Enzymatic stability of 2′‐ethylcarbonate‐linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapy

In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2′-ethylcarbonate-linked paclitaxel (TAX-2′-Et) in the sera of several different species including humans. TAX-2′-Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX-2′-Et hydrolysis, a CES isolated from rabbit liver (Ra-CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX-2′-Et with a half-life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16 h−1) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p-nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX-2′-Et was 5.1 × 10−2 and 0.15 h−1, respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX-2′-Et. The amount of TAX produced accounted for only 8–11% of the decrease in TAX-2′-Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5 h incubation with human or rabbit serum. Ra-CES enzyme converted TAX-2′-Et to TAX with Vmax and Km of 74.7±13.8 nmol/min/mg protein and 8.8±2.8 µM, respectively. These results indicate that TAX-2′-Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species-dependent hydrolysis of TAX-2′-Et. Although human serum allows the slow release of TAX, TAX-2′-Et is expected to reduce the side-effects of TAX. The Ra-CES enzyme is capable of hydrolysing TAX-2′-Et, which may be beneficial for the development of a TAX-2′-Et/enzyme therapy strategy for ovarian cancer. Copyright © 2008 John Wiley & Sons, Ltd.