用人全血评价5-脂氧合酶抑制剂系统的建立

Francis J. Sweeney, James D. Eskra, Thomas J. Carty
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引用次数: 32

摘要

描述了一种可靠的系统,用于评估5-脂氧合酶(5-LO)途径抑制剂,利用钙离子载体,A-23187和酵母细胞壁(YCW)刺激的人全血。在开发该系统的过程中,我们已经证明白三烯B4 (LTB4)和5-羟基二碳四烯酸(5-HETE)可以从全血中定量回收,并且可以以±129%的准确度和精度(标准偏差)进行测量。通过将LTB4/5-HETE的产生正常化到中性粒细胞数量,可以最小化供者之间LTB4/5-HETE水平的明显差异。通过增加离子载体浓度,降低了不同供体间LTB4/5-HETE生成的变异性。离子载体刺激产品生产的动力学显示1-4分钟的滞后,这取决于离子载体的浓度。用5 μg/ml细胞松弛素b预处理血液可以消除这种滞后性。同样,酵母细胞壁刺激后产物形成的动力学表现出滞后性,这一滞后性可以通过事先调理YCW来缩短。在该系统中,LTB4代谢为20-OH-LTB4和20-COOH-LTB4的量约为20%。已知的5-LO通路抑制剂苯尼酮、去甲二氢愈创木酸和纳唑特罗姆的半最大抑制点分别为0.4、1.5和9 μg/ml。总之,我们相信该试验为预测有效抑制人类细胞LTB4/5-HETE合成/释放所需的全身药物水平提供了指导。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Development of a system for evaluating 5-lipoxygenase inhibitors using human whole blood

A reliable system for evaluating 5-lipoxygenase (5-LO) pathway inhibitors employing human whole blood stimulated by the calcium ionophore, A-23187, and yeast cell walls (YCW) is described.

In developing this system, we have shown that leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) can be recovered quantitatively from whole blood, and can be measured with accuracy and a precision (standard deviation) of ± 129% Apparent differences in LTB4/5-HETE levels between donors can be minimized by normalizing the LTB4/5-HETE production to neutrophil number. Variability in LTB4/5-HETE production among different donors was reduced by increasing the ionophore concentration. The kinetics of ionophore stimulated product production display a 1–4 min lag which is dependent on ionophore concentration. The lag is removed by pretreatment of blood with 5 μg/ml cytochalasin B. Likewise, the kinetics of product formation after stimulation with yeast cell walls demonstrated a lag period, which could be shortened by prior opsonization of the YCW. The amount of LTB4 metabolism to 20-OH-LTB4 and 20-COOH-LTB4 in this system is approximately 20%. Phenidone, nordihydroguaiaref acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 μg/ml, respectively. In summary, we believe that this assay offers a guide for predicting systemic levels of drug needed to be achieved for effective inhibition of cellular LTB4/5-HETE synthesis/release in humans.

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