Vol. 1, /525-1536, Dee,nher 1995 Clinical Cancer Research 1525 Effect of Ifosfamide Treatment on Glutathione and Glutathione Conjugation Activity in Patients with Advanced Cancers’ Titia M T. Mulders,2 H. Jan Keizer, ever, whether a 35 % decrease is sufficient to increase tumor Jan Ouwerkerk, E. A. van der Velde, sensitivity toward (other) cytostatics remains uncertain. Douwe D Breimer, and Gerard J. Mulder3 INTRODUCTION Divisions of Toxicology and Pharmacology, LeidenlAmsterdam Center for Drug Research IT. M. T. M.. D. D. B.. G. J. M.]. and Ifosfamide is an alkylating oxazaphosphorine nitrogen Division of Medical Statistics IE. A. v. d. V.1, Leiden University, and mustard with activity against a wide range of tumors both in Division of Clinical Oncology, University Hospital H J. K.. J. 0.1, adults and children. It is a prodrug that requires biotransforma- Leiden, the Netherlands tion by cytochrome P450 isoenzyrnes to become cytotoxic. This metabolic process results in the formation of the active bifunc- ABSTRACT tional alkylating metabolite, isophosphoramide mustard, and Several studies have suggested that the glutathione/ acrobein, a metabolite believed to contribute to the hemorrhagic glutathione S-transferase (GSH/GST) system is involved in cystitis associated with oxazaphosphorine treatment. Ifosfamide resistance of tumors toward ifosfamide and other cytostatic can also undergo side chain dealkylation, resulting in the for- agents. Besides, ifosfamide metabolites (in vitro) as well as mation of 2- and 3-dechboroethylifosfarnide and equimolar ifosfamide treatment (in vivo) have been shown to decrease amounts of the highly reactive chloroacetaldehyde ( 1-4). High cellular GSH availability. In the present study, the in vivo plasma levels of chboroacetaldehyde have been suggested to be effects of three different ifosfamide treatment schedules on associated with ifosfamide-related neurotoxicity (5. 6). the GSH/GST system were studied in patients with ad- Resistance to cytostatic therapy is a common clinical prob- vanced cancers (n 24): continuous i.v. infusions of 1304) lem in the treatment of cancer. A wide variety of mechanisms mg/m2 daily for 10 days and 5000 mg/m2/day for 24 h, as has been implicated in the aetiobogy of drug resistance, includ- well as a 4-h infusion of 3000 mg/m2 daily for 3 days. The ing detoxification by the GSH/GST4 system. The relationship GSHJGST system was characterized by administering bro- between drug resistance and this system has been studied ex- misoval, a probe drug to assess GSH conjugation activity in tensively in human cancer cell lines and tumor tissues (e.g., vivo, as well as by daily monitoring of GSH concentrations in 7-13). However. evidence for involvement of the GSH/GST blood cells and plasma. Bromisoval pharmacokinetics was system in drug resistance in patients in vito is limited. Such assessed before and at the end of the ifosfamide treatment. studies are generally restricted to the assessment of GSH content Blood cell GSH levels decreased significantly (P < 0.05) (tumors, blood cells) on GST profile/activity of tumors of pa- during the 3- and 10-day ifosfamide treatment schedules; tients (14). the 24-h treatment had no effect. The ifosfamide treatment So far, several in vitro studies have provided evidence for schedules had only minimal effects on bromisoval pharma- the involvement of GSH and GSTs in the detoxification of cokinetics. Assuming that the kinetics of the probe drug metabolites derived from ifosfarnide and its structural analogue provide an accurate reflection of enzyme activity, this sug- cycbophospharnide. The ifosfarnide-derived 4-hydroxy interme- gests that GST activity remains unchanged. Because GSH diate and especially chloroacetaldehyde depleted intracellular conjugation of bromisoval enantiomers requires both GST GSH and inhibited the GST-catalyzed conjugation of b-chloro- activity and GSH availability, these results also indicate 2,4-dinitrobenzene (15). Depletion of cellular GSH levels was that, despite the 35% decrease in GSH in blood cells of two also demonstrated for the 4-hydroxy metabolite of cycbophos- patient groups, the GSH availability of the cancer patients phamide (16). Furthermore, GSH conjugation of 4-hydroxycy- was not rate-limiting for GSH conjugation of bromisoval cbophospharnide and phosphoramide mustard was shown to be enantiomers. If GSH levels in blood cells reflect those in catalyzed by cytosolic GSTs, in particular GSTA1-1 ( 17. 