Comparative metabolism of cyclophosphamide and ifosfamide in the mouse using UPLC-ESI-QTOFMS-based metabolomics

Abstract

Ifosfamide (IF) and cyclophosphamide (CP) are common chemotherapeutic agents. Interestingly, while the two drugs are isomers, only IF treatment is known to cause nephrotoxicity and neurotoxicity. Therefore, it was anticipated that a comparison of IF and CP drug metabolites in the mouse would reveal reasons for this selective toxicity. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), and the results analyzed by multivariate data analysis. Of the total 23 drug metabolites identified by UPLC-ESI-QTOFMS for both IF and CP, five were found to be novel. Ifosfamide preferentially underwent N-dechloroethylation, the pathway yielding 2-chloroacetaldehyde, while cyclophosphamide preferentially underwent ring-opening, the pathway yielding acrolein (AC). Additionally, S-carboxymethylcysteine and thiodiglycolic acid, two downstream IF and CP metabolites, were produced similarly in both IF- and CP-treated mice. This may suggest that other metabolites, perhaps precursors of thiodiglycolic acid, may be responsible for IF encephalopathy and nephropathy.

Fig. 1

Metabolic activation and transport of IF and CP in vivo. (A) Rational design of CP based upon its supposed metabolism in tumor tissue versus a contemporary view of CP metabolism. (B) A contemporary view of IF metabolism, including the N-dechloroethylation reactions that lead to two-carbon metabolites and ultimately SCMS and TDGA.

Fig. 2

Identification of urinary IF and CP metabolites through LC-MS-based metabolomics. (A) Scores plot of an OPLS model from control and IF-treated mice. Each point represents an individual mouse urine (B) Scores plot of a OPLS model from control and CP-treated mice. Each point represents an individual mouse urine (C) OPLS loadings S-plot of urinary ions from control and IF-treated mice. Each point represents a urinary ion (D) OPLS loadings S-plot of chemical ions from control and CP-treated mice. Each point represents a urinary ion.

Fig. 3

Trend plots of ions from novel metabolites in the treatment groups. (A) Novel IF metabolites with m/z values of 259.0171 (F5) and 453.0584 (F11). (B) Novel CP metabolites with m/z values 213.0198 (P11), 217.0511 (P12), and 455.0781 (P9). Note the absence of suspected drug metabolites in the vehicle-treated group. Metabolite codes correspond to those in Fig. 4.

Fig. 4

Tandem MS and chemical structures of novel CP and IF metabolites. (A) Iminoifosfamide (F5). (B) 4-Hydroxyifosfamide glucuronide (F11). (C) Dechloroethylketocyclophosphamide (P11). (D) Dechloroethylalcophosphamide (P12). (E) Alcophosphamide glucuronide (P9). Note the chlorine isotope ratios depending on the presence of a one (3:1 ratio) or two (9:6:1) chlorine atoms.

Fig. 5

Relative quantitation of urinary metabolites from different groups of metabolic pathways in mouse urine following the treatment of IF and CP. Except for unchanged IF and CP, there were significant differences in these metabolites with similar chemical structures from IF and CP. NS, not significant. Note that the β-elimination of acrolein (IF>CP; P<0.05) and glucuronidation (CP>IF; P<0.01) pathways were very minor compared to other pathways, but nevertheless showed statistically significant differences.

Fig. 6

The amount (μmol/24h) and % dose excreted for SCMC and TDGA in 0-24 h mouse urines following treatment with IF and CP. (A) The μmol/24h of SCMC from IF- and CP-treated mice. (B) Percent dose excretion of SCMC from IF- and CP-treated mice. (C) The μmol/24h of TDGA from IF- and CP-treated mice. (D) Percent dose excretion of TDGA from IF- and CP-treated mice. N.S. means not significant. Note the small amounts of SCMC and TDGA excreted in blank (control) 0-24 h mouse urines.

Fig. 7

Major in vivo IF metabolic pathways showing the enzyme systems that are believed to produce each metabolite. Boxed structures represent novel metabolites.

Fig. 8

Major in vivo CP metabolic pathways showing the enzyme systems that are believed to produce each metabolite. Boxed structures represent novel metabolites.