Proteasome inhibition measurements: Clinical application

Background: PS-341, a selective inhibitor of the proteasome, currently is u nder evaluation as an anticancer agent in multiple phase I clinical trials. In animal-model studies, PS-341 was rapidly removed from the vascular comp artment and distributed widely, quickly approaching the limits of detection . An accurate pharmacodynamic assay has been developed as an alternative or complement to pharmacokinetic measurements. Methods: Fluorogenic kinetic assays for both the chymotryptic and tryptic a ctivities of the proteasome have been optimized for both whole blood and bl ood cells. Using the ratio of these activities and the catalytic mechanism of the proteasome, we developed a novel method of calculating percentage of inhibition, using two structurally unrelated inhibitors (PS-341 and lactac ystin). Results: This ratio method was demonstrated to be sensitive (detection limi t of 13% inhibition with 10 mu g of cell lysate), specific to the proteasom e (PS-341 provides >98% inhibition), accurate (112% analyte recovery), and precise (0% +/- 5% inhibition at 0 nmol/L PS-341 and 74.5% +/- 1.7% inhibit ion at 200 nmol/L PS-341). Using these assays, we found that both erythrocy tes and leukocytes contain proteasome at 3 mu mol/L. Pharmacodynamic result s for PS-341 obtained from the whole-blood ratio method were comparable to those using leukocytes determined by another method. Conclusions: The described assay provides a reliable method for studying th e pharmacodynamics of proteasome inhibitors and is now in use in concurrent phase I clinical trials with PS-341. (C) 2000 American Association for Cli nical Chemistry.