The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, PR China; The Liver Center of Fujian Province, Fujian Medical University, Fuzhou 350025, PR China; Precision Pharmacy Laboratory, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, PR China.
Precision Pharmacy Laboratory, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, PR China.
J Pharm Biomed Anal. 2024 Nov 15;250:116389. doi: 10.1016/j.jpba.2024.116389. Epub 2024 Jul 30.
Immunosuppressive drugs (ISDs) are given to avoid the allograft rejection after transplantation. The concentrations of ISDs should be closely monitored owing to their wide inter-individual variability in its pharmacokinetics and narrow therapeutic window. Currently, the whole blood concentration measurement is the major approach of therapeutic drug monitoring of clinical ISDs in organ transplantation. Its correlation with the efficacy of ISDs remains elusive. While the acute rejection after transplantation may occur even when whole-blood ISDs concentrations are within the target range. Since the site of action of ISDs are within the lymphocyte, direct measurement of drug exposure in target cells may more accurately reflect the clinical efficacy of ISDs. Although several methods have been developed for the peripheral blood mononuclear cells (PBMCs) extraction and drug concentration measurement, the complex pre-processing has limited the study of the relationship between intracellular ISDs concentrations and the occurrence of rejection. In this study, the extraction of ISDs in PBMCs was carried out by the liquid-liquid extraction with low temperature purification, without centrifugation. The lower limit of quantitation were 0.2 ng/mL for cyclosporine A, tacrolimus and sirolimus, 1.0 ng/mL for mycophenolic acid, and the within-run and between-run coefficient of variations were both less than 12.4 %. The calibration curves of mycophenolic acid had a linear range (ng/mL): 1.0-128.0 (r = 0.9992). The calibration curves of other three ISDs had a linear range (ng/mL): 0.2-20.48 (r > 0.9956). A total of 157 clinical samples were analyzed by the UPLC-MS/MS for ISDs concentration in blood or plasma ([ISD]) and the concentration within PBMCs ([ISD]). Although there was strong association between [ISD] and [ISD], the large discrepancies between concentration within [ISD] and [ISD] were observed in a small proportion of clinical samples. The developed method with short analysis time and little amounts of blood sample can be successfully applied to therapeutic drug monitoring of ISDs in PBMCs for analysis of large numbers of clinical samples and is helpful to explore the clinical value of ISDs concentration in PBMCs.
免疫抑制药物（ISDs）用于避免移植后移植物排斥。由于其药代动力学的个体间变异性很大，治疗窗狭窄，因此应密切监测 ISD 的浓度。目前，全血浓度测量是器官移植中临床 ISD 治疗药物监测的主要方法。其与 ISD 疗效的相关性仍不清楚。尽管全血 ISD 浓度在靶范围内，移植后仍可能发生急性排斥反应。由于 ISD 的作用部位在淋巴细胞内，直接测量靶细胞内的药物暴露可能更准确地反映 ISD 的临床疗效。尽管已经开发了几种用于外周血单个核细胞（PBMCs）提取和药物浓度测量的方法，但复杂的预处理限制了细胞内 ISD 浓度与排斥反应发生之间关系的研究。在这项研究中，通过低温净化的液液萃取法从 PBMCs 中提取 ISD，无需离心。环孢素 A、他克莫司和西罗莫司的定量下限均为 0.2ng/mL，霉酚酸为 1.0ng/mL，批内和批间变异系数均小于 12.4%。霉酚酸的校准曲线呈线性范围（ng/mL）：1.0-128.0（r=0.9992）。其他三种 ISD 的校准曲线呈线性范围（ng/mL）：0.2-20.48（r>0.9956）。通过 UPLC-MS/MS 分析了 157 份临床样本，以测定血液或血浆中的 ISD 浓度（[ISD]）和 PBMCs 中的浓度（[ISD]）。虽然 [ISD] 和 [ISD] 之间存在很强的相关性，但在一小部分临床样本中观察到 [ISD] 内浓度与 [ISD] 之间存在较大差异。该方法分析时间短，所需血样量少，可成功应用于 PBMCs 中 ISD 的治疗药物监测，分析大量临床样本，有助于探讨 PBMCs 中 ISD 浓度的临床价值。
