Chen C L, Malaviya R, Chen H, Liu X P, Uckun F M
Department of Pharmaceutical Sciences, Hughes Institute, St. Paul, MN 55113, USA.
J Chromatogr B Biomed Sci Appl. 1999 Apr 30;727(1-2):205-12. doi: 10.1016/s0378-4347(99)00047-x.
The novel quinazoline derivative 4-(4'-hydroxyphenyl)amino-6,7-dimethoxyquinazoline (WHI-P131) has recently been identified as a potent mast cell inhibitor capable of preventing IgE/antigen induced cutaneous as well as systemic fatal anaphylaxis in mice. Here we describe a sensitive high-performance liquid chromatography (HPLC)-based quantitative detection method for measurement of WHI-P131 levels in plasma as well as in target mast cells. The average extraction recovery for WHI-P131 was 88.4% for plasma and 75.7% for RBL-2H3 mast cell lysates. Good linearity (r>0.999) was observed throughout the concentration range of 0.1-20 microM in plasma and 0.01-5 nmol in 5 x 10(6) cells (0.5-238 microM per cell) for WHI-P131. Intra- and inter-assay variabilities were <7% and the lowest detection limit of WHI-P131 was 0.05 microM in plasma and 0.005 nmol in 5 million cells, respectively, at a signal-to-noise ratio of approximately 2. The practical utility of this new HPLC method was confirmed in a pilot pharmacokinetic study in BALB/c mice as well as in a cellular drug uptake and disposition study in RBL-2H3 mast cells. After intraperitoneal administration of a non-toxic 40 mg/kg bolus dose of WHI-P131, the estimated maximum plasma concentration was 92.7 microM, which is approximately 1-log higher than the effective in vitro mast cell inhibitory concentrations of WHI-P131. The drug absorption was rapid with an absorption half-life of only 2.9 min and the estimated time to reach the maximum plasma concentration was 8.3 min. WHI-P131 was cleared with an apparent systemic clearance rate of 2586 ml/h/kg and an elimination half-life of 1.8 h. An intracellular exposure level (AUC) of 55 microM x h was obtained after in vitro treatment of RBL-2H3 mast cells with WHI-P131 at a 33.6 microM final concentration in culture medium. The availability of the described quantitative HPLC detection method for WHI-P131 provides the basis for further development of WHI-P131 as an anti-allergic drug through detailed pharmacodynamic studies in preclinical animal models.
新型喹唑啉衍生物4-(4'-羟基苯基)氨基-6,7-二甲氧基喹唑啉(WHI-P131)最近被鉴定为一种有效的肥大细胞抑制剂，能够预防小鼠中IgE/抗原诱导的皮肤以及全身性致命过敏反应。在此，我们描述了一种基于高效液相色谱(HPLC)的灵敏定量检测方法，用于测定血浆以及靶肥大细胞中WHI-P131的水平。WHI-P131在血浆中的平均提取回收率为88.4%，在RBL-2H3肥大细胞裂解物中的平均提取回收率为75.7%。在血浆中0.1 - 20 microM以及在5×10(6)个细胞中0.01 - 5 nmol(每细胞0.5 - 238 microM)的浓度范围内，WHI-P131均呈现出良好的线性关系(r> 0.999)。批内和批间变异均<7%，WHI-P131在血浆中的最低检测限为0.05 microM，在500万个细胞中的最低检测限为0.005 nmol，信噪比约为2。这种新的HPLC方法的实际效用在BALB/c小鼠的初步药代动力学研究以及RBL-2H3肥大细胞的细胞药物摄取和处置研究中得到了证实。腹腔注射无毒的40 mg/kg推注剂量的WHI-P131后，估计的最大血浆浓度为92.7 microM，比WHI-P131的有效体外肥大细胞抑制浓度高约1个对数。药物吸收迅速，吸收半衰期仅为2.9分钟，估计达到最大血浆浓度的时间为8.3分钟。WHI-P131的清除表现为表观全身清除率为2586 ml/h/kg，消除半衰期为1.8小时。在培养基中用终浓度为33.6 microM的WHI-P131对RBL-2H3肥大细胞进行体外处理后，获得的细胞内暴露水平(AUC)为55 microM×h。所描述的用于WHI-P131的定量HPLC检测方法的可用性为通过临床前动物模型中的详细药效学研究进一步开发WHI-P131作为抗过敏药物提供了基础。
