Individual variation in the activation and inactivation of metabolic pathways of cyclophosphamide.

BACKGROUND: Carboxyphosphamide is an inactive metabolite of cyclophosphamide, which is a widely used antineoplastic drug. Deficiencies in the production of this metabolite have been reported. Such deficiencies would have important consequences for therapeutic and toxic effects of oxazaphosphorines like cyclophosphamide. PURPOSE: This study further investigates the variability in cyclophosphamide metabolism and carboxyphosphamide recovery in urine. METHODS: The 24-hour urinary metabolic profile of cyclophosphamide was investigated in 17 Turkish patients receiving doses of 100-1080 mg orally or by short intravenous infusion. Urine samples were assayed quantitatively for cyclophosphamide and its principal metabolites (phosphoramide mustard, 4-ketocyclophosphamide, carboxyphosphamide, and dechloroethylcyclophosphamide) with combined thin-layer chromatography-photography-densitometry. The amount of each metabolite excreted in 24 hours was expressed as a percentage of the dose. RESULTS: Recovery of drug and metabolites varied greatly among individuals (range, 0.01%-13.56% of dose). In particular, the amount of carboxyphosphamide varied over a thousandfold range and was undetectable in urine from four patients. The patients were classified by phenotype as demonstrating low or high carboxylation. Those with low carboxylation excreted less than 0.2% of the cyclophosphamide dose as carboxyphosphamide, while those with high carboxylation excreted 0.8%-13.6% (median, 1.81%). No association was observed between carboxylation phenotype and patient age, sex, disease, or concomitant therapy, although the three lifetime nonsmokers all showed poor carboxylation. No correlation was observed between the percent of dose excreted as any of the other metabolites and that excreted as carboxyphosphamide. There was a statistically significant inverse correlation between the combined recovery of carboxyphosphamide and phosphoramide mustard and the dose of prednisolone administered. CONCLUSIONS: These data confirm an earlier observation of a phenotypic deficiency of carboxyphosphamide excretion in British patients treated with cyclophosphamide. This deficiency may arise from a polymorphism in the enzyme aldehyde dehydrogenase. Carboxylation phenotype may have important implications for both the therapeutic effect and toxicity of cyclophosphamide.     

Metabolism of high doses of cyclophosphamide

Summary The excretion of cyclophosphamide and the enzymatically derived metabolites 4-ketocyclophosphamide and carboxyphosphamide has been measured in four patients after the administration of cyclophosphamide (5 g). At this dose the enzymes responsible for the biotransformation and detoxification of cyclophosphamide are not saturated. In two patients the metabolite profile was unaffected by a previous high dose of cyclophosphamide and in one patient a small primary dose did not alter metabolism.     

The effect of cimetidine on cyclophosphamide metabolism in rabbits

Summary Six female rabbits were given 20 mg/kg cyclophosphamide (containing 100 Ci [³H-chloroethyl]-cyclophosphamide) alone or 1 h following 100 mg/kg cimetidine. Serial plasma and urine specimens were collected and levels of cyclophosphamide and its metabolites (4-hydroxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard, and carboxyphosphamide) were measured. 4-Ketocyclophosphamide was the major metabolite present in rabbit plasma and urine, with lesser amounts of 4-hydroxycyclophosphamide, carboxyphosphamide, and phosphoramide mustard also being identified. Cimetidine pretreatment resulted in prolongation of cyclophosphamide's half-life from 24.3±7.3 to 33.5±9.5 min (mean ± SD;P=0.036) but did not significantly alter the AUC_{0–8 h} for the latter drug. Cimetidine pretreatment resulted in a significantly greater AUC_{0–8 h} for 4-hydroxycyclophosphamide (189.4±77 vs 364.6±126.7 mol min/l^–1;P=0.016) as compared with control values. A higher AUC_{0–8 h} value for phosphoramide mustard (53.7±69.2 vs 95.7±34.7 mol min/l^–1) was also observed after cimetidine dosing but the difference was not significant (P=0.21). Kinetics of 4-ketocyclophosphamide and carboxyphosphamide were not significantly affected by cimetidine treatment. Cimetidine was added to hepatic microsomes isolated from phenobarbital-treated rabbits; it did not inhibit cyclophosphamide's metabolism in vitro, suggesting that its in vivo effect may be mediated through mechanisms other than cytochrome P-450 inhibition. Cimetidine pretreatment increases exposure to cyclophosphamide and its major activated metabolite, 4-hydroxycyclophosphamide. Potentiation rather than inhibition of cyclophosphamide's pharmacodynamic effect is to be predicted when cimetidine is given concomitantly with the former. Alterations in hepatic blood flow or mechanisms other than microsomal inhibition by cimetidine may explain this potentiation.Supported in part by the Department of Veteran Affairs and grant CA-49186 from the National Institutes of Health (NIH)Department of Clinical Pharmacology, Sun Yat-sen University of Medical Sciences, Guangzhou, People's Republic of China     

