New ifosfamide analogs designed for lower associated neurotoxicity and nephrotoxicity with modified alkylating kinetics leading to enhanced in vitro anticancer activity.

Ifosfamide is a well known prodrug for cancer treatment with cytochrome P450 metabolism. It is associated with both antitumor activity and toxicities. Isophosphoramide mustard is the bisalkylating active metabolite, and acrolein is a urotoxic side product. Because acrolein toxicity is limited by coadministration of sodium mercaptoethanesulfonate, the incidence of urotoxicity has been lowered. Current evidence suggests that chloroacetaldehyde, a side-chain oxidation metabolite, is responsible for neurotoxicity and nephrotoxicity. The aim of our research is to prevent chloroacetaldehyde formation using new enantioselectively synthesized ifosfamide analogs, i.e., C7,C9-dimethyl-ifosfamide. We hypothesize that reduced toxicogenic catabolism may induce less toxicity without changing anticancer activity. Metabolite determinations of the dimethyl-ifosfamide analogs were performed using liquid chromatography and tandem mass spectrometry after in vitro biotransformation by drug-induced rat liver microsomes and human microsomes expressing the main CYP3A4 and minor CYP2B6 enzymes. Both human and rat microsomes incubations produced the same N-deschloroalkylated and 4-hydroxylated metabolites. A coculture assay of 9L rat glioblastoma cells and rat microsomes was performed to evaluate their cytotoxicity. Finally, a mechanistic study using (31)P NMR kinetics allowed estimating the alkylating activity of the modified mustards. The results showed that C7,C9-dimethyl-ifosfamide exhibited increased activities, although they were still metabolized through the same N-deschloroalkylation pathway. Analogs were 4 to 6 times more cytotoxic than ifosfamide on 9L cells, and the generated dimethylated mustards were 28 times faster alkylating agents than ifosfamide mustards. Among these new ifosfamide analogs, the 7S,9R-enantiomer will be assessed for further in vivo investigations for its anticancer activity and its toxicological profile.

Pharmacokinetic-pharmacodynamic relationships of imatinib and its main metabolite in patients with advanced gastrointestinal stromal tumors.

PURPOSE: This study explored factors affecting the pharmacokinetic variability of imatinib and CGP 74588, and the pharmacokinetic-pharmacodynamic correlations in patients with advanced gastrointestinal stromal tumors. EXPERIMENTAL DESIGN: Thirty-five patients with advanced gastrointestinal stromal tumors received 400 mg of imatinib daily. Six blood samples were drawn: before intake, during 1- to 3- and 6- to 9-hour intervals after intake on day 1, and before intake on days 2, 30, and 60. Plasma imatinib and CGP 74588 concentrations were quantified by reverse-phase high-performance liquid chromatography coupled with tandem mass spectrometry, and analyzed by the population pharmacokinetic method (NONMEM program). The influence of 17 covariates on imatinib clearance (CL) and CGP 74588 clearance (CLM/fm) was studied. These covariates included clinical and biological variables and occasion (OCC = 0 for pharmacokinetic data corresponding to the first administration, or OCC = 1 for the day 30 or 60 administrations). RESULTS: The best regression formulas were: CL (L/h) = 7.97 (AAG/1.15)(-0.52), and CLM/fm (L/h) = 58.6 (AAG/1.15)(-0.60) x 0.55(OCC), with the plasma alpha1-acid glycoprotein (AAG) levels indicating that both clearance values decreased at a higher AAG level. A significant time-dependent decrease in CLM/fm was evidenced with a mean (+SD) CGP 74588/imatinib area under the curve (AUC) ratio of 0.25 (+/-0.07) at steady state, compared with 0.14 (+/-0.03) on day 1. Hematologic toxicity was correlated with pharmacokinetic variables: the correlation observed with the estimated unbound imatinib AUC at steady-state (r = 0.56, P < 0.001) was larger than that of the total imatinib AUC (r = 0.32, NS). CONCLUSIONS: The plasma AAG levels influenced imatinib pharmacokinetics. A protein-binding phenomenon needs to be considered when exploring the correlations between pharmacokinetics and pharmacodynamics.

Liquid chromatography-mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium.

