Background: Tenofovir disoproxil fumarate (TDF) is an oral prodrug and a nucleotide analog
used for treating human immunodeficiency virus (HIV) and hepatitis B virus (HBV). A growing subset of
TDF-treated HIV(+) individuals experienced drug-associated acute renal failure, suggesting drug-related
nephrotoxicity. Impurities in pharmaceutical drugs generally present a risk, and no obvious benefit.
Method: In an effort to determine whether the impurities also contribute to the nephrotoxicity, seven impurities
were isolated and identified by using nuclear magnetic resonance (NMR) and mass spectra (MS).
Human renal organic anion transporter 1 (hOAT1) localized in the kidney was the major transporter of
tenofovir and presumably mediates its accumulation in the renal proximal tubules. On the basis of the
Guidance for Industry document published by the United States Food and Drug Administration (U. S.
FDA), we conducted an uptake assay and performed inhibition analysis using the Madin-Darby canine
kidney cells stably expressing human OAT1 (MDCK-hOAT1), before analyzing our data by liquid chromatography
tandem mass spectrometry (LC-MS/MS).
Results: The uptake of the newly detected impurity RS5 was significantly higher than that of TDF. The 3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that RS5 was more
cytotoxic than TDF. The structure-affinity relationships indicated that 9-methyl functioned as a detoxification
Conclusion: Our findings provide useful information for understanding the structure and toxicity of TDF
and its impurities, and for assessing the clinical applications of TDF.