[Identification of glufosfamide metabolites in rats].

AIM: To elucidate the metabolic pathway of glufosfamide in rats. METHODS: In this study, a liquid chromatography-tandem mass spectrometric method was developed and applied to characterize the metabolites of glufosfamide in rat urine, after an i.v. administration of 50 mg x kg(-1). The analysis was performed under two ionization modes in two different chromatographic systems, separately. To make sure that the compounds detected in rat urine were metabolites or degradation products, the stability of glufosfamide, isophosphoramide mustard (M1), and the degradation products of M1 in urine were investigated. RESULTS: In positive ionization mode, besides glufosfamide, two metabolites, isophosphoramide mustard and monoaziridinyl derivative of isophosphoramide mustard, were detected. In negative ionization mode, only glufosfamide itself was detected, while derivatives of isophosphoramide mustard have no response in such condition. CONCLUSION: Glufosfamide was mainly unchanged excreted in urine, and two metabolites were detected as isophosphoramide mustard and monoaziridinyl derivative of isophosphoramide mustard.

Excretion kinetics of ifosfamide side-chain metabolites in children on continuous and short-term infusion.

Ifosfamide (IFO) requires metabolic activation by hydroxylation of the ring system to exert cytotoxic activity. A second metabolic pathway produces the cytostatically inactive metabolites 2-dechloroethyl-ifosfamide (2-D-IFO) and 3-dechloroethyl-ifosfamide (3-D-IFO) under release of chloroacetaldehyde. This side-chain metabolism has been suggested to be involved in CNS- and renal toxicity. The total urinary excretion of ifosfamide and its metabolites was investigated during 23 cycles in 22 children at doses ranging from 400 mg/m2 to 3 g/m2. The kinetics of the excretion were compared following short-term and continuous ifosfamide infusion at a dosage of 3 g/m2. IFO and side-chain metabolites were analyzed by gas chromatography, the active metabolites by indirect determination of acrolein (ACR) and IFO mustard (IFO-M) with the NBP test. 59+/-15% of the applied dose could be recovered in the urine, 23+/-9% as unmetabolized IFO. The main metabolite was 3-D-IFO (14+/-4%) followed by isophosphoramide mustard (IFO-M) (13+/-4%) and 2-D-IFO (8+/-3%). Neither the total amount recovered nor the excretion kinetics of ifosfamide and side-chain metabolites showed obvious schedule dependency. The excretion kinetics of side-chain metabolites as well as unmetabolized IFO were nearly superimposable on short-term and continuous infusion. Even after 1-hour infusion there was a lag of 3 - 6 hours until dechloroethylation became relevant. Therefore, differences in toxicity and efficacy cannot be explained by an influence of the application time on the metabolic profile of ifosfamide.

The pharmacokinetics and metabolism of ifosfamide during bolus and infusional administration: a randomized cross-over study.

In a randomized cross-over trial, 11 patients received ifosfamide (IFOS) in 21-day cycles, which alternated between 3 g m(-2) x (2 or 3) days given as a 1-h bolus doses, or the same total dose as a continuous infusion. Patients who received four or more cycles also alternated between two cycles on dexamethasone 4 mg 8 hourly for 3 days starting 8 h before IFOS, and two cycles off dexamethasone. A total of 34 patient cycles were studied and serum and urinary levels of IFOS, 2 dechloroethylifosfamide (2DC), 3 dechloroethylifosfamide (3DC), carboxyifosfamide (CX) and isophosphoramide mustard (IPM) were measured by thin-layer chromatography. No significant differences could be detected in the areas under the curve (AUCs) of serum concentration, nor in the proportion of IFOS or its metabolites found in the urine. There was no significant effect of dexamethasone on IFOS metabolism. These results indicate that there is no identifiable pharmacokinetic basis for insistence on either bolus or infusional methods of IFOS administration.

Cyclophosphamide metabolism in children.

The alkylating agent cyclophosphamide is a prodrug which is metabolized in vivo to produce both therapeutic and toxic effects. Cyclophosphamide metabolism was investigated in 36 children with various malignancies. Concentrations of cyclophosphamide and its principal metabolites were measured in plasma and urine using a quantitative high-performance TLC method. The results indicated a high degree of inter-patient variation in metabolism. In contrast to previous adult studies on urinary metabolites, plasma carboxyphosphamide concentrations did not support the existence of polymorphic metabolism. Plasma concentrations of dechlorethylcyclophosphamide and carboxyphosphamide were correlated in individual patients, suggesting that the activity of both aldehyde dehydrogenase and cytochrome P450 enzyme(s) determine carboxyphosphamide production in vivo. The presence of ketocyclophosphamide in plasma was strongly associated with dexamethasone pretreatment and was also accompanied by a high clearance of the parent drug. Interpatient differences in metabolism reflect individual levels of enzyme expression and may contribute to variation in clinical effect.

