ABSTRACT
The role of polymorphic CYP2B6 in cyclophosphamide (CPA) bioactivation was investigated in human liver microsomes. A total of 67 human liver specimens were first genotyped with respect to the CYP2B6*5 and CYP2B6*6 variant alleles. CYP2B6 apoprotein levels in 55 liver microsomal preparations were assessed by immunoblotting. 4-Hydroxy-CPA and hydroxy-bupropion were quantified by using HPLC and LC-MS, respectively. 7-Ethoxy-4-trifluoromethyl coumarin O-deethylase activity was measured fluorometrically. The frequencies of CYP2B6*5 and CYP2B6*6 mutant alleles were 9.0 and 16.4%, respectively. CYP2B6 protein expression was detected in 80% of the samples, with a large variation (0.003–2.234, arbitrary units). There was a high correlation between CYP2B6 apoprotein content and CPA 4-hydroxylation (n=55, r=0.81, P<0.0001). When based on the CYP2B6 apoprotein levels, the *6 carriers had significantly higher CPA 4-hydroxylation (P<0.05). CPA 4-hydroxylation also correlated significantly with other CYP2B6-specific reactions (n=20, P<0.0001). Vmax and Km for CPA 4-hydroxylation in recombinant CYP2B6 enzyme were 338 nmol/min/nmol enzyme and 1.4 mM, respectively. CYP2B6 showed much higher in vitro intrinsic clearance than previously observed in recombinant CYP2C19 and CYP2C9 variants in yeast expression system. Our results demonstrate that the polymorphic CYP2B6 is a major enzyme in the bioactivation of CPA. Moreover, we identified a strong impact of CYP2B6*6 on CPA 4-hydroxylation.
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References
Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP . Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 1994; 270: 414–423.
Mimura M, Baba T, Yamazaki H, Ohmori S, Inui Y, Gonzalez FJ et al. Characterization of cytochrome P-450 2B6 in human liver microsomes. Drug Metab Dispos 1993; 21: 1048–1056.
Yamano S, Nhamburo PT, Aoyama T, Meyer UA, Inaba T, Kalow W et al. cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 1989; 28: 7340–7348.
Ekins S, Wrighton SA . The role of CYP2B6 in human xenobiotic metabolism. Drug Metab Rev 1999; 31: 719–754.
Ekins S, Vandenbranden M, Ring BJ, Gillespie JS, Yang TJ, Gelboin HV et al. Further characterization of the expression in liver and catalytic activity of CYP2B6. J Pharmacol Exp Ther 1998; 286: 1253–1259.
Roy P, Yu LJ, Crespi CL, Waxman DJ . Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab Dispos 1999; 27: 655–666.
Yang TJ, Krausz KW, Shou M, Yang SK, Buters JT, Gonzalez FJ et al. Inhibitory monoclonal antibody to human cytochrome P450 2B6. Biochem Pharmacol 1998; 55: 1633–1640.
Chang TK, Weber GF, Crespi CL, Waxman DJ . Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res 1993; 53: 5629–5637.
Code EL, Crespi CL, Penman BW, Gonzalez FJ, Chang TK, Waxman DJ . Human cytochrome P4502B6: interindividual hepatic expression, substrate specificity, and role in procarcinogen activation. Drug Metab Dispos 1997; 25: 985–993.
Faucette SR, Hawke RL, Lecluyse EL, Shord SS, Yan B, Laethem RM et al. Validation of bupropion hydroxylation as a selective marker of human cytochrome P450 2B6 catalytic activity. Drug Metab Dispos 2000; 28:1222–1230.
Weber GF, Waxman DJ . Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes. Biochem Pharmacol 1993; 45: 1685–1694.
Granvil CP, Madan A, Sharkawi M, Parkinson A, Wainer IW . Role of CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)- and (S)-ifosfamide in human liver microsomes. Drug Metab Dispos 1999; 27: 533–541.
Nakayama H, Okuda H, Nakashima T, Imaoka S, Funae Y . Nicotine metabolism by rat hepatic cytochrome P450s. Biochem Pharmacol 1993; 45: 2554–2556.
Imaoka S, Yamada T, Hiroi T, Hayashi K, Sakaki T, Yabusaki Y et al. Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. Biochem Pharmacol 1996; 51: 1041–1050.
Heyn H, White RB, Stevens JC . Catalytic role of cytochrome P4502B6 in the N-demethylation of S- mephenytoin. Drug Metab Dispos 1996; 24: 948–954.
Ono S, Hatanaka T, Hotta H, Satoh T, Gonzalez FJ, Tsutsui M . Specificity of substrate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 1996; 26: 681–693.
Kobayashi K, Abe S, Nakajima M, Shimada N, Tani M, Chiba K et al. Role of human CYP2B6 in S-mephobarbital N-demethylation. Drug Metab Dispos 1999; 27: 1429–1433.
Stresser DM, Kupfer D . Monospecific antipeptide antibody to cytochrome P-450 2B6. Drug Metab Dispos 1999; 27: 517–525.
Oda Y, Hamaoka N, Hiroi T, Imaoka S, Hase I, Tanaka K et al. Involvement of human liver cytochrome P4502B6 in the metabolism of propofol. Br J Clin Pharmacol 2001; 51: 281–285.