A specific and sensitive quantitative assay has been developed using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantitation of the antitumor drug ifosfamide (IFM) and its two metabolites, N2-deschloroethylifosfamide (N2-DCE-IFM) and N3-deschloroethylifosfamide (N3-DCE-IFM) in microsomal medium. The analytes and the internal standard (cyclophosphamide) were isolated by ethylacetate extraction from rat liver microsomes. They were analysed on a Nucleosil C18 HD column (125 mm x 4 mm, 5 microm) using a step gradient with the mobile phase (2 mM ammonium formate and methanol). The HPLC-ESI-MS method used selected ion monitoring of ions m/z 199.1 Th and m/z 261.1 Th and was validated in the concentrations ranges of 100-5000 ng/mL for IFM and 50-2500 ng/mL for its N-deschloroethylated metabolites (DCE-IFM) with good accuracy and precision (CV less than 15%). The low limits of quantitation (LLOQ) were found at 50 ng/mL for N-deschloroethylated metabolites and at 100 ng/mL for the parent drug (IFM). The method was applied for the determination of ifosfamide and its N-deschloroethylated metabolites in rat microsomal incubations.

Quantitative liquid chromatographic determination of sanguinarine in cell culture medium and in rat urine and plasma.

Sanguinarine is a quaternary benzo[c]phenanthridine alkaloid, extracted from the argemone oil, which produced severe human intoxications. To investigate the sanguinarine biotransformation, we develop a simple extraction process and a high performance liquid chromatographic separation coupled to a sensitive fluorometric detection of sanguinarine in cell culture medium, as well as in rat urine and plasma. After extraction with an acidified organic solvent, sanguinarine elution is performed within 15 min on a Nucleosil C18 column with a gradient using 0.2% formic acid/water/acetonitrile as mobile phase. Extracted and standard sanguinarine are characterized by mass spectrometry. The extraction recovery of sanguinarine is about 80% in cell culture medium and in rat urine, but lower in plasma. This convenient high performance liquid chromatography (HPLC) method allows to quantify sanguinarine over concentrations ranged 10-2000 ng ml(-1). The limit of fluorometric detection is 0.5 ng. Under these conditions, the lower limit of quantification of sanguinarine is 50 ng ml(-1) in cell culture medium and in rat urine and 100 ng ml(-1) in rat plasma. This analytical HPLC method is specific, linear and reproducible in all media and is suitable for quantitative determination of sanguinarine in biological fluids.

Metabolism of sanguinarine in human and in rat: characterization of oxidative metabolites produced by human CYP1A1 and CYP1A2 and rat liver microsomes using liquid chromatography-tandem mass spectrometry.

The quaternary benzo[c]phenanthridine alkaloid, sanguinarine (SA), has been detected in the mustard oil contaminated with Argemone mexicana, which produced severe human intoxications during epidemic dropsy in India. Today, SA metabolism in human and in rat has not yet been fully elucidated. The goal of this study is to investigate the oxidative metabolites of SA formed during incubations with rat liver microsomes (RLM) and recombinant human cytochrome P450 (CYP) and to tentatively identify the CYP isoforms involved in SA detoxification. Metabolites were analyzed by liquid chromatography combined with electrospray ionization-tandem mass spectrometry. Up to six metabolites were formed by RLM and their modified structure has been proposed using their mass spectra and mass shifts from SA (m/z 332). The main metabolite M2 (m/z 320) resulted from ring-cleavage of SA followed by demethylation, whereas M4 (m/z 348) is oxidized by CYP in the presence of NADPH. The diol-sanguinarine metabolite M6 (m/z 366) formed by RLM might derive from a putative epoxy-sanguinarine metabolite M5 (m/z 348). M4 and M6 could be detected in rat urine as their respective glucuronides. 5,6-Dihydrosanguinarine is the prominent derivative formed from SA in cells expressing no CYP. Oxidative biotransformation of SA was investigated using eight human CYPs: only CYP1A1 and CYP1A2 displayed activity.

Synthesis and biological evaluation of two glycerolipidic prodrugs of didanosine for direct lymphatic delivery against HIV.

Novel glycerolipidic prodrugs of didanosine and didanosine monophosphate designed to by-pass the hepatic first pass metabolism were synthesized and tested for their cytotoxicity and anti-HIV-1 activity. Formulation as liposomes of dipalmitoylphosphatidylcholine was elaborated. A simple quantitative HPLC-UV method was developed and validated, and ESI-MS was used for qualitative purpose. These two prodrugs exhibited promising biological activities against HIV-1 in in vitro infected cell culture.

Induction of glutathione synthesis explains pharmacodynamics of high-dose busulfan in mice and highlights putative mechanisms of drug interaction.

