 from the strips. De-endothelialized strips released much smaller amounts of prostaglandins and contracted slightly to ACh. The endothelium-dependent vasodilation was resistant to cyclooxygenase inhibition by indomethacin, flurbiprofen and diclofenac. However, it could be reversed by six different inhibitors of lipoxygenase (nordihydroguairetic acid, phenidone, eicosatetraynoic acid, nafazatrom, compound BW 755C and caffeic acid). BW 755C and caffeic acid, a selective inhibitor of 5-lipoxygenase, had comparatively weak effects on the relaxation. Eicosatetraynoic acid, which probably does not inhibit C-5-lipoxygenase, completely reversed the effect of ACh. It is concluded that ACh relaxes strips of rabbit aorta by a mechanism in involving a non-cyclooxygenase metabolite of arachidonic acid of endothelial origin. This compound is probably not a product of C-5-lipoxygenase.

Although the platelets of the mouse are refractory to the direct effects of platelet-activating-factor (PAF), tail vein injection of 10-150 micrograms/kg PAF produces lethal anaphylactic shock. Sensitivity varies with strain and source: Swiss Webster mice show a range of sensitivity and DBA/2 (complement C5-deficient) mice are very resistant. At lethal doses of PAF, animals show labored respiration and general depression; death occurs within 15-45 min. Dexamethasone administered at least 1.5 hr prior consistently protects, whereas the cyclooxygenase inhibitors do not. Antihistamines, adrenergic antagonists, and methysergide have no effect, but cyproheptadine is partially protective at near lethal doses. Calcium entry blockers and calcium chelators, tetracycline and chlortetracycline are partially protective at very high doses consistent with non-specific effects on calcium dependent processes. The arachidonic acid lipoxygenase inhibitors BW755c, phenidone, nordihydroguaiaretic acid and diphenyldisulfide provide nearly complete protection after oral administration of 50-200 mg/kg. Phosphodiesterase inhibitors and dapsone are also effective orally. The leukotriene antagonist FPL55712 administered intraperitoneally (10 mg/kg) 5 min. prior to PAF challenge provides almost complete protection. PAF-induced mortality in the mouse represents a small animal model of systemic anaphylaxis particularly useful for the systemic testing of arachidonic acid lipoxygenase inhibitors and leukotriene antagonists.