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[Cancer Research 59, 4720-4724, September 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 4720-4724, September 15, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

A Search for New Metabolites of N,N',N''-Triethylenethiophosphoramide

Maria J. van Maanen1, Inge M. Tijhof, J. Mirjam A. Damen, Cees Versluis, Jantien J. Ketteness van den Bosch, Albert J. R. Heck, Sjoerd Rodenhuis and Jos H. Beijnen

Departments of Pharmaceutical Analysis [M. J. v. M., I. M. T., J. H. B.] and Biomolecular Mass Spectrometry [J. M. A. D., C. V., J. J. K. v. d. B., A. J. R. H.], University of Utrecht, 3584 CA Utrecht; The Netherlands Cancer Institute/Slotervaart Hospital, 1066 EC Amsterdam [M. J. v. M., J. H. B.]; and The Netherlands Cancer Institute/Antoni van Leeuwenhoek Hospital, 1066 CX Amsterdam [S. R.], the Netherlands


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N''-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a ß-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95°C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54–100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The alkylating agent thioTEPA2 has been applied in cancer therapy now for >40 years (1) . Because of its broad spectrum of antitumor activity and its relative lack of extramedullary toxicity, thioTEPA is recently being used in high-dose combination regimens for breast cancer, ovarian cancer, and other solid tumors (2) . Despite the extensive use of thioTEPA, only a part of the metabolic profile is known. The formation of the metabolite TEPA was first reported by Mellet and Woods (3) . TEPA is formed in the liver after oxidative desulfuration, mediated by specific cytochrome P450 enzymes, including IIB1 and IIC11 (4 , 5) . Previous studies describe the cellular metabolic pathway wherein thioTEPA liberates aziridine, which is hydrolyzed to ethanolamine and is incorporated into phosphatidylethanolamine (6 , 7) . It was not until 1985 that sensitive methods were developed to determine the levels of thioTEPA and TEPA simultaneously in biological samples (8 , 9) . TEPA rapidly appears in plasma after thioTEPA infusion (10 , 11) and has a half-life that is 2–7 times longer than that of thioTEPA (12 , 13) . Urinary excretion of thioTEPA and TEPA represented only 5% of the administered dose, whereas urinary alkylating activity accounts for 25% of the administered dose (11 , 12) . These findings suggest the presence of metabolites other than TEPA. A metabolic study with 32P-labeled TEPA in rats showed that, after 24 h, 50% of the drug was excreted unchanged and ~40% was excreted as phosphate (14) . In a study with 14C-labeled thioTEPA in humans, recovery of total urinary 14C radioactivity in a patient receiving i.v. thioTEPA was 63%, of which 0.2% was excreted unchanged thioTEPA (15) . In vitro studies on the stability of thioTEPA in urine at various pHs show decomposition of the drug at decreasing pH, but no loss of alkylating activity was observed. In urine that had been adjusted to pH 4 with HCl, conversion of thioTEPA to ß-chloroethyl moieties was seen. In summary, it is well known now that thioTEPA is extensively metabolized, and the metabolites are also alkylating products. However, apart from TEPA, no other metabolic products have been identified until now. This study was set up to elucidate the structure(s) of additional thioTEPA metabolites.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Chemicals
thioTEPA was obtained from Cyanamid Benelux (Etten-Leur, the Netherlands). N-Acetylcysteine, GSH, and NBP were obtained from Sigma Chemical Co. (St. Louis, MO). All other chemicals used were analytical grade, unless otherwise stated.

Synthesis and Structure Identification of Reference Compounds
TEPA.
Here, we used a slightly modified version of the procedure for the synthesis of TEPA described by Craig and Jackson (14) . In a two-necked round-bottomed flask equipped with a magnetic stirring bar and a dropping funnel, 3.99 g (93 mmol) of aziridine and 10.01 g (99 mmol) of triethylamine were dissolved in 50 ml of dry benzene under dry nitrogen. The flask was cooled in an ice bath, and a solution of 4.60 g (30 mmol) of phosphorusoxychloride in 20 ml of benzene was added dropwise with vigorous stirring. After completion of the addition (~45 min), the cooling bath was removed, and stirring was continued for 24 h. The white precipitate was removed by filtering through a glass filter (G4). The reaction flask and precipitate were thoroughly washed with benzene. The combined filtrate and washings were concentrated under reduced pressure, followed by removal of remaining traces of volatile compounds in an oil pump vacuum at room temperature. There was a yield of 4.48 g of a slightly yellow oil. Distillation in a microdistillation apparatus gave a main fraction of 3.53 g (68%) of a colorless oil boiling at 91–93°C/0.04 mm. The structure and purity of the reaction product were determined by 1H NMR and GC-MS. GC-MS, using chemical ionization, showed the protonated molecular ion [M+H]+ at m/z 174. A fragment ion at m/z 131 corresponds to the loss of an aziridine ring. The 1H-NMR spectrum showed a single doublet at 2.18 ppm, JPH = 14.4 Hz. The deuterated chloroform signal at 7.26 ppm was used as the reference line.

