29. Advanced Applications of the Triple Quadropole/Linear Ion Trap Mass Spectrometer and High Performance Liquid Chromatography for Identification and Quantitative Measurement of HIV Prophylactic Drug Metabolites in Macaque Blood and Tissue
Tue 12:24 PM - PosterSplash Track 3
Zsuzsanna Kuklenyik
*Zsuzsanna Kuklenyik, #Angela Holder, #Chou-Pong Pau, #Ae S. Youngpairoj, #Qi Zheng, #Mian-er J. Cong, #Wutyi Aung, #Gerardo Garcia-Lerma, #Walid Heneine, *James L. Pirkle, *John R. Barr

* Division of Laboratory Sciences National Center for Environmental Health Centers for Disease Control and Prevention
# Division of HIV/AIDS Prevention Intervention National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention Centers for Disease Control and Prevention
In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs is a promising novel strategy to prevent HIV transmission. Emtricitabine (FTC) and tenofovir (TFV) are HIV reverse transcriptase inhibitors that are being considered for PrEP because of their favorable safety profiles and long intracellular half-lives. The intracellular metabolite that is able to inhibit HIV transcription is the triphosphorylated anabolite of these drugs. The concentrations of the triphosphate metabolites depends both on the efficacy of the drug delivery into the target cells and on the rate of subsequent metabolite depletion. To aid the design of effective intervention strategies on macaques we developed high performance liquid chromatography mass spectrometry methods for monitoring of both cellular drug delivery and intracellular metabolism.

The application of a hybrid triple quadrupole/linear ion trap mass spectrometer and coupled HILIC/anion exchange HPLC separations allowed both identification and simultaneous quantitative measurement of FTC and TFV pro-drugs (TDF and GS7430) simultaneously with their mono- di- and tri-phosphorylated metabolites (MP, DP, and TP). Limits of detections were in the range of 0.1-1 ng/sample. In addition to quantitative measurements, the presence of FTC and TFV metabolites in macaque samples and in thermally degraded solutions of metabolite standards and pro-drugs were monitored by scanning for precursors of structure specific collision fragments of TFV (176, 270 and 288 m/z) and FTC (130 m/z). The presence of the metabolites was confirmed by information dependent enhanced product ion scans using both the triple quadruple and linear ion trap functionalities of the MS instrument. We also developed sensitivity and selectivity enhanced reverse phase HPLC methods for monitoring low ng/mL levels of FTC and TFV in plasma, and coupled weak anion exchange/ion pair method for low pg/million cell detection of TFC-TP and TFV-DP metabolites in peripheral blood mononuclear cells.

These methods were applied for the evaluation of the efficacy of event-driven PrEP with Truvada (FTC and TDF combination) in macaques in relation to drug pharmacokinetics in blood and tissues (rectal, vaginal, cervical) and lymph nodes collected from orally or vaginally dosed macaques.

Our findings in Truvada dosed macaques suggest that the rapid FTC penetration in rectal tissues and the long TFV-DP persistence likely complement each other to maximize the effectiveness of PrEP.