The metabolism of HIV RT inhibitors : Biochemical and clinical studies

Abstract: HIV infection is currently treated with a combination of nucleoside analogues and protease inhibitors. Nucleoside analogues inhibit HIV- I reversed transcriptase (RT), a vital enzyme in the life cycle of HIV. Nucleoside analogues are phophorylated by host-cell enzymes to their respective triphosphate. Azidothymidine (AZT) is phosphorylated to AZTMP by TK1, to AZTDP by dTMPK, and finally to AZTTP, the pharmacological active metabolite, by NDPK. There are large differences in clinical effect and adverse events in AZT treated patients. The intracellular metabolism of nucleoside analogues including AZT, and the responsible enzymes was studied in cells from healthy individuals and HIV-I infected patients. The intracellular anabolism of AZT was investigated in PHA stimulated peripheral blood mononuclear cells (PBMC). There was a block in the anabolism of AZT at the thymidylate level. AZTMP constituted 96% while the active metabolite AZTTP only 2% of total intracellular metabolites. This block increased with higher extracellular concentration of AZT. The T1/2 of AZTTP was 3.5 h compared to I h for AZT in serum. There was a large inter- and intraindividual variation in the levels of phosphorylation. In order to elucidate the reason for this large variation the activity of thymidine kinase (TK I ) and thymidylate kinase (dTMPK) was determined in extracts from PBMC. The same large variation was noted also for the enzyme activities. Furthermore, a clear decrease of TKI and dTMPK activity was noted in cell extracts from HIV-I infected patients compared to cell extracts from healthy individuals. This downregulation was not due to a different extent of cell stimulation as measured by flow cytometry. The level of dCK activity, a cell cycle independent enzyme, was not decreased in cell extracts from HIV-I infected patients. The amount of TK 1 polypeptide, determined immunologically, was directly related to the enzyme activity. To further clarify the mechanism for the different enzyme activity, a quantitative PCR method for TKI cDNA was developed. We compared AZT phosphorylation in PBMC from healthy persons and HIV infected subjects and found that TKI and dTMPK which phosphorylated d4T and AZT were decreased in cells from HIV I infected subjecs. dTMPK activity was quantified in different HIV negative cells and tissues. Three levels of thymidylate kinase activity were found, the lowest in cells without proliferative capacity e.g. brain tissue. A medium level in normal and stimulated cells and a high level in several malignant cells and Iymphoid tissue. These results have implications for antiretroviral treatment since Iymphoid cells are a reservoir for HIV-I during the asymtomatic period of the disease. dTMPK activity with AZTMP as substrate was only 0.5% as compared to when dTMP was used as substrate. Foscarnet acts as a non-phosphorylated product analogue for RT, in contrast to nucle oside analogues which are substrate analogues. The clinical effect of foscarnet was determined in a group of asymtomatic HIV infected patients. Patients without CMV infection were treated with foscarnet (50mg/kg bodyweight x 3, iv.). There was a good antiretroviral effect measured by plasma HIV-I RNA levels.