Comprehensive genotoxicity and carcinogenicity assessment of molnupiravir

Abstract

Molnupiravir is registered or authorized in several countries as a 5-d oral coronavirus disease 2019 treatment for adults. Molnupiravir is a prodrug of the antiviral ribonucleoside β-D-N4-hydroxycytidine (NHC) that distributes into cells, where it is phosphorylated to its pharmacologically active ribonucleoside triphosphate (NHC-TP) form. NHC-TP incorporates into severe acute respiratory syndrome coronavirus 2 RNA by the viral RNA-dependent RNA polymerase, resulting in an accumulation of errors in the viral genome, leading to inhibition of viral replication and loss of infectivity. The potential of molnupiravir to induce genomic mutations and DNA damage was comprehensively assessed in several in vitro and in vivo genotoxicity assays and a carcinogenicity study, in accordance with international guideline recommendations and expert opinion. Molnupiravir and NHC induced mutations in vitro in bacteria and mammalian cells but did not induce chromosome damage in in vitro or in vivo assays. The in vivo mutagenic and carcinogenic potential of molnupiravir was tested in a series of in vivo mutagenicity studies in somatic and germ cells (Pig-a Assay and Big Blue® TGR Mutation Assay) and in a carcinogenicity study (transgenic rasH2-Tg mouse), using durations of exposure and doses exceeding those used in clinical therapy. In vitro genotoxicity results are superseded by robustly conducted in vivo studies. Molnupiravir did not increase mutations in somatic or germ cells in the in vivo animal studies and was negative in the carcinogenicity study. The interpretation criteria for each study followed established regulatory guidelines. Taken together, these data indicate that molnupiravir use does not present a genotoxicity or carcinogenicity risk for patients.

Keywords: antiviral; carcinogenicity; genotoxicity; molnupiravir; mutagenicity; β-D-N4-hydroxycytidine (NHC).

Fig. 1.

Major routes of molnupiravir metabolism.

Fig. 2.

In vitro (a) and in vivo (b) micronucleus assay for chromosome breakage and chromosome loss after treatment with molnupiravir. Positive controls: CP, MMC, VIN. The dotted line represents the upper 95% CI of the historical vehicle control distribution. CI, confidence interval; CP, cyclophosphamide; MMC, mitomycin C; MN, micronucleated; PCE, polychromatic erythrocyte; S9, Aroclor 1254-induced rat liver; VIN, vinblastine.

Fig. 3.

Molnupiravir (a/b) and β-D-N4-hydroxycytidine (c/d) Ames assay results without (a and c) and with (b and d) S9 metabolic activation. All strains were assessed up to either 5,000 µg/plate or toxicity-limited concentrations. S9, Aroclor 1254-induced rat liver.

Fig. 4.

Pig-a mutation assay in rats after 28-d treatment with molnupiravir. Mutation frequency in reticulocytes (a) and mutation frequency in red blood cells (b). Positive control: ENU 20 mg/kg administered on 6 nonconsecutive days; *P ≤ 0.05. Dotted line represents upper 95% CL of the historical vehicle control study mean distribution. CL, confidence limit; ENU, N-Nitroso-N-ethylurea; HC, historical control; PC, positive control.

Fig. 5.

Big Blue^® Transgenic Rat Gene Mutation Assay in somatic cells after 28-d treatment with molnupiravir. Mutation frequency in bone marrow (a) and mutation frequency in liver cells (b). The dotted line represents the upper 95% CL of the historical vehicle control study mean distribution. Positive control: ENU 20 mg/kg administered on 6 nonconsecutive days (ENU); *P ≤ 0.05. CL, confidence limit; ENU, N-Nitroso-N-ethylurea; HC, historical control; PC, positive control.

Fig. 6.

Germ cell development over a 28-d exposure window and 70-d treatment-free periods accounts for the spermatogenic cycle kinetics in rats and maximizes exposure in undifferentiated spermatogonia (stem cells). The full line denotes the developmental trajectory of spermatogonia that received a full 28-d exposure to molnupiravir into sperm that were collected from the cauda epididymis at the end of the treatment-free period.

Fig. 7.

Big Blue^® Transgenic Rat Gene Mutation Assay in sperm from cauda epididymis after 28-d treatment and a 70-d treatment-free period. The vehicle was methylcellulose, 1% (wt/vol), in deionized water. The dotted line represents the upper 95% CL of the historical vehicle control study mean distribution. Positive control: ENU dose was 20 mg/kg administered on days 1, 2, 3, 12, 19, and 26. CL, confidence limit; ENU, N-Nitroso-N-ethylurea; HC, historical control; PC, historical positive control.