Human Carboxylesterase-2 Hydrolyzes the Prodrug of Gemcitabine (LY2334737) and Confers Prodrug Sensitivity to Cancer Cells

Abstract

Purpose: The oral prodrug of gemcitabine LY2334737 is cleaved systemically to gemcitabine; the mechanism responsible for hydrolysis is unknown. LY2334737 cytotoxicity was tested in the NCI-60 panel; mining of microarray expression data identified carboxylesterase (CES) as a top hydrolase candidate. Studies examined whether CES is responsible for hydrolysis and whether cellular CES expression confers prodrug sensitivity.Experimental Design: Human recombinant CES isozymes were assayed for LY2334737 hydrolysis. Stable CES-overexpressing HCT-116 transfectants and a SK-OV-3 knockdown were prepared. Cell lines were tested for drug sensitivity and CES expression by quantitative real time-PCR (qRT-PCR), Western blotting, and immunohistochemical staining. Bystander cytotoxicity studies were conducted with GFP-tagged PC-3 cells as the reporter cell line. Therapeutic response of the HCT-116 transfectants was evaluated in xenografts.Results: Of 3 human CES isozymes tested, only CES2 hydrolyzed LY2334737. Five cell lines that express CES2 responded to LY2334737 treatment. LY2334737 was less cytotoxic to a SK-OV-3 CES2 knockdown than parental cells. The drug response of CES2-transfected HCT-116 cells correlated with CES2 expression level. Bystander studies showed statistically greater PC-3-GFP growth inhibition by LY2334737 when cells were cocultured with CES2 and not mock transfectants. Oral treatment of xenograft models with 3.2 mg/kg LY2334737 once a day for 21 days showed greater tumor growth inhibition of CES2 transfectant than the mock transfectant (P 0.001).Conclusions: CES2 is responsible for the slow hydrolysis of LY2334737. Because intact prodrug circulates at high plasma levels after oral LY2334737 administration, improved response rates may be observed by tailoring LY2334737 treatment to patients with CES2 tumor expression.

P-Glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain and Blood Disposition of the mTOR Inhibitor Everolimus (Afinitor) in Mice

“…Upon oral everolimus administration, local concentrations of everolimus might be high especially in the intestine, and possibly surpass the K i of approximately 20 mmol/L toward CES2. CES2 is for instance thought to be a main enzyme responsible for the conversion of irinotecan to SN-38 in humans (38), and for hydrolysis of a prodrug of gemcitabine (39). Coadministration of everolimus with ester (pro-) drugs affected by carboxylesterases, including the 5-FU anticancer prodrug capecitabine (40), should thus be assessed very carefully.…”

Efficacy of Low-Dose Oral Metronomic Dosing of the Prodrug of Gemcitabine, LY2334737, in Human Tumor Xenografts

“…LY2334737 is cleaved only by the CES2 isozyme (11). Certain tumor types express elevated CES2 levels; these include colon tumors, NSCLC, pleural mesothelioma tumors, hepatic, renal, and ovarian cancers (40)(41)(42).…”

“…HCT-116 xenograft studies (11,27) included treatment with LY2334737 administered by oral gavage either as the free base in a vehicle of 1% sodium carboxymethylcellulose, 0.5% sodium lauryl sulfate, 0.05% antifoam, or as the salt of LY2334737 in a vehicle of 0.1 mol/L Na3P04, pH 6; the dose is reported as the free base. Gemcitabine.HCl was administered intraperitoneally (0.2 mL) every third day for a total of 4 doses.…”

“…Oral LY2334737 is absorbed intact and cleaved slowly resulting in high plasma levels of circulating prodrug and prolonged, lower levels of circulating gemcitabine emulating a slow gemcitabine infusion (9,10). Recently, carboxylesterase 2 (CES2), expressed at high levels in the liver, kidney, and intestine, was identified as the hydrolase responsible for LY2334737 cleavage (11,12). In vitro, LY2334737 is cytotoxic to CES2-expressing cells capable of intracellular prodrug hydrolysis and more efficacious in a xenograft model expressing the CES2 transgene (11).…”

“…Recently, carboxylesterase 2 (CES2), expressed at high levels in the liver, kidney, and intestine, was identified as the hydrolase responsible for LY2334737 cleavage (11,12). In vitro, LY2334737 is cytotoxic to CES2-expressing cells capable of intracellular prodrug hydrolysis and more efficacious in a xenograft model expressing the CES2 transgene (11). "Metronomic dosing" refers to administration of chemotherapy at frequent intervals (e.g., daily) with low, minimally toxic doses (13).…”