Hyperforin is a dual inhibitor of cyclooxygenase-1 and 5-lipoxygenase

The acylphloroglucinol derivative hyperforin is the major lipophilic constituent in the herb Hypericum perforatum (St. John's wort). The aim of the present study was to investigate if hyperforin as well as extracts of H. perforatum can suppresses the activities of 5-lipoxygenase (5-LO) and cyclooxygenases (COX), key enzymes in the formation of proinflammatory eicosanoids from arachidonic acid (AA). In freshly isolated human polymorphonuclear leukocytes stimulated with Ca(2+) ionophore A23187, hyperforin inhibited 5-LO product formation with IC(50) values of about 1-2 microM, in the absence or presence of exogenous AA (20 microM), respectively, being almost equipotent to the well-documented 5-LO inhibitor zileuton (IC(50) = 0.5-1 microM). Experiments with purified human 5-LO demonstrate that hyperforin is a direct 5-LO inhibitor (IC(50) approximately 90 nM), acting in an uncompetitive fashion. In thrombin- or ionophore-stimulated human platelets, hyperforin suppressed COX-1 product (12(S)-hydroxyheptadecatrienoic acid) formation with an IC(50) of 0.3 and 3 microM, respectively, being about 3- to 18-fold more potent than aspirin. At similar concentrations, hyperforin suppressed COX-1 activity in platelets in presence of exogenous AA (20 microM) as well as in cell-free systems. Hyperforin could not interfere with COX-2 product formation and did not significantly inhibit 12- or 15-LO in platelets or leukocytes, respectively. We conclude that hyperforin acts as a dual inhibitor of 5-LO and COX-1 in intact cells as well as on the catalytic activity of the crude enzymes, suggesting therapeutic potential in inflammatory and allergic diseases connected to eicosanoids.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.     

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 activity in 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.     

Licofelone,a dual lipoxygenase-cyclooxygenase inhibitor,downregulates polymorphonuclear leukocyte and platelet function

Polymorphonuclear leukocytes are strongly implicated in the pathogenesis of inflammatory disease. Polymorphonuclear leukocyte recruitment at sites of inflammation, mainly sustained by the beta2-integrins, is followed by the synthesis and release of inflammatory mediators, such as leukotrienes, proteolytic enzymes and reactive oxygen species. Functional and metabolic interactions between polymorphonuclear leukocytes and platelets can contribute to and exacerbate the process. The effects of the dual 5-lipoxygenase and cyclooxygenase inhibitor licofelone ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid) were studied on arachidonic acid transcellular metabolism occurring between polymorphonuclear leukocytes and platelets. The formation of leukotriene C(4), a leukotriene A(4)-derived metabolite, by mixed polymorphonuclear leukocyte/platelet suspensions stimulated with 10 microM A23187 was inhibited by licofelone with an IC(50) of 3.8 +/- 0.07 microM. The formation of 5,12-di-hydroxy-eicosatetraenoic acid (HETE) was abolished at concentrations > or = 10 microM. Licofelone also inhibited the generation of reactive oxygen species by polymorphonuclear leukocytes stimulated with 1 microM n-formyl-methionyl-leucyl-phenylalanine (fMLP), 10 nM complement fraction 5a (C5a) and 1 microM platelet activating factor (PAF) with IC(50)s of 24.4 +/- 0.6, 11.0 +/- 1.5 and 11.7 +/-1.2 microM; elastase release induced by the three agonists was inhibited with IC(50)s of 12.2 +/- 2.2, 23.5 +/- 8 and 2.6 +/- 1 microM, respectively. Homotypic polymorphonuclear leukocyte aggregation induced by fMLP, C5A and PAF was inhibited by licofelone with IC(50)s of 23.7 +/- 4.8, 15.6 +/- 3.4 and 15.4 +/- 4 microM, respectively. The present study extends the anti-lipoxygenase and anti-cyclooxygenase activities of licofelone to the production of arachidonic acid metabolites generated as a consequence of polymorphonuclear leukocyte-platelet transcellular metabolism and to polymorphonuclear leukocyte responses relevant to the pathogenesis of inflammation. The coexistence within the same molecule of a wide spectrum of anti-inflammatory properties is of interest.     

Inhibitors of cyclooxygenase-2 and 5-lipoxygenase

This patent describes a series of cyclooxygenase-2 (COX-2) inhibitors that are also potent 5-lipoxygenase (5-LO) inhibitors. The molecules are hybrid structures containing fragments of known COX-2 and 5-LO inhibitors. One compound is reported to have in vivo activity in the rat paw oedema model.     

