Development of a system for evaluating 5-lipoxygenase inhibitors using human whole blood.
A reliable system for evaluating 5-lipoxygenase (5-LO) pathway inhibitors employing human whole blood stimulated by the calcium ionophore, A-23187, and yeast cell walls (YCW) is described. In developing this system, we have shown that leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) can be recovered quantitatively from whole blood, and can be measured with accuracy and a precision (standard deviation) of +/- 12%. Apparent differences in LTB4/5-HETE levels between donors can be minimized by normalizing the LTB4/5-HETE production to neutrophil number. Variability in LTB4/5-HETE production among different donors was reduced by increasing the ionophore concentration. The kinetics of ionophore stimulated product production display a 1-4 min lag which is dependent on ionophore concentration. The lag is removed by pretreatment of blood with 5 micrograms/ml cytochalasin B. Likewise, the kinetics of product formation after stimulation with yeast cell walls demonstrated a lag period, which could be shortened by prior opsonization of the YCW. The amount of LTB4 metabolism to 20-OH-LTB4 and 20-COOH-LTB4 in this system is approximately 20%. Phenidone, nordihydroguaiaretic acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 micrograms/ml, respectively. In summary, we believe that this assay offers a guide for predicting systemic levels of drug needed to be achieved for effective inhibition of cellular LTB4/5-HETE synthesis/release in humans.
D001094:Arachidonate 5-Lipoxygenase / Q000097:blood; D001091:Arachidonate Lipoxygenases / Q000037:antagonists & inhibitors*; D001095:Arachidonic Acids / Q000097:blood; D000001:Calcimycin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D003571:Cytochalasin B / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000097:blood; D066298:In Vitro Techniques; D007700:Kinetics; D017572:Leukotriene A4; D007975:Leukotriene B4 / Q000097:blood; D016859:Lipoxygenase Inhibitors; D011863:Radioimmunoassay; D013056:Spectrophotometry, Ultraviolet; D015002:Yeast, Dried / Q000494:pharmacology
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D017572:Leukotriene A4; D016859:Lipoxygenase Inhibitors; D007975:Leukotriene B4; D000001:Calcimycin; D003571:Cytochalasin B; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D004533:Egtazic Acid; D001091:Arachidonate Lipoxygenases; D001094:Arachidonate 5-Lipoxygenase