monochloroTEPA.
Synthesis of monochloroTEPA was carried out by incubation of TEPA with 1 M sodium chloride in 25 mM sodium phosphate buffer (pH 8) for 2 h at 80°C. The monochloroTEPA was separated from TEPA by SPE with a C18 cartridge, and its identity was established by GC-MS, using electron impact ionization. Molecular ions [M]+ were at m/z 209 and 211, in a 3:1 ratio. Fragmentations were as follows: m/z 160, loss of chloromethyl moiety; m/z 174, loss of chlorine; and m/z 131, loss of chloroethylamine.

thioTEPA-Mercapturate.
The synthesis of thioTEPA-mercapturate was carried out by incubation of thioTEPA with a 10-fold molar excess of N-acetylcysteine in 28 mM sodium carbonate buffer (pH 11) at 90°C for 15 min. thioTEPA-mercapturate was isolated by HPLC. The identity was established by FAB-MS-MS, showing a protonated molecular ion [M+H]+ at m/z 353, and two major fragment ions at m/z 310 and m/z 267, corresponding to subsequent loss of two aziridine moieties.

monoglutathionylthioTEPA.
MonoglutathionylthioTEPA was obtained by incubation of thioTEPA with a 10-fold molar excess of GSH in 28 mM sodium carbonate buffer pH 10 at 80°C for 30 min monoglutathionylthioTEPA was isolated with HPLC. The formation of monoglutathionylthioTEPA was confirmed with FAB-MS and LC-MS, using electrospray ionization, showing a protonated molecular ion [M+H]+ at m/z 497.

TEPA-Mercapturate and monoglutathionylTEPA.
The synthesis and isolation of TEPA-mercapturate and monoglutathionylTEPA was carried out in the same way as described for thioTEPA. The structure was confirmed by LC-MS, using electrospray ionization, showing a protonated molecular ion [M+H]+ at m/z 337 for TEPA-mercapturate and at m/z 481 for monoglutathionylTEPA.

Sample Collection
To determine the total alkylating activity and search for metabolites of thioTEPA, we used the urine of a 52-year-old female patient receiving carboplatin and thioTEPA. The patient suffered from stage IV mammary carcinoma and was pretreated with two courses of 5-fluorouracil (500 mg/m2), epidoxorubicin (120 mg/m2), and cyclophosphamide (500 mg/m2; Ref. 16 ). Next, three 4-day courses of cyclophosphamide (1000 mg/m2), thioTEPA (40 mg/m2, twice daily), and carboplatin (265 mg/m2) were planned (CTC regimen; Ref. 17 ). The patient developed hemorrhagic cystitis during course 2. Therefore, only carboplatin and thioTEPA were administered in course 3. Urine was collected during course 3, in which the dosing regimen was as follows: carboplatin (265 mg/m2) and thioTEPA (40 mg/m2 twice daily) on days 1–4. Urine was collected over the course of 24 h (stored at 4°C) on each day of thioTEPA administration (days 1–4) and on the day after the last administration (day 5). After collection, samples were stored at -80°C. Before analysis, the pH of the urine was measured. At a later stage, urine samples from two patients treated with the CTC regimens (17) were analyzed to verify whether the same metabolites were also present in these individuals.

thioTEPA, TEPA, and Total Alkylating Activity Analysis
thioTEPA and TEPA concentrations were measured by GC as described previously (18) . The total alkylating activity was determined with a modified colorimetric method with NBP, as described by Friedman and Boger (19) . To 1 ml of urine, 100 µl of a 5% (w/v) NBP solution in acetone and 300 µl of 25 mM sodium acetate buffer (pH 4.6) were added. The mixture was placed in a thermostatically controlled water bath at 95°C. After 30 min, the solution was cooled in ice to room temperature, and 1 ml of acetone and 0.5 ml of 0.25 M NaOH were added successively. The solution was mixed, and the extinction was measured at 600 nm, 1 min after the addition of NaOH with a Double Beam Spectrophotometer 100-60 (Hitachi, Tokyo, Japan). Standard curves of TEPA in urine were used for quantification, and alkylating activity was expressed as TEPA alkylating activity equivalents.