Pharmacokinetics and metabolism of cyclophosphamide in paediatric patients

Summary The pharmacokinetics and metabolism of cyclophosphamide were studied in nine paediatric patients. Plasma samples were obtained from eight subjects and urine was collected from six children during a 24-h period after drug administration. Cyclophosphamide and its major metabolites phosphoramide mustard (PM), carboxyphosphamide (CX), dechloroethylcyclophosphamide (DCCP) and 4-ketocyclophosphamide (KETO) were determined in plasma and urine using high-performance thin-layer chromatography-photographic densitometry (HPTLC-PD). Cyclophosphamide (CP) was nearly, if not completely, cleared from plasma by 24 h after its administration. The plasma half-life of CP ranged from 2.15 to 8.15 h; it decreased following higher doses and was shorter than that previously reported for adult patients. Both the apparent volume of distribution (0.49±1.4 l/kg) and the total body clearance (2.14±1.4 l m^–2 h^–1) increased with increasing dose. Renal clearance ranged between 0.12 and 0.58 l/h (mean, 0.43±0.19l/h). Between 5.4% and 86.1% of the total delivered dose was recovered as unchanged drug in the urine. The major metabolites identified in plasma and urine were PM and CX. One patient appeared to be deficient in CX formation. This study suggests that there is interpatient variability in the pharmacokinetics and metabolism of CP in paediatric patients. The shorter half-life and higher clearance as compared with adult values indicate faster CP metabolism in children.M.J.T. was supported by a grant from the Fondo de Investigacion Sanitaria, Ministerio de Sanidad y Consumo, Spain. This work was also supported in part by grants from the North of England Cancer Research Campaign, North of England Children's Cancer Research Fund, ASTA Werke Germany, and the Wellcome Trust.     

Quantitation by gas chromatography-chemical ionization mass spectrometry of cyclophosphamide, phosphoramide mustard, and nornitrogen mustard in the plasma and urine of patients receiving cyclophosphamide therapy

Unambiguous and sensitive methods based on gas chromatography-chemical ionization mass spectrometry have been developed to quantitate cyclophosphamide and two alkylating and cytotoxic metabolites, phosphoramide mustard and nornitrogen mustard. The levels of these materials have been determined in the plasma and urine of five patients receiving cyclophosphamide, 60 or 75 mg/kg i.v. Peak plasma levels of phosphoramide mustard of 50 to 100 nmoles/ml were found at 3 hr after cyclophosphamide administration. Variable levels of nornitrogen mustard were found in the plasma. This product may be arising in part from the decomposition of other metabolites during sample storage and preparation.     

A cyclophosphamide/DNA phosphoester adduct formed in vitro and in vivo

The antitumor activity of cyclophosphamide is thought to be due to the alkylating activity of phosphoramide mustard, a metabolite of cyclophosphamide. Reaction of 2'-deoxyguanosine 3'-monophosphate and phosphoramide mustard resulted in the formation of several adducts that could be detected by high performance liquid chromatography (HPLC). One of these adducts, isolated and purified by HPLC, could be detected by 32P postlabeling. This product was identified by UV, nuclear magnetic resonance, and mass spectrometry and by acid, base, and enzymatic hydrolysis to be 2'-deoxyguanosine 3'-monophosphate 2-(2-hydroxyethyl)aminoethyl ester. A combination of HPLC fractionation of digested DNA and 32P postlabeling was used to detect this adduct in calf thymus DNA incubated in vitro with metabolically activated cyclophosphamide and in DNA from the liver of mice treated with cyclophosphamide. In these DNA samples the adduct occurred at a level of 1/10(5) and 1/3 x 10(7) nucleotides, respectively.     

