El-Serafi Ibrahim, Afsharian Parvaneh, Moshfegh Ali, Hassan Moustapha, Terelius Ylva
Experimental Cancer Medicine (ECM), Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden.
Cancer Center of Karolinska (CCK), Department of Oncology-Pathology, Karolinska Institutet, Solna, Stockholm, Sweden.
PLoS One. 2015 Nov 6;10(11):e0141979. doi: 10.1371/journal.pone.0141979. eCollection 2015.
Cyclophosphamide is commonly used as an important component in conditioning prior to hematopoietic stem cell transplantation, a curative treatment for several hematological diseases. Cyclophosphamide is a prodrug activated mainly by cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in cyclophosphamide kinetics has been reported in several studies.
Hydroxylation of cyclophosphamide was investigated in vitro using three microsomal batches of CYP2B6*1 with different ratios of POR/CYP expression levels. Twenty patients undergoing hematopoietic stem cell transplantation were also included in the study. All patients received an i.v. infusion of cyclophosphamide (60 mg/kg/day, for two days) as a part of their conditioning. Blood samples were collected from each patient before cyclophosphamide infusion, 6 h after the first dose and before and 6 h after the second dose. POR gene expression was measured by mRNA analysis and the pharmacokinetics of cyclophosphamide and its active metabolite were determined.
A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent Km for CYP2B6.1 was almost constant (3-4 mM), while the CLint values were proportional to the POR/CYP ratio (3-34 μL/min/nmol CYP). In patients, the average expression of the POR gene in blood was significantly (P <0.001) up-regulated after cyclophosphamide infusion, with high inter-individual variations and significant correlation with the concentration ratio of the active metabolite 4-hydroxy-cyclophosphamide/cyclophosphamide. Nine patients were carriers for POR*28; four patients had relatively high POR expression.
This investigation shows for the first time that POR besides CYP2B6 can influence cyclophosphamide metabolism. Our results indicate that not only CYPs are important, but also POR expression and/or activity may influence cyclophosphamide bioactivation, affecting therapeutic efficacy and treatment related toxicity and hence on clinical outcome. Thus, both POR and CYP genotype and expression levels may have to be taken into account when personalizing treatment schedules to achieve optimal therapeutic drug plasma concentrations of cyclophosphamide.
环磷酰胺是造血干细胞移植预处理中的常用重要成分，造血干细胞移植是几种血液系统疾病的根治性治疗方法。环磷酰胺是一种前体药物，主要在肝脏中由细胞色素P450 2B6（CYP2B6）激活。多项研究报道了环磷酰胺动力学存在高度的个体间和个体内差异。
使用三批具有不同POR/CYP表达水平比例的CYP2B6*1微粒体在体外研究环磷酰胺的羟基化。该研究还纳入了20例接受造血干细胞移植的患者。所有患者作为预处理的一部分接受静脉输注环磷酰胺（60mg/kg/天，共两天）。在环磷酰胺输注前、首剂后6小时以及第二剂前和第二剂后6小时从每位患者采集血样。通过mRNA分析测量POR基因表达，并测定环磷酰胺及其活性代谢物的药代动力学。
发现环磷酰胺的体外内在清除率与POR/CYP比例之间存在强相关性。CYP2B6.1的表观Km几乎恒定（3 - 4mM），而CLint值与POR/CYP比例成正比（3 - 34μL/min/nmol CYP）。在患者中，环磷酰胺输注后血液中POR基因的平均表达显著上调（P<0.001），个体间差异很大，并且与活性代谢物4 - 羟基环磷酰胺/环磷酰胺浓度比显著相关。9例患者是POR*28携带者；4例患者的POR表达相对较高。
本研究首次表明，除CYP2B6外，POR也可影响环磷酰胺代谢。我们的结果表明，不仅细胞色素P450很重要，而且POR的表达和/或活性可能影响环磷酰胺的生物活化，从而影响治疗效果和治疗相关毒性，进而影响临床结局。因此，在制定个性化治疗方案以达到环磷酰胺最佳治疗药物血浆浓度时，可能必须同时考虑POR和CYP的基因型及表达水平。
