In an effort to identify inhibitors of the SARS-CoV-2 main protease (Mpro) which cleaves to polyproteins to form structural proteins that are vital for viral replication, investigators developed 7 different investigational COVID-19 oral antiviral candidates that inhibit Mpro. They selected compound 6, Paxlovid (PF-07321332/ritonavir), for further testing since it could be easily scaled up synthetically, had increased solubility allowing for a simple formulation vehicle in support of preclinical toxicology, and also was less likely to epimerize at the P1 stereocenter. These findings were published in Science.

In fluorescence resonance energy transfer (FRET) Mpro assays, Paxlovid demonstrated potent inhibition of Mpro from all coronaviruses known to infect humans (6, 7, and 30), including beta-coronaviruses SARS-CoV-2, SARS-CoV-1, HKU1, OC43, Middle East respiratory syndrome (MERS), and alpha-coronaviruses 229E and NL63. Of note, the investigators observed no inhibitory effects against several mammalian cysteine, serine, and aspartyl proteases at the highest concentration of Paxlovid tested.

The investigators found that Paxlovid demonstrated in vitro viral inhibition in human adenocarcinoma-derived alveolar basal epithelial (A549) cells expressing ACE2, as well as in differentiated normal human bronchial epithelial (dNHBE) cells. In addition, Paxlovid had potent in vitro antiviral activity against SARS-CoV-1, MERS, and human coronavirus 229E in cytopathic effect (CPE) assays.


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To evaluate the in vivo antivral activity of Paxlovid, investigators used a mouse-adapted SARS-CoV-2 model. Mice treated with either a 300- or 1000-mg/kg dose of Paxlovid twice daily were found to have a decreased risk for weight loss compared with vehicle-treated mice. On analysis of lung titers 4 days postinfection, control mice (n=12) developed a severe respiratory infection and mice treated with Paxlovid had significantly decreased viral loads.

Histopathologic analysis and immunostaining of lungs from mice infected with SARS-CoV-2 MA10 showed that Paxlovid limits cellular infiltration and confers increased protection against lung tissue damage caused by viral replication compared with vehicle-treated mice. Additionally, immunohistochemical analysis showed that Paxlovid inhibits viral replication in a dose-dependent manner.

Intravenous Paxlovid was found to have moderate plasma clearance, with an elimination half-life of 5 hours in rats and less than 1 hour in monkeys. In addition, the oral bioavailability of Paxlovid was increased in rats (range, 34-50%) compared with monkeys (8.5%). After analyzing the drug-metabolizing enzyme effects of Paxlovid, the investigators found that the compound was significantly inhibited (>82%) by the selective CYP3A4/5 inhibitor ketoconazole.

The investigators noted that Paxlovid had a favorable off-target selectivity profile in a broad panel of G protein-coupled receptors, kinases, transporter and phosphodiesterase enzyme inhibitor screens, and it showed no activity against cardiac ion channels Kv1.1, Cav1.2, or Nav1.5. In addition, Paxlovid was found to be neither mutagenic nor clastogenic in vitro genetic toxicity assays, and an in vivo rat micronucleus assay was negative. Paxlovid was well tolerated in both rats and monkeys, with no adverse events observed at the highest doses tested (600 mg/kg/day in monkeys and 1000 mg/kg/day in rats).

In an ongoing randomized, double-blind, placebo-controlled single ascending dose study, investigators assessed the safety, tolerability, and pharmacokinetics of Paxlovid, alone and in combination with ritonavir (RTV), in healthy adults. They found that PF-07321332 alone was safe and well-tolerated, and co-administration with RTV, a potent CYP3A4 inactivator, led to a significant increase in plasma concentrations.

Additionally, 12 hours after the first dose of Paxlovid 250 mg, oral plasma concentrations were significantly increased compared with the SARS-CoV-2 antiviral EC90 value. The investigators noted that this finding leads to “increasing confidence in achieving robust pan-coronavirus antiviral activity [in clinical settings],” with Paxlovid.

Disclosure: Some author(s) declared affiliations with biotech, pharmaceutical, and/or device companies. Please see the original reference for a full list of disclosures.

Reference

Owen DR, Allerton CMN, Anderson AS, et al. An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19Science. Published online November 2, 2021. doi:10.1126/science.abl4784