Novel anti-inflammatory plant labdanes: Comparison ofin vitro properties with aspirin and indomethacin

Two purified plant products were obtained from anti-inflammatory extracts of the Spanish herbSideritis javalambrensis: ent-13-epi-12-acetoxy-manoyl oxide (=manoyl oxide F1) andent-8-hydroxy-labda-13 (16), 14-dien (=labdane F2). They were evaluated for possible anti-inflammatory actionsin vitro, and were compared with aspirin, sodium salicylate and indomethacin. Neither of the natural products affected superoxide generation or scavenging and they did not affect granular enzyme secretion from activated human and rat neutrophils. The compounds were not toxic to the cells at up to 10^–4 M. However, both F1 and F2 inhibited thromboxane B₂ and leukotriene B₄ generation by A23187-treated rat peritoneal leukocytes, suppressing leukotriene generation at 10^–5 to 10^–4 M and thromboxane B₂ at 10^–4 M. Labdane F2 also inhibited human secretory synovial phospholipase A₂ activity at 10^–3 M, a property not shared by manoyl oxide F1. We conclude that these two natural products interact with the eicosanoid system, perhaps at the phospholipase level, but do not interfere with the other tested leukocyte functions or with reactive oxygen species, and are essentially non-toxic at the doses used.     

Differential effects of malotilate on 5-, 12- and 15-lipoxygenase in human ascites cells

Conclusions These effects of malotilate on eicosanoid formation differ from those of known lipoxygenase inhibitors such as BW 755C (IC₅₀ of 5-lipoxygenase 35 M, 12-lipoxygenase >100 M and 15-lipoxygenase 1.2 M), nordihydroguiaretic acid (IC₅₀ of 5-lipoxygenase 1.4 M, 12-lipoxygenase 26 M and 15-lipoxygenase 1 M) and ketoconazole (5-lipoxygenase 28 M, 12-lipoxygenase not affected and 15-lipoxygenase increased) [5]. The differential effects of malotilate on the 5-, 12- and 15-lipoxygenases and also on the generation of the compounds of the cyclooxygenase, have not previously been reported. The suppression of leukotriene productionin vitro occurred at concentrations found following normal therapeutic dosesin vivo. Inhibition of the production of the chemotactic substance LTB₄ and the vasoconstrictive TxA₂ provide a possible explanation for the useful effects of this drug on liver necrosis and liver fibrosis.     

Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.     

Eicosanoid production by density-defined human peritoneal macrophages during inflammation

Density-defined macrophages isolated from fluids of patients with liver cirrhosis mainly generated the 5-lipoxygenase products leukotriene B₄ (LTB₄, 16%) and 5-hydroxy-eicosatetraenoic acid (5-HETE, 24%) and the cyclooxygenase products 12-hydroxyheptadecatrienoic acid (HHT, 22%) and thromboxane B₂ (TXB₂, 18%). The synthesis of eicosanoids was linear with the maturity of the macrophage subpopulations, suggesting that eicosanoid production is increased inin-vivo activated macrophages.

     

Differential effects of putative lipoxygenase inhibitors on arachidonic acid metabolism in cell-free and intact cell preparations

The effects of nordihydroguairetic acid (NDGA), 3-amino-1-trifluoromethyl-)-phenyl-2-pyrazoline (BW755c), eicostatetraynoic acid (ETYA), phenidone, quercetin, and indomethacin (INDO) on the synthesis of 15-hydroxyeicosatatetraenoic acid (15-HETE) from soybean 15-lipoxygenase, leukotriene B₄ (LTB₄ from 5-lipoxygenase, and prostaglandin E₂ (PGE₂ from cyclooxygenase enzymes of rat neutrophils and mouse peritoneal macrophages were investigated. All of the drugs caused a dose-related inhibition of increased oxygen consumption by soybean 15-lipoxygenase in the presence of arachidonic acid and the rank order of potency was phenidone BW755c > ETYA > quercetin > NDGA > indomethacin. The reduction in oxygen consumption correlated with a reduction of 15-HETE formation as identified by high-performance liquid chromatography. Apart from indomethacin, these drugs were also effective against the rat neutrophil 5-lipoxygenase, although the rank order of potency did not correlate with that obtained with soybean 15-lipoxygenase. Furthermore, in both A23187-activated rat neutrophils and zymosan-activated mouse peritoneal macrophages the synthesis of prostaglandins was inhibited by all of these drugs. In the neutrophils, the rank order of potency was INDO > ETYA > BW755c > quercetin > NDGA > phenidone, whereas in mouse peritoneal macrophages, the order was INDO > ETYA > BW755c > NDGA > quercetin > phenidone. These results suggest that putative lipoxygenase inhibitors exhibit both qualitative and quantitative differences in their effects on both lipoxygenases and cyclooxygenases.     

