Department of Pharmacokinetics, Dynamics & Metabolism, Pfizer Global Research & Development, 10628 Science Center Drive, San Diego, CA 92121, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Oct 1;879(27):2860-5. doi: 10.1016/j.jchromb.2011.08.009. Epub 2011 Aug 16.
In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 μL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated "small volume" sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing "small volumes" was developed. The automated PPT method was achieved by extracting and processing 10 μL out of a minimum sample volume of 15 μL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton "small volume" method platform. Analyses of a 10 μL plasma aliquot from 15 μL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C(18) Extend, 1.0 × 50 mm, 3.5 μm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90-107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0-1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1mg/kg and oral administration at 2mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T(1/2)) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 μL of plasma from 15 μL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.
在肿瘤治疗领域，小鼠是用于功效研究的主要动物模型。通常，由于动物体型小，需要将样本分开进行其他测量，如生物标志物分析，因此用于小鼠药代动力学（PK）和药代动力学/药效学（PK/PD）研究的总血浆样品体积通常小于 20 μL。因此，需要进行自动化的“小体积”样品处理来进行定量生物分析。为了支持一种名为 PD 0332991 的肿瘤候选药物的 PK 和 PK/PD 研究，开发了一种使用 Hamilton MicroLab Star（内华达州里诺市）的自动化专用蛋白沉淀（PPT）方法，该药物是一种特定的周期蛋白依赖性激酶 4（CDK-4）抑制剂，目前正在开发中。该自动化 PPT 方法通过从最小 15 μL 血浆样品体积中提取和处理 10 μL 来实现。Agilent Technologies, Inc.（特拉华州威尔明顿）的 96 孔浅孔板是自动化 Hamilton“小体积”方法平台中首选的实验室设备。通过自动化和手动 PPT 方法对来自 15 μL 血浆研究样品的 10 μL 血浆等分试样进行分析。所有血浆样品均使用 Sciex API 4000 三重四极杆质谱仪与 Eksigent Express HT Ultra HPLC 系统进行定量。通过使用 Agilent 微流路 C（18）扩展，1.0×50mm，3.5μm 柱，流速为 0.150mL/min，总运行时间为 1.8min，实现色谱分离。标准和 QC 浓度水平的准确度和精密度均在 90-107%和<14%之间。校准曲线在 1.0-1000ng/mL 的动态范围内呈线性。在雌性 C3H 小鼠中以 1mg/kg 静脉给药和以 2mg/kg 口服给药后，进行了 PD 0332991 的 PK 研究。AUC、Vd、Cl、T(1/2)和生物利用度（F%）等 PK 值显示，自动化 Hamilton 和手动 PPT 方法之间的差异小于 11%。结果表明，自动化 Hamilton PPT 方法可以准确、精确地从 15 μL 或更大体积的血浆样品中分配 10 μL 血浆。专用的 Hamilton PPT 方法适用于支持发现研究的微采样 PK 和 PK/PD 样品的常规血浆样品分析。
