Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants

Galvin Vredenburg, Shalenie den Braver-Sewradj, Barbara M A van Vugt-Lussenburg, Nico P E Vermeulen, Jan N M Commandeur, J Chris Vos

Research output: Contribution to JournalArticleAcademicpeer-review

Abstract

Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

Original languageEnglish
Pages (from-to)182-92
Number of pages11
JournalToxicology Letters
Volume232
Issue number1
DOIs
Publication statusPublished - 5 Jan 2015

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Ifosfamide
Cyclophosphamide
Cytochrome P-450 Enzyme System
Chemical activation
Pharmaceutical Preparations
Hydroxylation
Enzyme Therapy
Prodrugs
Genes
Metabolic Activation
Liver Microsomes
Enzymes
Cytotoxicity
Metabolites
Drug-Related Side Effects and Adverse Reactions
Metabolism
Liver
Antineoplastic Agents
Byproducts
Toxicity

Keywords

  • Activation, Metabolic
  • Antineoplastic Agents, Alkylating
  • Bone Neoplasms
  • Cell Line, Tumor
  • Cell Survival
  • Cyclophosphamide
  • Cytochrome P-450 CYP2B6
  • Dose-Response Relationship, Drug
  • Genotype
  • Humans
  • Hydroxylation
  • Ifosfamide
  • Kinetics
  • Microsomes, Liver
  • Mutation
  • Osteosarcoma
  • Phenotype
  • Journal Article

Cite this

Vredenburg, Galvin ; den Braver-Sewradj, Shalenie ; van Vugt-Lussenburg, Barbara M A ; Vermeulen, Nico P E ; Commandeur, Jan N M ; Vos, J Chris. / Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants. In: Toxicology Letters. 2015 ; Vol. 232, No. 1. pp. 182-92.
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abstract = "Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.",
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Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants. / Vredenburg, Galvin; den Braver-Sewradj, Shalenie; van Vugt-Lussenburg, Barbara M A; Vermeulen, Nico P E; Commandeur, Jan N M; Vos, J Chris.

In: Toxicology Letters, Vol. 232, No. 1, 05.01.2015, p. 182-92.

Research output: Contribution to JournalArticleAcademicpeer-review

TY - JOUR

T1 - Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants

AU - Vredenburg, Galvin

AU - den Braver-Sewradj, Shalenie

AU - van Vugt-Lussenburg, Barbara M A

AU - Vermeulen, Nico P E

AU - Commandeur, Jan N M

AU - Vos, J Chris

N1 - Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

PY - 2015/1/5

Y1 - 2015/1/5

N2 - Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

AB - Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

KW - Activation, Metabolic

KW - Antineoplastic Agents, Alkylating

KW - Bone Neoplasms

KW - Cell Line, Tumor

KW - Cell Survival

KW - Cyclophosphamide

KW - Cytochrome P-450 CYP2B6

KW - Dose-Response Relationship, Drug

KW - Genotype

KW - Humans

KW - Hydroxylation

KW - Ifosfamide

KW - Kinetics

KW - Microsomes, Liver

KW - Mutation

KW - Osteosarcoma

KW - Phenotype

KW - Journal Article

U2 - 10.1016/j.toxlet.2014.11.005

DO - 10.1016/j.toxlet.2014.11.005

M3 - Article

VL - 232

SP - 182

EP - 192

JO - Toxicology Letters

JF - Toxicology Letters

SN - 0378-4274

IS - 1

ER -