Effect of auranofin on eicosanoids and protein kinase C in human neutrophils

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB₄ formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipiddependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B₄ (LTB₄) in FMLP-stimulated neutrophils, the ID₅₀ was approximately 4.5 g/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB₄ nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB₄ production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.     

A comparison of the anti-inflammatory activity of selective 5-lipoxygenase inhibitors with dexamethasone and colchicine in a model of zymosan induced inflammation in the rat knee joint and peritoneal cavity

Intraperitoneal and intra-articular (knee joint) injection of zymosan in the rat caused two phases of increased vascular permeability, a rapid increase (0.25–0.5 h) and a secondary increase (2–3 h) which was temporally associated with the onset of leukocyte infiltration. Intraperitoneal injection of zymosan led to a single peak of eicosanoid production (LTB₄, C₄, D₄, E₄ and 6-oxo-PGF₁ ) which was maximal at 0.125–0.25 h. Intra-articular injection led to an initial peak of LTB₄ production (maximal at 0.25 h) and a secondary peak of LTB₄ and PGE₂ production (maximal at 3 h). Oral administration of the 5-lipoxygenase (5-LO_ inhibitors phenidone, BW A4C (N-hydroxy-N-[3-(3-phenoxyphenyl)-2-propenyl] acetamide), A63162 (N-hydroxy-N-[1-(4-(phenylmethoxy) phenyl)ethyl] acetamide and ICI 207 968 (2-[3-pyridylmethyl]-indazolinone inhibited LTB₄ production in A23187 stimulation bloodex vivo. The glucocorticosteroid dexamethasone had no effect in this model. The initial phase of increased vascular permeability in the peritoneal cavity and LTB₄ production was dose dependently inhibited by the 5-LO inhibitors phenidone, BW A4C, A63162, and ICI 207 968 but not by dexamethasone or colchicine. The initial phase of increased permeability in the joint was unaffected by phenidone, BW A4C, dexamethasone or colchicine. However the latter two drugs inhibited the later phase of increased permeability and leukocyte infiltration in the joint and peritoneal cavity. These results demonstrate that zymosan induces eicosanoid productionin vivo but the relative importance of these mediators varies depending on the inflammatory site. Dexamethasone had a similar profile of anti-inflammatory activity to colchicine suggesting that inhibition of cell infiltration is the major mechanism of its therapeutic effect. No evidence for dexamethasone induced inhibition of eicosanoid production could be found suggesting that inhibition of the production or activity of other mediators is more important.     

CGS 22745: A selective orally active inhibitor of 5-lipoxygenase

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) synthesis in guinea pig leukocytes (IC₅₀=0.6M). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB₄ in blood (IC₅₀'s of 4.3, 0.56 and 3.2 M for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB₄ formationex vivo after 5 min. Its effective biological half-life was >160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibitedex vivo A23187-stimulated LTB₄ formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.     

