12th International HIV Drug Resistance Workshop


10–14 June 2003, Cabo del Sol, Los Cabos, Mexico


IN VITRO CROSS RESISTANCE PROFILE OF RO033-4649 AGAINST A PANEL OF MULTIPLY-SUBSTITUTED PROTEASE INHIBITOR-RESISTANT VIRUSES: ROLE OF COMMON PROTEASE RESISTANCE MUTATIONS

Antivir Ther. 2003; 8:S21 (abstract no. 18)

G Heilek-Snyder1, A Kohli1, N Cammack1 and N Parkin2
1Roche, Palo Alto, Calif.; and 2Virologic, Inc., South San Francisco, Calif., USA

BACKGROUND: RO033-4649 is a potent, highly selective HIV-1 protease inhibitor (PI) with activity against PI-resistant viruses and a promising pharmacokinetic profile.

METHODS: Susceptibility to RO033-4649 and to amprenavir, indinavir, nelfinavir, saquinavir and lopinavir was tested in single cycle (PhenoSense™) and live HIV-1 virus assays. The fold change (FC) in IC50 from NL4-3 reference was calculated and analysed using correlation matrices to investigate relationships between protease mutations and FC, either as a continuous or dichotomous variable. FC values between groups of viruses were compared using the Mann-Whitney non-parametric test. Virus isolates were passaged in MT-4 cells in the presence of RO33-4649 and the emergence of resistant populations was analysed by sequencing of the outgrowing virus isolates.

RESULTS: In a panel of 49 clinical isolates 39 and 28 samples showed high-level resistance (FC>20) to ≥3 or ≥4 PIs, respectively. The number of samples with FC<5 and between 5 and 20 were 11 and 30 for RO033- 4649, 11 and 26 for amprenavir, 0 and 17 for indinavir, 0 and 5 for nelfinavir, 3 and 2 for saquinavir, and 0 and 12 for lopinavir. Isolates with RO033-4649 FC>20 (n=9) carried on average 14 protease mutations, with 2.67 primary mutations (primary mutations at positions 30, 46, 48, 50, 82, 84, 90. There was a significant positive correlation between RO033-4649 FC and the presence of mutations at positions 10, 33, 46, 54, 54, 71 and 73. For example, isolates containing mutations at position 73 (C, S or T) had a median FC of 17 compared to 7.6 for isolates lacking such mutations (P=0.003). A negative effect was observed in the five samples with V82F or T: compared to samples without a mutation at position 82, median FC was lower (3.9 vs 13.5, P=0.035). The two isolates with an I50V mutation also had low RO033-4649 FC (median three-fold). Cross resistance between pairs of PIs showed the highest degree of correlation with nelfinavir (r2=0.63) and lowest with amprenavir (r2=0.15). Both HXB2 and a clinical isolate G34 (M46L, G48V, I62V, L63P, T74S, V77I, V82A) were used in passaging studies at increasing drug concentrations. No mutation arose in the wild-type HXB2 culture after 11 passages, and in the clinical isolate, V32I and later I54V appeared under increasing drug pressure (120 nM and 480 nM, respectively).

CONCLUSIONS: (1) An average of 14 mutations are required before RO033-4649 demonstrates a loss of susceptibility greater than 20-fold. (2) In a background of multiple (10–18) resistance mutations and polymorphisms, clinical samples carrying G73C, S or T show reduced susceptibility to RO033-4649. (3) In a background of seven pre-existing resistance mutations, V32I and I54V arose under drug pressure, confirming the requirement of multiple mutations to confer reduced susceptibility to RO033-4649.

PRESENTING AUTHOR: G Heilek-Snyder

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2003-07-08
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