High exposures to bioactivated cyclophosphamide are related to the occurrence of veno-occlusive disease of the liver following high-dose chemotherapy

We investigated whether the occurrence of veno-occlusive disease of the liver (VOD) may be associated with individual variations in the pharmacokinetics of high-dose cyclophosphamide. Patients received single or multiple courses of cyclophosphamide (1000 or 1500 mg m-2 day-1), thiotepa (80 or 120 mg m-2 day-1) and carboplatin (265-400 mg m-2 day-1) (CTC) for 4 consecutive days. The area under the plasma concentration-time curves (AUCs) were calculated for cyclophosphamide and its activated metabolites 4-hydroxycyclophosphamide and phosphoramide mustard based on multiple blood samples. Possible relationships between the AUCs and the occurrence of VOD were studied. A total of 59 patients (115 courses) were included. Four patients experienced VOD after a second CTC course. The first-course AUC of 4-hydroxycyclophosphamide (P=0.003) but not of phosphoramide mustard (P=0.101) appeared to be predictive of the occurrence of VOD after multiple courses. High exposures to bioactivated cyclophosphamide may lead to increased organ toxicity.     

Pharmacokinetics of trofosfamide and its dechloroethylated metabolites

 To contribute to effective and safe outpatient treatment, we investigated the metabolism of trofosfamide (Trofo) after oral administration. We analyzed Trofo metabolism in 15 patients aged from 3 to 73 years who were treated with 150 or 250 mg/m² Trofo in combination with etoposide. Serum samples were collected with 13 patients after oral administration, and Trofo and its dechloroethylated metabolites were quantified by gas chromatography. Urine samples were collected from five patients and analyzed by same method. Ifosfamide (Ifo) was the main metabolite in serum and urine (AUC_Trofo:AUC_Ifo 1:13), whereas cyclophosphamide (Cyclo) was formed in smaller amounts (AUC_Ifo:AUC_Cyclo 18:1). Ifo and Cyclo were further oxidized in the chloroethyl side chains to form 2- and 3-dechloroethylifosfamide in varying quantities. The urinary excretion of Trofo and its dechloroethylated metabolites amounted to about 10% of the total dose. Our results confirm former in vitro observations about the metabolism of Trofo. The main side-chain metabolites Ifo and Cyclo can be further activated by oxidation and formation of their respective phosphoramide mustards. Hence, Trofo is an interesting agent for oral chemotherapy. Received 21 July 1996 / Accepted: 11 November 1996     

Alkylating properties of phosphoramide mustard.

The relative alkylating activities of two of the cytotoxic metabolites of cyclophosphamide, phosphoramide mustard and nornitrogen mustard, have been studied at pH 4.6 and 7.4. The products formed on alkylation of ethanethiol by these metabolites have been identified, confirming that phosphoramide mustard undergoes alkylation reactions as an intact molecule. Deuterated analogs of the two metabolites have been synthesized, namely N,N-bis(2,2-dideutero-2-chloroethyl)-phosphorodiamidic acid and N,N-bis(2,2-dideutero-2-chloroethyl)amine and used to determine that alkylation proceeds directly via an aziridinium intermediate rather than a direct SN2 displacement of the chlorine atom.     

Isophosphoramide mustard, a metabolite of ifosfamide with activity against murine tumours comparable to cyclophosphamide.

