Fig 1.
Conceptual design of evaluation matrix for antiviral activity and cytotoxicity of drug combinations in vitro.
Cytotoxicity (upper left) is expressed as cell viability; Antiviral activity (lower left) is expressed as 100% or <100% inhibition of virus replication; LIC100 is the lowest drug concentration that completely inhibits virus replication; fold activity increase (fold changes in LIC100) and area of synergy are shown. The lower left well in each panel represents DMSO-containing medium with no drugs as control. 3D representations of the results are shown in the right part of the figure where synergy is only detected for activity, not for cytotoxicity.
Fig 2.
Antiviral activity of drug combinations.
The model analyzes all the possible combinations of three drugs in a three-dimensional diagram (pyramid). Experimental outcomes: (left) synergistic effect, that is a pyramid up; (center) no interaction, that is no pyramid; (right) antagonistic effect, that is a pyramid down.
Table 1.
Synergistic antiviral activity of RDV-based cocktails.
Fig 3.
Analyses of RDV and AZI combination.
(A) RDV and AZI lowest concentrations required for 100% inhibition of SARS-CoV-2 (LIC100) are lowered by an average of 4- and 12-fold, respectively, when the drugs are combined in vitro. (B) RDV antiviral activity in vitro vs. exposure in vivo in human plasma (PK data was adapted from [9]). RDV monotherapy in vitro has a LIC100 (upper arrow) that cannot be consistently reached in the plasma with standard I.V. dosages. Combining RDV with AZI reduces in vitro LIC100 (lower arrow) down to levels that can be consistently maintained in the plasma for at least 1 hour. (C) Antiviral activity of AZI in vitro vs. exposure in vivo in human plasma after I.V. administration (adapted from [12]). AZI monotherapy in vitro has a LIC100 (upper arrow) that cannot be reached in the plasma with standard (e.g., 1, 2, 4 grams) I.V. dosages. Combining AZI with RDV reduces the in vitro LIC100 (lower arrow) down to levels that can be maintained in the plasma for up to 8 hours. (D) Antiviral activity of AZI in vitro vs. exposure in vivo in human tissues after oral administration (calculated through Pfizer’s azithromycin PK Simulator [https://azpksim.pfizer.com]). AZI monotherapy in vitro has a LIC100 (upper arrow) that cannot be reached in the lung after per os administration of 500 mg AZI once daily for 7 days. Combining AZI with RDV reduces in vitro LIC100 (lower arrow) down to levels that can be maintained in the lung for up to 21 days.
Table 2.
Complete inhibition of SARS-CoV-2 replication in the lung is achievable by RDV + AZI and RDV + IVM, but not by RDV monotherapy.
Fig 4.
Analyses of RDV and IVM combination.
(A) RDV and IVM lowest concentrations required for 100% inhibition of SARS-CoV-2 (LIC100) are lowered by an average of 6- and 13-fold, respectively, when the drugs are combined in vitro. (B) RDV antiviral activity in vitro vs. exposure in vivo in human plasma (PK data was adapted from [9]). RDV monotherapy in vitro has a LIC100 (upper arrow) that cannot be consistently reached in the plasma with standard I.V. dosages. Combining RDV with IVM reduces in vitro LIC100 (lower arrow) to levels that can be consistently maintained in the plasma for at least 1.5 hours. (C) Antiviral activity of IVM in vitro vs. exposure in vivo in the plasma and the lung (adapted from [11]). IVM monotherapy in vitro has a LIC100 (upper arrow) that can be reached neither in the plasma nor in the lung with standard oral dosages. Combining IVM with RDV reduces the in vitro LIC100 (lower arrow) to levels that can be maintained in the plasma for 16 hours and in the lung for 30 hours (blue dotted line). Both LIC100 values and plasma concentrations are expressed as ng/mL.
Table 3.
Increased activity and broadened Therapeutic Index (TI) by combining RDV with either AZI or IVM.
Upper Table: Comparison of the effect of drug combinations on antiviral activity and cytotoxicity. Lower Table: TI is calculated here as the ratio between the highest HCC0 and the lowest LIC100 as monotherapy and among all RDV-based combinations.
Fig 5.
Mechanism of synergy between RDV and AZI.
RDV and AZI synergize both at entry and post-entry level, synergy being optimal with continuous treatment. (A) Drugs were added only at the time of infection; (B) Drugs were added only after infection; (C) Drugs were added throughout the experiment. In all cases Vero E6 cells were seeded at Day 0, infection was performed at Day 1 and readout was measured at Day 4. The results of the Mac Synergy II, Synergy Plot 99.9% analyses are shown in the right part of each panel. Tx = treatment.