Division of Pharmacotherapy and Experimental Therapeutics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Chimerix, Inc., Durham, NC, USA.
Biomed Chromatogr. 2021 May;35(5):e5061. doi: 10.1002/bmc.5061. Epub 2021 Jan 14.
Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d and C N -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C (50 × 2.1 mm, 3.5 μm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 μm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.
布立昔洛韦（BCV）是一种核苷酸类似物西多福韦（CDV）的脂类缀合物，正在被开发为治疗天花的医疗对策。BCV 对包括 BK 和 JC 病毒在内的双链 DNA 病毒具有活性。在这里，我们验证了定量检测小鼠肾、脑和脾组织匀浆中 BCV 及其具有药理活性的部分二磷酸西多福韦（CDV-PP）的程序。组织匀浆后，通过与稳定的同位素标记内标 BCV-d 和 C N -CDV-PP 的蛋白质沉淀从组织中提取 BCV 和 CDV-PP。然后，使用 Waters Xterra MS C（50×2.1mm，3.5μm 粒径）柱进行反相色谱法分析 BCV，使用 Thermo BioBasic AX（50×2.1mm，5μm 粒径）柱进行阴离子交换色谱法分析 CDV-PP。通过 AB Sciex API-5000 三重四极杆质谱仪在正离子模式下进行电喷雾电离进行检测。BCV 和 CDV-PP 的校准曲线在 1.00-1,000ng/ml 匀浆和 0.050-50.0ng/ml 匀浆范围内呈线性。这些方法是根据美国食品和药物管理局（FDA）的行业指南进行验证的，可用于描述两种分析物的组织药理学，以推进其临床前开发。