Extractions
For SPE, cartridges with reversed-phase, normal-phase, and ion-exchange packings (Supelco, Bellefonte, PA) with a sorbent volume of 100 mg were used. Liquid-liquid extraction was done with organic solvents of various polarities. Analysis of the extracts was carried out with GC-NPD (18) . Structure determination was carried out with GC-MS, as described previously (18) .

Fractionation of Alkylating Activity and Structure Determination
Forty-five ml of urine were concentrated with C18-SPE cartridges containing 300 mg of packing material. Urine was divided into portions of 10–15 ml, and each portion was loaded separately on a cartridge. After a washing step with 200 µl of water, the components were eluted with 4 times with 500 µl of 60% methanol. The eluate of the cartridges was collected and evaporated under a nitrogen stream at room temperature to dryness. The residue was reconstituted in 400 µl of water. A volume of 100 µl was injected into the HPLC system (Thermo Separation Products, Fremont, CA), equipped with a LiChrospher 100 RP-18 (5-µm) column (125 x 4 mm internal diameter; Merck, Darmstadt, Germany). Water was used as mobile phase at a flow of 1 ml/min, and fractions of 2 ml were collected. Each fraction was evaporated under a stream of nitrogen at room temperature to dryness, followed by reconstitution in 1 ml of water. The alkylating activity was determined as described above. Fractions containing alkylating activity were evaporated under a nitrogen stream at room temperature to dryness separately, reconstituted in 50 µl of methanol, and analyzed by FAB-MS and FAB-MS-MS. MS measurements were carried out with a JMS-SX/SX102A tandem mass spectrometer (Jeol, Tokyo, Japan) operating at 10 kV and equipped with a Jeol FAB gun set at 10-mA emission current and producing a beam of 6-keV Xe atoms. Mass spectra were obtained by scanning MS 1, whereas product-ion spectra of selected precursor ions were acquired by scanning MS 2 in the linked B/E scan mode using a collision cell in the third field-free region of the instrument with air as the collision gas. The pressure of the collision gas was adjusted to obtain a 50% intensity of the main beam.

Quantification of the Found Metabolites
The metabolite monochloroTEPA was analyzed as described previously for thioTEPA and TEPA in urine (18) . The analysis of thioTEPA-mercapturate was carried out with LC-MS. A mobile phase of 17.5% acetonitrile in a 10 mM ammonium acetate buffer (pH 4.8) was delivered at a flow rate of 0.2 ml/min. LC-MS measurements were carried out with a VG Platform, equipped with an electrospray ionization source operating in the positive ion mode (Fisons Instruments, Beverly, MA). Nitrogen was used as drying gas and nebulizing gas at flow rates of 400 and 15 liters/h, respectively. The source temperature was set at 120°C, and the cone voltage was set at 30 V. For thioTEPA-mercapturate and sulfadiazine, which was used as internal standard, mass to charge (m/z) ranges of 350–360 and 255–265 were scanned, respectively. An aliquot of 90 µl urine was mixed with 10 µl of a 100 µg/ml solution of sulfadiazine. Standard curves of thioTEPA-mercapturate in urine were used for quantification and the method proved to be linear in the concentration range of 1–25 µg/ml. The accuracy and within- and between-run precisions were within the accepted criteria of <=15% (20) .


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
thioTEPA, TEPA, and Total Alkylating Activity Analysis
Table 1Citation shows the total amount of thioTEPA, TEPA, and the total alkylating activity excreted over 24 h, relative to the administered dose, for each day of urine collection. Less than 1% of the total dose was excreted as thioTEPA. The total excretion of TEPA was ~8 times higher than that of thioTEPA. The urinary alkylating activity was 16.0% of the total dose, in terms of TEPA equivalents, and was on the same order of magnitude as that reported by others (11 , 12) . The relative alkylating activity of thioTEPA toward TEPA was 88% at a concentration of 125 µM. Carboplatin was also assessed for alkylating activity but gave no absorption at 600 nm. Therefore, the total urinary alkylating activity exceeded the sum of the amounts of thioTEPA and TEPA, indicating the presence of other metabolites with alkylating activity.


