The dependence of human T-lymphocyte migration on the 5-lipoxygenation of endogenous arachidonic acid

Human T lymphocytes in modified Boyden migration chambers exhibited a chemokinetic response, but no detectable chemotaxis, to 5(S),12(R)-dihydroxyeicosa-6, 14-cis-8,10-trans-tetraenoic acid (leukotriene B₄) and to 5(S)-hydroxyeicosatetraenoic acid (5-HETE), which are 5-lipoxygenase products of arachidonic acid, and failed to respond to either leukotriene C₄ or 11-HETE. Concentrations of lymphocyte-derived 15-HETE and of 15-hydroperoxyeicosatetraenoic acid that inhibited the 5-lipoxygenation of endogenous arachidonic acid suppressed significantly both random migration and the chemokinetic responses of T lymphocytes to concanavalin A and -thioglycerol. That the suppressive effect of 15-HETE on T lymphocyte chemokinesis was attributable in part to the inhibition of 5-lipoxygenase was further suggested by the ability of exogenous 5-hydroperoxyeicosatetraenoic acid to restore chemokinetic responsiveness to lymphocytes that had been preincubated with 15-HETE.     

Antiinflammatory effects of second-generation leukotriene B4 receptor antagonist,SC-53228: Impact upon Leukotriene B4- and 12(R)-HETE- mediated events

Leukotriene B₄ (LTB₄) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE] are proinflammatory products of arachidonic acid metabolism that have been implicated as mediators in a number of inflammatory diseases. When injected intradermally into the guinea pig, LTB₄ and 12(R)-HETE elicit a dose-dependent migration (chemotaxis) of neutrophils (PMNs) into the injection sites as assessed by the presence of a neutrophil marker enzyme myeloperoxidase. SC-41930 {7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxyl]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid}, a first-generation LTB₄ receptor antagonist, inhibited the chemotactic actions of LTB₄ when given orally with an ED₅₀ value of 1.7 mg/kg. The second-generation LTB₄ receptor antagonist, SC-53228 [(+)-(S)-7-(3-{2-(cyclopropylmethyl)-3-methoxy-4-[(methylamino)carbonyl]phenoxy} propoxy)-3,4-dihydro-8-propyl-2H-1-benzopyran-2-propanoic acid], inhibited LTB₄-induced chemotaxis when given intragastrically with an ED₅₀ value of 0.07 mg/kg. Furthermore, SC-53228 inhibited 12(R)-HETE-induced granulocyte chemotaxis with an oral ED₅₀ value of 5.8 mg/kg. When dosed orally over a range of 0.03–100 mg/kg, SC-53228 gaveC _max plasma concentrations of 0.015–41.1g/ml. SC-53228 inhibited LTB₄-primed membrane depolarization of human neutrophils with an IC₅₀ value of 34 nM. As a potent LTB₄ receptor antagonist, SC-53228 may well have application in the medical management of disease states such as asthma, rheumatoid arthritis, inflammatory bowel disease, contact dermatitis, and psoriasis, in which LTB₄ and/or 12(R)-HETE are implicated as inflammatory mediators.     

Differential effects of monohetes (monohydroxyeicosatetraenoic acids) on arachidonic acid metabolism in glycogen-elicited rat polymorphonuclear leukocytes

We investigated the effects of monohydroxyeicosatetraenoic acids (monoHETEs) on lipoxygenase- and cyclooxygenase-catalyzed reactions in glycogen-elicited rat PMNs challenged with A23187 and exogenous [¹⁴C]arachidonic acid. A23187 (10M) stimulated a 10-, 4-, 1.7- and 1.8-fold increase in the synthesis of radiolabeled 5-HETE, LTB₄, TxB₂, and PGE₂ by rat PMNs. Addition of 5-HETE, 5 lactone-HETE, 12-HETE, and 15-HETE led to a dose-related reduction in [¹⁴C]5-HETE and [¹⁴C]LTB₄ synthesis by these cells. These monoHETEs also inhibited [¹⁴C]TxB₂ synthesis, but only 5-HETE and 5 lactone-HETE inhibited the synthesis of [¹⁴C]PGE₂. Both 12-HETE and 15-HETE failed to reduce the formation of [¹⁴C]PGE₂. These results suggest that monoHETEs differ significantly in their effects on arachidonic acid metabolism in rat PMNs and may play a role in modulating the synthesis of both lipoxygenase and cyclooxygenase products.     