[Determination of glyciphosphoramide and its metabolite in plasma and the pharmacokinetics in rats after oral administration].

Glyciphosphoramide (GPA) is one of the anticancer agents belonging to the group of phosphoramide mustard. It has apparent antitumor effects in some animal models and in clinical trials against breast cancer, lymphosarcoma, uterocervical cancer and cancerous ulcer with good results. In this paper, the determination procedure of GPA and its metabolite using nitrobenzylpyridine (NBP) method is reported. The absorbance of the coloured products from the reaction of hydrolyzed or metabolized GPA with NBP was measured at 570 nm and 564 nm, respectively. The linearity of the reaction for GPA and its metabolite was established over the range of 6.25-100 micrograms/ml water or plasma. The plasma of rats and mice was found to be able to metabolize GPA to form alkylating agent (s) which react with NBP, but that of rabbits cannot. The plasma concentration-time curve of metabolite obtained after oral administration of GPA (100 mg/kg) in rats was shown to fit a two compartment open model with the following parameters: T1/2 beta = 44.5 min, T1/2 alpha = 3.16 min, T1/2 Ka = 2.14 min, T1/2Km = 0.0644 min, Tmax = 7.57 min, Cmax = 55.8 micrograms/ml, AUC = 2827. 39 micrograms/ml.min, K21 = 0.09663/min, K10 = 0.03535/min, K12 = 0.1030/min, Vc = 1.00 L/kg, Vd = 2.07 L/kg, CLt = 2.12 L/h. Kidney was found to be the main organ for GPA metabolite elimination. About one fourth of the given dose was excreted in urine within 24 h with the main portion excreted in the first 2 h.

Phenotypically deficient urinary elimination of carboxyphosphamide after cyclophosphamide administration to cancer patients.

The 0-24-h urinary metabolic profile of cyclophosphamide was investigated in a series of 14 patients with various malignancies receiving combination chemotherapy including i.v. cyclophosphamide. This was accomplished using combined thin-layer chromatography-photography-densitometry, which can quantitate cyclophosphamide and its four principal urinary metabolites (4-ketocyclophosphamide, nor-nitrogen mustard, carboxyphosphamide, and phosphoramide mustard). Recovery of drug-related metabolites was 36.5 +/- 17.8% (SD) dose, the most abundant metabolites being phosphoramide mustard (18.5 +/- 16.1% dose) and unchanged cyclophosphamide (12.7 +/- 9.3% dose). The most variable metabolite was carboxyphosphamide, with five patients excreting 0.3% dose or less. These patients were termed low carboxylators (LC) and could be distinguished from high carboxylators (HC) by a carboxylation index (relative percentage as carboxyphosphamide multiplied by 10). Mean carboxylation indices for the LC and HC phenotypes were 3.4 +/- 2.6 and 151 +/- 115, respectively. There were no associations between patient age, sex, body weight, tumor type, or concomitant drug therapy and carboxylation phenotype. Neither 4-ketocyclophosphamide nor nor-nitrogen mustard excretion differed between LC and HC phenotypes; however, HC patients had a greater excretion of cyclophosphamide (46.4 +/- 15.5 relative percentage) than LC patients (19.4 +/- 12.6%). The DNA cross-linking cytotoxic metabolite phosphoramide mustard was elevated more than 2-fold in the LC (76.5 +/- 13.9%) compared with the HC (33.0 +/- 12.2%) phenotype. It is concluded that these data represent the first evidence of a defect in cyclophosphamide metabolism, and it is proposed that this arises from a hitherto unrecognized aldehyde dehydrogenase genotype.

Isolation, identification and determination of cyclophosphamide and two of its metabolites in urine of a multiple sclerosis patient by high pressure liquid chromatography and field desorption mass spectrometry.

The off-line combination of high pressure liquid chromatography and field desorption mass spectrometry has been used for the simultaneous isolation, identification and determination of cyclophosphamide and two of its metabolites, 4-ketocyclophosphamide and carboxyphosphamide in urine from a patient suffering from multiple sclerosis. Cyclophosphamide and its metabolites were separated using reverse phase liquid chromatography. Field desorption mass spectrometry was employed for identification and quantification. The technique applied needs no derivatization for analysis. The limits of detection by field desorption mass spectrometry for 1, 2 and 3 are factor of about 4 X 10(3)-10(5) lower than those of a common variable ultraviolet detector. Quantitative determination was carried out using the method of stable isotope dilution with deuterated analogues of 1, 2 and 3. In a pilot study, the ratio of 1:2:3 was determined to 1:0.02:0.6. One ml of urine is sufficient for simultaneous analysis of the three compounds. The typical analysis time, including separation by liquid chromatography and field desorption measurement, is about 30 minutes.