Background: The triphosphate of AL-8112, a nucleoside analog, is a potent and specific inhibitor of RSV replication in vitro. AL-8176, a prodrug of AL-8112, is in development to treat RSV infection. AL-8176 has demonstrated a rapid decline and clearance of RSV RNA compared to placebo in a challenge study in healthy volunteers (HV) infected with a clinical strain of RSV. Generally, antiviral nucleoside analogs exhibit higher barriers to the emergence of viral resistance compared to other compound classes. The resistance profile of RSV after exposure to AL-8112 is unknown.
Objectives: To characterize the development of viral resistance to AL-8112 in vitro and in vivo.
Methods: In vitro, RSV A2 was passaged in HEp-2 cells with increasing concentrations of AL-8112 or a fusion inhibitor (FI). In the challenge study, nasal wash samples were collected before, during, and after dosing. Prespecified viral load criteria to identify resistance and breakthrough were established. Using viral population sequencing, amino acid (aa) sequences were compared to the subject's first available RSV-positive sample and the reference RSV sequence.
Results: After 35 passages, AL‑8112 exhibited a >50-fold shift in activity compared to the control passaged virus in an antiviral assay. In contrast, resistance to an RSV FI developed after 4 passages. Sequencing of the genome after passaging with AL-8112 revealed 4 aa substitutions in the RSV polymerase domain; M628L, A789V, L795I and I796V (quad mutant). Clonal sequencing of 94 clones revealed that 93.6% of the genomes contained all 4 aa substitutions. Engineering the quad mutant into the RSV mini-genome system showed a 39-fold shift of the mutant vs. wild-type polymerase.
In the challenge study, no subjects met the prespecified criteria for resistance or breakthrough; consequently we evaluated 17 AL-8176- and 12 placebo- treated subjects for evidence of viral resistance. Analysis of the RSV polymerase coding domain did not detect the aa variants associated with in vitro resistance to AL-8112 or identify any new resistance-associated variants.
Conclusion: In vitro, AL-8112 demonstrated a higher barrier to resistance compared to an RSV FI. Further, we observed no emergence of resistance to AL-8112 in an RSV human challenge model.
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