Limits...
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

Michaelis–Menten curves and Hanes-Woolf plots for 4-OH-Cyclophosphamide enzyme kinetics for different batches of CYP2B6.1.In 2 independent experiments, Cyclophosphamide (at different concentrations) was incubated with 3 batches of Escherichia coli microsomes containing cDNA-expressed CYP2B6.1 (See Table 1) with different POR/CYP ratios. The CYP concentration per mg protein and the protein concentration per mL incubation were similar between batches. The 4-OH-Cy formation was measured using HPLC with fluorescence detector. The results shown are the averages obtained with each of the three batches. Fitting the data to Michaelis-Menten kinetics gave an apparent intrinsic clearance, CLint (Vmax/Km), of 3.1, 25.1, 33.7 μL/min/nmol CYP for Batch 1, Batch 2 and Batch 3, respectively. The intrinsic clearance thus increased with increasing POR/CYP ratio. See also Fig 2 and Table 3.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4636385&req=5

pone.0141979.g001: Michaelis–Menten curves and Hanes-Woolf plots for 4-OH-Cyclophosphamide enzyme kinetics for different batches of CYP2B6.1.In 2 independent experiments, Cyclophosphamide (at different concentrations) was incubated with 3 batches of Escherichia coli microsomes containing cDNA-expressed CYP2B6.1 (See Table 1) with different POR/CYP ratios. The CYP concentration per mg protein and the protein concentration per mL incubation were similar between batches. The 4-OH-Cy formation was measured using HPLC with fluorescence detector. The results shown are the averages obtained with each of the three batches. Fitting the data to Michaelis-Menten kinetics gave an apparent intrinsic clearance, CLint (Vmax/Km), of 3.1, 25.1, 33.7 μL/min/nmol CYP for Batch 1, Batch 2 and Batch 3, respectively. The intrinsic clearance thus increased with increasing POR/CYP ratio. See also Fig 2 and Table 3.

Mentions: The enzyme kinetics for 4-OH-Cy formation with microsomes containing human cDNA-expressed CYP2B6.1 and various ratios of POR/CYP was determined under linear conditions. Fitting the data from two independent experiments (each with the three different batches of microsomes at a constant concentration of 40 pmol CYP/mL and Cy concentration up to 10 mM) to Michaelis-Menten kinetics, the apparent Km for CYP2B6.1 was similar for the 3 batches (3–4 mM). However, the apparent maximum rate of Cy 4-hydroxylation (Vmax) increased with the POR/CYP ratio, ranging from 12.6 to 99.0 nmol/min/nmol CYP (Fig 1). The CYP concentrations and the protein concentrations were almost identical in the different batches, indicating that the the electron transfer by POR from NADPH to CYP may be rate limiting and that the POR/CYP ratio thus may be important for the Cy kinetics in vitro.


Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation.

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

Michaelis–Menten curves and Hanes-Woolf plots for 4-OH-Cyclophosphamide enzyme kinetics for different batches of CYP2B6.1.In 2 independent experiments, Cyclophosphamide (at different concentrations) was incubated with 3 batches of Escherichia coli microsomes containing cDNA-expressed CYP2B6.1 (See Table 1) with different POR/CYP ratios. The CYP concentration per mg protein and the protein concentration per mL incubation were similar between batches. The 4-OH-Cy formation was measured using HPLC with fluorescence detector. The results shown are the averages obtained with each of the three batches. Fitting the data to Michaelis-Menten kinetics gave an apparent intrinsic clearance, CLint (Vmax/Km), of 3.1, 25.1, 33.7 μL/min/nmol CYP for Batch 1, Batch 2 and Batch 3, respectively. The intrinsic clearance thus increased with increasing POR/CYP ratio. See also Fig 2 and Table 3.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141979.g001: Michaelis–Menten curves and Hanes-Woolf plots for 4-OH-Cyclophosphamide enzyme kinetics for different batches of CYP2B6.1.In 2 independent experiments, Cyclophosphamide (at different concentrations) was incubated with 3 batches of Escherichia coli microsomes containing cDNA-expressed CYP2B6.1 (See Table 1) with different POR/CYP ratios. The CYP concentration per mg protein and the protein concentration per mL incubation were similar between batches. The 4-OH-Cy formation was measured using HPLC with fluorescence detector. The results shown are the averages obtained with each of the three batches. Fitting the data to Michaelis-Menten kinetics gave an apparent intrinsic clearance, CLint (Vmax/Km), of 3.1, 25.1, 33.7 μL/min/nmol CYP for Batch 1, Batch 2 and Batch 3, respectively. The intrinsic clearance thus increased with increasing POR/CYP ratio. See also Fig 2 and Table 3.
Mentions: The enzyme kinetics for 4-OH-Cy formation with microsomes containing human cDNA-expressed CYP2B6.1 and various ratios of POR/CYP was determined under linear conditions. Fitting the data from two independent experiments (each with the three different batches of microsomes at a constant concentration of 40 pmol CYP/mL and Cy concentration up to 10 mM) to Michaelis-Menten kinetics, the apparent Km for CYP2B6.1 was similar for the 3 batches (3–4 mM). However, the apparent maximum rate of Cy 4-hydroxylation (Vmax) increased with the POR/CYP ratio, ranging from 12.6 to 99.0 nmol/min/nmol CYP (Fig 1). The CYP concentrations and the protein concentrations were almost identical in the different batches, indicating that the the electron transfer by POR from NADPH to CYP may be rate limiting and that the POR/CYP ratio thus may be important for the Cy kinetics in vitro.

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