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  		Table 1 Excreted amount of thioTEPA, TEPA, and alkylating activity relative to the administered dose, calculated as thioTEPA equivalents

 
Extractions
Little is known about the metabolic profile of thioTEPA, beside oxidative desulfuration to TEPA. Degradation studies of thioTEPA in buffered media show its ability to form chloro and hydroxyl adducts (21, 22, 23) ; in addition, the in vitro formation of GSH conjugates has been described (24) . It was expected that, with the use of a wide polarity range of extraction solvents as used in our extraction procedure, both hydrophilic and lipophilic compounds would be extracted. Analysis of the extracts was carried out by GC-NPD because the presence of endogenous compounds in the residues of the extracts was prohibitive for the use of HPLC with UV detection at 205 nm. Low wavelength detection is required because of lack of specific absorption of thioTEPA.

GC-NPD analysis of extracts obtained by reversed-phase SPE and liquid-liquid extraction with ethyl acetate, chloroform, and mixtures of chloroform and 1-propanol showed the presence of thioTEPA (retention time, 4.4 min), TEPA (retention time, 3.7 min), and an additional peak at 5.7 min (Fig. 1Citation ; the peak with retention time 5.5 min is the internal standard diphenylamine). GC-MS analysis of the unknown peak showed a molecular ion at m/z 209 with an isotope at 211 (Fig. 2)Citation . The ratio 3:1 for m/z 209 and 211 indicates the presence of a chlorine atom. The mass spectrum shows loss of chloromethyl (m/z 160), chlorine (m/z 174; same mass as protonated TEPA), and chloroethylamine (m/z 131); m/z 42 originates from an aziridine group; and m/z 91 is a background signal. These results indicate the presence of monochloroTEPA in the urine samples. GC-NPD analysis of TEPA incubated with 1 M sodium chloride at pH 5 showed the formation of two degradation products with retention times of 5.7 and 8.9 min. GC-MS analysis of the degradation products showed that these compounds are the monochloro (Fig. 2B)Citation and the dichloro derivatives of TEPA (data not shown), respectively. The dichloro derivative of TEPA was not found in urine. The reference molecule monochloroTEPA was assessed for alkylating activity, resulting in absorption at 600 nm, with a relative alkylating activity of 98% toward TEPA at a concentration of 125 µM. For thioTEPA, conversion in urine adjusted with HCl to pH 4.0 at 37°C in vitro to a ß-chloroethyl moiety has been reported (25) . However, we did not found these chloro adducts in our urine samples. The formation of chloro adducts of thioTEPA strongly depends on pH and chloride concentration (23) . The pH of the urine samples used ranged from 6.0 to 7.1, at which thioTEPA proved to be stable (23 , 25) . The urine samples used for this study were collected over 24 h (stored at 4°C) and, after collection, were stored at -80°C, which precludes in vitro formation of monochloroTEPA.



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  		Fig. 1. GC chromatogram of a urine sample, collected prior to (A) and on day 4 (B) after drug administration, extracted with a SPE C8 cartridge: 3.7 min, TEPA; 4.4 min, thioTEPA; 5.5 min, internal standard (diphenylamine); and 5.7 min, possible metabolite of thioTEPA.

 


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  		Fig. 2. GC/MS of the metabolite of thioTEPA (5.7-min retention time peak in Fig. 1Citation ) extracted with SPE C8 cartridge (A) and of monochloroTEPA formed after incubation of TEPA with sodium chloride (B).

 
Fractionation of Alkylating Activity and Structure Determination
Fraction collection of the concentrated urine with HPLC and alkylating activity determination resulted in a profile as depicted in Fig. 3Citation . The major part of the alkylating activity eluted between 8 and 12 ml. TEPA eluted after 18 ml and gave a broad tailing peak, resulting in the low alkylating activity of the fractions after 17 ml (Fig. 3, A)Citation . monochloroTEPA and thioTEPA eluted after 40 and >80 ml, respectively (Fig. 3, B and CCitation , respectively). FAB-MS analysis of the fraction eluting between 8 and 12 ml showed the presence of a compound with a protonated molecular ion [M+H]+ at m/z 353. This ion was selected for FAB-MS-MS measurements, which resulted in a spectrum with two major fragment ions at m/z 310 and 267 (Fig. 4A)Citation . The ion at m/z 310 corresponds to loss of an aziridine molecule, also observed in the fragmentation pattern of thioTEPA. Loss of a second aziridine results in an ion at m/z 267. A corresponding fragment was not seen for thioTEPA.



