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

Abstract

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 x 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 V(max) and K(m) of 74.7+/-13.8 nmol/min/mg protein and 8.8+/-2.8 microM, 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.

Organic anion transporting polypeptide 2-mediated uptake of paclitaxel and 2′-ethylcarbonate-linked paclitaxel in freshly isolated rat hepatocytes

“…Therefore, fresh rat hepatocytes may clarify uptake properties of paclitaxel and its derivatives including prodrugs. Furthermore, slight amounts (1–5% of dose) of paclitaxel are produced from TAX‐2′‐Et in rat and human liver microsomes; however, rat serum provides rapid enzymatic hydrolysis of TAX‐2′‐Et 6 . The in‐vitro disposition profile of TAX‐2′‐Et in human serum is similar to that observed in rabbit serum.…”

“…To overcome these drawbacks, a number of investigators have developed paclitaxel prodrugs and conjugates based on the chemical modification of the hydroxyl groups at position 7 of the baccatin core and/or position 2′ of the paclitaxel side chain 2–4 . In our previous studies, 2′‐ethylcarbonate‐linked paclitaxel (TAX‐2′‐Et; Figure 1) showed a low sensitivity to hydrolytic enzymes in human serum, and circumvented P‐glycoprotein (P‐gp)‐mediated efflux of paclitaxel in paclitaxel‐resistant cells, demonstrating one of the most important factors of gene‐directed enzyme prodrug therapy (GDEPT) strategies that allow the tumour cell‐specific cytotoxicity of paclitaxel 5,6 …”

Detection and Quantification of Carboxylesterase 2 Activity by Capillary Electrophoresis

“…Although BuChE and AcChE did not hydrolyze 4-MUBA, some biological samples may contain other enzymes that could metabolize this substrate. For this reason, we evaluated BNPP, an irreversible inhibitor specific for CESs. , Taking advantage of these two cheap and commercially available inhibitors, the relative activity of CES2 can be determined in any biological sample. BNPP also absorbs at 350 nm, and therefore classical spectrophotometric enzymatic assays cannot be used.…”

“…The proposed methodology should be applicable to a wide variety of samples with esterase activity. For example, the analysis and determination of hCE-1 exclusive activity would only imply the replacement of loperamide by a specific inhibitor of this enzyme such as the partially noncompetitive inhibitor 27-hydroxycholesterol . For the analysis of the activities of other esterases, appropriate inhibitors should be used.…”

Novel Nanoassemblies Composed of Squalenoyl−Paclitaxel Derivatives: Synthesis, Characterization, and Biological Evaluation

“…The release of paclitaxel from the bioconjugates following incubation in serum attributed to the hydrolysis caused by the esterases activity of serum. Indeed, the paclitaxel 2′-esters and carbonates demonstrated to be good substrates for these enzymes (43).…”