Busulfan is an example of a drug eliminated through glutathione S-transferase (GST)-catalyzed conjugation with reduced glutathione (GSH). We studied the pharmacokinetics and toxicity of busulfan in C57BL6 mice in correlation with liver GST activity and GSH synthesis by accurate determination of precursors, namely, gamma-glutamyl-cysteine and cysteine. A significantly lower incidence of acute toxicity was observed in mice receiving busulfan 16.5 mg/kg twice a day compared with animals receiving 33 mg/kg once a day. In both cases, a total dose of 132 mg/kg was administered over 4 days. The difference in toxicity was explained by pharmacokinetics since a strong induction of clearance was observed only in animals treated twice daily. Induction of metabolism was correlated with an increase in liver cysteine content and enhanced glutathione synthesis rate, whereas GST activity was unchanged. To our knowledge, this is the first time that in vivo flux of GSH synthesis has been shown to be closely related to a drug plasma clearance and toxicity. These results allow hypothesizing that GSH liver synthesis may directly influence busulfan clearance in humans with possible implications in the occurrence of hepatic veno-occlusive disease.

Pharmacokinetics, metabolism, and routes of excretion of intravenous irofulven in patients with advanced solid tumors.

Irofulven is currently in Phase 2 clinical trials against a wide variety of solid tumors and has demonstrated activity in ovarian, prostate, gastrointestinal, and non-small cell lung cancer. The objectives of this study were to determine its pharmacokinetics and route of excretion and to characterize its metabolites in human plasma and urine samples after a 30-min i.v. infusion at a dose of 0.55 mg/kg in patients with advanced solid tumors. Three patients were administered i.v. 100 microCi of [14C]irofulven over a 30-min infusion on day 1 of cycle 1. Serial blood and plasma samples were drawn at 0 (before irofulven infusion) and up to 144 h after the start of infusion. Urine and fecal samples were collected for up to 144 h after the start of infusion. The mean urinary and fecal excretion of radioactivity up to 144 h were 71.2 and 2.9%, respectively, indicating renal excretion was the major route of elimination of [14C]irofulven. The C(max), AUC(0-infinity), and terminal half-life values for total radioactivity were 1130 ng-Eq/ml, 24,400 ng-Eq . h/ml, and 116.5 h, respectively, and the corresponding values for irofulven were 82.7 ng/ml, 65.5 ng . h/ml, and 0.3 h, respectively, suggesting that the total radioactivity in human plasma was a result of the metabolites. Twelve metabolites of irofulven were detected in human urine and plasma by electrospray ionization/tandem mass spectrometry. Among these metabolites, the cyclopropane ring-opened metabolite (M2) of irofulven was found, and seven others were proposed as glucuronide and glutathione conjugates.

In vitro metabolism of ethoxidine by human CYP1A1 and rat microsomes: identification of metabolites by high-performance liquid chromatography combined with electrospray tandem mass spectrometry and accurate mass measurements by time-of-flight mass spectrometry.

Ethoxidine (N-methyl-12-ethoxy-2,3,8,9-tetramethoxybenzo[c]phenanthridinium methylsulfonate salt) is a synthetic 2-methoxy-12-ethoxy derivative of the natural alkaloid fagaronine. This new inhibitor of DNA-topoisomerase I is considered as a potential antitumor agent with higher in vitro activity than fagaronine. In order to further improve the efficiency of ethoxidine, its in vitro biotransformation by hepatic monooxygenases and the structures of its metabolites were investigated by high-performance liquid chromatography (HPLC) combined with electrospray ionization tandem mass spectrometry (ESI-MS/MS) and accurate mass measurement by time-of-flight mass spectrometry (TOFMS). When ethoxidine was incubated with BNF-treated rat liver microsomes or with cells expressing different recombinant human cytochrome P450, the same four ethoxidine metabolites (m(1)-m(4)) were detected and were formed exclusively by CYP1A1. The structures of these metabolites were assigned from ESI-MS/MS mass spectra and compared with those of ethoxidine derivatives. Accurate mass measurements of in-source ESI-TOFMS fragment ions exhibited successive neutral losses of C(2)H(4) and CO for ethoxidine and its metabolites. Whereas a 15 Da loss (methyl radical) was observed for the metabolites m(1)-m(4) containing a quaternary ammonium group, a 16 Da loss (methane) was observed for ethoxidine and could have resulted from the presence of two methoxy groups at adjacent positions (C-2 and C-3). The proposed oxidative modifications of ethoxidine were further confirmed by determination of the number of exchangeable hydrogen atoms and by the proposed elemental compositions of the metabolites based on accurate mass measurements by TOFMS. Two major metabolites resulted from O-demethylation of ethoxidine; one was tentatively identified as 12-ethoxyfagaronine (m(3)) and the second as an O-demethylated ethoxidine isomer (m(4)). Two polar metabolites were shown to be O-demethylated (m(1)) and hydroxylated (m(2)) derivatives of 12-ethoxyfagaronine. When 12-ethoxyfagaronine was incubated under the same conditions as ethoxidine, m(2) was formed, thus supporting the proposal that 12-ethoxyfagaronine is the primary oxidative product of ethoxidine.