Differential mechanisms of tenofovir and tenofovir disoproxil fumarate cellular transport and implications for topical preexposure prophylaxis

Ekaterina Taneva, Kerry Crooker, Sung Hyun Park, Jonathan T. Su, Adina Ott, Natalia V. Cheshenko, Igal Szleifer, Patrick F. Kiser, Bruce Frank, Pedro M M Mesquita, Betsy Herold

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Abstract

Intravaginal rings releasing tenofovir (TFV) or its prodrug, tenofovir disoproxil fumarate (TDF), are being evaluated for HIV and herpes simplex virus (HSV) prevention. The current studies were designed to determine the mechanisms of drug accumulation in human vaginal and immune cells. The exposure of vaginal epithelial or T cells to equimolar concentrations of radiolabeled TDF resulted in over 10-fold higher intracellular drug levels than exposure to TFV. Permeability studies demonstrated that TDF, but not TFV, entered cells by passive diffusion. TDF uptake was energy independent but its accumulation followed nonlinear kinetics, and excess unlabeled TDF inhibited radiolabeled TDF uptake in competition studies. The carboxylesterase inhibitor bis-nitrophenyl phosphate reduced TDF uptake, suggesting saturability of intracellular carboxylesterases. In contrast, although TFV uptake was energy dependent, no competition between unlabeled and radiolabeled TFV was observed, and the previously identified transporters, organic anion transporters (OATs) 1 and 3, were not expressed in human vaginal or T cells. The intracellular accumulation of TFV was reduced by the addition of endocytosis inhibitors, and this resulted in the loss of TFV antiviral activity. Kinetics of drug transport and metabolism were monitored by quantifying the parent drugs and their metabolites by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Results were consistent with the identified mechanisms of transport, and the exposure of vaginal epithelial cells to equimolar concentrations of TDF compared to TFV resulted in ∼40-fold higher levels of the active metabolite, tenofovir diphosphate. Together, these findings indicate that substantially lower concentrations of TDF than TFV are needed to protect cells from HIV and HSV-2.

Original languageEnglish (US)
Pages (from-to)1667-1675
Number of pages9
JournalAntimicrobial Agents and Chemotherapy
Volume60
Issue number3
DOIs
StatePublished - Mar 1 2016

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ASJC Scopus subject areas

  • Pharmacology (medical)
  • Pharmacology
  • Infectious Diseases

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Differential mechanisms of tenofovir and tenofovir disoproxil fumarate cellular transport and implications for topical preexposure prophylaxis. / Taneva, Ekaterina; Crooker, Kerry; Park, Sung Hyun; Su, Jonathan T.; Ott, Adina; Cheshenko, Natalia V.; Szleifer, Igal; Kiser, Patrick F.; Frank, Bruce; Mesquita, Pedro M M; Herold, Betsy.

In: Antimicrobial Agents and Chemotherapy, Vol. 60, No. 3, 01.03.2016, p. 1667-1675.

Research output: Contribution to journalArticle

Taneva, Ekaterina ; Crooker, Kerry ; Park, Sung Hyun ; Su, Jonathan T. ; Ott, Adina ; Cheshenko, Natalia V. ; Szleifer, Igal ; Kiser, Patrick F. ; Frank, Bruce ; Mesquita, Pedro M M ; Herold, Betsy. / Differential mechanisms of tenofovir and tenofovir disoproxil fumarate cellular transport and implications for topical preexposure prophylaxis. In: Antimicrobial Agents and Chemotherapy. 2016 ; Vol. 60, No. 3. pp. 1667-1675.
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abstract = "Intravaginal rings releasing tenofovir (TFV) or its prodrug, tenofovir disoproxil fumarate (TDF), are being evaluated for HIV and herpes simplex virus (HSV) prevention. The current studies were designed to determine the mechanisms of drug accumulation in human vaginal and immune cells. The exposure of vaginal epithelial or T cells to equimolar concentrations of radiolabeled TDF resulted in over 10-fold higher intracellular drug levels than exposure to TFV. Permeability studies demonstrated that TDF, but not TFV, entered cells by passive diffusion. TDF uptake was energy independent but its accumulation followed nonlinear kinetics, and excess unlabeled TDF inhibited radiolabeled TDF uptake in competition studies. The carboxylesterase inhibitor bis-nitrophenyl phosphate reduced TDF uptake, suggesting saturability of intracellular carboxylesterases. In contrast, although TFV uptake was energy dependent, no competition between unlabeled and radiolabeled TFV was observed, and the previously identified transporters, organic anion transporters (OATs) 1 and 3, were not expressed in human vaginal or T cells. The intracellular accumulation of TFV was reduced by the addition of endocytosis inhibitors, and this resulted in the loss of TFV antiviral activity. Kinetics of drug transport and metabolism were monitored by quantifying the parent drugs and their metabolites by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Results were consistent with the identified mechanisms of transport, and the exposure of vaginal epithelial cells to equimolar concentrations of TDF compared to TFV resulted in ∼40-fold higher levels of the active metabolite, tenofovir diphosphate. Together, these findings indicate that substantially lower concentrations of TDF than TFV are needed to protect cells from HIV and HSV-2.",
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