The COVID‐19 Medicine Remdesivir Is Therapeutically Activated by Carboxylesterase‐1, and Excessive Hydrolysis Increases Cytotoxicity

TO THE EDITOR:

The pandemic of coronavirus disease 2019 (COVID‐19) has become a global health crisis with the death toll of over 3 million people. So far, there are limited options to treat COVID‐19. Remdesivir was granted emergency‐use authorization earlier, and full use recently. Remdesivir was originally developed against Ebola viral infection and has since been shown to exert a broad antiviral activity against as many as seven viral families.^{( 1 )} However, serious adverse events and mortality remained high even with remdesivir among patients with COVID‐19.^{( 2}, ³, ^{4 )}

We have made concerted efforts to address the safety issues, given the promise that remdesivir is effective against a broad spectrum of viruses with pandemic potentials now and future.^{( 1 )} Specifically, we have been addressing the issues in the context of metabolism. Remdesivir is an ester prodrug and undergoes hydrolysis, most likely by carboxylesterase‐1 (CES1). Consistent with this assumption, human pooled liver samples (n = 27) efficaciously hydrolyzed remdesivir, and the hydrolysis was significantly correlated with the expression of CES1 but not CES2, although there were several outliers (Fig. 1A). The involvement of CES1 in the hydrolysis was further confirmed by recombinant CES1 (Fig. 1B). To test whether hydrolysis has toxicological significance, CES1 stably transfected cells (HEK293T) were treated with remdesivir (1 µM) for 72 hours, and the cytotoxicity was monitored. As controls, the parent and vector‐transfected cells were included. As shown in Fig. 1C, overexpression of CES1 significantly increased remdesivir cytotoxicity. The cell viability decreased by 39% (P < 0.05), and some cells were morphologically abnormal such as rounding and shrinking (Fig. 1C, images on right side).

FIG. 1.

Hydrolysis of remdesivir by human liver S9 fractions and by recombinant CES1 or CES2 and hydrolysis‐based cytotoxicity. (A) Hydrolysis of remdesivir by human‐liver S9 fractions. Remdesivir (1 µM) was incubated at 37°C for 0 or 60 minutes with individual human S9 fraction (0.1 µg/µL) (n = 26) The reactions were terminated by two volumes of termination buffer (acetonitrile and methanol: 50:50) containing the internal standard tenofovir (adenine‐13C[U]) (438 ng/mL). The reaction mixtures were subjected to centrifugation at 4°C for 15 minutes to remove the proteins, and the supernatants were analyzed for the decrease of remdesivir at mass transition of m/z 603.2 to 200 and internal standard at 293.2 to 181.1 by liquid chromatography with tandem mass spectrometry (TSQ Fortis). The quantification was determined with the standard curve generated with remdesivir. To specify the level of CES1 or CES2, S9 fractions (0.5 µg) were analyzed by western blotting and normalized with the level of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). The correlation of hydrolysis by S9 fractions was made by SPSS software (IBM, Armonk, NY). Potential outliers are indicated by arrows. (B) Hydrolysis of remdesivir by recombinant CES1 or CES2. Reactions were set up as previously with cell lysates (0.005 µg/µL) from cells transfected with CES1, CES2, or the corresponding vector. The expression of CES1 and CES2 in the lysates was confirmed by western blotting. (C) Implicated cytotoxicity of excessive hydrolysis of remdesivir cells (parent, nontransfected HEK293T; vector, vector‐transfected HEK293T; and CES1, CES1 transfected HEK293T‐CES1) were seeded as 8,000 per well in a 96‐well plate. After overnight, cells were treated with remdesivir (1 µM) for 72 hours and analyzed for proliferation using a Cell Proliferation kit (Sigma‐Aldrich, St. Louis, MO). The spectrophotometric absorbance of sample was measured using a microplate reader (Varioskan Lux; Thermo Fisher Scientific, Waltham, MA). To verify the expression of CES1, cell lysates (6.25 µg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the expression of CES1 and GAPDH was analyzed by western blotting (×250 magnification). All experiments were performed in triplicate. *P < 0.05. **P < 0.01.

It is interesting that CES1‐based hydrolysis contributes to both activation and cytotoxicity of remdesivir. This is of significance, as remdesivir, although delivering benefits, has been linked to severe adverse effects.^{( 2}, ³, ^{4 )} Added to the concern is that remdesivir is marketed as intravenous infusion, which delivers transient high systemic concentrations. As a result, the infusion formulation is inheritably related to the development of cytotoxicity. Furthermore, patients with COVID‐19 receive many other medicines that may interact with remdesivir through CES1. For example, dexamethasone, a glucocorticoid established to induce CES1,^{( 5 )} likely increases the cytotoxicity in the cells/organs with high exposure to remdesivir. Once again, remdesivir is effective against a broad spectrum of viruses with pandemic potentials.^{( 1 )} Our study concludes that considerations should be made to maximize its efficacy and minimize cytotoxicity on such factors as formulation, delivery route, dosage regimens, drug–drug interactions, and CES1 polymorphism.