Characterization of effect ofN-formyl-methionyl-leucyl-phenylalanine on leukotriene synthesis in human polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNLs) were exposed toN-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) in the presence or absence of exogenous arachidonic acid. Analysis of incubation mixtures by high-performance liquid chromatography showed that f-Met-Leu-Phe stimulated the synthesis of 5-hydroxy-eicosatetraenoic acid (HETE) and of leukotriene B₄ (LTB₄) which was rapidly metabolized into 20-OH-LTB₄ and 20-COOH-LTB₄. The stimulatory effect of f-Met-Leu-Phe was dose and time dependent. The tripeptide showed maximum stimulatory activity at the concentration of 1 M and after 20–30 min of incubation. Addition of arachidonic acid to the f-Met-Leu-Phe-stimulated PMNLs resulted in an increase in the synthesis of LTB₄ and 5-HETE. Pretreatment of the PMNLs with cytochalasin B strongly potentiated (up to six-fold) the stimulatory effect of f-Met-Leu-Phe on 5-lipoxygenase product synthesis, whereas cytochalasin B alone or with arachidonic acid had no significant effect. The tripeptide did not increase the synthesis of plateletderived 12-HETE, and 12-hydroxyheptadecatrienoic acid, or of PMNL-derived 15-HETE, suggesting that its action was selective for PMNL 5-lipoxygenase. The present data indicate that f-Met-Leu-Phe causes the release of arachidonic acid from cellular lipids and activates the 5-lipoxygenase.     

Platelet activating factor and tracheobronchial respiratory glycoconjugate release in feline and human explants: Involvement of the lipoxygenase pathway

It has been suggested that platelet activating factor (PAF) may participate in many aspects of bronchial asthma, including stimulation of mucus secretion. Feline tracheal and human bronchial explant production of respiratory glycoconjugates (RGC) in response to platelet activating factor (PAF) was investigatet, in order to differentiate the actions of this putative mediator on mucus secretion. PAF caused a dose-dependent increase in RGC release in concentrations ranging from 100–0.5 M during a 1–2 hours incubation with either feline or human explants, and the effect was inhibited by the PAF receptor antagonists Ro 19-3704. Several lines of evidence suggest that PAF enhances RGC release indirectly through stimulation of the production of lipoxygenase metabolites of arachidonic acid. 1) Incubation of 10 M PAF together with arachidonic acid (100 g/ml) enhances PAF's stimulatory effect on RGC release in cats. 2) The cyclooxygenase inhibitor ibuprofen (65 and 420 M) either failed to effect or slightly enhanced PAF induced RGC release in both species. 3) The combined cyclooxygenase and lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) as well as the putatively specific 5-lipoxygenase inhibitor L-651, 392 (both at 50 M) inhibited the response to PAF in both species. 4) The putative LTD₄ receptor antagonists (L-660, 711, 100 M) slightly reduced the PAF secretory response in human bronchi. We conclude that PAF causes specific receptor mediated RGC release. This response is indirectly mediated through the generation of lipoxygenase metabolite formation including 5-lipoxygenase pathway metabolites.Parts of this report has previously been published as abstract in: Clin. Res., 36(3), A257 (1988); J. Allergy Clin. Immunol., 83, 274 (1989); and FASEB J., 3(3), A609 (1989).     

Oxatomide inhibits the release of bronchoconstrictor arachidonic acid metabolites (iLTC4 and PGD2) from rat mast cells and guinea-pig lung

The effect of oxatomide, an orally active antiallergic drug, on immunoreactive LTC₄ (iLTC₄) production has been studied in rat peritoneal exudate cells (PEC) and guinea-pig lung fragments using the calcium ionophore A23187 and specific antigen in vitro. Oxatomide (10^–5 M) inhibited iLTC₄ release by 70% with A23187 from rat PEC, and by 48% with antigen from guinea-pig lung. Oxatomide is supposed to affect the biosynthesis pathway of leukotrienes, because oxatomide inhibits 5-lipoxygenase from guinea-pig peritoneal leukocytes with an IC₅₀ 17 M. Oxatomide also depressed the release of PGD₂ from rat peritoneal mast cells stimulated by A23187 (IC₅₀ 4.2 M). The effects of oxatomide on iLTC₄ and PGD₂ release were more potent than other antiallergic drugs (DSCG, ketotifen, tranilast).     