Isophosphoramide mustard was synthesized and was found to demonstrate activity essentially comparable to cyclophosphamide and ifosfamide against L1210 and P388 leukaemia. Lewis lung carcinoma, mammary adenocarcinoma 16/C, ovarian sarcoma M5076, and colon tumour 6A, in mice and Yoshida ascitic sarcoma in rats. At doses less than, or equivalent to, the LD10, isophosphoramide mustard retained high activity against cyclophosphamide-resistant L1210 and P388 leukaemias, but was less active against intracerebrally-implanted P388 leukaemia while cyclophosphamide produced a 4 log10 tumour cell reduction. It was also less active (one log10 lower cell kill) than cyclophosphamide against the B16 melonoma. Metabolism studies on ifosfamide in mice identified isophosphoramide mustard in blood. In addition, unchanged drug, carboxyifosfamide, 4-ketoifosfamide, dechloroethyl cyclophosphamide, dechloroethylifosfamide, and alcoifosfamide were identified. The latter 4 metabolites were also identified in urine from an ifosfamide-treated dog. In a simulated in vitro pharmacokinetic experiment against L1210 leukaemia in which drugs were incubated at various concentrations for various times, both 4-hydroxycyclophosphamide and isophosphoramide mustard exhibited significant cytoxicity at concentration times time values of 100-1000 micrograms X min ml-1, while acrolein was significantly cytotoxic at 10 micrograms X min ml-1. Treatment of mice with drug followed by L1210 cells demonstrated a shorter duration of effective levels of cytotoxic activity for isophosphoramide mustard and phosphoramide mustard in comparison with cyclophosphamide and ifosfamide. Isophosphoramide mustard and 2-chloroethylamine, a potential hydrolysis product of isophosphoramide mustard and carboxyifosfamide, were less mutagenic in the standard Ames test than the 2 corresponding metabolites of cyclophosphamide [phosphoramide mustard and bis(2-chloroethyl)amine].     

DNA cross-linking and single-strand breaks induced by teratogenic concentrations of 4-hydroperoxycyclophosphamide and phosphoramide mustard in postimplantation rat embryos

Postimplantation rat embryos (Day 10) were exposed in vitro to teratogenic concentrations of 4-hydroperoxycyclophosphamide, an activated form of cyclophosphamide, and phosphoramide mustard, the major teratogenic metabolite of cyclophosphamide. Following a 5-h exposure to these agents, drug-induced DNA damage was assessed by alkaline elution. Both drugs induced detectable DNA cross-linking at teratogenic concentrations. Alkaline elution combined with proteinase K digestion indicated that approximately half of the DNA cross-linking was DNA-DNA cross-linking and the other half was DNA-protein cross-linking. In addition to DNA cross-linking, phosphoramide mustard produced DNA strand breaks and/or alkaline labile sites. However, 4-hydroperoxycyclophosphamide did not produce detectable DNA strand breaks or alkaline labile sites. Our data also indicate that the induction of abnormal morphogenesis by 4-hydroperoxycyclophosphamide and phosphoramide mustard is correlated with drug-induced DNA cross-linking.     

Thermodynamic analysis of the reaction of phosphoramide mustard with protector thiols

The systemic use of thiol-containing uroepithelial protecting agents, e.g., N-acetylcysteine (NAC) or mesna, in conjunction with the alkylating agent cyclophosphamide is predicated on the assumption that the toxic metabolic by-products will be consumed by thiol without diminishing the cytotoxicity of the active alkylating intermediate, phosphoramide mustard. Studies in murine tumor systems have been with either a single dose or two equally divided doses of thiol, administered within 30 min of the addition of cyclophosphamide, without an observed adverse effect on antitumor activity; however, the relatively short serum half-life of thiol relative to alkylating agent in humans weakens the clinical relevance of these results. This study presents a thermodynamic model for the chemical reaction of phosphoramide mustard with either NAC or mesna. The gas phase thermodynamic parameters for these reactions, enthalpy (H) and entropy (S), were calculated using the semiempirical quantum mechanical method AM1 and were used to predict the free energy (delta G) for these processes. For the reaction of phosphoramide mustard with NAC or mesna, delta G = +3.82 and 2.29 kcal/mol, respectively. In the absence of enzyme catalysis, these results suggest that such reactions are not favored. In order to assess the validity of this gas phase thermodynamic model, the cellular cytotoxicity of phosphoramide mustard in the presence or absence of either NAC or mesna was studied using CCRF-CEM cells in culture. In these experiments the 50% effective dose of phosphoramide mustard was 1.7 micrograms/ml; this result was unchanged in the presence of 10 micrograms/ml concentration of either thiol. This study supports the conclusion that phosphoramide mustard and protector thiols are compatible.     

