In vitro selection and characterization of HIV-1 with reduced susceptibility to PMPA

9-(2-phosphonomethoxypropyl)adenine (PMPA) has demonstrated remarkable anti -simian immunodeficiency virus (SIV) activity in macaque models of SIV infe ction and transmission prevention. Recently, PMPA and its oral prodrug, bis -POC PMPA, have also shown potent antihuman immunodeficiency virus type 1 ( HIV-1) activity in Phase I clinical studies. In vitro experiments were perf ormed to address the resistance properties of PMPA. After eight passages in increasing concentrations of PMPA, HIV-1(IIIB) was able to grow in the pre sence of 2 mu M PMPA, fivefold above the IC50 of PMPA for wild-type parenta l virus. Sequence analysis of the reverse transcriptase (RT) genes from fou r of 15 RT clones demonstrated the presence of a K65R substitution in RT an d recombinant HIV expressing the K65R RT mutation showed a threefold to fou rfold increase in IC50 value for PMPA as compared to wild-type. Additional experiments demonstrated that viruses expressing other nucleoside-associate d RT resistance mutations all showed wild-type or <threefold reduced suscep tibility to PMPA in vitro. Interestingly, lamivudine-resistant viruses expr essing the M184V RT mutation showed wild-type to slightly increased suscept ibility to PMPA in vitro and addition of the M184V mutation to HIV with the K65R mutation resulted in reversion to wild-type susceptibility for PMPA. In agreement with the cell culture findings, Escherichia coil-expressed K65 R Ri showed fivefold reduced susceptibility to PMPA diphosphate, the active moiety of PMPA. Furthermore, in combination experiments, PMPA with hydroxy urea showed synergistic inhibition of HIV replication in vitro. The potent antiretroviral activity and favourable resistance profile of PMPA and bis-P OC PMPA are being further investigated in ongoing clinical trials.