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  		Fig. 3. Alkylating activity of urine after extraction with SPE, using a C8 cartridge and fractionation with HPLC: 17 min, TEPA (A); 40 min, monochloroTEPA (B); 80 min, thioTEPA (C).

 


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  		Fig. 4. FAB-MS-MS spectrum of the metabolite of thioTEPA, isolated with HPLC after SPE extraction with a C8 cartridge (precursor ion m/z 353; A) and of thioTEPA-mercapturate, synthesized by incubation of thioTEPA with N-acetylcysteine for 30 min in 28 mM sodium carbonate buffer (pH 11) at 95°C (precursor ion, m/z 353; B).

 
A possible metabolic pathway of thioTEPA is GSH conjugation. The ability of thioTEPA to form such conjugates in vitro was demonstrated by Dirven et al. (24) . They found that incubation of thioTEPA with GSH results in formation of monoglutathionylthioTEPA and diglutathionylthioTEPA and that this reaction is catalyzed by GSTs (24) . The formation of monoglutathionylthioTEPA in MCF-7 breast cancer cells and the influence of GST-P1–1, the most abundant GST in human tumors, was demonstrated by Cnubben et al. (26) . However, the mass of monoglutathionylthioTEPA is 496 Da and exceeds that of the metabolite. GSH conjugates may convert in separate stages to mercapturic acids, which are conjugates of N-acetylcysteine (27 , 28) . For example, fotemustine (29) and 1,3-butadiene (30) are metabolized by mercapturic acid biosynthesis. The mass of the unknown metabolite was the same as the mass calculated for thioTEPA-mercapturate. HPLC analysis of reference thioTEPA-mercapturate with water as mobile phase gave a product with an identical retention time as the metabolite. FAB-MS-MS analysis of the protonated molecular ion at m/z 353 of the reference molecule thioTEPA-mercapturate (Fig. 4B)Citation resulted in a fragmentation pattern that was both qualitatively and quantitatively identical to that of the metabolite. The reference thioTEPA-mercapturate molecule was assessed for alkylating activity, resulting in absorption at 600 nm, with a relative alkylating activity of 68% toward TEPA at a concentration of 125 µM. From these data, we concluded that the unknown metabolite is thioTEPA-mercapturate. monoglutathionylthioTEPA is probably the precursor of thioTEPA-mercapturate and its presence in urine was investigated. In HPLC, reference monoglutathionylthioTEPA gave a peak eluting at 3.7 min. monoglutathionylthioTEPA was assessed for alkylating activity, resulting in absorption at 600 nm. No alkylating activity, however, was observed in urine at 3.7 min (Fig. 3)Citation . The protonated molecular ion [M+H]+ with m/z 497 of monoglutathionylthioTEPA could not be detected in the fraction from 3 to 5 ml with FAB-MS measurements. Considering the structural resemblance of TEPA with thioTEPA, we investigated whether TEPA forms conjugates with GSH and N-acetylcysteine. HPLC analysis of the references TEPA-mercapturate and monoglutathionylTEPA resulted in a peak eluting after 1.1 or 2.1 min, respectively. Both references TEPA-mercapturate and monoglutathionylTEPA were assessed for alkylating activity, resulting in absorption at 600 nm. However, no alkylating activity was observed in urine after 1 min (Fig. 3)Citation . Also, with FAB-MS, the protonated molecular ion [M+H]+ of TEPA-mercapturate (m/z 337) or monoglutathionylTEPA (m/z 481) could not be detected in the fraction between 1 and 3 ml.

In Fig. 5Citation , the proposed metabolic pathway of thioTEPA is presented.



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  		Fig. 5. Proposed metabolic pathway of thioTEPA, in which A is thioTEPA, B is TEPA, C is monochloroTEPA, D is the GSH conjugate of thioTEPA, E is thioTEPA-cysteinate, and F is thioTEPA-mercapturate. Metabolites B, C, and F were found in urine of patients receiving thioTEPA and structures were confirmed with mass spectrometry.