Effect of manoalide on human 5-lipoxygenase activity

The marine natural product manoalide (MLD) has been described to inactivate phospholipase A₂(PLA₂) from several sources as well as to inhibit synthesis of eicosanoids in human polymorphonuclear leukocytes (HPMNL). MLD also reduces chemically-induced inflammation in vivo. In this investigation we have examined the effect of MLD on A23187-induced generation of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) in HPMNL as well as 5-lipoxygenase (5-LO) activity from HPMNL sonicated preparations. In the intact cell system, MLD inhibited with similar potency biosynthesis of LTB₄ and TXB₂ (IC₅₀ 1.7 and 1.4 M, respectively). In order to discern if inhibition of 5-LO is involved in the effect of MLD, we examined the action of this compound on 5-LO activity from 10,000×g and 100,000×g supernatants of sonicated HPMNL homogenates. The enzymatic activity was not affected at concentrations of MLD up to 50 M. These data indicate that MLD is not a direct inhibitor of 5-LO activity from HPMNL and support the hypothesis that its antiinflammatory action could be related with a reduction of eicosanoid biosynthesis via inhibition of PLA₂.accepted by I. Ahnfelt-Rønne     

Role of corticosterone in modulation of eicosanoid biosynthesis and antiinflammatory activity by 5-lipoxygenase (5-LO) and cyclooxygenase (CO) inhibitors

The effectiveness of 5-lipoxygenase (LO) and dual LO/cyclooxygenase (CO) inhibitors when administered by the topical or oral routes was significantly decreased in corticosterone depleted (adrenalectomized, Adx) mice as compared to sham mice in the mouse arachidonic acid (AA) induced ear edema model. In contrast, rat carrageenan paw edema was inhibited similarly in sham and Adx animals by 5-LO and dual 5-LO/CO inhibitors. Supplementation of cortisol levels (100 g/dl) in human whole blood for 2 hr increased the observed inhibition of LTB₄ biosynthesis by A-64077, WY-50,295 tromethamine and naproxen while having no effect on thromboxane B₂ (TXB₂) biosynthesis. Thus, corticosteroids may have a permissive effect, by modulating 5-LO inhibitor, effects on mouse AA induced ear edema and human blood leukocytes.     

The effect of adherence on the generation of reactive oxygen species by human neutrophilic granulocytes

Human neutrophilic granulocytes (PMN) suspended in protein containing salt solution or adherent on protein coated nylon fibers were tested for the production of H₂O₂ and O ₂ ^– in response to various PMN stimulants. Upon stimulation with the chemotactic factors formyl-methionyl-leucyl-phenylalanine, C5a and platelet activating factor, the non-chemotactic ionophore A23187, and the chemotaxis inhibitors tumor necrosis factor (TNF) and lymphotoxin (TNF ) adherent PMN produced considerably more reactive oxygen metabolites than suspended cells. The relative amounts of the two metabolites varied with the stimulus and its concentration, TNF and TNF favoring H₂O₂ production, C5a eliciting more O ₂ ^– than H₂O₂ and the other active stimulants being in between. Leukotriene B₄ and a novel monocytederived chemotaxin were inactive in releasing either oxygen derivative from adherent or suspended PMN. The data indicate that attachment of PMN to endothelial cells or to connective tissue substances can strongly enhance its ability to respond to a given stimulus with the production of reactive oxygen metabolites. The findings may in part explain the priming phenomenon since many PMN-priming mediators increase the cells' adherence.     

Tumour necrosis factor production in a rat airpouch model of inflammation: Role of eicosanoids

TNF is a potent cytokine which can induce many of the pathological changes associated with inflammatory disease.In vitro studies have demonstrated that 5-lipoxygenase products promote the production of TNF by activated macrophages, suggesting that 5-lipoxygenase inhibitors may have therapeutic utility for the treatment of inflammatory conditions. A rat airpouch model of inflammation has been used to investigate the relationship between eicosanoid generation and TNF productionin vivo. Injection of zymosan into the airpouch caused a time-dependent stimulation of TNF production which preceded leukotriene generation by at least 30 minutes. Injection of LPS into the airpouch also stimulated TNF production but not leukotriene generation. The selective 5-lipoxygenase inhibitors, ICI207968, A64077 and BWA4C, and the 5-lipoxygenase translocation inhibitor MK886, decreased leukotriene generation but enhanced TNF production. Taken together, these results do not support a role for 5-lipoxygenase products in the regulation of TNF productionin vivo.     

