Potential contribution of cytochrome P450 2B6 to hepatic 4-hydroxycyclophosphamide formation in vitro and in vivo

Abstract

Results from retrospective studies on the relationship between cytochrome P450 (P450) 2B6 (CYP2B6) genotype and cyclophosphamide (CY) efficacy and toxicity in adult cancer patients have been conflicting. We evaluated this relationship in children, who have faster CY clearance and receive different CY-based regimens than adults. These factors may influence the P450s metabolizing CY to 4-hydroxycyclophosphamide (4HCY), the principal precursor to CY's cytotoxic metabolite. Therefore, we sought to characterize the in vitro and in vivo roles of hepatic CYP2B6 and its main allelic variants in 4HCY formation. CYP2B6 is the major isozyme responsible for 4HCY formation in recombinant P450 Supersomes. In human liver microsomes (HLM), 4HCY formation correlated with known phenotypic markers of CYP2B6 activity, specifically formation of (S)-2-ethyl-1,5-dimethyl-3,3-diphenyl pyrrolidine and hydroxybupropion. However, in HLM, CYP3A4/5 also contributes to 4HCY formation at the CY concentrations similar to plasma concentrations achieved in children (0.1 mM). 4HCY formation was not associated with CYP2B6 genotype at low (0.1 mM) or high (1 mM) CY concentrations potentially because CYP3A4/5 and other isozymes also form 4HCY. To remove this confounder, 4HCY formation was evaluated in recombinant CYP2B6 enzymes, which demonstrated that 4HCY formation was lower for CYP2B6.4 and CYP2B6.5 compared with CYP2B6.1. In vivo, CYP2B6 genotype was not directly related to CY clearance or ratio of 4HCY/CY areas under the curve in 51 children receiving CY-based regimens. Concomitant chemotherapy agents did not influence 4HCY formation in vitro. We conclude that CYP2B6 genotype is not consistently related to 4HCY formation in vitro or in vivo.

Fig. 1.

Schematic of CY metabolism.

Fig. 2.

Inhibition of cyclophosphamide 4-hydroxylation by specific chemical inhibitors and the total normalized rate assessed in pooled human liver microsomes. The rate of 4HCY formation was determined in Supersomes (5 pmol) after a 10-min incubation with 0.1 mM CY. A, the percentage of total normalized rate (as determined in Materials and Methods) is plotted and compared with the percentage of inhibition obtained from specific chemical inhibition. As described under Materials and Methods, 4HCY formation in pooled HLM (0.08 mg) was measured after 10-min incubation. The concentration of specific chemical inhibitors were clopidogrel (1 μM), montelukast (1 μM), NBN (5 μM), sulfaphenazole (10 μM), TAO (100 μM), and ketoconazole (1 μM). The term “All four” is used to abbreviate the addition of clopidogrel, NBN, sulfaphenazole, and TAO. B, data are displayed as percentage reduction in control (no inhibitor) activity. Each data point is mean ± S.D. of four replicates.

Fig. 3.

Association of cyclophosphamide 4-hydroxylation with phenotypic markers of CYP2B6 and CYP3A4 activity in human liver microsomes. Correlation of the hydroxybupropion formation with 1-hydroxymidazolam formation (A; r = 0.58, p < 0.0001, n = 47). B–D, correlation of 4HCY formation with 1-hydroxymidazolam formation (B; r = 0.69, p < 0.0001, n = 37), hydroxybupropion formation (C; r = 0.96, p < 0.0001, n = 31), or S-EDDP formation (D; r = 0.68, p < 0.01, n = 13). The substrate concentrations used were 0.1 mM CY, 8 μM midazolam, 75 μM bupropion, and 1 μM S-methadone. Each data point is the mean of duplicate samples.

Fig. 4.

Variation of cyclophosphamide 4-hydroxylation by CYP2B6 genotype at low (0.1 mM; A) and high (1 mM; B) CY concentrations in human liver microsomes. A, 4HCY formation using 0.1 mM CY (p = 0.43). B, 4HCY formation using 1 mM CY (p = 0.42). Each data point is the mean of duplicate samples with the horizontal line representing the median value for each CYP2B6 genotype. Each reaction mixture contained 0.08 mg of HLM and was incubated for 30 min at 37°C. Incubations performed with HLM. HLM from donors <18 years of age (n = 2) are denoted by open triangles.

Fig. 5.

Kinetic parameters of CY 4-hydroxylation in purified CYP2B6 enzymes. CY (0.1–20 mM) was incubated with reconstituted systems (10 pmol) containing CYP2B6.1 (●), CYP2B6.4 (■), CYP2B6.5 (▾), or CYP2B6.6 (▴) for 10 min at 37°C. Data points are mean ± S.D. of four replicates.

Fig. 6.

CY clearance (A) and ratio of 4HCY/CY AUCs (B) by CYP2B6 genotype in children receiving a conventional dose of CY. A, CY clearance (l · h⁻¹ · m²⁻¹) for each CYP2B6 genotype (p = 0.57). B, ratio of 4HCY/CY AUC for each CYP2B6 genotype (p = 0.37). Pharmacokinetic samples were drawn immediately before CY administration, upon completion of the 30-min infusion, and at 2.5, 4.5, 6.5, 22.5, and 24 h after the start of the first CY dose. Children that received fluconazole concomitant with CY are denoted by open triangles. All others are denoted by filled circles. Genomic DNA was extracted from blood and genotyped for the most common CYP2B6 variants as described under Materials and Methods. Horizontal lines represent the median value for each group; the Kruskal-Wallis test was used to determine significance.