Wade Calcutt M, Lee Wooin, Puzanov Igor, Rothenberg Mace L, Hachey David L
Mass Spectrometry Research Center and Department of Biochemistry, Vanderbilt University Medical Center, Nashville, TN 37232-8575, USA.
J Mass Spectrom. 2008 Jan;43(1):42-52. doi: 10.1002/jms.1268.
SJG-136 1,1'-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyr- rolo[2,1-c][1,4]benzodiazepin-5-one]] (NSC 694501), is a bifunctional pyrrolobenzodiazepine (PBD) dimer that forms selective, irreversible, interstrand DNA cross-links via exocyclic N2 atoms of two guanine bases, with a preference for 5'PuGATCPy binding sites. SJG-136 is highly cytotoxic in human tumor cells in vitro and in human tumor xenograft models in vivo at subnanomolar concentrations and is currently in anticancer phase I clinical trials in the United Kingdom and United States. To support correlative pharmacokinetics studies, a highly sensitive HPLC-MS/MS assay was developed and validated for the reliable quantitation of SJG-136 in human plasma, using the structurally similar PBD dimer DSB-120 as an internal standard. Chemical reduction of SJG-136 to its corresponding amine (SJG-136-H(4), M + Hm/z 561) improved HPLC peak resolution and sensitivity by minimizing complications that arose from the reactivity of the labile imine moieties. Plasma samples were processed by protein precipitation and centrifugal membrane dialysis; components were separated by HPLC using an Agilent Rapid Resolution HT 1.8 mm (2.1 mm x 50 mm) analytical column. The total analysis time from injection to injection was 11 min. Electrospray MS/MS detection of SJG-136-H(4) was based on the selected reaction monitoring (SRM) transition M + Hm/z 561 --> 301. The analytical response ratio was linearly proportional to the plasma concentration of SJG-136 over the nominal concentration range of 25 pg/ml to 250 ng/ml, with a coefficient of determination of r > or = 0.999. The intrarun absolute %RE was < or =19.6, 14.2, and 14.0% at 0.056, 2.83, and 56.3 ng/ml, respectively. The corresponding %RSD was < or =14.9%, 9.01, and 4.59%. The interday %RSD was < or =2.72, 3.46, and 5.20%. The lower and upper limits of quantitation were 0.056 and 56 ng/ml, respectively; recovery of SJG-136 from plasma was > or = 62% across the validated concentration range. The sensitivity of the validated assay was sufficient to detect SJG-136 in human subjects for up to 6 h after intravenous administration of 6 microg/m(2), the starting dose of an NCI-sponsored dose escalation study.
SJG - 136，即1,1'-[[(丙烷 - 1,3 - 二基)二氧基]双[(11aS)-7 - 甲氧基 - 2 - 亚甲基 - 1,2,3,11a - 四氢 - 5H - 吡咯并[2,1 - c][1,4]苯并二氮杂䓬 - 5 - 酮]]（NSC 694501），是一种双功能吡咯并苯并二氮杂䓬（PBD）二聚体，它通过两个鸟嘌呤碱基的环外N2原子形成选择性、不可逆的链间DNA交联，优先结合5'PuGATCPy位点。SJG - 136在体外人肿瘤细胞和体内人肿瘤异种移植模型中，在亚纳摩尔浓度下具有高度细胞毒性，目前正在英国和美国进行抗癌I期临床试验。为支持相关药代动力学研究，开发并验证了一种高灵敏度的HPLC - MS/MS测定法，以可靠地定量人血浆中的SJG - 136，使用结构相似的PBD二聚体DSB - 120作为内标。将SJG - 136化学还原为其相应的胺（SJG - 136 - H(4)，M + Hm/z 561），通过最小化不稳定亚胺部分反应性引起的并发症，提高了HPLC峰分辨率和灵敏度。血浆样品通过蛋白沉淀和离心膜透析进行处理；使用安捷伦快速分离HT 1.8 mm（2.1 mm×50 mm）分析柱通过HPLC分离各成分。从进样到出样的总分析时间为11分钟。SJG - 136 - H(4)的电喷雾MS/MS检测基于选择反应监测（SRM）转换M + Hm/z 561 --> 301。在25 pg/ml至250 ng/ml的标称浓度范围内，分析响应比与SJG - 136的血浆浓度呈线性比例，测定系数r≥0.999。在0.056、2.83和56.3 ng/ml时，批内绝对%RE分别≤19.6%、14.2%和14.0%。相应的%RSD分别≤14.9%、9.01%和4.59%。批间%RSD分别≤2.72%、3.46%和5.20%。定量下限和上限分别为0.056和56 ng/ml；在验证的浓度范围内，SJG - 136从血浆中的回收率≥62%。经过验证的测定法的灵敏度足以在静脉注射6 μg/m(2)（美国国立癌症研究所赞助的剂量递增研究的起始剂量）后的长达6小时内检测人体受试者中的SJG - 136。