 
Quantification of the Found Metabolites
monochloroTEPA.
The metabolite monochloroTEPA was quantified in the same way as described previously for thioTEPA and TEPA (18) . monochloroTEPA was synthesized by incubation of TEPA with sodium chloride. The monochloro compound was separated from TEPA with SPE. The eluted fractions, however, always contained >20% TEPA; therefore, TEPA was used as standard for quantification of monochloroTEPA in urine. The total amount of monochloroTEPA excreted during 24 h was 1.06 mg (0.81–1.18 mg), accounting for only 0.76% (0.58–0.84%) of the administered dose of thioTEPA.

thioTEPA-Mercapturate.
GC analysis of a thioTEPA-mercapturate solution in methanol resulted in a broad peak at 8 min. thioTEPA was formed probably due to thermal instability of thioTEPA-mercapturate. Lowering the injector temperature or on-column injection resulted in even broader peaks and can be explained by the polar character of the metabolite. HPLC was, therefore, used for the analysis of thioTEPA-mercapturate. The low specific absorption of thioTEPA-mercapturate required detection at low wavelength (205 nm). Liquid-liquid extraction of a thioTEPA-mercapturate solution in water performed with ethyl acetate, chloroform, or mixtures of 1-propanol in chloroform resulted in recoveries of <10%. Reduction of the pH to 4 or 5 improved recoveries to 50% but were still not satisfactory. With ion exchange SPE of a thioTEPA-mercapturate solution in water, the recovery improved to 80%. The eluate of ion exchange SPE of thioTEPA-mercapturate in urine, however, contained large amounts of endogenous compounds, which interfered with the detection of thioTEPA-mercapturate. With the use of LC-MS, no extraction procedure was necessary, and detection of thioTEPA-mercapturate was more selective.

The total amount of thioTEPA-mercapturate excreted was 32.1 mg (22.6–37.4 mg), which is 12.3% (8.6–14.4%) of the administered dose, calculated as thioTEPA. The sum of the alkylating activity of thioTEPA and its metabolites in the patient receiving only thioTEPA and carboplatin accounts for 74% (54–100%) of the total alkylating activity. Table 2Citation shows the total excreted amount of thioTEPA and its metabolites relative to the administered dose of two other patients treated with the CTC regimen and the patient receiving thioTEPA and carboplatin. Only 10–22% of the administered dose was recovered. The results show that, apart from TEPA, monochloroTEPA, and thioTEPA-mercapturate, still other metabolites with alkylating activity are present. Because during conversion of GSH conjugates to N-acetylcysteine conjugates, GSH, cysteinylglycine, and cysteine conjugates are formed (Fig. 5)Citation , we tried to find these conjugates of thioTEPA in urine, but without success. These conjugates, if formed, are probably mostly excreted in the bile or as intermediate metabolites rapidly converted into thioTEPA-mercapturate (28) .


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  		Table 2 Excreted amount of thioTEPA and its metabolites relative to the total dose, calculated as thioTEPA equivalents

 
Conclusion
Our search for new metabolites of thioTEPA resulted in the identification of two compounds, monochloroTEPA and thioTEPA-mercapturate. Whereas TEPA was assumed to be the main metabolite of thioTEPA, the excreted amount of thioTEPA-mercapturate is higher. With these results, the difference between the total alkylating activity and the total amount of thioTEPA and excreted metabolites is decreased from 25–85 to 0–50%. In addition to the found metabolites, still other compounds with alkylating activity are present.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Pharmaceutical Analysis, University of Utrecht, Sorbonnelaan 16, 3584 CA Utrecht, the Netherlands. Back

2 The abbreviations used are: thioTEPA, N,N',N''-triethylenethiophosphoramide; TEPA, N,N',N''-triethylenephosphoramide; GSH, glutathione; NBP, p-nitrobenzylpyridine; NMR, nuclear magnetic resonance; GC, gas chromatography; MS, mass spectrometry; monochloroTEPA, N,N'-diethylene-N''-2-chloroethylphosphoramide; HPLC, high-performance liquid chromatography; FAB, fast atom bombardment; MS-MS, tandem MS; NPD, nitrogen phosphorus detection; SPE, solid-phase extraction; LC, liquid chromatography; GST, GSH S-transferase. Back