Species differences in the pattern of eicosanoids produced by inflamed and non-inflamed tissue

The synthesis of¹⁴C labelled arachidonic acid metabolites was measured in colonic tissues obtained from mice, rats, guinea pigs, rabbits, piglets and in colonic biopsies from humans during colonoscopy. The main eicosanoids formed after stimulation with calcium ionophore A23187 were: in humans, 15-hydroxy-eicosatetraenoic acid (15-HETE); in mice, 12-HETE; in rats, 12-HETE, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 6-keto-prostaglandine F₁ (6kPGF₁); in guinea pigs, PGD₂; in rabbits, 6kPGF₁, PGE₂ and 15-HETE; and in pigs PGE₂ and 12-HETE. In inflamed 15-HETE production was increased in man, HHT and 12-HETE production in rats and overall eicosanoid production in mice.     

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.     

The effect of RG 12525 on leukotriene D4-mediated pulmonary responses in guinea pigs

RG 12525 (5-(2-[4-quinolin-2-yl)methoxy] phenoxymethyl)benzyl tetrazole) is under investigation as a specific inhibitor of leukotriene D₄ (LTD₄). The present studies examine the effect of orally administered RG 12525 on LTD₄ mediated pulmonary responses in three separate guinea pig models. The compound inhibited antigen-induced mortality in the systemic anaphylaxis model with an ED₅₀ (95% confidence interval)=2.2 (0.8–6.4) mg/kg. In this model, the activity half-life of RG 12525 was shown to be 6.5 hours and the compound offered significant protection within 15 minutes of administration. RG 12525 also protected against LTD₄-induced bronchoconstriction in a model measuring changes in pulmonary function with an ED₅₀=0.6 (0.4–1.0) mg/kg. The same level of activity was observed in a similar model which monitored changes in pulmonary function in response to exogenous antigen in actively-sensitized guinea pigs. Together, these data indicate that RG 12525 is a potent, orally active LTD₄ antagonist which possesses the requisite profile for potential clinical development.     

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.     

Pharmacological characterization of WAY-121,520: A potent anti-inflammatory indomethacin-based inhibitor of 5-lipoxygenase (5-LO)/phospholipase A2 (PLA2)

WAY-121,520 inhibited human synovial fluid PLA₂ (HSF-PLA₂) (IC₅₀=4 M) using arachidonic acid-labeledE. coli as substrate. Further biochemical characterization of WAY-121,520 demonstrated potent inhibition of 5-lipoxygenase (5-LO) activity in the murine macrophage (LTC₄, IC₅₀=4nM) and rat PMN (LTB₄, IC₅₀=10 nM) and an ability to antagonize LTD₄ binding to isolated guinea-pig trachea (pK _B=6.0).In vivo anti-inflammatory activity was noted in murine TPA-induced (ED₅₀=91 g/ear) and arachidonic acid-induced (66% inhibition at 400 g/ear) ear edema and in leukotriene-dependent antigen-induced bronchoconstriction in the guinea pig (73% inhibition at 50 mg/kg, p.o.). WAY-121,520 represents a novel series of indomethacin-based inhibitors of PLA₂ with anti-inflammatory activity resulting from a combination of biochemical activities (inhibition of 5-LO and PLA₂ and LTD₄ antagonism). This agent may provide added therapeutic efficacy over more selective inhibitors.     

Pharmacological profile of a novel,orally active leukotriene B4 antagonist,SM-15178

SM-15178, a new hydroxyacetophenone derivative, was evaluated to determine its antiinflammatory activity and antagonistic activity against leukotriene B₄ (LTB₄). SM-15178 inhibited [³H]LTB₄ binding to its receptors on human neutrophils (IC₅₀=0.30M). It inhibited LTB₄-induced chemotaxis of human neutrophils (IC₅₀ =0.72M) with little inhibitory effect against C5a or FMLP-induced chemotaxis at concentrations up to 30M. The compound alone did not cause human neutrophil chemotaxis at concentrations up to 10M. LTB₄-induced chemotaxis of mouse and rat neutrophils and guinea pig eosinophils was also inhibited by the compound, with IC₅₀ values of 0.55, 0.52, and 0.58 M, respectively. In an in vivo study, SM-15178, given orally, significantly prevented LTB₄-induced transient leukopenia. It also suppressed LTB₄-induced bronchoconstriction in the guinea pig almost completely when given orally at a dose of 40 mg/kg. Furthermore, orally given SM-15178 suppressed arachidonic acid-induced neutrophil infiltration in mouse ears and Arthus reaction-induced paw edema in the mouse in a dose-dependent manner. These results suggest that SM-15178 is a selective and orally active LTB₄ antagonist and that it might be effective for the treatment of some types of inflammatory diseases.     

