ZRX1, the first EGFR inhibitor-capecitabine based combi-molecule, requires carboxylesterase-mediated hydrolysis for optimal activity

Abstract

Capecitabine, an orally available prodrug of 5-FU, requires activation by carboxylesterase (CES) enzymes present in the liver to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR). The deamination of the latter by cytidine deaminase gives 5'-deoxy-5-fluorouridine ribose (5'-DFUR). Finally, the conversion of 5'-DFUR to the cytotoxic drug 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP). Accordingly, it was surmised that events associated with an increase of TP levels should enhance the potency of capecitabine and its metabolites. EGFR inhibition was found to be one such event. The observed synergy between gefitinib and 5'-DFUR has inspired the design of single molecules capable of acting as prodrugs of both an EGFR inhibitor and 5-FU. Here, we report on the synthesis and characterization of one such molecule, ZRX1, that consists of an acetylated 5'-DFCR moiety linked to a quinazoline inhibitor of EGFR through an alkyl dicarbamate spacer that requires CES activation to generate the two active metabolites. Our results showed that ZRX1 was ineffective as an intact molecule. However, when CES was present, ZRX1 induced an increase in EGFR inhibition, TP expression, DNA damage and apoptosis. ZRX1 was, at least, 3-fold more potent than capecitabine and 5'-DFUR and recapitulated the effects of the combination treatments. LC-MS analysis showed that in the presence of CES, ZRX1 is metabolized into a mixture of bioactive quinazoline derivatives and 5'-DFCR derived metabolites. Our results in toto, suggest that capecitabine-based EGFR targeting combi-molecules of the same type than ZRX1, have the potential to induce stronger growth inhibitory potency than capecitabine, 5'-DFUR or single EGFR inhibitors and equivalent potency when compared with combinations of EGFR inhibitors + 5'-DFUR.

Alisma genus: Phytochemical constituents, biosynthesis, and biological activities

“…Anti‐colon cancer drug CPT‐11 can be metabolize by CES 2 that is a subtype of CES, to produce the active metabolite SN‐38. The excessive accumulation of the latter in the intestinal tract results in severe diarrhea, therefore, the inhibition on CES 2 to reduce this side effect has become a research topic (Ait‐Tihyaty, Rachid, Larroque‐Lombard, & Jean‐Claude, 2013; Aizawa et al, 2003; Humerickhouse, Lohrbach, Li, Bosron, & Dolan, 2000; Small et al, 2010). 5 β ,29‐Dihydroxy alisol A ( 59 ), obtained from A. orientale , inhibited CES 2 with an IC 50 value of 29.2 μM, and the underlying mechanism between 59 and CES 2 was studied by molecular docking, all of which revealing the presence of hydrogen bond interactions with Glu77, Ser80, IIe446, and Lys447 and a lowest binding free energy of −3.94 kcal/mol (Wang et al, 2019).…”

Evidence for Shikonin acting as an active inhibitor of human carboxylesterases 2: Implications for herb–drug combination

“…Compared with hCE1, hCE2 is expressed mainly in the gastrointestinal tract and at a relatively low level in the liver. hCE2 preferentially catalyzes substrates with a large alcohol group and a small acyl group, such as antitumor drugs irinotecan (CPT‐11; Hatfield et al, ), capecitabine (Ait‐Tihyaty, Rachid, Larroque‐Lombard, & Jean‐Claude, ), the anticoagulant prasugrel (Williams et al, ), and antiandrogen flutamide (Goda et al, ).…”

“…However, the clinical use of CPT‐11 is limited by its side effects, especially life‐threatening delayed diarrhea that is mainly induced by the cytotoxicity of SN‐38 in the intestinal epithelial cells (S. J. Chen et al, ; Fujita, Kubota, Ishida, & Sasaki, ). Capecitabine, another antitumor prodrug, is metabolized first by hCE2 and then by other enzymes to 5‐fluorouracil, which is cytotoxic and frequently causes intestinal mucositis (Ait‐Tihyaty et al, ).…”

“…Chen et al, 2013;Fujita, Kubota, Ishida, & Sasaki, 2015). Capecitabine, another antitumor prodrug, is metabolized first by hCE2 and then by other enzymes to 5-fluorouracil, which is cytotoxic and frequently causes intestinal mucositis (Ait-Tihyaty et al, 2013).…”

Pilot study investigating the prognostic significance of thymidine phosphorylase expression in patients with metastatic breast cancer: a single institution retrospective analysis

“… 24 We and others have shown that inhibition of the epidermal growth factor receptor with the clinical drug gefitinib leads to an increase in TP levels in BC cells. 25 – 27 It has also been demonstrated that high tumoral TP activity may serve as an indicator of response to fluoropyrimidine therapies. 28 Due to the contradictory role of TP reported in the literature, targeting TP as a rationale for anticancer therapy or establishing its use as a biomarker remains unclear.…”