Molnupiravir Active Metabolite–N4 hydroxycytidine causes Cytotoxicity to Mammalian CellsIn Vitro

Abstract ID 93714 Poster Board 212 Since COVID-19 pandemic, many old and new antiviral drugs have been used for treatment of the disease including molnupiravir. N⁴-hydroxycytidine (NHC), the active form of molnupiravir, incorporates into SARS-CoV-2 RNA leading to false base-pairing and lethal mutations to the virus. Many had concerns about the risk of non-specific mutation in host cells, as suggested in previous published studies, causing toxicity to the patients. The study determined NHC cytotoxic effects on human lung large cell carcinoma (COR-L23) and hepatoblastoma (HepG2) cell lines by using sulforhodamine B (SRB) colorimetric assay. The cells were seeded in 96-well plates and treated with NHC (0.1-30 μM) for 3, 5, and 10 days. The media and NHC solutions were replaced every 3 days. Dose-response curves were obtained, and the half-maximal inhibitory concentration (IC₅₀) values were calculated by using GraphPadPrsim9. Data were presented as mean±SD. NHC induced cytotoxicity to COR-L23 cells in concentration-(0.1-30μM), and time-(3-10 days) dependent manners (pconcentration = 0.0007, ptime = 0.0485, two-way ANOVA). On the other hand, HepG2 cells showed cytotoxicity in response to NHC treatment in concentration-(0.1-30μM), but not time-(3-10 days) dependent manner, (pconcentration < 0.0001, ptime = 0.1984, two-way ANOVA). IC₅₀ values of NHC were lower in COR-L23 than that in HepG2 at 3 days (6.76±0.63 vs 19.16±2.99μM, p = 0.0230), 5 days (4.56±1.16 vs 12.40±2.00μM, p = 0.0145), but not at 10 days of treatment (5.91±0.97 vs 15.41±10.62μM, p = 0.4611, one-way ANOVA). The data suggest that COR-L23 is more susceptible to NHC-induced cytotoxicity compared to HepG2. Moreover, the cytotoxic effects of NHC in these two cancer cells were compared to that effect in a normal cell line-- human keratinocyte cells (HaCaT) in recently published data. At 3 days of treatment, 30μM NHC induced a maximal reduction in cell viability to 23.21±0.37, 49.31±3.56, and 52.98±10.91% for HaCaT, COR-L23 and HepG2, respectively (p = 0.0272), and IC₅₀ values were 4.40±0.09 vs 6.76±0.63 vs 19.16±2.99, respectively (p = 0.0001, one-way ANOVA). At 5 days of treatment, HaCaT and COR-L23, but not HepG2, showed similar degree of decreased cell viability (26.64±1.19, 14.67±0.61, 62.29±18.86%) in response to 30μM NHC. IC₅₀ values were 5.82±0.91, 4.56±1.16, 12.40±2.00μM, respectively (p = 0.0011). At 10 days of NHC treatment, all 3 cell types exhibited similar reduction of cell viability. IC₅₀ values were 5.41±0.88, 5.91±0.97, 15.41±10.62μM, respectively (p=0.1627). Taken together, the active metabolite of molnupiravir, NHC exerted robust cytotoxicity to normal human keratinocyte cells, but less cytotoxicity to lung and liver cancer cells. Lung cancer cells (COR-L23) were more susceptible to NHC-induced cytotoxicity compared to liver cancer cells (HepG2). Previous study reported that therapeutic dose of 800 mg molnupiravir every 12 hours for 5 days resulting in serum concentration of NHC at 10.8 μM, which relatively relates to NHC IC₅₀ values obtained in the current work. We would recommend that usage of molnupiravir must be used under consideration and supervision, particularly in patients with impaired xenobiotic clearance until future investigations. Moreover, the therapeutic response and toxicity mediated by molnupiravir could possibly be different in patients with cancer. Financial support This project is supported by Faculty of Medicine, Thammasat university. Grant agreement number ทป.2-36/2565 (to SS).