Intracellular concentrations of antiretroviral drugs in peripheral blood mononuclear cells (PBMCs) are an important determinant of therapeutic success. In vitro data indicate that efavirenz induces several ATP-binding cassette (ABC) transporters, and pharmacogenetic studies found an association between ABCB1(C3435T) and efavirenz exposure and between this polymorphism and improved virological outcomes. We therefore aimed to clarify whether efavirenz also induces ABC transporters in vivo in PBMCs and whether intracellular concentrations might be altered after induction. Twelve healthy individuals received multiple oral doses of efavirenz over 14 days (400 mg once daily). Blood samples were drawn on study days 1 (single dose) and 14 (multiple dose), and efavirenz concentrations were analyzed by liquid chromatography-tandem mass spectrometry. Expression of P glycoprotein (P-gp) and of the multidrug resistance-associated proteins 1 and 2 as well as P-gp activity was analyzed in PBMCs on day 1 and day 14 using real-time reverse transcription-PCR (RT-PCR) and rhodamine 123 efflux. Although a clear autoinduction could be confirmed by a significant decrease of efavirenz exposure from day 1 to day 14, efavirenz did not change expression of the ABC transporters or P-gp activity in PBMCs. Moreover, intracellular concentrations of efavirenz were 1.3-to 1.8-fold higher than the corresponding plasma concentrations, and the intracellular/plasma concentration ratio remained constant during the treatment and did not correlate with ABC transporter expression or function. In conclusion, our study confirmed that intracellular concentrations of efavirenz are independent from these efflux transporters and demonstrated for the first time that the transporters are not induced in PBMCs in vivo after 2 weeks of treatment with efavirenz.

The ability of different stationary phases developed for the analysis of polar compounds (ZIC-HILIC, ZIC-pHILIC and Zorbax SB-Aq) to separate isoniazid, its metabolites (acetylisonazid, pyridoxal isonicotinoyl hydrazone, pyridoxal isonicotinoyl hydrazone 5-phosphate), pyridoxine, pyridoxal and pyridoxal 5-phosphate under MS compatible conditions was systematically investigated using HPLC-UV. The mobile phase strength, pH and buffer concentration were modified to assess their impact on the retention of these compounds. The best available separation of the compounds was achieved using 1 mM ammonium formate (pH≈6) and ACN (20:80, v/v) on ZIC-HILIC and employing 5 mM ammonium formate (pH 3.0) and ACN (40:60, v/v) on ZIC-pHILIC. A gradient profile using 0.5 mM ammonium formate (pH≈6) and MeOH (0-12 min: 10% MeOH, 12-15 min: 10-50% MeOH, 15-35 min: 50% MeOH, 35.0-35.2 min: 50-10% MeOH, 35.2-45.0 min: 10% MeOH) provided the best separation of the compounds on Zorbax SB-Aq. Subsequent LC-MS analysis demonstrated that ZIC-HILIC is useful for the analysis of pyridoxine, pyridoxal and pyridoxal isonicotinoyl hydrazone. However, the chromatographic conditions developed for the analysis of the compounds on Zorbax SB-Aq are capable of achieving the best separation of all compounds in this study with the higher sensitivity for most of the analytes.

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This paper deals with the search for optimal conditions for the identification of Transkarbam 12 (T 12) as a substance used for acceleration of transdermal penetration. Such substances are used in cases when drugs do not pass through the skin barrier under normal conditions. Other advantages are that they do not irritate the digestive system, provide continuous administration to an organism, and reduce fluctuations of drug concentration in blood. TLC and HPLC were used for identification. In the case of TLC, Silufol UV254 was used as stationary phase and the mobile phase consisted of chloroform, ethanol, and acetic acid. Detection was performed with iodine vapour. In the case of HPLC, the following three chromatographic columns were tested for the analysis of T 12: Silasorb SPH C 18, Silasorb SPH nitrile, and LiChrosorb Si-60. Because of the absence of any chromophore in the structure of T 12, work was performed on the derivatized compound. Detection was carried out at 230 nm. Quantification was studied on LiChrosorb Si-60 and the linearity, precision, and accuracy were evaluated.

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