Covalent CES2 Inhibitors Protect against Reduced Formation of Intestinal Organoids by the Anticancer Drug Irinotecan

Abstract

Background: Irinotecan is widely used to treat various types of solid and metastatic cancer. It is an ester prodrug and its hydrolytic metabolite (SN-38) exerts potent anticancer activity. Irinotecan is hydrolyzed primarily by carboxylesterase-2 (CES2), a hydrolase abundantly present in the intestine such as the duodenum. We have identified several potent and covalent CES2 inhibi¬tors such as remdesivir and sofosbuvir. Remdesivir is the first small molecule drug approved for COVID-19, whereas sofosbuvir is a paradigm-shift medicine for hepatitis C viral infection. Irinotecan is generally well-tolerated but associated with severe/life-threatening diarrhea due to intestinal accu¬¬mula¬tion of SN-38. Objective: This study was to test the hypothesis that remdesivir and sofosbuvir protect against irinotecan-induced epithelial injury associated with gastrointestinal toxicity. Methods: To test this hypothesis, formation of organoids derived from mouse duodenal crypts, a robust cellular model for intestinal regeneration, was induced in the presence or absence of irinotecan +/- pretreatment with a CES2 drug inhibitor. Results: Irinotecan profoundly inhibited the formation of intestinal organoids and the magnitude of the inhibition was greater with female crypts than their male counterparts. Consistently, crypts from female mice had significantly higher hydrolytic activity toward irinotecan. Critically, remdesivir and sofosbuvir both reduced irinotecan hydrolysis and reversed irinotecan-reduced formation of organoids. Human duodenal samples robustly hydrolyzed irinotecan, stable CES2 transfection induced cytotoxicity and the cytotoxicity was reduced by CES2 drug inhibitor. Conclusion: These findings establish a therapeutic rationale to reduce irinotecan-gastrointestinal injury and serve as a cellular foundation to develop oral formulations of irinotecan with high safety.

Regulation of human hydrolases and its implications in pharmacokinetics and pharmacodynamics

“…Different from CES1, CES2 is mainly expressed in the small intestine and also expressed to a lesser extent in the kidney, liver, heart, brain, and testis (Figure 2) (Satoh et al, 2002;Taketani et al, 2007;Sanghani et al, 2009). CES2 substrates include acebutolol, aspirin, flupirtine, flutamide, heroin, irinotecan, gemcitabine (Pratt et al, 2013), doxazolidine (Barthel et al, 2009), molnupravir (Shen et al, 2022), and capecitabine (Quinney et al, 2005) (Table 1). Similar to CES1, CES2 expression and activity can be regulated by both genetic and nongenetic factors.…”

Viral target and metabolism‐based rationale for combined use of recently authorized small molecule COVID‐19 medicines: Molnupiravir, nirmatrelvir, and remdesivir

“…Carboxylesterases constitute a large class of serine hydrolases with high catalytic efficiency. In humans, CES1 and CES2 are two c arboxyl es terases with known pharmacological and toxicological significance 106–109 . These two carboxylesterases, on the other hand, differ in their substrate specificity and tissue distribution 110–112 .…”

“…Remdesivir, on the other hand, is hydrolytically activated, and the hydrolysis is catalyzed by CES1 96 . At the same time, remdesivir is a covalent inhibitor of CES2, 97 a hydrolase that therapeutically activates molnupiravir 98,106 . It has been shown that the expression of CES1 is inversely correlated with the inhibition of CES2, suggesting that remdesivir but not its hydrolytic metabolite is a CES2 inhibitor.…”

“…In humans, CES1 and CES2 are two carboxylesterases with known pharmacological and toxicological significance. [106][107][108][109] These two carboxylesterases, on the other hand, differ in their substrate specificity and tissue distribution. [110][111][112] CES1 preferably hydrolyzes esters with an acyl moiety relatively larger than its alkoxy moiety, 96 and opposite is true with CES2.…”

“…96 At the time, remdesivir is a covalent inhibitor of CES2, 97 a hydrolase that therapeutically activates molnupiravir. 98,106 It has been shown that the expression of CES1 is inversely correlated with the inhibition of CES2, suggesting that remdesivir but not its hydrolytic metabolite is a CES2 inhibitor. Nevertheless, copresence of remdesivir would likely impact the hydrolytic activation of molnupiravir.…”