18). tumors/other tissues, the present results indicate that ifosf- Acrolein, a metabolite derived from both ifosfamide and cycbo- amide may be used clinically to decrease GSH levels. How- phosphamide. could be inactivated by human class a. p.. and in particular class ii GST isoenzymes. either by conjugation with GSH or by covalent binding to the enzymes in the absence of GSH ( 19). In vivo. Lind et a!. ( 1 5) demonstrated that an infusion Received 7/10/95; accepted 8/2/95. I Supported in part by ASTA Medica Aktiengesellschaft (Frankfurt am Main, Germany) and the Dutch ‘Praeventiefonds” (Project 28-1962, Den Haag. the Netherlands). 2 Present address: NV Organon, Medical Research and Development Unit, Molenstnaat 1 10, P. 0. Box 20, 5340 BH Oss, the Netherlands. 4 The abbreviations used are: GSH, glutathione: GSSG, glutathione 3 To whom requests for reprints should be addressed. at Division of disulfide (oxidized glutathione): GST, glutathione S-transferase: GSTM, Toxicology, Leiden/Amstendam Center for Drug Research. Leiden Uni- class p. glutathione S-transfenases: Hb, hemoglobin: HPLC, high pen- versity. Wassenaarseweg 72, P. 0. Box 9503, 2300 RA Leiden. the fonmance liquid chromatography: Cl11. clearance of formation of men- Netherlands. capturate: AUC, area under the curve: Cl/F, clearance/bioavailability. Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. 1526 Ifosfamide and Glutathione in Cancer Patients of ifosfamide in a patient resulted in a 70% decrease of GSH in ment period, mesna infusion was continued at a lower dose for lymphocytes. 1 day. For the prolonged ifosfamide (and mesna) infusions, Based on the above-mentioned in vitro and in vivo data, it external infusion pumps (CADD-I pump) were used, which was hypothesized that changes in the GSH/GST system may were connected to a Port-A-Cath (Pharmacia Deltec Inc., Woer- affect tumor resistance toward ifosfamide. Furthermore, GSH den, the Netherlands) iv. device. The ifosfarnide and mesna conjugation of ifosfamide (metabolites) may also lead to con- solutions were supplied by the Leiden University Hospital Phar- sumption of GSH, thereby making ifosfamide a potentially macy. GSH-lowering cytostatic agent, which may be used in combi- nation with (an)other cytostatic agent(s)-for which involve- Characterization of GSH Conjugation ment of the GSHJGST system in resistance is a major contrib- Characterization of the GSHJGST system was performed uting factor-with the aim to improve the efficacy of the by administering the racemic drug bromisoval as well as by cytostatic treatment. daily assessment of GSH (plasma and blood cells) and 0550 In the present study, the in vivo effects of ifosfamide (blood cells) concentrations. In addition, the GSTM 1 phenotype treatment on the GSHJGST system were studied in patients with of all patients was determined. advanced cancers. Three different ifosfamide treatment sched- Bromisoval. Brornisoval pharmacokinetics (plasma ules were studied, i.e., a continuous iv. infusion of 1 300 mg/rn2 pharmacokinetics of the bromisoval enantiomers and urinary daily for 10 days or 5000 mg/m2/day for 24 h and a 4-h infusion excretion of their corresponding mercapturates) was assessed on of 3000 mg/rn2 daily for 3 days. The GSH/GST system of the day prior to the start of the ifosfamide infusion (day 0), the patients in vivo was characterized by administering 2-bro- last day of the 3 (3000 mg/rn2 daily)-. and 1 0 ( 1 300 mg/rn2 moisovalerylurea (bromisoval), a probe drug to assess GSH daily)-day ifosfamide treatment period, and the day after the conjugation activity in vivo (20, 21), as well as by daily moni- 24-h (5000 mg/m2/day) ifosfamide infusion. On both bromi- toring of GSH (plasma and blood cells) and GSSG (blood cells) soval study days, patients received a capsule containing 600 rng concentrations. (2.69 mmol) racemic brornisoval after an overnight fast. The brornisoval (Dutch Pharmacopoeia) capsules were supplied by PATIENTS AND METHODS the Leiden University Hospital Pharmacy. Patients refrained The study protocol was approved by the Ethics Review from all food and drinks (except plain mineral water ad libitum) Board of the Leiden University Hospital (Leiden, the Nether- until 3 h after brornisoval administration. To ensure sufficient lands). The study was an open trial. Twenty-four patients par- urine production, the patients were asked to drink regularly. ticipated after each gave oral informed consent before study Patient 102 (24-h ifosfamide treatment) did not receive bromi- entry. soval because this patient refused to participate in the brorni- soval study on the ifosfamide pretreatment day. Patient 304 Patients (3-day ifosfamide treatment) did not receive bromisoval because Patients of either sex, 75 years old or less, with histologi- this patient had a jejunal fistula. cally proven advanced cancer and for whom no standard therapy Blood and Urine Sampling. On the ifosfamide pretreat- was available were eligible. Furthermore, patients had to have ment day