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Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation.

El-Serafi I, Afsharian P, Moshfegh A, Hassan M, Terelius Y - PLoS ONE (2015)

Bottom Line: A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Cancer Medicine (ECM), Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden.

ABSTRACT

Introduction: 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.

Materials and methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Up-regulation of POR mRNA during Cy conditioning.The gene expression of POR was significantly (p < 0.001, t-test) up-regulated 24 hours after the first Cy infusion and 6 hours after the second dose of Cy, compared to pre-Cy conditioning. The inter-individual variation in relative POR expression increased from 4.3-fold before Cy conditioning to 9.9-fold before the 2nd dose of Cy and to 16.5-fold by the end of the treatment. The high variation in up-regulation of POR gene expression during Cy treatment may contribute to the high inter-individual variations in Cy kinetics reported in patients, possibly in part due to different inducibility of the polymorphic forms of POR. Circles in the figure are for four patients with POR*28 polymorphism.
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pone.0141979.g003: Up-regulation of POR mRNA during Cy conditioning.The gene expression of POR was significantly (p < 0.001, t-test) up-regulated 24 hours after the first Cy infusion and 6 hours after the second dose of Cy, compared to pre-Cy conditioning. The inter-individual variation in relative POR expression increased from 4.3-fold before Cy conditioning to 9.9-fold before the 2nd dose of Cy and to 16.5-fold by the end of the treatment. The high variation in up-regulation of POR gene expression during Cy treatment may contribute to the high inter-individual variations in Cy kinetics reported in patients, possibly in part due to different inducibility of the polymorphic forms of POR. Circles in the figure are for four patients with POR*28 polymorphism.

Mentions: The average expression level of the POR gene was significantly (p <0.001, t-test) up-regulated 24 h and 30 h after the first Cy infusion (i.e. before and 6 h after the second infusion), compared to the levels before conditioning. However, a high inter-individual variation in POR gene expression after Cy infusion was observed, with a difference in individual relative POR expression of 4.3-fold before Cy conditioning increasing to 9.9-fold after 24 h and 16.5-fold by the end of the treatment, i.e. the expression levels increased in some patients more than others. The average expression levels after Cy treatment increased by 2.2 fold compared to the average before the start of the treatment (Fig 3).


Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation.

El-Serafi I, Afsharian P, Moshfegh A, Hassan M, Terelius Y - PLoS ONE (2015)

Up-regulation of POR mRNA during Cy conditioning.The gene expression of POR was significantly (p < 0.001, t-test) up-regulated 24 hours after the first Cy infusion and 6 hours after the second dose of Cy, compared to pre-Cy conditioning. The inter-individual variation in relative POR expression increased from 4.3-fold before Cy conditioning to 9.9-fold before the 2nd dose of Cy and to 16.5-fold by the end of the treatment. The high variation in up-regulation of POR gene expression during Cy treatment may contribute to the high inter-individual variations in Cy kinetics reported in patients, possibly in part due to different inducibility of the polymorphic forms of POR. Circles in the figure are for four patients with POR*28 polymorphism.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4636385&req=5

pone.0141979.g003: Up-regulation of POR mRNA during Cy conditioning.The gene expression of POR was significantly (p < 0.001, t-test) up-regulated 24 hours after the first Cy infusion and 6 hours after the second dose of Cy, compared to pre-Cy conditioning. The inter-individual variation in relative POR expression increased from 4.3-fold before Cy conditioning to 9.9-fold before the 2nd dose of Cy and to 16.5-fold by the end of the treatment. The high variation in up-regulation of POR gene expression during Cy treatment may contribute to the high inter-individual variations in Cy kinetics reported in patients, possibly in part due to different inducibility of the polymorphic forms of POR. Circles in the figure are for four patients with POR*28 polymorphism.
Mentions: The average expression level of the POR gene was significantly (p <0.001, t-test) up-regulated 24 h and 30 h after the first Cy infusion (i.e. before and 6 h after the second infusion), compared to the levels before conditioning. However, a high inter-individual variation in POR gene expression after Cy infusion was observed, with a difference in individual relative POR expression of 4.3-fold before Cy conditioning increasing to 9.9-fold after 24 h and 16.5-fold by the end of the treatment, i.e. the expression levels increased in some patients more than others. The average expression levels after Cy treatment increased by 2.2 fold compared to the average before the start of the treatment (Fig 3).

Bottom Line: A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found.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.

View Article: PubMed Central - PubMed

Affiliation: Experimental Cancer Medicine (ECM), Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden.

ABSTRACT

Introduction: 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.

Materials and methods: 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.

Results: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus