New test to measure intracellular levels of nucleosides

Simon Collins, HIV i-Base

Grassi and colleagues from the Pharmacology and Immunology Unit of CEA (French Atomic Energy Commission) presented several examples of results from their research into developing an assay to measure intracellular levels of nucleoside triphosphates in PBMCs using liquid chromatography coupled with mass spectrometry (LC/MS/MS).

Unlike protease and NNRT inhibitors, plasma levels of nucleoside analogues do not generally or usefully correlate with intracellular levels and measuring intracellular levels are not particularly easy even in a research setting.

Simpler assays to monitor these levels are much needed. In addition to individual monitoring, they would enable a more thorough understanding of the importance of both drug and food interactions, drug dosing and the safety of changes to once-daily dosing and the comparison of new formulations.

From a patient perspective, the ability to measure intracellular levels more easily would have clarified the confusion over the various formulations of ddI and changing recommendations for the interaction with food, and more recently the three-way interactions between ddI, food and tenofovir.

Validation for the direct LC/MS/MS is within international guidelines of 15% in routine conditions. The assay also allows simultaneous quantification of several nucleotides. They are explained in detail in published articles in Anal Chem 74(16):4220-4227 (2002) and Rapid Commun Mass Spectrom 16:555-565 (2002).

Using the assay to examine intracellular behaviour of d4T and ddI, they report half-lives of d4T-TP at around seven hours and >24 hours for ddA-TP (the intracellular metabolite of ddI) and report a weak correlation between intracellular levels of each in treatment naïve patients using d4T and ddI together.

This team also outlined again their research that generated significant attention at the Glasgow Conference last November and recently published in March 7th issue of AIDS. When measuring levels of AZT-TP when developing this test, they unexpectedly discovered significant levels of d4T triphosphate in 10 d4T-naïve patients who were taking AZT-containing combinations. This may provide insight into the complicated cross-resistance between the two drugs. How and why d4T-TP should be detected at all is still unexplained, and it is unclear whether it is formed as part of the process of AZT phosphorylation or as a breakdown product of AZT-TP.

It is hoped that further development of the assay will lead to new and exciting pharmacological research and add to our knowledge of how to most effectively use many of the oldest and most essential HIV drugs that continue to provide the backbone for current treatment.

Reference:

Grassi J, Becher F, Pruvost A et al. New light on the intracellular pharmacology of NRTIs in HIV-infected patients. 4th Intl Workshop on Clinical Pharmacology of HIV Therapy, Cannes 27th-29 March 2003. Abs 52 P7.1.

Comment

Regarding the biological plausibility of in vivo AZT to d4T conversion may certainly be possible, as AZT and d4T are structurally very closely related and it would only require a single reduction reaction to chemically convert AZT to d4T.

If this reduction reaction could occur enzymatically in vivo this might explain the findings. What was surprising is the amount of d4T triphospate detected in some of the patients. This may also account for some of the cross toxicities seen with these agents.