Guinea pig whole blood 5-lipoxygenase assay: utility in the assessment of potential 5-lipoxygenase inhibitors.
Guinea pigs are widely used in the study of the role of leukotrienes in airway pathophysiology. Extensive research efforts have utilized this species in the development of potential therapeutic agents associated with inhibition of leukotriene production (e.g. 5-lipoxygenase inhibitors and 5-lipoxygenase-activating protein antagonists) for the treatment of acute bronchospasm in asthma. We now report, for the first time, an ex vivo whole blood 5-lipoxygenase assay in guinea pigs which should prove useful in the future development of leukotriene biosynthesis inhibitors. Addition of 150 microM arachidonic acid (AA) to heparinized whole blood for 5 min prior to the stimulation with 20 micrograms/mL A23187 resulted in a 10-fold increase in leukotriene B4 (LTB4; 11.36 +/- 1.55 ng/mL) above basal (0.96 +/- 0.29 ng/mL) within 10 min. To further validate the utility of the assay, we utilized the 5-lipoxygenase inhibitor BW A4C. Pretreatment of guinea pig whole blood with BW A4C in vitro prior to stimulation resulted in a concentration-dependent inhibition of LTB4 production (IC50 = 229 nM), whereas thromboxane B2 (TXB2) production was unaffected. Likewise, when BW A4C was administered to guinea pigs intravenously (3 mg/kg), we observed a rapid and marked (approximately 90%) reduction in whole blood LTB4 production which returned to control (pre-drug values) by 5 hr. In contrast, TXB2 production was unaffected over the same experimental time period. In summary, we have described a whole blood assay which can discriminate 5-lipoxygenase inhibitors both in vitro and in vivo. Furthermore, this assay system will be of use in determining the potency, efficacy, selectivity, and pharmacodynamic properties of 5-lipoxygenase inhibitors in guinea pigs.
Spaethe
S M
SM
Department of Pulmonary Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
Snyder
D W
DW
Pechous
P A
PA
Clarke
T
T
VanAlstyne
E L
EL
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000097:blood*; D016718:Arachidonic Acid / Q000494:pharmacology; D044482:Benzeneacetamides; D000001:Calcimycin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D006168:Guinea Pigs; D006877:Hydroxamic Acids / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D007213:Indomethacin / Q000494:pharmacology; D007975:Leukotriene B4 / Q000032:analysis*; D016859:Lipoxygenase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D013929:Thromboxane B2 / Q000032:analysis*
D044482:Benzeneacetamides; D006877:Hydroxamic Acids; D006893:Hydroxyeicosatetraenoic Acids; D016859:Lipoxygenase Inhibitors; C056614:N-(3-phenoxycinnamyl)acetohydroxamic acid; D007975:Leukotriene B4; D016718:Arachidonic Acid; D000001:Calcimycin; D013929:Thromboxane B2; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001094:Arachidonate 5-Lipoxygenase; D007213:Indomethacin