Court MH, Duan SX, Hesse LM, Venkatakrishnan K, Greenblatt DJ . Cytochrome P-450 2B6 is responsible for interindividual variability of propofol hydroxylation by human liver microsomes. Anesthesiology 2001; 94: 110–119.
Chang TK, Yu L, Maurel P, Waxman DJ . Enhanced cyclophosphamide and ifosfamide activation in primary human hepatocyte cultures: response to cytochrome P-450 inducers and autoinduction by oxazaphosphorines. Cancer Res 1997; 57: 1946–1954.
Hassan M, Svensson US, Ljungman P, Bjorkstrand B, Olsson H, Bielenstein M et al. A mechanism-based pharmacokinetic-enzyme model for cyclophosphamide autoinduction in breast cancer patients. Br J Clin Pharmacol 1999; 48: 669–677.
Ariyoshi N, Miyazaki M, Toide K, Sawamura Y, Kamataki T . A single nucleotide polymorphism of CYP2b6 found in Japanese enhances catalytic activity by autoactivation. Biochem Biophys Res Commun 2001; 281: 1256–1260.
Lang T, Klein K, Fischer J, Nussler AK, Neuhaus P, Hofmann U et al. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics 2001; 11: 399–415.
Sladek NE, Doeden D, Powers JF, Krivit W . Plasma concentrations of 4-hydroxycyclophosphamide and phosphoramide mustard in patients repeatedly given high doses of cyclophosphamide in preparation for bone marrow transplantation. Cancer Treat Rep 1984; 68: 1247–1254.
Yule SM, Boddy AV, Cole M, Price L, Wyllie R, Tasso MJ et al. Cyclophosphamide pharmacokinetics in children. Br J Clin Pharmacol 1996; 41: 13–19.
May-Manke A, Kroemer H, Hempel G, Bohnenstengel F, Hohenlochter B, Blaschke G et al. Investigation of the major human hepatic cytochrome P450 involved in 4- hydroxylation and N-dechloroethylation of trofosfamide. Cancer Chemother Pharmacol 1999; 44: 327–334.
Ren S, Yang JS, Kalhorn TF, Slattery JT . Oxidation of cyclophosphamide to 4-hydroxycyclophosphamide and deschloroethylcyclophosphamide in human liver microsomes. Cancer Res 1997; 57: 4229–4235.
Chang TK, Yu L, Goldstein JA, Waxman DJ . Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-ILE359 allele as low-Km catalysts of cyclophosphamide and ifosfamide activation. Pharmacogenetics 1997; 7: 211–221.
von Bahr C, Groth CG, Jansson H, Lundgren G, Lind M, Glaumann H . Drug metabolism in human liver in vitro: establishment of a human liver bank. Clin Pharmacol Ther 1980; 27: 711–725.
Yasar U, Tybring G, Hidestrand M, Oscarson M, Ingelman-Sundberg M, Dahl ML et al. Role of CYP2C9 polymorphism in losartan oxidation. Drug Metab Dispos 2001; 29: 1051–1056.
Livak KJ . Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 1999; 14: 143–149.
Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES et al. 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 2000; 28: 655–661.
Griskevicius L, Meurling L, Hassan M . Simple method based on fluorescent detection for the determination of 4- hydroxycyclophosphamide in plasma. Ther Drug Monit 2002; 24: 405–409.
Sladek NE . Metabolism of oxazaphosphorines. Pharmacol Ther 1988; 37: 301–355.
Huitema AD, Kerbusch T, Tibben MM, Rodenhuis S, Beijnen JH . Reduction of cyclophosphamide bioactivation by thioTEPA: critical sequence-dependency in high-dose chemotherapy regimens. Cancer Chemother Pharmacol 2000; 46: 119–127.
Rae JM, Soukhova NV, Flockhart DA, Desta Z . Triethylenethiophosphoramide is a specific inhibitor of cytochrome P450 2B6: implications for cyclophosphamide metabolism. Drug Metab Dispos 2002; 30: 525–530.
Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, Negishi M . The repressed nuclear receptor CAR responds to phenobarbital in activating the human CYP2B6 gene. J Biol Chem 1999; 274: 6043–6046.
Gervot L, Rochat B, Gautier JC, Bohnenstengel F, Kroemer H, de Berardinis V et al. Human CYP2B6: expression, inducibility and catalytic activities. Pharmacogenetics 1999; 9: 295–306.
Acknowledgements
We thank Associate Professor Gunnel Tybring and technicians Ann-Louise Hagbjörk, Margareta Lind, and Margareta Rais for their technical help. This study was supported by the Swedish Cancer Society, Swedish Science Council (MRC 04496), Swedish Children Cancer Society, and Stockholm's Cancer Foundation.
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Xie, HJ., Yasar, Ü., Lundgren, S. et al. Role of polymorphic human CYP2B6 in cyclophosphamide bioactivation. Pharmacogenomics J 3, 53–61 (2003). https://doi.org/10.1038/sj.tpj.6500157
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DOI: https://doi.org/10.1038/sj.tpj.6500157
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