leukotriene-b4 and nafazatrom

The effect of oral nafazatrom (Bay g6575, 2 X 3 g) or placebo on inhaled antigen challenge was assessed in a double-blind study. In four subjects antigen challenge resulted in an immediate fall of 93.2 +/- 3.36% in airflow at 40% of vital capacity (Vp40) and a 45.85 +/- 4.95% reduction in forced partial expiratory volume at one second (FEV1). Neither nafazatrom nor placebo had any effect on baseline lung function or that after challenge. Leukotriene B4 was generated by ex vivo stimulus of blood with ionophore A23187, and quantified by high performance liquid chromatography (h.p.l.c.)-radioimmunoassay. No inhibition of LTB4 formation occurred ex vivo following oral nafazatrom, although addition of 10(-5) M nafazatrom to blood in vitro significantly inhibited LTB4 release. Peak plasma nafazatrom levels during the study ranged from 3.3 X 10(-7) M to 1.47 X 10(-6) M which are below the concentration (10(-5) M) at which significant 5-lipoxygenase inhibition occurs in vitro. Oral nafazatrom is ineffective as a 5-lipoxygenase inhibitor in man, probably because of poor bioavailability after administration.

Topics: Adult; Allergens; Bronchi; Calcimycin; Chromatography, High Pressure Liquid; Female; Fibrinolytic Agents; Humans; Leukotriene B4; Male; Pyrazoles; Pyrazolones; Radioimmunoassay; Respiratory Function Tests; Vital Capacity

The purpose of this study was to determine the role, if any, of Leukotriene B4 (LTB4) in Platelet Activating Factor (PAF)-induced aggregation of rat polymorphonuclear leucocytes (PMNs). Exposure of rat PMNs to 10(-7) M PAF resulted in the release of 4.5 +/- 0.7 ng/10(7) cells of LTB4 measured by radioimmunoassay. However, the maximum aggregation of PMNs achieved by exposure to LTB4 (10(-7)M) was only 50% of that produced by maximally aggregating concentrations of PAF (10(-7)M). 5-Lipoxygenase inhibitors, BW755c and Nafazatrom at concentrations that completely abolished LTB4 synthesis inhibited the aggregation induced by PAF only by 40% and 50% respectively. Furthermore, desensitisation experiments revealed that the aggregatory response of PMNs to PAF was only partially refractory to prior treatment with LTB4 whereas the aggregatory response to LTB4 was completely refractory to prior treatment with PAF. These results suggest that PAF-induced aggregation of rat PMNs is in part mediated by LTB4 and in part directly by an as yet unidentified mechanism.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Cell Aggregation; In Vitro Techniques; Leukotriene B4; Lipoxygenase Inhibitors; Male; Neutrophils; Platelet Activating Factor; Pyrazoles; Pyrazolones; Rats; Rats, Inbred Strains

We have evaluated the biosynthesis, characterization and inhibition of Leukotriene (LT) B4 in unstimulated and in A23187-stimulated human whole blood. LTB4 was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37 degrees C for 60 min produced only trace amounts of LTB4 (0.16 +/- 0.05 ng/ml, mean +/- SD, n = 3). LTB4-like immunoreactivity (ir-LTB4) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrelated to cyclooxygenase or lipoxygenase activity. Incubation of human whole blood with A23187 (2-10 microM) resulted in a concentration-dependent stimulation of LTB4 production. At 10 microM A23187, ir-LTB4 was 18 +/- 2.4 ng/ml (mean +/- SEM, n = 28). In A23187-stimulated serum samples, LTB4 concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added in vitro caused a dose-dependent inhibition of A23187-stimulated ir-LTB4 production with an IC50 of 17 microM.

Topics: Aspirin; Calcimycin; Chromatography, Thin Layer; Cross Reactions; Fibrinolytic Agents; Humans; Immune Sera; Kinetics; Leukotriene B4; Pyrazoles; Pyrazolones; Radioimmunoassay

