title: Glutathione trapping of reactive drug metabolites produced by biomimetic metalloporphyrin catalysts.
abstract: BACKGROUND
  Metalloporphyrins can be useful in the production of drug metabolites, as they enable easier production of oxidative metabolites usually produced by the cytochrome P450 enzymes. Our aim was to test metalloporphyrin-based biomimetic oxidation (BMO) methods for production and S-glutathione trapping of reactive drug metabolites in addition to phase I metabolites.
  
  
  METHODS
  Clozapine, ticlopidine and citalopram were selected as model compounds. These were incubated with the BMO assay and the incubations were analyzed with high-resolution liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (LC/MS/MS). Additionally, incubations with human liver S9 fraction were performed to compare the results with the BMO assay.
  
  
  RESULTS
  Six glutathione conjugates were identified for clozapine from the S9 incubation, while the BMO assay produced four of these. Four out of the five phase I metabolites produced by S9 were detected using the BMO assay. For ticlopidine, four glutathione conjugates were detected from the S9 incubation, but none of these were observed using the BMO assay. Eight of the nine phase I metabolites produced by S9 incubation were detected in the BMO assay. As expected, no glutathione conjugates were detected for citalopram, and the same three phase I metabolites were detected in both S9 and BMO incubations.
  
  
  CONCLUSIONS
  Differences in formation of GSH-trapped reactive metabolites by BMO assay between clozapine and ticlopidine are probably due to different reactive intermediates and reaction mechanisms. The reactive intermediate of clozapine, the nitrenium ion was generated, but the reactive intermediates of ticlopidine, S-oxide and epoxide, were not detected from the incubations. However, the results show that for selected cases the use of biomimetic assays can be used to produce high amounts of S-glutathione conjugates identical to those from liver subfraction incubations, on a scale that is relevant for purification and subsequent identification by NMR spectroscopy; which is often difficult using incubations with liver subfractions.
authors.affiliation: Department of Chemistry, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland.
authors.forename: Toni
authors.initials: T
authors.lastname: Lassila
authors.affiliation: Department of Chemistry, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland.
authors.forename: Sampo
authors.initials: S
authors.lastname: Mattila
authors.affiliation: Institute of Biomedicine, Department of Pharmacology and Toxicology and Medical Research Center Oulu, P.O. Box 5000, 90014, University of Oulu, Finland.
authors.forename: Miia
authors.initials: M
authors.lastname: Turpeinen
authors.affiliation: Admescope Ltd, Typpitie 1, 90620, Oulu, Finland.
authors.forename: Ari
authors.initials: A
authors.lastname: Tolonen
mesh_terms: D015283:Citalopram / Q000737:chemistry / Q000378:metabolism*; D003024:Clozapine / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism; D008862:Microsomes, Liver / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D053719:Tandem Mass Spectrometry; D013988:Ticlopidine / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008665:Metalloporphyrins; D015283:Citalopram; D005978:Glutathione; D003024:Clozapine; D013988:Ticlopidine