Development of a system for evaluating 5-lipoxygenase inhibitors using human whole blood

Abstract

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 ± 129% 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 μg/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, nordihydroguaiaref acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 μg/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.