The subcutaneous sponge implant model of acute inflammation in the rat has been evaluated as a suitable test system for evaluating the potential anti-inflammatory efficacy of 5-lipoxygenase inhibitors. The inflammatory parameters measured were exudate volume and leukocyte recruitment. Specific radioimmunoassays were used to measure (1) 5-lipoxygenase (LPO) and cyclo-oxygenase (CO) activity in exudate leukocytes stimulated ex vivo with A23187, and (2) the LTB4 and PGE2 content of inflammatory exudate. The NSAIDs flurbiprofen and indomethacin inhibited cell recruitment, exudate volume and CO activity with ED50S of approximately 1 mg per kg p.o. but failed to inhibit LPO activity at 10 mg per kg p.o. Nafazatrom (Bayer 6575), quercetin and NDGA, which inhibit LPO activity in vitro, were inactive against all parameters when dosed at 100 mg per kg p.o. The "mixed inhibitors" BW755C and phenidone were approximately equipotent inhibitors of LPO activity but BW755C was 10 times more potent than phenidone against CO activity. BW755C was also greater than 10 times more potent at inhibiting cell recruitment and exudate volume than phenidone suggesting that the anti-inflammatory efficacy of the mixed inhibitors reflect their potency against CO rather than LPO activity. Time course studies demonstrated that the inhibitor effects of BW755C and phenidone on leukocyte recruitment reflected a reduction in the PGE2 but not the LTB4 content of the inflammatory exudate. Polyester sponges soaked in high concentrations of LTB4 caused only a modest (2-fold) increase in leukocyte recruitment whilst physiological levels were inactive. The results taken together suggest that CO products make a major contribution to leukocyte recruitment in this model whilst the LPO product LTB4 has little role. This model therefore is of little value for evaluating the anti-inflammatory efficacy of 5-lipoxygenase inhibitors. Moreover, the rat would appear to be unsuitable for evaluating the role of LTB4 in acute inflammation.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Acute Disease; Animals; Blood Proteins; Calcimycin; Chemotaxis, Leukocyte; Cyclooxygenase Inhibitors; Dinoprostone; Dose-Response Relationship, Drug; Flurbiprofen; Indomethacin; Inflammation; Leukocytes; Leukotriene B4; Lipoxygenase Inhibitors; Male; Prostaglandins E; Pyrazoles; Pyrazolones; Rats; Rats, Inbred Strains; Skin

A sensitive RIA for LTB4 (which cross-reacts 60% with 6-trans LTB4) has been developed with a minimal detectable mass of 7.4 X 10(-15) mole. The properties of a second RIA more specific for 6-trans LTB4 are also described. The latter has utility in the measurement of the enzymatic and non-enzymatic transformations of LTA4 and in the characterisation of inhibitors of LTA4 epoxide hydrolase. Conditions for the direct RIA of immunoreactive LTB4 in human plasma are described. Addition of calcium ionophore, A23187, to human blood increased basal immunoreactive LTB4 levels from less than 100pg ml-1 to 259 +/- 23ng ml-1 (mean +/- SEM, n = 16). Extraction and RPHPLC confirmed that greater than 90% of immunoreactive LTB4 co-eluted with synthetic and [3H]LTB4. Pharmacological characterisation of immunoreactive eicosanoids revealed that in vitro the NSAIDs: aspirin, indomethacin, flurbiprofen and benoxaprofen did not inhibit LTB4 but inhibited TXB2, consistent with cyclo-oxygenase inhibition whereas the prototype mixed inhibitor BW755c inhibited both LTB4 and TXB2. This experimental paradigm may be used in the clinical measurement of the bio-availability of novel agents that inhibit eicosanoid biosynthesis.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Antibodies; Antibody Specificity; Calcimycin; Catechols; Chromatography, High Pressure Liquid; Cross Reactions; Dose-Response Relationship, Drug; Female; Humans; Leukotriene B4; Male; Masoprocol; Pyrazoles; Pyrazolones; Rabbits; Radioimmunoassay; Thromboxane B2; Time Factors

Three grams of nafazatrom (Bay g 6575), given orally to healthy male volunteers in a single dose, significantly reduce the formation of leukotriene B4 in polymorphonuclear leukocytes. LTB4 synthesis fell from 57.1 +/- 17.0 ng/10(7) PMNL, mean +/- S.D., in control to 34.3 +/- 14.4 ng/10(7) PMNL 3 hr after nafazatrom (2 P less than 0.001). In vitro, nafazatrom inhibited LTB4 formation in human PMNL in a dose dependent manner. At 1 microM nafazatrom LTB4 formation was reduced to 65% of the control value. Nafazatrom had no effect on the excretion of 2,3-dinor-6-keto-PGF1 alpha and 2,3-dinor-TXB2, the major urinary metabolites of endogenously synthesized PGI2 and TXA2, respectively. Serum levels of TXB2 in clotted whole blood also remained unchanged. The inhibitory effect of nafazatrom on leukotriene biosynthesis in human PMNL suggests a therapeutic potential of this drug in processes like allergy and chronic inflammation, where leukotrienes play a pathogenetic role.

Topics: Administration, Oral; Adult; Epoprostenol; Fibrinolytic Agents; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Male; Neutrophils; Pyrazoles; Pyrazolones