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.     

Thein vivo andin vitro activity of AHR-13268D,a new antiallergic/antihistaminic agent

AHR-13268D (4-[3-[4-[Bis(4-flurophenyl)hydroxymethyl]-1-piperidinyl]propoxy]benzoic acid, sodium salt) is a potent, long-acting water soluble, antiallergic and antihistaminic agent. AHR-13268D protects sensitive guinea pigs from collapse induced by aerosolized antigen; 1, 5, and 24 h ED₅₀s in the test were 0.27, 0.25, 0.93 mg/kg, PO, respectively. AHR-13268D was also active when given as an aerosol, the 1 h ED₅₀=0.29%. In the rat passivefoot anaphylaxis test, AHR-13268D was slightly more active (1.55 times) than AHR-5333B when given orally 1 h prior to challenge and equipotent to cromolyn when given intravenously immediately prior to challenge. AHR-13268D displayed potent, long-acting antihistaminic activity in naive guinea pigs; the 1, 5, and 24 h oral ED₅₀s being in the range of 0.3 mg/kg. AHR-13268D (10 to 20 mg/kg, PO) attenuated the skin responses to ascaris antigen in sensitive dogs and did not alter the EEG pattern or sleep/wake patterns of cats at doses in vast excess of its antihistaminic activity.In vitro, AHR-13268D was a potent inhibitor of histamine release from rat peritoneal mast cells (IC₅₀=0.51 nM) and was as potent as the reference 5-LO inhibitor phenidone in inhibiting antigen-induced contractions of guinea pig ileum in the presence of pyrilamine, atropine, and imidazole (IC₅₀300 M). AHR-13268B was bioavailable (88%) from capsules or from oral solutions.     

Pharmacological characterization of WAY-121,520: A potent anti-inflammatory indomethacin-based inhibitor of 5-lipoxygenase (5-LO)/phospholipase A2 (PLA2)

WAY-121,520 inhibited human synovial fluid PLA₂ (HSF-PLA₂) (IC₅₀=4 μM) using arachidonic acid-labeledE. coli as substrate. Further biochemical characterization of WAY-121,520 demonstrated potent inhibition of 5-lipoxygenase (5-LO) activity in the murine macrophage (LTC₄, IC₅₀=4nM) and rat PMN (LTB₄, IC₅₀=10 nM) and an ability to antagonize LTD₄ binding to isolated guinea-pig trachea (pK_B=6.0).In vivo anti-inflammatory activity was noted in murine TPA-induced (ED₅₀=91 μg/ear) and arachidonic acid-induced (66% inhibition at 400 μg/ear) ear edema and in leukotriene-dependent antigen-induced bronchoconstriction in the guinea pig (73% inhibition at 50 mg/kg, p.o.). WAY-121,520 represents a novel series of indomethacin-based inhibitors of PLA₂ with anti-inflammatory activity resulting from a combination of biochemical activities (inhibition of 5-LO and PLA₂ and LTD₄ antagonism). This agent may provide added therapeutic efficacy over more selective inhibitors.     

Endogenous LXA4 Circuits Are Determinants of Pathological Angiogenesis in Response to Chronic Injury

Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A₄ receptors (ALX) constitute a LXA₄ circuit that is a key feature of inflammatory resolution. LXA₄ analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA₄ circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA₄, respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA₄, but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA₄ circuit as an endogenous regulator of pathological angiogenesis.Formation of a new functional microvasculature, neovascularization, is a fundamental response to ischemia and a salient feature of wound healing. The primary function of newly formed blood vessels is to increase tissue oxygen tension and delivery of essential nutrients and effector cells to restore normal function. However, aberrant neovascularization is a hallmark feature of chronic inflammation and is associated with numerous pathological conditions that include diabetic retinopathy, Crohn''s Disease, atherosclerosis, and cancer.^1–3The growth of microvessels from existing vessels, angiogenesis, is tightly controlled by a range of angiogenic factors and inhibitors; a circuit that is highly evolved in avascular tissues such as the cornea. The vascular endothelial growth factor (VEGF) family of angiogenic factors and their receptors are key mediators of this process, which has led to the recent development and clinical use of anti-VEGF strategies for the treatment of pathological neovascularization in the retina, colon cancer, and lung cancer.^2–5 Many insights into the endogenous role of the VEGF network have been gained by using the cornea,^5,6 which maintains an immune-privileged and avascular state despite expression of VEGF-A and its immediate proximity to the vasculature.⁷ Specifically, recent findings have demonstrated that avascularity of the cornea requires expression of a soluble VEGF receptor-1 (sFLT1), which traps VEGF-A.⁸ In addition, the receptor for VEGF-C/VEGF-D, namely VEGF receptor-3 (VEGFR-3, FLT4), is a critical regulator of inflammatory neovascularization.^9–11 FLT4 is of special of interest because its essential expression during development becomes restricted primarily to lymph vessels and activation of this endothelial receptor is a critical step in initiating lymphangiogenesis. However, FLT4 expression is also up-regulated in microvessels of tumors and wounds, and in macrophages and in addition is constitutively expressed in corneal epithelial cells.^3,10–13 A recent report demonstrates that FLT4 is highly expressed in angiogenic sprouts and is a critical regulator of sustained heme-angiogenesis,¹² which underscores the potential key role of this receptor in pathological neovascularization.Inflammation is intimately associated with neovascularization especially during wound healing and ischemic injury. Lipid autacoids are some of the earliest signals that are released in response to injury or insult. In this regard, the 15-lipoxygenase pathway^14,15 is of interest as it is one of the most inducible genes in macrophages and highly expressed in mucosal and corneal epithelial cells. Macrophages and epithelial cells are important regulators of angiogenesis, especially in avascular tissue such as the cornea.^3,10,16 Macrophages have a central and well-documented role in angiogenesis, especially in tumors and inflammatory neovascularization. In the cornea, a well-established model tissue for studying inflammatory neovascularization, VEGF-A recruits macrophages, the major cell type to generate VEGF, which drives inflammatory heme and lymphangiogenesis. Corneal epithelial cells constitutively express the receptor for VEGF-C/VEGF-D (ie, FLT4), which has been proposed as a critical pathway for regulating inflammatory neovascularization.¹⁰Human 15-LOX (ALOX15 and ALOX15B) generate 15S-hydroxyeicosatetraenoic acid (HETE) and mouse 12/15-LOX (Alox15) generates 15S-HETE and 12S-HETE from arachidonic acid. 15-HETE and 12-HETE have been shown to induce proliferation, migration, and tube formation in endothelial cells.¹⁷ More importantly, 15-HETE is a key intermediate in the formation of the well-studied anti-inflammatory mediator lipoxin A₄ (LXA₄) that is generated via the rate-limiting enzyme 5-lipoxygenase (5-LOX). A body of work^18–24 has established that the anti-inflammatory actions, which are associated with the up-regulation of 15-LOX and/or 15S-HETE formation are mediated by LXA₄ and its G-protein coupled receptor ALX. Recent reports have demonstrated that stable analogs of LXA₄ inhibit VEGF induced angiogenic responses in endothelial cells.^25–27 These metabolically stable analogs are mimetics of aspirin-triggered LXA₄, an endogenous isomer whose synthesis can be triggered by aspirin-acetylated cyclooxygenase-2 rather than 15-LOX. This 15-epi-isomer of LXA₄ resists metabolic inactivation and mediates it bioactions, like LXA₄, via the ALX receptor. The intimate link between inflammation and angiogenesis and the ability of LXA₄²⁸ and analogs of 15-epi LXA₄^25–27 to inhibit VEGF-A induced angiogenic responses in vitro points toward a potential role of endogenous LXA₄ circuits in pathological angiogenesis. However, the endogenous role of 15-LOX in the regulation of angiogenesis remains controversial. Reports have demonstrated that the enzyme or its products promote or inhibit angiogenic responses in in vitro studies.^29–32 More importantly, the in vivo role of the 15-LOX pathway or the LXA₄ circuit in pathological neovascularization remains to be clearly defined. To this end, we assessed the role the 15-LOX pathway and LXA₄ circuit in chronic injury-induced inflammatory neovascularization.Here, we report that inflammatory neovascularization and up-regulation of the VEGF circuit correlate with changes in both 15-LOX (Alox15) and LXA₄ receptor (ALX) expression and temporally defined 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key enzymes in the formation of LXA₄, led to amplified neovascularization and expression of VEGF-A and FLT4 in the avascular cornea during chronic injury. LXA₄, but not 15S-HETE, attenuated expression of VEGF-A and FLT4 and the angiogenic response, which provides evidence that selective autacoids from the prominent 15-LOX pathway have an endogenous role in limiting pathological neovascularization.     

