Uptake of pivaloyloxymethyl butyrate into leukemic cells and its intracellular esterase-catalyzed hydrolysis

Pivaloyloxymethyl butyrate (AN-9), a butyric acid (BA) prodrug, exhibited l ow toxicity and significant anticancer activity in vitro and in vivo. The p urpose of this study was to elucidate the basis for AN-9 increased anticanc er activity compared to BA, by studying the uptake of BA and AN-9 into the cells. Methods: The uptake rate and level of [C-14]-AN-9 and [C-14]-BA, lab eled on the carboxylic moiety of BA, into HL-60 and MEL leukemic cell lines was measured. The cells were filtered and the retained radioactivity was d etermined. The dependence of the uptake on the activity of cellular esteras es and membrane fluidity was investigated. Results: The uptake level in cel ls incubated with [C-14]-AN-9 increased rapidly, peaked after 30 min in MEL and 1 h in HL-60 cells, and declined thereafter. This decline could be att ributed to the hydrolysis of AN-9 by cellular esterases and catabolism of t he released BA to CO2. In cells pretreated with an esterase inhibitor and i ncubated with [C-14]-AN-9, the reduction of radioactivity was less precipit ous. In cells exposed to [C-14]-BA, the intracellular radioactivity level w as low and unaffected by treatment with an esterase inhibitor. The uptake o f [C-14]-AN-9 decreased significantly at 4 degreesC compared to that at 37 degreesC. Conclusion: The higher potency of AN-9 compared to BA could be at least partially attributed to the more rapid uptake of the lipophilic AN-9 and the release of BA in the cells.