MALDI Mass Spectrometry Imaging Reveals Heterogenous Distribution of Tenofovir and Tenofovir‐Diphosphate in Human Colorectal Tissue

Abstract

Tenofovir (TFV) disoproxil fumarate is currently used orally in combination with other antiretroviral drugs for HIV treatment and pre‐exposure prophylaxis. Tenofovir disoproxil fumarate is a prodrug that undergoes diester hydrolysis to TFV, which upon phosphorylation to the nucleotide triphosphate analog TFV‐diphosphate (TFV‐DP) competitively inhibits HIV reverse transcriptase. In addition, TFV is under investigation as a topical microbicide for application to mucosal tissues that are routes of sexual transmission of HIV, such as colorectal tissue, in order to reduce the likelihood of infection upon exposure to HIV. In order to be efficacious as a microbicide it is necessary for a drug to be present in sufficient concentrations in the relevant tissues and over intervals of time; however, the disposition of TFV in mucosal tissues is not fully understood. With this in mind, we developed a Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) approach in order to determine the spatial distribution of TFV and TFV‐DP in colorectal tissue. For this study, colorectal biopsies were obtained from healthy volunteers (n=4) who received TFV‐containing enemas, and these biopsies were embedded with OCT medium and sectioned into 20 micron slices. Using α‐cyano‐4‐hydroxycinnamic acid as the matrix, both TFV and TFV‐DP were detected in these samples as their protonated molecular ions, [M+H]+ ions at m/z 288.0861 ± 5 ppm and 448.0188 ± 5 ppm, respectively, in positive ion mode. Confirmation of the detection of TFV and TFV‐DP in the colorectal biopsies was performed by comparing the ionization patterns to their authentic standards that yielded [M+H]+ ions at m/z 288.0851 and 448.0169, respectively. Interestingly, the MALDI MS ion images of TFV and TFV‐DP revealed heterogeneity in the distribution of both TFV and the pharmacologically active metabolite, TFVDP. In addition, a time dependence in the abundance of TFV and TFV‐DP was evident, as the intensities of both the TFV and TFV‐DP ions were greater in biopsies collected 6 hours following treatment as compared to those collected at 24 hours. In order to confirm that the patterns of heterogeneity that we observed in the spatial distributions of TFV and TFV‐DP were specific to these molecules, we simultaneously imaged the spatial localization of three different lipid species, namely PC (34:1), PC (16:0/OH) and PC (36:2) at m/z 760.5856 ± 5 ppm, 496.3403 ± 5 ppm and 786.6012 ± 5 ppm, respectively. The homogeneous distribution of these phospholipids suggested that there is specificity in the observed TFV and TFV‐DP distribution in colorectal tissue. In conclusion, the above results demonstrate the heterogeneity in the spatial distribution of both TFV and TFV‐DP in colorectal tissue. Further, our results provide important insights into the disposition of topical TFV in colorectal tissue.Support or Funding InformationThis work was funded by NIH grants U19AI113127, UM1 AI068613 and R01AI128781.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.