Effect of CI-922, a potential new antiallergy agent,on arachidonic acid metabolism in vitro

CI-922 (3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo[3,2-b]indole-2-carboxamide, 1,2-ethanediamine, 21), an antiallergy compound, was tested for its effects on arachidonic acid metabolism, and its potency was compared to that of proxicromil, BW-755C, indomethacin, and nordihydroguaiaretic acid (NDGA). CI-922 was both a relatively potent and selective inhibitor of 5-HETE and LTB₄ formation in human leukocytes, being equipotent to BW-755C and about fourfold more potent than proxicromil. However, CI-922 was rather weak in inhibiting the formation of PGE₂ in bovine seminal vesicles. The parallel inhibition of 5-HETE and LTB₄ formation by CI-922 suggests that either direct or indirect inhibition of the 5-lipoxygenase pathway may explain, at least in part, its antiallergy properties.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

FPL 62064, a topically active 5-lipoxygenase/cyclooxygenase inhibitor

FLP 62064 [N-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-3-amine] is a dual inhibitor of prostaglandin synthetase and 5-lipoxygenase. The compound had anti-inflammatory activityin vivo in a number of models. It inhibited peritoneal inflammation induced by immune-complex when given locally. When applied to the skin, FPL 62064 inhibited UV irradiation-induced erythema and PGE₂ formation in the guinea pig and also oedema formation and eicosanoid production in the mouse ear produced by arachidonic acid. Co-injected with arachidonic acid in rabbit skin, FPL 62064 inhibited oedema and eicosanoid formation.     

Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophilsin vitro. Evidence of formation of antiinflammatory compounds

Enzymatic transformation of then-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) by the 5-lipoxygenase (LO) enzyme results in the formation of leukotrienes (LTs) including leukotriene B₄ (LTB₄), which is a potent mediator of inflammation. The purpose of the present study was to determine the effect of othern-6 fatty acids on the formation of LTB₄ by human neutrophils and to determine if thesen-6 fatty acids themselves may be transformed into products with antiinflammatory capacity. Purified neutrophils isolated from heparinized human venous blood were incubated with A23187 (5 M) and different concentrations (0–100 M) of then-6 fatty acids linoleic acid (LA) and dihomo-gammalinolenic acid (DGLA). LO products were determined by use of quantitative reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry. The formation of LTB₄ was dose dependently inhibited by both LA (IC₅₀=45 M) and DGLA (IC₅₀=40M). This inhibition of LTB₄ formation was associated with a dose dependent increase in the formation of the respective 15-LO products of LA (13-hydroxy-octadecadienoic acid; 13-HODE) and DGLA (15-hydroxy-eicosatrienoic acid; 15-HETrE). To determine whether these 15-LO products themselves might inhibit LTB₄ formation, neutrophils were incubated with 13-HODE and 15-HETrE. Both 15-LO products lead to a dose-dependent inhibition of LTB₄ formation (IC₅₀=7.5 M and IC₅₀=0.2 M). For comparison the 15-LO product of AA, 15-hydroxy-eicosatetraenoic acid (15-HETE), also inhibited LTB₄ formation (IC₅₀=0.75 M). The results show that the addition of LA and DGLA to neutrophils results in an inhibition of LTB₄ formation and simultaneously to the formation of 13-HODE and 15-HETrE, that also inhibits LTB₄ formation. Therefore, dietary supplementation or topical application of LA and DGLA or preferentially their respective 15-LO products, may have a therapeutic effect in inflammatroy diseases in which LTs are suspected to play a pathogenic role.     

