A comprehensive understanding of thioTEPA metabolism in the mouse using UPLC-ESI-QTOFMS-based metabolomics

Abstract

ThioTEPA, an alkylating agent with anti-tumor activity, has been used as an effective anticancer drug since the 1950s. However, a complete understanding of how its alkylating activity relates to clinical efficacy has not been achieved, the total urinary excretion of thioTEPA and its metabolites is not resolved, and the mechanism of formation of the potentially toxic metabolites S-carboxymethylcysteine (SCMC) and thiodiglycolic acid (TDGA) remains unclear. In this study, the metabolism of thioTEPA in a mouse model was comprehensively investigated using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) based-metabolomics. The nine metabolites identified in mouse urine suggest that thioTEPA underwent ring-opening, N-dechloroethylation, and conjugation reactions in vivo. SCMC and TDGA, two downstream thioTEPA metabolites, were produced from thioTEPA from two novel metabolites 1,2,3-trichloroTEPA (VII) and dechloroethyltrichloroTEPA (VIII). SCMC and TDGA excretion were increased about 4-fold and 2-fold, respectively, in urine following the thioTEPA treatment. The main mouse metabolites of thioTEPA in vivo were TEPA (II), monochloroTEPA (III) and thioTEPA-mercapturate (IV). In addition, five thioTEPA metabolites were detected in serum and all shared similar disposition. Although thioTEPA has a unique chemical structure which is not maintained in the majority of its metabolites, metabolomic analysis of its biotransformation greatly contributed to the investigation of thioTEPA metabolism in vivo, and provides useful information to understand comprehensively the pharmacological activity and potential toxicity of thioTEPA in the clinic.

Fig. 1.

Identification of thioTEPA metabolites in urine and serum using UPLC-ESI-QTOFMS-based metabolomics. (A) Scores plot of an OPLS model and OPLS loadings S-plot of urinary ions from control and thioTEPA-treated mice. Each point represents an individual mouse urine sample (top) and a urinary ion (bottom). ThioTEPA and its metabolites are labeled in the S-plot (I, II, IIa, III, IV, IVa, IVb, V, VI, VII, and VIII). (B) Scores plot of an OPLS model and OPLS loadings S-plot of serum ions from control and thioTEPA-treated mice. Each point represents an individual mouse serum sample (top) and a serum ion (bottom). ThioTEPA and its metabolites are labeled in the S-plot (I, II, III, and IV). The t[1] and t[2] correspond to principal components 1 and 2, respectively. The p(corr)[1]P values represent the interclass difference and p[1]P values represent the relative abundance of the ions. TDGA and SCMC were best detected in negative ion mode (ESI-) and therefore they are not shown in the OPLS loadings S-plot of positive ions (ESI+).

Fig. 2.

Trend plots of ions from thioTEPA metabolites in the control and thioTEPA-treated mice urine after treatment for 24 h. (A) Metabolite II with m/z value of 174.0797⁺. (B) Metabolite IV with m/z value of 353.0854⁺. (C) Metabolite V with m/z value of 311.0776⁺. (D) Metabolite VI with m/z value of 246.0326⁺. (E) Metabolite VII with m/z value of 282.0105⁺. (F) Metabolite VIII with m/z value of 220.0175⁺. Note the absence of putative drug metabolites in the control group. Metabolite codes correspond to those in Fig. 1. Obs ID represents the mouse urine sample and XVar values represent the relative abundance of the ions.

Fig. 3.

Tandem MS and chemical structures of thioTEPA metabolites. (A) TEPA (II). (B) MonochloroTEPA (III). (C) ThioTEPA-mercapturate (IV). (D) 1,2-DichloroTEPA (VI). (E) 1,2,3-TrichloroTEPA (VII). (F) DechloroethyltrichloroTEPA (VIII). Note the chlorine isotope ratios depending on the presence of one (3:1), two (9:6:1) or three (27:27:9:1) chlorine atoms.

Fig. 4.

Urinary excretion of thioTEPA and its metabolites from 0 to 24 h following the treatment of thioTEPA (n = 4). (A) Percent dose excretion of unchanged thioTEPA (I) at different time points. (B) Relative urinary excretion of thioTEPA metabolites II, III, and IV at different time points. (C) Relative urinary excretion of thioTEPA (I) and its metabolites V, VI, VII, and VIII at different time points.

Fig. 5.

The amount for SCMC and TDGA in 24 h mice urine following the treatment of thioTEPA (50 mg/kg, approximately 1 mg of thioTEPA), CAA (2 mg/kg, approximately 0.04 mg of CAA), and SCMC (100 mg/kg, approximately 2 mg of SCMC). (A) The μmol/24 h of SCMC from control and thioTEPA-treated mice. (B) The μmol/24 h of TDGA from control and thioTEPA-treated mice. (C) The fold change of SCMC from control and CAA-treated mice. (D) The fold change of TDGA from control and CAA-treated mice. (E) The fold change of TDGA from control and SCMC-treated mice. Significant differences (P < 0.05) in control and thioTEPA-treated, CAA-treated, or SCMC-treated group.

Fig. 6.

The concentration-time profiles of thioTEPA and its metabolites in serum after intraperitoneal administration at 50 mg/kg of thioTEPA (approximately 1 mg of drug) in mice (n = 4). (A) Pharmacokinetic process of thioTEPA (I) in mouse (from 0.08 to 10 h). (B) Relative peak area-time profiles of thioTEPA metabolites II, III, IV, VI, and VII in mouse (from 0.08 to 10 h).

Fig. 7.

Serum chemistry of thioTEPA- and TDGA-treated mice. (A) Serum amylase in thioTEPA treatment and control mice. (B) Serum glucose in thioTEPA treatment and control mice. (C) Serum amylase in TDGA treatment and control mice. (D) Serum glucose in TDGA treatment and control mice. Significant differences (P < 0.05) in thioTEPA or TDGA treatment and control group.

Fig. 8.

Major in vivo ThioTEPA metabolic pathways. Boxed structures represent novel metabolites.