Received 3/30/99. Accepted 7/22/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Sykes M., Karnovsky D., Phillips F., Burchenal J. Clinical studies of triethylenephosphoramide and diethylenephosphoramide compounds with nitrogen mustard-like activity. Cancer (Phila.), 6: 142-148, 1953.
  2. Van der Wall E., Beijnen J. H., Rodenhuis S. High-dose chemotherapy regimens for solid tumors. Cancer Treat. Rev., 21: 105-132, 1995.[Medline]
  3. Mellet L. B., Woods L. A. The comparative physiological disposition of thioTEPA and TEPA in the dog. Cancer Res., 20: 524-532, 1960.
  4. Chang T. K. H., Chen G., Waxman D. J. Modulation of thioTEPA antitumor-activity in-vivo by alteration of liver cytochrome P450-catalyzed drug-metabolism. J. Pharmacol. Exp. Ther., 274: 270-275, 1995.[Abstract/Free Full Text]
  5. Ng S. F., Waxman D. J. Biotransformation of N, N', N''-triethylenethiophosphoramide: oxidative desulfuration to yield N, N', N''-triethylenephosphoramide associated with suicide inactivation of a phenobarbital-inducible hepatic P-450 monooxygenase. Cancer Res., 50: 464-471, 1990.[Abstract/Free Full Text]
  6. Egorin M. J., Snyder S. W., Pan S., Daly C. Cellular transport and accumulation of thioTEPA in murine, human and avian cells. Cancer Res., 49: 5611-5617, 1989.[Abstract/Free Full Text]
  7. Egorin M. J., Snyder S. W., Wietharn B. E. Effects of ethanolamine and choline on thiotepa cellular accumulation and cytotoxicity in L1210 cells. Cancer Res., 50: 4322-4327, 1990.[Abstract/Free Full Text]
  8. Egorin M. J., Cohen B. E., Kohlhepp E. A., Gutierrez P. L. Gas-liquid chromatographic analysis of N, N',N''-triethylenethiophosphoramide and N, N',N''-triethylenephosphoramide in biological samples. J. Chromatogr., 343: 196-202, 1985.[Medline]
  9. McDermott B. J., Double J. A., Bibby M. C., Wilman D. E. V., Loadman P. M., Turner R. L. Gas chromatographic analysis of triethylenephosphoramide and triethylenethiophosphoramide in biological specimen. J. Chromatogr., 338: 335-345, 1985.[Medline]
  10. Masters J. R. W., McDermott B. J., Harland S., Bibby M. C., Loadman P. M. ThioTEPA pharmacokinetics during intravesical chemotherapy: the influence of dose and volume of instillate on systemic uptake and dose rate to the tumour. Cancer Chemother. Pharmacol., 38: 59-64, 1996.[Medline]
  11. Cohen B. E., Egorin M. J., Kohlhepp E. A., Aisner J., Gutierrez P. L. Human plasma pharmacokinetics and urinary excretion of thioTEPA and its metabolites. Cancer Treat. Rep., 70: 859-864, 1986.[Medline]
  12. Hagen B., Neverdal G., Walstad R. A., Nilsen O. G. Long-term pharmacokinetics of thio-TEPA, TEPA and total alkylating activity following i.v. bolus administration of thio-TEPA in ovarian cancer patients. Cancer Chemother. Pharmacol., 25: 257-262, 1990.[Medline]
  13. Hagen B. Pharmacokinetics of thio-TEPA and TEPA in the conventional dose-range and its correlation to myelosuppressive effects. Cancer Chemother. Pharmacol., 27: 373-378, 1991.[Medline]
  14. Craig A. W., Jackson H. The metabolism of 32P-labelled triethylenephosphoramide in relation to its anti-tumour activity. Br. J. Pharmacol., 10: 321-325, 1955.
  15. Mellet L. B., Hodgson M. D., Woods L. A. Absorption and fate of 14C-labeled N, N', N''-triethylenethiophosphoramide (thio-TEPA) in humans and dogs. J. Lab. Clin. Med., 60: 818-825, 1962.
  16. Van der Wall E., Richel R. J., Kusumanto Y. H., Rutgers E., Schornagel J. H., Schaake-Koning C. C. E., Peterse J. L., Rodenhuis S. Feasibility study of FEC-chemotherapy with dose-intensive epirubicin as initial treatment in high-risk breast cancer. Ann. Oncol., 4: 791-792, 1993.[Abstract/Free Full Text]
