Cancer Chemother Pharmacol (1985) 15: 63-65 ancer hemotherapyand harmacology © Sprmger-Verlag 1985 Evidence for electrophilic properties of N2-methyl-9-hydroxy ellipticinium acetate (Celiptium) from human biliary metabolites Jean Bernadou l*, Bernard Monsarrat 1, Henri Roche 2, Jean-Pierre Armand 2, Claude Paoletti 3, and Bernard Meunier 1 Laboratoire de Pharmacologic et de Toxicologic Fondamentales du CNRS, 205 route de Narbonne, F-31400 Toulouse, France 2 Centre Claudius Regaud, 20-24, rue de Pont Saint-Pierre, F-31052 Toulouse, France 3 Laboratoire de Biochimie et d'Enzymologie, INSERM U140, CNRS LA147, Institut Gustave Roussy, F-94800 Villejuif, France Summary. The human biliary metabolism of the antitumor agent N2-methyl-9-hydroxyellipticinium acetate is de- scribed. Three major compounds have been identified by high-performance liquid chromatography and comparison with synthetic reference derivatives: the unchanged drug, the O-glucuronide conjugate and the cysteinyl-ellipticini- um adduct. The latter one is the expected detoxification compound of an intermediate electrophilic quinone-imine derivative generated in vivo. This result provides a further evidence that hydroxylated forms of ellipticine derivatives might be activated by a biooxidation route. Introduction N~Methyl-9-hydroxyellipticinium (9-OH-NME) is one of the most efficient anticancer drugs in the ellipticine series and has consequently been used for clinical purposes [2, 6]. We recently reported that such a compound could be con- sidered as a stable reduced form of a p-quinone-imine structure [1]; an electrophilic center might be generated in vivo by an initial biooxidative transformation. This highly electrophilic form, when obtained in vitro, reacts with var- ious nucleophiles, including glutathione (GSH), to form adducts with a covalent C-S bond [8, 9]. Previously, we were able to isolate and identify from rats (i) a bile glutathione conjugate and (ii) a urine N-acetylcysteine conjugate as minor metabolites [7, 10]. This indicates that 9-OH-NME is susceptible to a minor metabolic pathway of activation and detoxification. In human urine, in addition to the glucuronide conju- gate of the drug, we have recently detected significant amounts of cysteine and N-acetylcysteine conjugates [10]. In this present work, we report the analysis of bile samples collected from a patient with external bile derivation treat- ed with Celiptium and the identification of a cysteine con- jugate. Case report A 36-year-old male patient being treated for a colon ade- nocarcinoma was evaluated in this study. He had a percu- taneous transhepatic drainage of the biliary tract as syrup- * Part of this work was done by J. B. as part of his preparation for the degree of Doctor of Medicine in the Centre Claudius Re- gaud Offprint requests to: B. Meunier tomatic treatment for neoplastic obstructive jaundice and had exhausted other conventional therapeutic measures. Ten days after insertion of the endoprothesis a clinical and biological improvement was obtained (bilirubin decreased from 254 to 78 lxM (normal = 3-20); alkaline phospha- tase decreased from 2,175 to 1,077 mU//ml (normal = 0- 260); transaminases returned to the normal levels; how- ever, GGT remained at a high level: 503 mU//ml (normal = 0-37); then the patient received 50 mg Celiptium in a 30-rain infusion. Bile samples were collected at time 0 (control) and at 1, 2, 3, 4, 5, 7, and 17 h after the beginning of the infusion. Urine was collected for the same period. All these samples were stored at - 18 °C. Materials and methods Glucuronide, cysteine, N-acetylcysteine, and glutathione- ellipticinium reference adducts were prepared according to the procedures previously described [3, 8, 9]. [~-Glucuronidase (E. coli type VII) was obtained from Sig- ma. Chromatographic analysis (HPLC) was performed as for human urine [10]. Results 1. Metabolic profile The chromatogram of bile for the 3 to 4 h period following drug administration (Fig. lb) indicates the presence of three new peaks compared with control bile (Fig. l a). Peaks I, II, and III are respectively identified as un- changed drug, glucuronide conjugate, and cysteine conju- gate; after the treatment of bile with [~-glucuronidase (Fig. lc) peak II disappeared and peak I increased, indi- cating that peak II represents the glucuronide conjugate of the administered drug. Since peak III has the same reten- tion time as the cysteine and glutathione reference conju- gates on a C18-~t-Bondapak Waters column, separation was achieved on an ultrasphere ODS Altex column [10]. Conse- quently, peak III was identified as the cysteine conjugate. 2. Kinetics of biliary excretion For each collection interval, the unchanged drug and its metabolites were quantified from linear calibration curves (peak areas vs concentrations) of bile samples spiked with known quantities of each reference compound. The biliary excretion of metabolites is shown by collection period in