Recombinant human secretory phospholipase A2 released thromboxane from guinea pig bronchoalveolar lavage cells: in vitro and ex vivo evaluation of a novel secretory phospholipase A2 inhibitor.
The primary objective of this study was to develop a functional assay that could provide rapid and reliable information on some pharmacologic characteristics of a novel inhibitor of human secretory phospholipase A2 (sPLA2). Guinea pig bronchoalveolar lavage (BAL) fluid, containing predominantly macrophages, eosinophils and epithelial cells, released thromboxane A2, as measured by thromboxane B2, in a concentration-dependent manner on exposure to recombinant human sPLA2 (rh-sPLA2). Similarly, n-formyl-L-methionyl-L-leucyl-L-phenylalanine (n-F-Met-Leu-Phe) or arachidonic acid also released this lipid mediator. Indomethacin, a cyclooxygenase inhibitor, blocked synthesis of thromboxane in response to these agents. p-Bromophenacylbromide-inactivated rh-sPLA2 was substantially less effective than the untreated enzyme in causing release of thromboxane. LY311727 is a potent indole-derived inhibitor of the isolated enzyme (IC50 = 23 nM). Incubation of this agent with BAL cells, just before addition of rh-sPLA2, reduced release of thromboxane with an IC50 = 1.8 x 10(-6) M. Specificity for sPLA2 was demonstrated in that LY311727, unlike indomethacin, did not reduce synthesis and subsequent release of thromboxane A2 in response to arachidonic acid. Using this technique as a basis, we determined whether LY311727 could sufficiently accumulate in lung after i.v. administration to inhibit rh-sPLA2-induced thromboxane A2 release from BAL cells. The compound, given i.v. to guinea pigs 5 min before collecting BAL fluid, produced a dose-dependent inhibition of rh-sPLA2 with an ED50 = 50 mg/kg. Thus, new in vitro and ex vivo assays were developed that permit functional evaluation of novel sPLA2 inhibitors. These techniques should serve as secondary assays for evaluation of human sPLA2 inhibitory activity from a chemical series and in addition provide initial data related to metabolic stability and distribution to the lung.
Fleisch
J H
JH
Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana, USA.
Armstrong
C T
CT
Roman
C R
CR
Mihelich
E D
ED
Spaethe
S M
SM
Jackson
W T
WT
Bobbitt
J L
JL
Draheim
S
S
Bach
N J
NJ
Dillard
R D
RD
Martinelli
M
M
Fouts
R
R
Snyder
D W
DW
D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D018893:Bronchoalveolar Lavage; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006168:Guinea Pigs; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008297:Male; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013928:Thromboxane A2 / Q000378:metabolism*; D013929:Thromboxane B2 / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007211:Indoles; C095553:LY 311727; D016718:Arachidonic Acid; D013929:Thromboxane B2; D013928:Thromboxane A2; D010741:Phospholipases A; D054467:Phospholipases A2