Flavocoxid, a dual inhibitor of cyclooxygenase-2 and 5-lipoxygenase, reduces pancreatic damage in an experimental model of acute pancreatitis

BACKGROUND AND PURPOSE

Acute pancreatitis is an autodigestive process resulting in acute inflammation of the pancreas. Accumulating evidence indicates the essential contribution of cyclooxygenase (COX)-2 and 5-lipoxygenase (5-LOX) to acute pancreatitis. We studied the effects of flavocoxid, a plant-derived dual inhibitor of COX-2 and 5-LOX, in a model of caerulein (CER)-induced acute pancreatitis.

EXPERIMENTAL APPROACH

Rats were given CER (80 µg·kg⁻¹ for each of four injections at hourly intervals) or vehicle (Sham-CER). Animals were then randomized to receive flavocoxid (20 mg·kg⁻¹ i.p.) or vehicle, 30 min after the first CER injection. Two hours after the last CER injection, we evaluated damage to the pancreas by histological methods; serum levels of amylase, lipase_, leukotriene (LT)B₄ and prostaglandin (PG)E₂; pancreatic expression of COX-2 and 5-LOX and tumour necrosis factor-α (TNF-α) gene expression by real-time polymerase chain reaction.

KEY RESULTS

Caerulein induced inflammatory changes in the pancreas and raised values of the other variables measured. In CER-treated animals, but not in those given saline, flavocoxid inhibited COX-2 and 5-LOX expression, reduced serum levels of lipase and amylase and the degree of pancreatic oedema. Treatment with flavocoxid blunted the increased pancreatic TNF-α mRNA expression, serum leukotriene B₄ and prostaglandin E₂ levels, and protected against histological damage in terms of vacuolization and leukocyte infiltration.

CONCLUSIONS AND IMPLICATIONS

Our results confirm the key role of both COX-2 and 5-LOX in the inflammatory response to acute pancreatitis. Flavocoxid may provide a potential therapeutic approach to the treatment of patients at high risk of developing this life-threatening condition.     

Styrylpyrazoles, styrylisoxazoles, and styrylisothiazoles. Novel 5-lipoxygenase and cyclooxygenase inhibitors.

A series of styrylpyrazoles, styrylisoxazoles, and styrylisothiazoles were prepared and found to be dual inhibitors of 5-lipoxygenase and cyclooxygenase in rat basophilic leukemia cells. Compounds from this series also were found to inhibit the in vivo production of LTB4 when dosed orally in rats. Among these compounds, di-tert-butylphenols 19 and 33 exhibit oral activity in various models of inflammation and, most importantly, are devoid of ulcerogenic potential.     

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 (ED50 approximately 0.25 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.     

Minocycline inhibits 5-lipoxygenase activation and brain inflammation after focal cerebral ischemia in rats

Aim： To determine whether the anti-inflammatory effect of minocycline on postischemic brain injury is mediated by the inhibition of 5-1ipoxygenase （5-LOX） expression and enzymatic activation in rats. Methods： Focal cerebral ischemia was induced for 30 min with middle cerebral artery occlusion, followed by reperfusion. The ischemic injuries, endogenous IgG exudation, the accumulation of neutrophils and macrophage/microglia, and 5-LOX mRNA expression were determined 72 h after reperfusion. 5-LOX metabolites （leukotriene B4 and cysteinyl leukotrienes） were measured 3 h after reperfusion. Results： Minocycline （22.5 and 45 mg/kg, ip, for 3 d） attenuated ischemic injuries, IgG exudation, and the accumulation of neutrophils and macrophage/microglia 72 h after reperfusion. It also inhibited 5-LOX expression 72 h after reperfusion and the production of leukotrienes 3 h after reperfusion. Conclusion： Minocycline inhibited postischemic brain inflammation, which might be partly mediated by the inhibition of 5-LOX expression and enzymatic activation.     

Minocycline protects PC12 cells from NMDA- induced excitotoxicity and inhibits 5 -lipoxygenase activation

Minocycline, a semi - synthetic tetracycline, possesses anti - inflammatory effects in a number of models of CNS diseases. We have reported that minocycline protects PC12 cells （a neuron -like cell line） against ischemic -like injury and inhibits in vitro ischemia- induced 5 -lipoxygenase （5 -LOX） activation. Since excitotoxicity is involved in ischemic injury and many other pathophysiological conditions, the purpose of this study was to determine whether minocycline has a neuroprotective effect on N - methyl - D - aspartate （NMDA） excitotoxicity in PC12 cells, if so,     

Diphenylene iodonium, an inhibitor of free radical formation, inhibits platelet aggregation.

Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25-2 microM) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents.     

A prenylflavone, artonin E, as arachidonate 5-lipoxygenase inhibitor

Several naturally occurring prenylflavones were tested for their inhibitory actions on arachidonate 5-lipoxygenase purified from porcine leukocytes. Of the compounds tested, artonin E (5'-hydroxymorusin) exhibited the most potent inhibition on arachidonate 5-lipoxygenase (IC50 = 0.36 microM). Arachidonate 12-lipoxygenase purified from porcine leukocytes and human platelets, 15-lipoxygenase from rabbit reticulocytes and fatty acid cyclooxygenase from bovine vesicular glands were inhibited by the compound only at higher concentrations (IC50 = 2.3, 11, 5.2 and 2.5 microM, respectively).     

Diphyllin acetylapioside, a 5-lipoxygenase inhibitor from Haplophyllum hispanicum

The topical anti-inflammatory activity of Haplophyllum hispanicum Spach is due to the presence of arylnaphthalene-type lignans acting as 5-lipoxygenase (5-LOX) inhibitors. The effects of the methanolic extract of this species on cellular systems have been determined by HPLC-DAD analysis. Diphyllin acetylapioside proved to be the main lignan endowed with 5-LOX inhibitory activity.     

The antiinflammatory action of guanabenz is mediated through 5-lipoxygenase and cyclooxygenase inhibition

Guanabenz (2,6-dichlorobenzylidene amino guanidine acetate), an α₂-agonist, possesses antiinflammatory substances, the effect of guanabenz on LT and Since leukotrienes (LT) and prostaglandins(PG) are proinflammatory substances, the effect of guanabenz on LT and PG synthesis by inflammatory cells was investigated. Guanabenz, but not clonidine, B-HT 920 or B-HT 933 inhibited zymosan-induced LTC₄ (IC₅₀=13 μM) and PGE₂ (IC₅₀=10.9 μM) synthesis with no concomitant reduction in zymosan phagocytosis or cell viability. Similarlyguanabenz reduced LTB₄ (IC₅₀=37.4 μM) and PGE₂ (IC₅₀=13.8 μM) synthesis by A23187-stimulated rat glycogen elicited neutrophils. Furthermore, guanabenz did not inhibit platelet 12-lipoxygenase or phospholipase A₂. In vivo, guanabenz was orally active against rat carrageenan paw edema and adjuvant arthritis (ED_50^s=9 and 10 mg/kg, respectively). Topically applied guanabenz reduced arachidonic acid (AA)- or tetradecanoyl phorbol acetate (TPA)-induced ear inflammation (ED_50^s:AAA-induced ear edema, 1.4 mg/ear; PMA-induced ear edema, 0.013 mg/ear). Therefore, the antiinflammatory activity of guanabenz may be due to its ability to inhibit the formation of 5-lipoxygenase and cyclooxygenase products.     

Effects of novel antifungal azole derivatives on the 5-lipoxygenase and cyclooxygenase pathway

5-Lipoxygenase and cyclooxygenase catalyze the first steps of the conversion of arachidonic acid into leukotrienes and prostanoids, respectively. Leukotrienes are important mediators of inflammation and thromboxanes are mainly involved in platelet functions. The effects of the new antimycotic drugs, itraconazole and fluconazole, on the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMNL), on the eicosanoid metabolism in platelets, platelet aggregation and on cyclooxygenase activity were investigated and compared with miconazole and ketoconazole. Itraconazole inhibited the formation of 5-lipoxygenase metabolites in human PMNL (IC50 = 2 x 10(-6) mol/l), while it had no effect on cyclooxygenase and platelet aggregation at concentrations up to 10(-4) mol/l and 10(-5) mol/l, respectively. Fluconazole was ineffective in all assays. Miconazole inhibited thromboxane synthase (IC50 = 1 x 10(-5) mol/l) and platelet aggregation (IC50 = 3 x 10(-5) mol/l). Ketoconazole was less active in this respect (IC50 for platelet aggregation = 2 x 10(-4) mol/l). All compounds did not affect cyclooxygenase activity in concentrations up to 10(-4) mol/l in the pure enzyme assay. These results indicate that among the tested azoles two compounds show remarkable effects. Miconazole inhibits thromboxane synthesis and itraconazole is a potent inhibitor of leukotriene formation which has only minor effects on the cyclooxygenase pathway. This finding is of considerable interest regarding the application of itraconazole during immunosuppressive therapy and suggests further studies of its potential antiphlogistic properties.     

