A rat air pouch model for evaluating the efficacy and selectivity of 5-lipoxygenase inhibitors.
The 5-lipoxygenase (5-LOX) pathway has been associated with a variety of inflammatory diseases including asthma, atherosclerosis, rheumatoid arthritis, pain, cancer and liver fibrosis. Several classes of 5-LOX inhibitors have been identified, but only one drug, zileuton, a redox inhibitor of 5-LOX, has been approved for clinical use. To better evaluate the efficacy of 5-LOX inhibitors for pharmacological intervention, a rat model was modified to test the in vivo efficacy of 5-LOX inhibitors. Inflammation was produced by adding carrageenan into a newly formed air pouch and prostaglandins produced. While macrophages and neutrophils are present in the inflamed pouch, little 5-LOX products are formed. Cellular 5-LOX activation was obtained by adding calcium ionophore (A23187) into the pouch thus providing a novel model to evaluate the efficacy and selectivity of 5-LOX inhibitors. Also, we described modifications to the in vitro 5-LOX enzyme and cell assays. These assays included a newly developed fluorescence-based enzyme assay, a 5-LOX redox assay, an ex vivo human whole blood assay and an IgE-stimulated rat mast cell assay, all designed for maximal production of leukotrienes. Zileuton and CJ-13,610, a competitive, non-redox inhibitor of 5-LOX, were evaluated for their pharmacological properties using these assays. Although both compounds achieved dose-dependent inhibition of 5-LOX enzyme activity, CJ-13,610 was 3-4 fold more potent than zileuton in all-assays. Evaluation of 5-LOX metabolites-by LC/MS/MS and ELISA confirmed that both compounds selectively inhibited all products downstream of 5-hydroperoxy eicosatetraenoic acid (5-HPETE), including 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxoETE), without inhibition of 12-lipoxygenase (12-LOX), 15-lipoxygenase (15-LOX), or cyclooxygenase (COX) products. In the rat air pouch model, oral dosing of CJ-13,610 and zileuton resulted in selective inhibition 5-LOX activity from pouch exudate and ex vivo rat whole blood with similar potency to in vitro assay. These data show that the rat air pouch model is a reliable and useful tool for evaluating in vivo efficacy of 5-LOX inhibitors and may aid in the development of the next generation of 5-LOX inhibitors, such as the non-redox inhibitors similar to CJ-13,610.
Zweifel
Ben S
BS
Pfizer Global Research & Development, Pfizer Inc., St. Louis, MO 63017, USA. ben.s.zweifel@pfizer.com
Hardy
Medora M
MM
Anderson
Gary D
GD
Dufield
Dawn R
DR
Pufahl
Robert A
RA
Masferrer
Jaime L
JL
D000388:Air; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000097:blood / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D000001:Calcimycin / Q000494:pharmacology; D002351:Carrageenan; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006918:Hydroxyurea / Q000031:analogs & derivatives* / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology*; D007073:Immunoglobulin E / Q000276:immunology; D007249:Inflammation / Q000139:chemically induced / Q000201:enzymology*; D007476:Ionophores / Q000494:pharmacology; D015289:Leukotrienes / Q000097:blood / Q000378:metabolism*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology*; D008297:Male; D008407:Mast Cells / Q000187:drug effects* / Q000201:enzymology / Q000276:immunology; D010084:Oxidation-Reduction; D051381:Rats; D011917:Rats, Inbred Lew; D015203:Reproducibility of Results; D013440:Sulfides / Q000494:pharmacology*; D053719:Tandem Mass Spectrometry
C496810:CJ 13610; D020536:Enzyme Activators; D007093:Imidazoles; D007476:Ionophores; D015289:Leukotrienes; D016859:Lipoxygenase Inhibitors; D013440:Sulfides; D007073:Immunoglobulin E; D000001:Calcimycin; D002351:Carrageenan; D001094:Arachidonate 5-Lipoxygenase; C063449:zileuton; D006918:Hydroxyurea