Pharmacogenetic and pharmacokinetic studies of cyclophosphamide : in cell, animal and human

Abstract

Cyclophosphamide (CPA) is widely used anti-cancer drug that has to be catalyzed by the cytochrome P450 (CYP) enzyme 2B6 as well as some other CYPs to exert its cytotoxic activity. A high degree of interindividual variation in CPA kinetics has been reported in both children and adult patients, which may be due to several factors such as concomitantly used drugs, variation in physiological status of individual patients and genetic polymorphism of the CYP enzymes. The general aim of the present thesis was to study the pharmacogenetic and pharmacokinetic aspects of the mutual interactions between CPA and CYPs, the role of polymorphic CYP enzymes on CPA bioactivation and the effect of CPA on CYP expression in order to better understand the mechanisms underlying the variation in CPA kinetics.

In the HL-60 cell line and its multi-drug resistant (MDR) phenotype HL-60R that was exposed to CPA, no measurable CYPs were induced by CPA exposure. this indicates that the CYP inducibility in cells is poor. CYP1B1, which is predominantly expressed in these cells, was suppressed after CPA treatment in a con centration-dependent manner. The beta-actin gene was suppressed when cells were exposed to CPA suggesting a possible mechanism that contributes to CPA cytotoxicity. The HL60R was shown to be sensitive to CPA treatment, demonstrating that the MDR phenotype is not involved in the mechanism of resistance to CPA.

Ciprofloxacin administration prior to cyclophosphamide in the rat resulted in a significant decrease in the metabolic rate of CPA as expressed as the ratio AUC 4-Hydroxy-cyclophosphamide/ AUC CPA (AUC4OH-CPA/AUC CPA). Moreover, a significant suppression of the gene expression of CYP2C11 and CYP3A1 was observed. These results may have a great clinical relevance when choosing antibiotic treatment to patients receiving CPA.

In rats treated with cyclophosphamide, the mRNAs specific for rat CYP2B1, 2B2, 3A2, 2C11, and proteins of CYP2B 1/2 and CYP3A were significantly induced after the administration of single intravenous dose of CPA. The microsomal activities of CYP2B, CYP3A and 2C11 were significantly increased accordingly. These results demonstrated that CPA has a marked inducing effect on CYP2B1, 2B2. 2C11, and 3A2. In particular, CPA showed a dramatic regulatory effect on CYP2B1 mRNA.

We have evaluated the role of the polymorphic human CYP2B6 in CPA bioactivation using sixty-seven human liver specimens that were genotyped with respect to the CYP2B6*5 and CYP2B6*6 allelic variation. We found that carriers of the *6 allele tended to have relatively lower protein expression compared to non-*6 carriers, and significant higher CPA 4-hydroxylation capacity (p<0.05). A significant correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation was observed (p<0.0001). CPA 4-hydroxylation significantly correlated with CYP2B6 specific reactions (p<0.0001). The results demonstrate that the polymorphic CYP2B6 is an important enzyme in the bioactivation of CPA and that the CYP2B6 *6 allele has a strong impact on CPA 4-hydroxylation.

The pharmacokinetics of CPA and its active 4-OH-CPA metabolite were determined in patients with hematological malignancies and related to the genotype of the CYP2B6, CYP2C9 and CYP2C19 genes. The interindividual variability in exposure to cycklophosphamide as expressed as the ratio of 4-OHCPA/CPA was 5.8-, 3.3- and 10.3- fold, respectively. In the population pharmacokinetic analysis the clearance contribution of the CYP2B6 G516T variant allele was about twice compared to the wild type gene while the genotype of CYP2C9 and CYP2C19 did not influence CPA clearance. A negative correlation was observed between bilirubin level and CPA bioactivation, showing the importance of the liver function for the metabolism of CPA.