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Tipranavir (PNU-140690) is a non-peptidic HIV-1 protease inhibitor of the 5,6-dihydro-4-hydroxy-2-pyrone sulfonamide class, which has resulted from structure-based design efforts at Pharmacia and Upjohn [1] In addition to having good potency against a variety of HIV-1 laboratory strains and clinical isolates, tipranavir is highly active against laboratory HIV-1 isolates that are resistant to the peptidomimetic protease inhibitor ritonavir [2,3] The drug is currently under clinical evaluation. Here we investigate the viral susceptibility for tipranavir of clinical HIV-1 isolates with reduced susceptibility to other substrate analogue protease inhibitors, in particular ritonavir, indinavir or saquinavir. Recombinant viruses were used for this study, created using the amplified pool of protease genes obtained from clinical samples of protease inhibitor-treated individuals. Samples of patients extensively treated (for more than 1 year) with saquinavir (hard capsule formulation, Invirase) were analysed as well as samples obtained from the same patients 8 weeks after switching to saquinavir (oral formulation, Fortovase) or indinavir. In addition, recombinant viruses were created with the amplified and cloned protease genes obtained from longitudinal samples of a patient treated with ritonavir (M. Nijhuis, et al., in press). RNA was first extracted and amplified in a (nested) reverse transcriptase–polymerase chain reaction [4–6] The pool of amplified protease genes were sequenced using dye primers and a Applied Biosystems International (Foster city, CA, USA) automated sequencer. The amplified products of the protease gene of the ritonavir-treated patient were cloned using the TA cloning system (Invitrogen Leek, The Netherlands), and these clones were sequenced using the taq dye deoxy terminator cycle sequencing kit (Applied Biosystems International). The same amplified and cloned fragments used for sequencing were used to generate recombinant viruses in order to determine the drug susceptibility [7,8] This assay was conducted with the HIV-1 recombinant stock virus at a multiplicity of infection of 0.002 and 0.006 in the presence of increasing drug concentrations [9] All IC50 concentrations indicated reflect the geometric mean of a drug susceptibility assay performed in duplicate. Significant changes in drug susceptibility are defined by at least a threefold change in drug susceptibility relative to the reference strain HIV-1 HXB2. The susceptibility for the protease inhibitors indinavir, ritonavir, saquinavir, and tipranavir was determined for the selected viral isolates (Table 1).Table 1: Phenotypic and genotypic analysis of clinical isolates treated with different protease inhibitors. Despite the presence of known primary and secondary protease inhibitor resistance mutations (e.g. protease positions 10, 20, 36, 63, 71, 82 and 90), none of the isolates showed reduced susceptibility to tipranavir. Recombinant viruses demonstrating up to 24-fold reduced susceptibility to ritonavir did not show a significant change in susceptibility to tipranavir. Therefore in this small study we did not observe cross-resistance to tipranavir for the 11 isolates with reduced susceptibility to other protease inhibitors [10] In addition, studies by Larder and coworkers [11] who tested 125 clinical isolates with varying degrees of cross-resistance to indinavir, ritonavir, nelfinavir and saquinavir, also showed that tipranavir is active against a large selection of resistant clinical samples. Other studies by Rusconi and coworkers [12] evaluated both indinavir and ritonavir-resistant isolates. They concluded that tipranavir retained a sustained antiviral activity against protease inhibitor multidrug-resistant clinical isolates. Tipranavir is a protease inhibitor of another class and with a different mechanism of inhibition than the substrate analogue protease inhibitors. Further studies are needed to investigate its possible resistance pattern and antiviral activity. However, our results are promising in the search for new antiviral compounds with a different resistance pattern. Nicole K.T. Back Albert van Wijk DaniMlle Remmerswaal Marion van Monfort Monique Nijhuis Rob Schuurman Charles A.B. Boucher