A SENSITIVE PLATELET ACTIVATION-BASED FUNCTIONAL ASSAY FOR THE ANTILEUKEMIC AGENT BRYOSTATIN-1

Bryostatin 1, a macrocyclic lactone activator of protein kinase C (PKC ) currently in phase I evaluation, is a biologic response modifier whi ch exhibits significant antitumor activity in several experimental sys tems. Clinical trials have been hampered by the absence of a sensitive assay for bryostatin 1 blood levels. The purpose of these studies was to exploit the exquisite sensitity of human platelets to bryostatin 1 -induced aggregation in order to develop an assay capable of detecting plasma bryostatin 1 levels in the nanomolar range. Addition of bryost atin 1 (5-100 nM) to platelet-rich plasma resulted in complete platele t aggregation. A highly linear relationship was observed between low b ryostatin 1 concentrations (i.e. 2-25 nM) and (i) reduction in the lag phase prior to aggregation and (ii) maximal rate of aggregation (R = 0.976). At higher bryostatin 1 concentrations (i.e. 10-100 nM), platel et aggregation was accompanied by detectable ATP release; both the ext ent and maximal rate of ATP secretion were highly linear functions of bryostatin 1 levels (R = 0.992). Bryostatin 1 concentrations in antico agulated human blood samples could also be determined by mixing platel et poor plasma obtained from such samples with normal platelet-rich pl asma. Notably, measurement of the delay in the aggregation lag phase p ermitted quantitation of bryostatin 1 concentrations of 5 nM or below. The capacity to detect bryostatin 1 plasma levels of 10 nM or lower s hould facilitate the conduct of pharmacokinetic and pharmacodynamic st udies in conjunction with ongoing phase 1 trials.