Pharmacokinetics of N-2-chloroethylaziridine, a volatile cytotoxic metabolite of cyclophosphamide, in the rat

Objectives: The objectives of this study were to characterize pharmacokinetics of N-2-chloroethylaziridine (CEA) in the rat model and assess the in vivo fraction of total clearance of phosphoramide mustard (PM) that furnished CEA to circulation. Methods: The disposition of CEA was investigated following separate intravenous (iv) administrations of PM, synthetic CEA, and their combination to the Sprague-Dawley rats. In addition, in rats receiving prodrug cyclophosphamide (CP), plasma concentrations of CP and its metabolites, 4-hydroxycyclophosphamide (HOCP), PM, and CEA, were simultaneously quantified using GC/MS and stable isotope dilution techniques. Results: Following iv administration of synthetic CEA, concentrations of CEA declined biexponentially with the mean terminal half-life and total body clearance of 47.5 min and 167 ml/min/kg, respectively. Urinary excretion of unchanged CEA was 0.164% of the administered dose. CEA was found to be the major circulating metabolite after iv administration of precursor PM to rats. The fraction of total clearance of PM that furnished CEA to circulation was estimated to be 100%, indicating virtually complete availability of the metabolite to circulation once formed. In rats administered with CP, PM exhibited the highest plasma and urinary concentrations compared to HOCP and CEA. Conclusions: For the first time, CEA was demonstrated to be an important in vivo metabolite of CP in the present study. In light of the poor permeability and in vivo stability of PM, the ultimate DNA alkylator, the findings obtained in this study suggested that CEA may contribute significantly to the overall antitumor activity of prodrug CP.     

Plasma pharmacokinetics of cyclophosphamide and its cytotoxic metabolites after intravenous versus oral administration in a randomized, crossover trial

Since cyclophosphamide is used by both oral and i.v. routes in the treatment of hematological and solid malignancies, we designed a randomized, crossover clinical trial to evaluate the pharmacokinetics of this anticancer agent after either administration route. Plasma levels of cyclophosphamide and its two cytotoxic metabolites, 4-hydroxycyclophosphamide and phosphoramide mustard, were determined in seven cancer patients randomly assigned to treatment initially with either orally or i.v. administered cyclophosphamide with a 30-day interim between alternate therapy courses. Oral treatment was used initially in five patients and i.v. treatment in two patients, and the pharmacokinetic parameter, area under the plasma disappearance curve, was determined for each metabolite in each patient for both routes of drug administration. Statistical comparison of area under the plasma disappearance curve values for this set of patients indicated no significant differences for either metabolite for oral versus i.v. drug treatment, suggesting equal efficacy for these two routes of cyclophosphamide administration.     

Binding of metabolites of cyclophosphamide to DNA in a rat liver microsomal system and in vivo in mice

The stability of phosphoramide mustard, a metabolite of cyclophosphamide was studied at pH 7.2 and 37 degrees C using 31P nuclear magnetic resonance. The phosphorus signal of phosphoramide mustard disappeared with a half-life of 8 min indicating rapid conversion to other species. The final product, inorganic phosphate, appeared with a half-life of 105 min indicating that phosphoramide mustard was easily dephosphoramidated. A rat liver microsomal system was used to study the binding of [chloroethyl-3H]cyclophosphamide to DNA. DNA was hydrolyzed in 0.1 N HCl:0.5 N NaCl at 80 degrees C for 20 min, conditions known to convert phosphoramide mustard to nornitrogen mustard with liberation of the phosphoramide residue. After such treatment three adducts were detected by high-performance liquid chromatography using several elution systems. They were all 7-substituted guanine adducts of nornitrogen mustard; two were monoalkylation products with an intact [N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine] or an hydroxylated mustard arm [N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine]; the third adduct was a cross-linked product [N,N-bis [2-(7-guaninyl)ethyl]-amine]. The relative abundance of these adducts depended on the length of the microsomal incubation. After 2 h, N-(2-chloroethyl)-N-[2-(guaninyl)ethyl]amine was the main product but after 6 h N-(2-hydroxyethyl)-N-[2-(7-guaninyl)ethyl]amine was most abundant, and at this time the cross-linked product represented 12% of the total adducts. The adducts in DNA depurinated readily and after 24 h at pH 7.0 and 37 degrees C 70% of them had been liberated. The rate of depurination was decreased in the presence of 0.5 N NaCl. After short-term depurination in 0.1 N HCl at 25 degrees C the primary alkylating species was phosphoramide mustard rather than nornitrogen mustard. In in vivo studies mice were given injections i.p. of 100 microCi of cyclophosphamide. Maximal levels of radioactivity had been incorporated into DNA between 2-7 h after injection; the specific activity of DNA from the kidney and lung exceeded that from the liver. While the level of radioactivity found in kidney DNA was rapidly reduced the rate of fall was lower in the lung. Between 24 and 72 h the specific activity of lung DNA exceeded that of kidney and liver DNA by a factor of 3:8. Lung is the principal target tissue for tumor formation in mice after an i.p. injection.     