Prostanoid generation in early stages of acute pancreatitis: A role for nitric oxide

The role of nitric oxide in eicosanoid and oxygen-free radical production in the early stages of sodium taurocholate-induced acute necrotizing pancreatitis has been studied. Male Wistar rats were divided into three groups: group I: control group, a volume of 0.1 ml/100 g body wt saline solution was injected at low pressure in the pancreatic duct; group II: acute pancreatitis was induced by administration of 3.5% sodium taurocholate; and group III: intravenous administration ofN ^G -nitro-l-arginine methyl esther (a nitric oxide synthase inhibitor) 5 min before induction of acute pancreatitis as stated for group II. At 5 and 60 min after induction of pancreatitis, blood and pancreas tissue samples were taken for assays. Increases in 6-keto PGF₁, TXB₂, PGE₂, PGF₂, and 12-HETE were observed in the pancreatic tissue. Lipoperoxidation was also enhanced and remained unaltered after nitric oxide inhibition. The fact that nitric oxide synthase inhibition could only reverse the increases in 6-keto PGF₁ and TXB₂ levels indicates that in acute pancreatitis endothelial and platelet eicosanoid generation is mediated through an nitric oxide-dependent mechanism. In contrast, nitric oxide appears to be not related with oxygen free radical damage associated with acute pancreatitis.     

Enzymes involved in the biosynthesis of leukotriene B4 and prostaglandin E2 are active in sebaceous glands

The expression of enzymes involved in leukotriene and prostaglandin signalling pathways, of interleukins 6 and 8 and of peroxisome proliferator-activated receptors in sebaceous glands of acne-involved facial skin was compared with those of non-involved skin of acne patients and of healthy individuals. Moreover, 5-lipoxygenase and leukotriene A₄ hydrolase were expressed at mRNA and protein levels in vivo and in SZ95 sebocytes in vitro (leukotriene A₄ hydrolase > 5-lipoxygenase), while 15-lipoxygenase-1 was only detected in cultured sebocytes. Cyclooxygenase-1 and cyclooxygenase-2 were also present. Peroxisome proliferator-activated receptors were constitutively expressed. Enhanced 5-lipoxygenase, cyclooxygenase 2 and interleukin 6 expression was detected in acne-involved facial skin. Arachidonic acid stimulated leukotriene B₄ and interleukin 6 release as well as prostaglandin E₂ biosynthesis in SZ95 sebocytes, induced abundant increase in neutral lipids and down-regulated peroxisome proliferator-activated receptor-, but not receptor-1 mRNA levels, which were the predominant peroxisome proliferator-activated receptor isotypes in SZ95 sebocytes. In conclusion, human sebocytes possess the enzyme machinery for functional leukotriene and prostaglandin pathways. A comprehensive link between inflammation and sebaceous lipid synthesis is provided.T. Alestas and R. Ganceviciene contributed equally to the study     

Activation of the human neutrophil secretory process with 5(S),12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid

