Investigation of Clozapine and Olanzapine Reactive Metabolite Formation and Protein Binding by Liquid Chromatography-Tandem Mass Spectrometry

Drug-induced
toxicity has, in many cases, been linked to oxidative
metabolism resulting in the formation of reactive metabolites and
subsequent covalent binding to biomolecules. Two structurally related
antipsychotic drugs, clozapine (CLZ) and olanzapine (OLZ), are known
to form similar nitrenium ion reactive metabolites. CLZ-derived reactive
metabolites have been linked to agranulocytosis and hepatotoxicity.
We have studied the oxidative metabolism of CLZ and OLZ as well as
two known metabolites of CLZ, desmethyl-CLZ (DCLZ), and CLZ-N-oxide (CLZ-NO), using in vitro rat liver
microsomal (RLM) incubations with glutathione (GSH) trapping of reactive
metabolites and liquid chromatography-high resolution tandem mass
spectrometry (LC-HRMS/MS). Reactive metabolite binding to selected
standard peptides and recombinant purified human proteins was also
evaluated. Bottom-up proteomics was performed using two complementary
proteases, prefractionation of peptides followed by LC-HRMS/MS for
elucidating modifications of target proteins. Induced RLM was selected
to form reactive metabolites enzymatically to assess the complex profile
of reactive metabolite structures and their binding potential to standard
human proteins. Multiple oxidative metabolites and several different
GSH adducts were found for CLZ and OLZ. Modification sites were characterized
on human glutathione S-transferase (hGST) alpha 1
(OLZ-modified at Cys112), hGST mu 2 (OLZ at Cys115), and hGST pi (CLZ,
DCLZ, CLZ-NO and OLZ at Cys170), human microsomal GST 1 (hMGST1, CLZ
and OLZ at Cys50), and human serum albumin (hSA, CLZ at Cys34). Furthermore,
two modified rat proteins, microsomal GST 1 (CLZ and OLZ at Cys50)
and one CYP (OLZ-modified, multiple possible isoforms), from RLM background
were also characterized. In addition, direct effects of the reactive
metabolite modifications on proteins were observed, including differences
in protease cleavage specificity, chromatographic behavior, and charge-state
distributions.