Effect of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and malignant blood cells to oxazaphosphorines

The ex vivo sensitivity of human multipotent and committed hematopoietic progenitor cells and several cultured human malignant blood cell lines to analogues of "activated" cyclophosphamide, namely, 4-hydroperoxycyclophosphamide and mafosfamide, and to phosphoramide mustard was quantified with and without concurrent exposure to an inhibitor of aldehyde dehydrogenase activity, namely, disulfiram, cyanamide, diethyldithiocarbamate, or ethylphenyl(2-formylethyl)phosphinate. Inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of 4-hydroperoxycyclophosphamide and mafosfamide toward all of the hematopoietic progenitors; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Potentiation of the cytotoxic action of mafosfamide toward cultured human malignant blood cells was minimal. Spectrophotometric assay revealed little NAD-linked aldehyde dehydrogenase activity present in the cultured human tumor cell lines as compared to that found in normal mouse liver or oxazaphosphorine-resistant L1210 cells. Cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major intermediate in cyclophosphamide bioactivation, to the relatively nontoxic acid, carboxyphosphamide. Thus, our findings indicate that human multipotent hematopoietic progenitor cells contain the relevant aldehyde dehydrogenase activity, the relevant activity is retained upon differentiation to progenitors committed to the megakaryocytoid, granulocytoid/monocytoid, and erythroid lineages, and the relevant activity may be lost or diminished upon transformation of hematopoietic progenitors to malignant cells.     

Plasma pharmacokinetics and urinary excretion of hexamethylene bisacetamide metabolites

In order to further understand the clinical toxicities of hexamethylene bisacetamide (HMBA) and to allow appropriate in vitro studies, we developed a suitable gas chromatographic assay and quantified plasma concentrations and urinary excretion of four metabolites which we had previously identified in urine of patients receiving 5-day HMBA infusions at 4.8-43.2 g/m2/day. 6-Acetamidohexanoic acid (AcHA) was the major plasma metabolite and reached steady state concentration (Css) by 24 h. AcHA Css increased from 0.12 +/- 0.02 (SD) mM at 4.8 g/m2/day to 0.72 mM at 43.2 g/m2/day. The Css AcHA:Css HMBA ratio decreased with increasing HMBA dosage. At dosages below 24 g/m2/day plasma Css of N-acetyl-1,6-diaminohexane (NADAH), the initial metabolite of HMBA, were below the limit of detection of our assay. With HMBA infusions of 24, 33.6, and 43.2 g/m2/day, Css of NADAH were 0.16 +/- 0.05, 0.14 +/- 0.06, and 0.19 +/- 0.04 mM, respectively. Css NADAH:Css HMBA ratios at 24, 33.6, and 43.2 g/m2/day were 0.18 +/- 0.06, 0.08 +/- 0.02, and 0.31 +/- 0.05, respectively. Plasma Css of 1,6-diaminohexane and 6-aminohexanoic acid were below the limit of detection of our assay. Each patient's urinary excretion of NADAH, AcHA, and 1,6-diaminohexane was consistent from day to day. The fraction of dose excreted in urine as AcHA was not affected by HMBA dosage and accounted for 12.7 +/- 3.9% of the daily dose. The percentage of daily HMBA dose accounted for by excretion of NADAH decreased with increasing HMBA dosage (10.8 +/- 6.0% at 4.8 g/m2/day to 4.2 +/- 1.2% at 33.6 g/m2/day). Urinary excretion of 1,6-diaminohexane always accounted for less than 3% of the daily dose. Our results indicate that: (a) plasma concentrations of AcHA alone cannot explain the degree of acidosis observed with toxic doses of HMBA; (b) NADAH is present in plasma at concentrations that we have found to cause differentiation in vitro; and (c) the probable rate-limiting step in HMBA metabolism is the initial deacetylation.     

