Department of Medicine, Baltimore, MD, 21287, USA; Johns Hopkins University School of Medicine, 4940 Eastern Avenue, Mason F. Lord Center Tower, Baltimore, MD, 21224, USA.
Department of Opthamology, Baltimore, MD, 21287, USA.
J Pharm Biomed Anal. 2018 Apr 15;152:248-256. doi: 10.1016/j.jpba.2018.02.011. Epub 2018 Feb 8.
The nucleotide reverse transcriptase inhibitor tenofovir (TFV) is widely administered in a disoproxil prodrug form (tenofovir disoproxil fumarate, TDF) for HIV management and prevention. Recently, novel prodrugs tenofovir alafenamide fumarate (TAF) and hexadecyloxypropyl tenofovir (CMX157) have been pursued for HIV treatment while minimizing adverse effects associated with systemic TFV exposure. Dynamic and sensitive bioanalytical tools are required to characterize the pharmacokinetics of these prodrugs in systemic circulation. Two parallel methods have been developed, one to combinatorially quantify TAF and TFV, and a second method for CMX157 quantification, in plasma.
KEDTA plasma was spiked with TAF and TFV, or CMX157. Following the addition of isotopically labeled internal standards and sample extraction via solid phase extraction (TAF and TFV) or protein precipitation (CMX157), samples were subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. For TAF and TFV, separation occurred using a Zorbax Eclipse Plus C18 Narrow Bore RR, 2.1 × 50 mm, 3.5 μm column and analytes were detected on an API5000 mass analyzer; CMX157 was separated using a Kinetex C8, 2.1 × 50 mm, 2.6 μm column and quantified using an API4500 mass spectrometer. Methods were validated according to FDA Bioanalytical Method Validation guidelines.
Analytical methods: were optimized for the multiplexed monitoring of TAF and TFV, and CMX157 in plasma. The lower limits of quantification (LLOQs) for TAF, TFV, and CMX157 were 0.03, 1.0, and 0.25 ng/mL, respectively. Calibration curves were generated via weighted linear regression of standards. Intra- and inter-assay precision and accuracy studies demonstrated %CVs ≤ 14.4% and %DEVs ≤ ± 7.95%, respectively. Stability and matrix effects studies were also performed. All results were acceptable and in accordance with the recommended guidelines for bioanalytical methods. Assays were also applied to quantify in vivo concentrations of prodrugs and TFV in a preclinical study post-rectal administration.
Sensitive, specific, and dynamic LC-MS/MS assays have been developed and validated for the multiplexed quantification TAF and TFV, as well as an independent assay for CMX157 quantification, in plasma. The described methods meet sufficient throughput criteria to support large research trials.
核苷酸逆转录酶抑制剂替诺福韦（TFV）以双异丙氧羰基前药形式（富马酸替诺福韦二异丙酯，TDF）广泛用于治疗 HIV 和预防 HIV。最近，新型前药替诺福韦艾拉酚胺富马酸盐（TAF）和十六烷氧基丙基替诺福韦（CMX157）已被用于治疗 HIV，同时将与全身 TFV 暴露相关的不良反应降至最低。需要动态且灵敏的生物分析工具来描述这些前药在全身循环中的药代动力学。已经开发了两种平行方法，一种用于组合定量测定 TAF 和 TFV，另一种用于定量测定 CMX157，均在血浆中进行。
用 TAF 和 TFV 或 CMX157 对 KEDTA 血浆进行了加标。加入同位素标记的内标物并通过固相萃取（TAF 和 TFV）或蛋白沉淀（CMX157）提取样品后，将样品进行液相色谱-串联质谱（LC-MS/MS）分析。对于 TAF 和 TFV，使用 Zorbax Eclipse Plus C18 窄孔 RR，2.1×50mm，3.5μm 柱进行分离，在 API5000 质谱仪上检测；CMX157 使用 Kinetex C8，2.1×50mm，2.6μm 柱进行分离，并在 API4500 质谱仪上定量。方法按照 FDA 生物分析方法验证指南进行了验证。
分析方法：优化了用于同时监测 TAF 和 TFV 以及 CMX157 在血浆中的方法。TAF、TFV 和 CMX157 的定量下限（LLOQ）分别为 0.03、1.0 和 0.25ng/mL。通过标准的加权线性回归生成校准曲线。日内和日间精密度和准确度研究表明，%CVs≤14.4%，%DEVs≤±7.95%。还进行了稳定性和基质效应研究。所有结果均符合生物分析方法建议的指南要求。该方法还应用于经直肠给药后进行的临床前研究中，以定量测定体内前药和 TFV 的浓度。
已经开发和验证了用于同时定量测定 TAF 和 TFV 以及用于独立定量测定 CMX157 的灵敏、特异和动态 LC-MS/MS 检测方法，这些方法具有足够的高通量标准，可支持大型研究试验。