Effect of a 5-lipoxygenase (5-LO)/cyclooxygenase (CO) inhibitor,WY-47, 288, on cutaneous models of inflammation

WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline) demonstrated topical antiinflammatory activity in several animal models of skin inflammation. Application of WY-47,288 to mouse ear surfaces inhibited arachidonic acid (ED₅₀=0.3 mg/ear) and tetradecanoylphorbol acetate (TPA)-induced inflammation (40% at 1 mg/ear). Administration of WY-47,288 (1 mg/ear) at 30 min and 5 h after TPA reduced ear edema and epidermal proliferation by 50%. WY-47,288 also inhibited oxazolone-induced contact hypersensitivity in mouse ears (ED₅₀=0.4 mg/ear) and UVB-induced guinea pig skin erythema (ED₅₀0.25 mg/spot). These antiinflammatory effects may be due to inhibition of 5-lipoxygenase (5-LO) and cyclooxygenase (CO) since the synthesis of 5-LO and CO products by rat neutrophils and mouse macrophages was dose-dependently reduced by WY-47,288. By contrast, WY-47,288 demonstrated no appreciable inhibition of 12-LO (rabbit platelet), 15-LO (soybean) or phospholipase A₂ (human platelet). Furthermore, no systemic adverse effects were observed after topical, parenteral or oral administration of WY-47,288, suggesting that WY-47,288 is a safe topical 5-LO/CO inhibitor for treating skin inflammation.Portions of this work were presented at the 1987 ASPET annual meeting in Honolulu, Hawaii.     

Biochemical and pharmacological properties of a new topical anti-inflammatory compound, 9-phenylnonanohydroxamic acid (BMY 30094)

Drugs which block the biosynthesis of leukotrienes and prostaglandins may have potential in the treatment of psoriasis and other skin diseases. The biochemical and anti-inflammatory activity of 9-phenylnonanohydroxamic acid (BMY 30094) is described. BMY 30094 inhibited human neutrophil 5-lipoxygenase with an IC₅₀ of 5.7 M. BMY 30094 also blocked human platelet cyclooxygenase and lipoxygenase with IC₅₀ values of 15.2 and 15.0 M, respectively. Topical application of this compound blocked arachidonic acid and 12-O-tetradecanoylphorbol ester-induced mouse skin inflammation with activity comparable to that observed for lonapalene. The topical ED₅₀ for BMY 30094 in the arachidonic acid-induced inflammation model is 2.2 moles/ear. In the sub-cutaneous carrageenan sponge assay in rats, BMY 30094 blocked LTB₄ and PGE₂ production and inhibited neutrophil migration. This compound would be a useful tool to determine the role of arachidonic acid metabolites in the etiology of inflammatory dermatoses.     

Eicosanoids and histamine mediate C5a-induced electrolyte secretion in guinea pig ileal mucosa

C5a is a biologically active polypeptide formed during the course of complement activation and is known to possess histamine-releasing and neutrophil chemotactic properties. In the present study, we have demonstrated that C5a can regulate electrolyte transport across guinea pig ileum, and we have investigated its mechanism of action. Segments of ileum stripped of longitudinal muscle were mounted in Ussing chambers (Krebs' buffer, 37°C, 95% O₂/5% CO₂) for monitoring short-circuit current (Isc). Serosal application of C5a evoked a transient increase in Isc with an EC₅₀ value of 5.0 nM indicating a potent effect. The C5a-induced increase in Isc was abolished by elimination of both Cl^– and HCO ₃ ^– from the Krebs' solution. Pretreatment with the cyclooxygenase inhibitor indomethacin (5M), the neurotoxin tetrodotoxin (0.5M) and the H₁ receptor antagonist pyrilamine (0.5M) reduced the effect of C5a, but the muscarinic antagonist atropine (0.5M) was without effect. C5a (100 nM) also evoked the release of histamine (measured by radioimmunoassay in the serosal bathing fluid) by 282% of the control value. In conclusion, in the guinea pig ileum C5a stimulates mucosal anion secretion by releasing histamine and cyclooxygenase products of arachidonic acid. The response is also mediated, in part, via non-chloinergic enteric nerves.