Effect of phenobarbital on plasma levels of cyclophosphamide and its metabolites in the mouse.

We have studied the quantitative pharmacokinetic differences of individual metabolites and unchanged cyclophosphamide (CPA) in control and phenobarbital-treated animals, using radiolabelled CPA together with thin-layer chromatography. On Day 0, one group was started on phenobarbital drinking water and one group stayed on regular acid water. P388 leukaemia, (10(6) cells i.p.) was administered to all mice on Day 8, and 2 days later both groups of mice were given i.p. CPA (200 mg/kg) with 14C-CPA (0.2 muCi per mouse). At 5--60 min after CPA administration, groups of 10 mice were killed and their blood collected for assay of parent compound and metabolites in plasma. Phenobarbital pretreatment reduced CPA and phosphoramide mustard CXT (concentration x time) by 66+% and 27+%, respectively. Assuming that phosphoramide mustard is both the ultimate cytotoxic form of CPA and the blood-transport form, the reduction of CPA by phenobarbital would predict a decreased therapeutic effect. The assay methods in this study will be used in the future to determine the importance of this potential drug interaction in man.     

A proposed mechanism of resistance to cyclophosphamide and phosphoramide mustard in a Yoshida cell line in vitro

Summary A Yoshida sarcoma cell line (Y_R/cyclo) showing decreased sensitivity to metabolically activated cyclophosphamide in vitro has been shown to be cross-resistant to phosphoramide mustard, the ultimate alkylating agent formed from cyclophosphamide. Resistance to these alkylating agents has been shown to be associated with increased activity of the glutathione S-transferase group of enzymes, and with elevated levels of glutathione, the cosubstrate of the enzyme. The resistant cell line shows lower levels of cellular damage, as measured by alkaline elution following treatment with phosphoramide mustard, than the parental (Ys) line. The mechanism of resistance is ascribed to increased deactivation of potentially damaging metabolites of cyclophosphamide by the glutathione S-transferase enzymes, resulting in decreased cellular damage in the resistant cell line.This work was supported by a grant from the Cancer Research Campaign     

Pharmacokinetics of cyclophosphamide and its metabolites in paediatric patients receiving high-dose myeloablative therapy

Introduction

In order to better understand the impact of high-dose on the pharmacokinetics and metabolism of cyclophosphamide, a pharmacological study was performed in children with malignant mesenchymal tumours with metastatic disease.

Methods

Patients received four courses of chemotherapy including two courses of cyclophosphamide. Plasma concentrations of cyclophosphamide and the metabolites 4-ketocyclophosphamide, dechloroethylcyclophosphamide and carboxyphosphamide were determined on days 1, 2 and 3 of each course. A population pharmacokinetic model for cyclophosphamide was developed using non-linear mixed effects modelling and metabolite AUC values were compared between days and courses.

Results

Data were available on 21 cyclophosphamide courses from 15 patients. A one compartment model, incorporating a term in surface area for both CL and V, best described cyclophosphamide pharmacokinetics. Typical CL and V on day 1 of treatment for a patient with a SA of 1.4 m² were 4.3 L/h and 28.5 L, respectively. On days 2 and 3 CL increased by 88% (95% CI, 72-105%) and 125% (95% CI, 108-145%) over day 1 levels; V increased by 14% (95% CI, 5-23%) on days 2 and 3. V tended to be larger for males than similarly sized females but no effect of age was found upon CL or V. Significant increases in metabolite AUCs were observed on days 2 and 3 compared to day 1 and a significant increase in CXCP AUC from course 1 to course 3.

Conclusion

Administration of high-dose cyclophosphamide over several days results in an increase in metabolism, possibly by induction of the activation pathway. This induction is effectively reversed following a four week period between cyclophosphamide doses. The degree of intersubject variation in cyclophosphamide elimination is largely accounted for by body surface area and is less than previously reported.