title: Characterization of two phase I metabolites of bendamustine in human liver microsomes and in cancer patients treated with bendamustine hydrochloride.
abstract: OBJECTIVE
  The metabolism of bendamustine (BM) hydrochloride, a bifunctional alkylator containing a heterocyclic ring, was investigated in vitro and in vivo for identification of cytochromes P450 (CYP) involved in the formation of two phase I metabolites, structural confirmation of these previously unidentified metabolites and assessment of their cytotoxic effect in relation to the parent compound.
  
  
  METHODS
  Potential metabolites of BM were synthesized and structurally characterized by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) analysis. In vitro metabolism of BM hydrochloride in human hepatic microsomes was conducted to identify the CYP450 isoenzymes involved in the oxidative metabolism of BM. Samples from cancer patients after treatment with BM hydrochloride and microsomal preparations were analyzed by LC-MS and HPLC with fluorescence detection. The cytotoxic effect of the metabolites was analyzed in several lymphoma cell lines and peripheral blood lymphocytes and compared with that of the parent compound using an MTT assay.
  
  
  RESULTS
  LC-MS as well as HPLC with fluorescence detection revealed hydroxylation of the methylene carbon at the C-4 position of the butanoic acid side chain and N-demethylation of the benzimidazole skeleton as the main metabolic pathways in human liver microsomes. Isoform-specific chemical inhibitors and correlation analysis pointed to CYP1A2 as the prominent enzyme in BM oxidation. The rate of formation for both metabolites correlated (r=0.931 and 0.933) with the activity of CYP1A2 and there were no other notable correlations with any of the other CYPs. In addition, both metabolites were identified in plasma, urine, and bile samples from cancer patients under BM hydrochloride therapy as shown by comparison with chromatograms obtained from the authentic reference standards. Cytotoxic activity observed for gamma-hydroxy BM was approximately equivalent to that obtained for the parental compound BM. N-demethyl BM displays five to tenfold less cytotoxic activity than BM.
  
  
  CONCLUSIONS
  The results indicate that CYP1A2-catalyzed N-dealkylation and gamma hydroxylation are the major routes for BM phase I metabolism producing two metabolites less or similarly toxic than the parent compound. In contrast to the metabolic pathways of the structurally related chlorambucil, no beta-oxidation of the butanoic acid side chain leading to enhanced toxicity was detected for BM.
authors.affiliation: Institut für Klinische Pharmakologie, Universität Leipzig, Härtelstr. 16-18, 04107, Leipzig, Germany. Jens.Teichert@medizin.uni-leipzig.de
authors.forename: Jens
authors.initials: J
authors.lastname: Teichert
authors.forename: Frank
authors.initials: F
authors.lastname: Baumann
authors.forename: Qi
authors.initials: Q
authors.lastname: Chao
authors.forename: Craig
authors.initials: C
authors.lastname: Franklin
authors.forename: Brandy
authors.initials: B
authors.lastname: Bailey
authors.forename: Lothar
authors.initials: L
authors.lastname: Hennig
authors.forename: Karel
authors.initials: K
authors.lastname: Caca
authors.forename: Konrad
authors.initials: K
authors.lastname: Schoppmeyer
authors.forename: Ulrich
authors.initials: U
authors.lastname: Patzak
authors.forename: Rainer
authors.initials: R
authors.lastname: Preiss
mesh_terms: D000368:Aged; D000069461:Bendamustine Hydrochloride; D001650:Bile Duct Neoplasms / Q000378:metabolism*; D001653:Bile Ducts, Intrahepatic / Q000378:metabolism; D002460:Cell Line; D018281:Cholangiocarcinoma / Q000188:drug therapy* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D009588:Nitrogen Mustard Compounds / Q000378:metabolism* / Q000627:therapeutic use; D012015:Reference Standards
chemical_list: D009588:Nitrogen Mustard Compounds; D003577:Cytochrome P-450 Enzyme System; D000069461:Bendamustine Hydrochloride