  17. Rodenhuis S., Westermann A., Holtkamp M. J., Nooijen W. J., Baars J. W., Van der Wall E., Slaper-Cortenbach I. C. M., Schornagel J. H. Feasibility of multiple courses of high-dose cyclophosphamide, thioTEPA and carboplatin for breast cancer or germ cell cancer. J. Clin. Oncol., 14: 1473-1483, 1996.[Abstract/Free Full Text]
  18. van Maanen R. J., van Ooijen R. D., Zwikker J. W., Huitema A. D. R., Rodenhuis S., Beijnen J. H. Determination of thioTEPA and its active metabolite TEPA in plasma and urine using capillary gas chromatography. J. Chromatogr. B, 719: 103-112, 1998.
  19. Friedman O. M., Boger E. Colorimetric estimation of nitrogen mustards in aqueous media: hydrolytic behavior of bis(ß-chloroethyl)amine, nor HN2. Anal. Chem., 33: 906-910, 1961.
  20. Braggio S., Barnaby R. J., Grossi P., Cugola M. A strategy for the validation of bioanalytical methods. J. Pharmacol. Biomed. Anal., 14: 375-388, 1996.
  21. Pyatigorskaya T. L., Zhilkova O. Y., Shelkovsky V. S., Arkhangelova N. M., Grizodub A. I., Sukhodub L. F. Hydrolysis of 1,1',1''-phosphinothioylidinetrisaziridine (thioTEPA) in aqueous solution. Biomed. Environ. Mass Spectrom., 14: 143-148, 1987.
  22. Zon G., Egan W., Stokes J. B. Observations of 1,1',1''-phosphinothioylidynetrisaziridine (thioTEPA) in acidic and saline media: a 1H-NMR study. Biochem. Pharmacol., 25: 989-992, 1976.[Medline]
  23. van Maanen M. J., Brandt A. C., Damen J. M. A., Beijnen J. H. Degradation study of thioTEPA in aqueous solutions. Int. J. Pharm., 179: 55-64, 1999.[Medline]
  24. Dirven H. A. A. M., Dictus E. L. J. T., Broeders N. L. H. L., Vanommen B., van Bladeren P. J. The role of human glutathione-S-transferase isoenzymes in the formation of glutathione conjugates of the alkylating cytostatic drug thioTEPA. Cancer Res., 55: 1701-1706, 1995.[Abstract/Free Full Text]
  25. Cohen B. E., Egorin M. J., Nayar M. S. B., Gutierrez P. L. Effects of pH and temperature on the stability and decomposition of N, N', N''-triethylenethiophosphoramide in urine and buffer. Cancer Res., 44: 4312-4316, 1984.[Abstract/Free Full Text]
  26. Cnubben N. H. P., Rommens A. J. M., Oudshoorn M. J., van Bladeren P. J. Glutathione-dependent biotransformation of the alkylating drug thioTEPA and transport of its metabolite monoglutathionylthioTEPA in human MCF-7 breast cancer cells. Cancer Res., 58: 4616-4623, 1998.[Abstract/Free Full Text]
  27. Gibson G. G., Skett P. Introduction to Drug Metabolism1-37, Chapman and Hall London 1986.
  28. Chasseaud L. F. Conjugation with glutathione and mercapturic acid excretion Arias I. M. Jakoby W. B. eds. . Glutathione: Metabolism and function, : 77-114, Raven Press New York 1976.
  29. Brakenhoff J. P. G., Commandeur J. N. M., Lamoree M. H., Dubbelaar A. C., van Baar B. L. M., Lucas C., Vermeulen N. P. E. Identification and quantitative determination of glutathione-related urinary metabolites of fotemustine, a new anti-cancer agent. Xenobiotica, 23: 935-947, 1993.[Medline]
  30. Elfarra A. A., Sharer J. E., Duesher R. J. Synthesis and characterization of N-acetyl-l-cysteine S-conjugates of butadiene monoxide and their detection and quantification in urine of rats and mice given butadiene monoxide. Chem. Res. Toxicol., 8: 68-76, 1995.[Medline]



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A. D. R. Huitema, M. Spaander, R. A. A. Mathot, M. M. Tibben, M. J. Holtkamp, J. H. Beijnen, and S. Rodenhuis
Relationship between exposure and toxicity in high-dose chemotherapy with cyclophosphamide, thiotepa and carboplatin
Ann. Onc., March 1, 2002; 13(3): 374 - 384.
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