15th International HIV Drug Resistance Workshop 13-17 June 2006, Sitges, Spain

IN VITRO SELECTION STRATEGY AND CHARACTERIZATION OF RESISTANCE TO A NOVEL HIGHLY POTENT HIV PROTEASE INHIBITOR SPI-256

Antivir Ther. 2006, 11:S30 (abstract no. 25)

E Afonina, S Gulnik, H Yokoe and J Erickson
Sequoia Pharmaceuticals, Inc., Gaithersburg, MD, USA

BACKGROUND: SPI-256 is highly active against WT-HIV with IC₅₀ values ≤2 nM in MT4 and PBMC cell based assays and ≤0.5 nM in the Phenosense assay; it retains high potency against MDR-HIV strains. The high potency of SPI-256 against primary PI mutations suggested that it may be difficult for WT HIV to evolve resistance against this PI. The aims of this study were to establish pathways of viral escape under SPI-256 selection pressure, to compare escape pathways starting from WT versus. MDR HIV genotypes, and to assess the impact of selected mutations on antiviral potency of SPI-256.

METHODS: In vitro selection experiments were performed by passaging WT-HXB2 HIV or MDR-HIV patient isolates (M4 and M7) in MT4 cells, in the presence of increasing concentrations of SPI-256. Both M4 and M7 contained numerous PI-resistance mutations, were sensitive to SPI-256 (IC₅₀s of 19 and 1 nM, respectively), and were highly cross-resistant to FDA approved PIs (IC₅₀ range 0.08-8 µM). Viral RNA was extracted from passaged virus and subjected to populational and clonal sequencing. Phenotypic characterization of selected mutant strains was done using a cell-based MTT assay.

RESULTS: Selection experiments were initiated using a drug concentration that was at the phenotypic IC₅₀ value, and drug concentrations were increased in small increments, often not more than 30%. About 136 days and 18 passages were necessary to establish a virus population growing at 90 nM of SPI-256 (45 × IC₅₀). Genotypic analysis of selected virus revealed the presence of L10F, M46I and I50V mutations in the protease gene with additional mutations in p7/p1 and p1/p6 cleavage sites. Selection of resistant virus starting from M4 and M7 viruses also required multiple passages: a 40-fold increase from the starting selection concentration was achieved after 50 days. Escape viruses from both the M4 and M7 selections contained I50V. Further passaging of WT virus led to the addition of I47V protease substitution. Mutant viruses selected using WT, M4 and M7 viruses were used for phenotypic susceptibility testing. SPI-256 retained nanomolar potency against all selected mutants (range 9-170 nM). The IC₅₀ of SPI-256 against the virus containing I50V and L10F, and the gag mutation, A431V increased ~5 fold compared to WT.

CONCLUSIONS: I50V appeared to be a common mutational escape pathway to SPI-256 for WT as well as highly divergent, MDR PI-resistant viruses. However, the selection of virus resistant to SPI-256 was a very slow process and required multiple passages with small incremental increases of concentration. These results support the potential use of SPI-256 in both first-line and salvage therapy regimens.

2006-06-13
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