Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation

Abstract

IntroductionCyclophosphamide 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.Materials and MethodsHydroxylation 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.ResultsA strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent K m 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.ConclusionsThis 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.

Differentiation‐associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic‐metabolizing activity of “luminal” muscle‐invasive bladder cancers

“…This study provides experimental and clinical evidence that CYP‐activity by normal urothelium is reliant on the differentiation‐dependent expression of POR, thereby defining the CYP‐capacity of different neoplastic programmes. POR abundance has been shown to influence CYP2B6‐mediated bioactivation of cyclophosphamide in patients and total CYP‐mediated metabolism in mice, and combined with this study suggests POR can be used as a biomarker of total CYP‐capacity in tissues. MIBC showed an overall suppression of POR; this was exemplified by the basal‐type T24 and SCaBER cell lines, which showed no EROD‐activity even though CYP1 transcripts were inducible (Supplementary Figure S11), suggesting an overall loss of functional CYP‐activity in basal MIBC.…”

“…CYP activity is driven by electron donation from the NADPH:CYP oxidoreductase (POR) and abundance of POR determines metabolic capacity in the CYP system. 13,14 POR is one member of the diflavin oxidoreductase family (nitric oxide synthase is the other) and is not specifically a reductase for the CYPs but can donate electrons to heme oxygenase among other enzymes (reviewed in Ref. 15 ).…”

“…Differentiated NHU cells from four donors showed the same trend of 1 μM ITE-induced EROD-activity peaking at 16 h and returning to control levels by 40 h. Results are presented as mean ± SD (n = 6 replicates per independent donor cell line) neoplastic programmes. POR abundance has been shown to influence CYP2B6-mediated bioactivation of cyclophosphamide in patients13 and…”

Population Pharmacokinetic, Pharmacogenetic, and Pharmacodynamic Analysis of Cyclophosphamide in Ethiopian Breast Cancer Patients

“…Indeed, having the same genotype, higher CYP2B6 enzyme activity in Ethiopians than Tanzanian is also reported previously (Ngaimisi et al, 2013). A previous study demonstrated that CYP2J2 expression was significantly up-regulated during CPA treatment and the bioactivation of the drug was significantly correlated to CYP2J2 expression (El-Serafi et al, 2015a). CYP2J2, expressed in high levels particularly in extra-hepatic organs such as the heart, intestine, and urinary bladder, may be responsible for the local CPA bio-activation and may explain CPA treatment-related toxicities (Hashizume et al, 2002;Matsumoto et al, 2002;Lee et al, 2010).…”

“…CPA is a prodrug and requires bioactivation in the liver to 4-hydroxy-cyclophosphamide (4-OH-CPA), which is subsequently converted to the ultimate alkylating metabolite, phosphoramide mustard, and acrolein, a urotoxic metabolite (Ekhart et al, 2008). Various cytochrome P450 (CYP) enzymes have been implicated to catalyze the 4-hydroxylation of CPA to 4-OH-CPA including CYP2B6 (Xie et al, 2003), CYP2C9, and CYP3A4/5 (Roy et al, 1999), CYP2C19 (Griskevicius et al, 2003;Timm et al, 2005), and CYP2J2 (El-Serafi et al, 2015a). These enzymes are genetically polymorphic and may contribute to interindividual variation in CPA metabolic disposition and clinical response including chemotherapy-induced toxicities (Nakajima et al, 2007;Zanger et al, 2007;Ekhart et al, 2008).…”