Exposure of human neutrophils to 5(S),12(R) dihydroxy-6, 14-cis-8, 10-trans-eicosatetraenoic acid (leukotriene B₄, LTB₄) resulted in a time- and concentration- (10^–9-10^–6 M) dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO). Enzyme extrusion was negligible if cells were not pretreated with cytochalasin B prior to exposure to LTB₄. A time-dependent deactivation of granule exocytosis was observed in neutrophils which were stimulated with LTB₄ prior to contact with cytochalasin B. LTB₄-induced enzyme release was markedly enhanced in the presence of extracellular calcium. Nevertheless, significant enzyme discharge occurred in the absence of extracellular calcium, and the percent of total activity released was not altered in the presence of EGTA. The calmodulin antagonist, trifluoperazine (TFP), and the intracellular calcium antagonist, 8-(N, N-dietliylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride (TMB-8), caused a dose-related inhibition of enzyme release from LTB₄-stimulated neutrophils. Degranulation was suppressed by the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), and the sulfhydryl reagents iodoacetic acid (IA) andN-ethylmaleimide (NEM). Sodium cyanide was inactive. Two inhibitors of transmethylation, 3-deazaadenosine (3-DZA) and L-homocysteine thiolactone (HCTL), alone or in combination, had no effect on LTB₄-elicited degranulation. The protein synthesis inhibitor, cycloheximide, was inactive, Neutrophils pretreated with LTR₄ or 5(S), 12(R), 20-trihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid (20-OH-LTB₄, an -oxidation metabolite of LTB₄) were desensitized to the subsequent exposure to LTB₄. Cross-desensitization was also demonstrated between LTB₄ and 20-OH-LTB₄. The stimulus specific nature of LTB₄-induced desensitization of neutrophil degranulation was demonstrated by the fact that cells exposed to 1-O-hexadecyl/octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) orN-formyl-methionyl-leucyl-phenylalanine (FMLP) were capable of inducing granule exocytosis from LTB₄-pretreated neutrophils. Enzyme release from LTB₄-treated cells was suppressed with the phospholipase inhibitor, 4-bromophenacyl bromide (4-BPB), the cyclooxygenase/lipoxygenase inhibitor, ETYA, and the 5-lipoxygenase inhibitor, U-60, 257. However, the cyclooxygenase inhibitor, flurbiprofen, exerted a weak suppressive effect on LTB₄-induced degranulation.     

Antiarthritic profile of BF-389 — A novel anti-inflammatory agent with low ulcerogenic liability

BF-389, dihydro-4-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2methyl-2H-1,2-oxazin-3(4H)-one, is a potent, orally active, antiarthritic and analesis agent with low ulcerogenic potential. A comparison of the activity profiles of BF-389 and naproxen showed similarities in: (1) suppression of developing and chronic adjuvant arthritis (AA); (2) maximal inhibitory response, as shown by theE(max0 values in the developing and established AA models; (3) inhibition of bone degenerative changes associated with chronic adjuvant arthritis; and (4) analgesic activity in the acetic acid and phenylquinone writhing assays. Though BF-389 has been shown to be a potent inhibitor of cyclooxygenase, IC₅₀=0.84±0.25 M against the production of PGE₂ in vitro, there is a great difference from most cyclooxygenase inhibitors; it also inhibits the 5-lipoxygenase enzyme. For BF-389, the IC₅₀ for invitro LTB₄ formation was found to be 3.65±1.19 M. The ulcerogenic potential of BF-389 was compared to that of naproxen using a five-dayin vivo ulcerogenic rat assay. The UD₅₀ for naproxen was found to be approximately 30 mg/kg/day, p.o. Based upon efficacy in the DEV AA and EST AA models, UD₅₀/ED₅₀ values for naproxen were estimated to be 0.7 and 1.9, respectively. For BF-389 the UD₅₀ was shown to be 520 (389–695) mg/kg/day, p.o., and the corresponding UD₅₀/ED₅₀ values were calculated to be 84 and 28, respectively, thus demonstrating the wide margin of safety between efficacy and ulcerogenicity in rats.     

Methoxyalkyl thiazoles: A novel series of potent,orally active and enantioselective inhibitors of 5-lipoxygenase

Methoxyalkyl thiazoles are novel 5-lipoxygenase inhibitors which are neither redox agents nor iron chelators and are exemplified by ICI211965 [1-(3-naphth-2-ylmethoxy)phenyl)-1-(thiazol-2-yl)propyl methyl ether]. ICI211965 potently inhibits LTC₄ synthesis in murine macrophages (IC₅₀=0.0085 M) and its selectivity with respect to cyclo-oxygenase (>5800) is greater than any previously reported lipoxygenase inhibitor. ICI211965 also selectively inhibits LTB₄ synthesis by human bloodin vitro (IC₅₀=0.45 M) and rat bloodex vivo (ED₅₀=10 mg/Kg, p.o.). Methoxyalkyl thiazoles exhibit a tight structure activity relationship and resolution of a chiral member of the series demonstrates that 5-lipoxygenase inhibition resides largely in one enantiomer. Methoxyalkyl thiazoles represent the first class of agents for which 5-lipoxygenase inhibition is mediated by specific, enantioselective interaction with the enzyme.