Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19

Abstract

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Fig. 1. Representative thermal shift binding data of PF-00835231 with SARS-CoV-2 3CL^pro.

X-ray structures of SARS CoV-2 3CL^pro apoenzyme (left) and SARS CoV-2 3CL^pro in complex with PF-00835231 (right). Representative of melting curves with and without PF-00835231 (n = 8, n = 16 respectively).

Fig. 2. Antiviral activity across cell lines and viruses.

A In vitro antiviral activity (EC₅₀), and cytotoxicity (CC₅₀) for PF-00835231 and PF-07304814 with and without the P-gp efflux inhibitor, CP-100356. n as shown reflects individual replicates (B) EC₅₀ values with PF-00835231 with increasing P-gp inhibitor in human lung and monkey kidney cell lines. A549-ACE2 human lung carcinoma data (red) as reported in ref. . Data are presented as mean±95% confidence interval as error bars.

Fig. 3. Measuring potential synergy between PF-00835231 and remdesivir in HeLa-ACE2 cells.

A (Top) Representative antiviral dose–response curves of PF-00835231 in combination with remdesivir against SARS-CoV-2. Serial dilutions of PF-00835231 with a range of fixed concentrations of remdesivir. Mean±SD from three technical replicates. Representative of three independent experiments. (Bottom) In vitro average absolute antiviral activity shift in 50% and 90% antiviral activity with fixed concentrations of remdesivir. Average from three technical replicates each of two different patient convalescent sera. Representative of three independent experiments. B (Top) A representative three-dimensional drug interaction landscape plotting Zero Interaction Potency synergy scores analyzed using Synergyfinder (median scores of three replicates). (Bottom) Average in vitro combination synergy scores from the three experiments using two different patients’ sera (shown separately).

Fig. 4. In vivo activity of PF-00835231 in mouse models of SARS-CoV infection.

A Study design for SARS-CoV-MA15 in vivo experiments. Infection with SARS-CoV-MA15 was on day 0. Treatment began on day 0 (B–D) or day 0, 1, or 2 post-infection (E). Lungs were harvested 4 days post-infection (n = 5 biologically independent animals per group). B Change in body weight of the mice starting on day 0. C Lung viral titers for the dose response of PF-00835231. Lung titers for treatment groups were compared to vehicle using a Kruskal–Wallis test for parts (C) and (E). Uncorrected Dunn’s test was used for multiple comparisons. (p-values: 30 mg/kg= 0.15, 100 mg/kg=0.0009, 300 mg/kg=0.0003). D Representative photomicrographs from the dose–response experiment in (C) of lung sections stained with H&E (scale bar = 50 µm) or with an anti-N antibody specific for SARS-CoV (rightmost column, scale bar = 100 µm). Lungs from infected/untreated mice (top row) displayed perivascular and interstitial inflammation (top, left), degeneration and desquamation of the bronchiolar epithelium (top, middle) and proteinaceous exudate in the alveolar space with interstitial inflammatory cells (top, right), all of which were not observed in uninfected/untreated mice (middle row), or in the infected mice treated with PF-00835231 at 300 mg/kg (bottom row). IHC shows the virus within the lung tissue in the infected and untreated sample (brown stain in top right). However, treatment with PF-00835231 prevented virus from populating the lung tissue (bottom right). All images are representative of two lung sections from each of five biologically independent animals per group. E Lung viral titers from mice treated with PF-00835231 (100 mg/kg, BID) starting on day 0 or delayed by 1 or 2 days (p-values: D0 = 0.0151, D + 1 = 0.08, D + 2 = 0.29). F Mice transduced with Ad5-hACE2 were infected with SARS-CoV-2 on day 0. Treatment with PF-00835231 (100 mg/kg, BID) or vehicle started on day 0. Viral titers were determined from lungs harvested on day 3 for n = 6 biologically independent animals per group. Lung titers for the treatment group were compared to vehicle using a parametric, two-tailed, unpaired t-test (p-value= 0.0006). LOD, limit of detection; LOQ, limit of quantification; I, infected, untreated; U, uninfected; V, vehicle-treated; IU, international units. Viral Titers represent Mean±SD; Weight change shown as Mean±SEM.

Fig. 5. PF-07304814 prodrug and PF-00835231 structures and dose considerations.

A Chemical structure of conversion of prodrug PF-07304814 to the active moiety PF-00835231 by alkaline phosphatase. B Dose feasibility matrix illustrating the ability to achieve higher target exposures with increasing solubility and the limitations of dosing PF-00835231. with dosing either aqueous PF-00835231, clinically formulated PF-00835231, or aqueous PF-07304814 (prodrug). The infeasible limit (red) is assumed to be 1 L per day with a 2x potential benefit with a Cyp inhibitor (orange). Any dose under that is considered feasible (green).

Fig. 6. PF-07304814 (prodrug) and PF-00835231 in vivo exposure summary.

A Rat, dog and monkey PK following IV administration of PF-07304814 (1.17 mg/kg) or PF-00835231 (2 mg/kg rat, 1 mg/kg dog and monkey) demonstrating high levels of PF-00835231 formed in vivo. (n = 2 or 3; individual data points plotted). B Predicted human PK parameters and measured protein binding for PF-07304814 and PF-00835231 used for human dose prediction. C Projected human systemic exposure profiles at the minimally efficacious dose of 500 mg/day of PF-07304814 delivered as a continuous IV infusion. The predicted unbound steady-state concentrations for the prodrug PF-07304814 (purple) and the active moiety PF-00835231 (blue) are 0.17 µM and 0.5 µM respectively. (NOAEL = No Observed Adverse Effect Level; C_eff = projected minimally efficacious concentration, Fu= unbound fraction; Clp= Plasma Clearance; Vdss= Volume of distribution steady state, Cssu = unbound steady-state concentration).