Reduction of renal ischemia-reperfusion injury in 5-lipoxygenase knockout mice and by the 5-lipoxygenase inhibitor zileuton

The role of 5-lipoxygenase (5-LOX) in the pathophysiology of renal ischemia/reperfusion (I/R) injury is not known. Here we investigate the effects of 1) the 5-LOX inhibitor zileuton and 2) 5-LOX gene knockout (5-LOX(-/-)) mice on renal dysfunction and injury caused by I/R of the kidney in mice. Wild-type mice treated with zileuton (3 mg/kg i.v.) or 5-LOX(-/-) mice were subjected to bilateral renal artery occlusion (30 min) followed by reperfusion (24 h). Plasma urea, creatinine, and aspartate aminotransferase (AST) were measured as markers of renal dysfunction and reperfusion injury. Kidneys were used for histological evaluation of renal injury. Renal myeloperoxidase activity was measured and used as an indicator of polymorphonuclear leukocyte (PMN) infiltration and renal expression of intercellular adhesion molecule-1 (ICAM-1) was determined using immunohistochemistry. Administration of zileuton before I/R significantly reduced the degree of renal dysfunction (urea, creatinine) and injury (AST, histology). In addition, zileuton reduced the expression of ICAM-1 and the associated PMN infiltration caused by I/R of the mouse kidney. Compared with wild-type mice, the degree of renal dysfunction, injury, and inflammation caused by I/R in 5-LOX(-/-) mice was also significantly reduced, confirming the pathophysiological role of 5-LOX in the development of renal I/R injury. We propose that 1) endogenous 5-LOX metabolites enhance the degree of renal injury, dysfunction, and inflammation caused by I/R of the kidney by promoting the expression of adhesion molecules, and 2) inhibitors of 5-LOX may be useful in the treatment of conditions associated with I/R of the kidney.     

A new 5-lipoxygenase selective inhibitor derived from Artocarpus communis strongly inhibits arachidonic acid-induced ear edema

Natural compounds isolated from the Indonesian plant, Artocarpus communis, inhibit 5-lipoxygenase of cultured mastocytoma cells. One of five compounds, AC-5-1, strongly inhibits 5-lipoxygenase with a half-inhibition dose of 5 +/- 0.12 X 10(-8) M. However, prostaglandin synthesizing activity is not inhibited until 10(-5) M. AC-5-1 is a highly selective inhibitor for 5-lipoxygenase. The AC-5-1 at 10(-5) M inhibits 96% of leukotriene C4 synthesis of mouse peritoneal cells facilitated by calcium-ionophore. Arachidonic acid-induced ear edema of mice, an in vivo inflammatory model, involving leukotriene induction, is strongly inhibited by AC-5-1 in a dose-dependent manner. The inhibition is the strongest of any inhibitors of 5-lipoxygenase reported previously. Since the natural compound AC-5-1 can selectively inhibit 5-lipoxygenase and affect in vivo inflammation, it will be interesting to investigate the role of leukotrienes on inflammation and other physiological processes.     

New approaches to the modulation of the cyclooxygenase-2 and 5-lipoxygenase pathways

The eicosanoid family comprises a number of biologically active lipid mediators involved in the regulation of inflammation and cancer cell growth. Eicosanoid biosynthesis is usually initiated by the release of arachidonic acid (AA) from membrane phospholipids in response to the interaction of a phospholipase-A(2) (PLA(2)) stimulus with a receptor on the cell membrane. The free released AA is subsequently metabolized by three major enzymatic pathways: the cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450-dependent pathways. The COX pathway transforms AA into prostaglandins (PGs) and is of particular clinical relevance because it is the main target for non-steroidal anti-inflammatory drugs (NSAIDs). Of interest, COX-2, one of the two COX isoforms, is primarily involved in inflammation and cancer and for this reason selective COX-2 inhibitors have been developed. The efficacy of these compounds is similar to that of traditional NSAIDs but with a lower risk of gastrointestinal toxicity and bleeding. On the other hand, emerging information has recognized the role of other AA metabolites derived from the 5-LO pathway, the leukotrienes (LTs), in mediating and maintaining inflammation. Consequently, drugs able to inhibit 5-LO are now included among the effective pharmacological therapies, especially in asthma and allergic inflammation. Moreover, COX-2 and 5-LO pathways appear to act in parallel in the regulation of cell proliferation and neo-angiogenesis and both COX-2 and 5-LO inhibitors are being investigated as potential anticancer drugs. This review article will update the progress achieved in the knowledge of COX-2 and 5-LO and discuss the emerging approaches for the pharmacological modulation of these pathways.