ABSTRACT:
Several studies had shown a complex picture regarding the -cross resistance to protease inhibitors (PIs) both in vitro and in vivo. In the present study we have examined the anti-HIV activity of a novel HIV-1 PI, tipranavir, on isolates derived from patients with multidrug resistance (MDR) to other PIs. The purpose of our experiments was to investigate the enotypic and in vitro phenotypic drug resistance of tipranavir (Pharmacia & Upjohn). We evaluated several drug-resistant isolates derived from patients who were undergoing anit-HIV-1 combination therapy. All the isolates were demonstrated to be both indinavir- and ritonavir-resistant. Several different clinical isolates and laboratory adapted strains of HIV-1 were used as controls. We investigated the sulfonamide-containing non-peptidic protease inhibitor tipranavir, indinavir, ritonavir and nelfinavir.
The virus-inhibitory concentrations of tipranavir were evaluated in PBMC. In all of the experiments, uninfected drug toxicity controls were maintained. Virus replication was measured in cell-free culture supernatant fluids by an HIV-1 p24 antigen ELISA. We carried out drug susceptibility tests using a fixed amount of infectious virus (1000 TCID50) to determine the IC50 and IC90, PCR assays for drug resistance mutations in plasma RNA, and direct sequencing of PCR products. Phenotypic resistance to PIs was invariably related to genotypic mutations. The substitutions among the amino acid residues of the protease included: L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I61V, A71V, V77I, V82A, I84V and L90M, variable combined.
All of the patients had developed a maximum degree of resistance to both indinavir, ritonavir and nelfinavir (IC > 0.1 µM). We also compared these mutations with few amino acid substitutions observed in only a small number of patients on tipranavir for 24 weeks. These changes included the following positions:I15V, E35D, N37D, R41K, D60E and A71T. Tipranavir potently suppressed viral replication of laboratory and clinical strains. Infections with IIIB, 14aPre and N70 HIV-1 strains were inhibited by an average drug IC90 concentration of 0.18±0.02 µM in multiple experiments. The average mean±SEM of the IC90 for the entire group of MDR isolates, derived from the mean of two culture-well supernatant p24 antigen values, appeared to be 0.56±0.11µM (range 0.310.86 µM). Tipranavir retained a sustained antiviral activity against PI MDR clinical isolates. Tipranavir might be useful in combination regimens with other antiretrovirals for patients who have already failed other PI-containing therapies.
