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.     

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.     

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.     

Comparative brain and plasma pharmacokinetics and anticancer activities of chlorambucil and melphalan in the rat

Summary Equimolar doses of chlorambucil and melphalan (both 10 mg/kg) were administered i.v. to anesthetized rats, and the plasma and brain concentrations of chlorambucil, its metabolites 3,4-dehydrochlorambucil and phenylacetic mustard, and melphalan were determined by high-performance liquid chromatography from 5 to 240 min therafter. Chlorambucil demonstrated a monophasic disappearance from plasma, with a half-life of 26 min. The compound was 99.6% plasma-protein-bound. Chlorambucil underwent -oxidation to yield detectable concentrations of 3,4-dehydrochlorambucil and substantial amounts of phenylacetic mustard in the plasma. Low concentrations of chlorambucil and phenylacetic mustard were detected in the brain. Calculated from the areas under the concentration-time curves, the brain: plasma concentration integral ratios of chlorambucil and phenylacetic mustard were 0.021 and 0.013, respectively. Melphalan demonstrated a biphasic disappearance from plasma, with half-lives of 1.9 and 78 min. The compound was approximately 86% plasma protein-bound. Low concentrations of melphalan were detected in the brain, and its brain: plasma ratio was 0.13. These data demonstrate that following the administration of chlorambucil and melphalan, only low concentrations of active drug are able to enter the brain. As a consequence, concentrations of both drugs that cause the complete inhibition of extracerebrally located tumor have no effect on those located within the brain. Further, the brain uptake of melphalan, although low, is greater than that of chlorambucil and its active metabolites, which coincides with its slightly greater intracerebral activity following the systemic administration of very high doses.     

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].     

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     

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.     

Comparative effects of cyclophosphamide, isophosphamide, 4-methylcyclophosphamide, and phosphoramide mustard on murine hematopoietic and immunocompetent cells.

The effects of equimolal doses of cyclophosphamide (CY), isophosphamide (IP), 4-methylcyclophosphamide (4-MCY), and phosphoramide mustard (PM) on murine hematopoietic spleen colonies and adoptively transferred antibody-forming cells in vivo were compared. Equimolal doses of the drugs produced significantly different effects. All the drugs exerted an increasing effect against the ability of adoptively transferred immunocompetent cells to produce a significant anti-sheep red blood cell titer as the length of time between cell transfer and drug administration was increased. The maximum effect was seen when a drug was given 48--72 hours after antigen and spleen cell transfer. CY and IP produced significantly greater immunosuppressive effects than did the other drugs at all times after cell transfer and at all doses administered. PM had the least immunosuppressive effect at each dose evaluated. Against hematopoietic spleen colonies, the cytotoxic effects of 4-MCY and PM were similar and, at most doses studied, significantly greater than the effect of either CY or IP. Inasmuch as PM is an active metabolite of CY, it appeared either that one of the prior metabolites of CY was responsible for this marked immunosuppressive effect or that due to differences in polarity, PM was differentially distributed within the two cell systems as compared to CY. The differences in hematopoietic effects among all drugs were much less than those seen against immunocompetent cells and were not dependent on time of drug administration.     

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.     

Pharmacokinetics of chlorambucil-tertiary butyl ester,a lipophilic chlorambucil derivative that achieves and maintains high concentrations in brain

Summary Equimolar doses of chlorambucil (10 mg/kg) and the lipophilic chlorambucil derivative, chlorambuciltertiary butyl ester (13 mg/kg), were given i. v. to rats. Plasma and brain concentrations of chlorambucil and its active metabolites, 3,4-dehydrochlorambucil and phenylacetic mustard, as well as of chlorambucil-tertiary butyl ester were then determined by HPLC between 2 and 240 min after drug administration. Chlorambucil demonstrated a monophasic disappearance from plasma following its administration, with a half-life of 28 min. Significant amounts of phenylacetic mustard were detected after 15 min, and this agent maintained high levels of active compounds in plasma throughout the study. Only low concentrations of chlorambucil and phenylacetic mustard were detected in brain between 2 and 120 min. Following equimolar chlorambucil-tertiary butyl ester administration, it rapidly disappeared from plasma, with a half-life of approximately 2 min, and maintained low plateau concentrations between 15 and 120 min after treatment. It was not detected thereafter, although significant amounts of chlorambucil and phenylacetic mustard were detected throughout the study. Significant amounts of chlorambucil-tertiary butyl ester entered and remained within the brain, achieving a peak concentration at 15 min and disappearing thereafter with a half-life of 37 min. Low levels of chlorambucil and phenylacetic mustard were also detected. Calculated from the areas under the concentration vs time curves of total active compounds derived from chlorambucil and chlorambucil-tertiary butyl ester in brain and plasma, the brain: plasma concentration integral ratios were 0.018 and 0.68, respectively. Following equimolar doses of chlorambucil and chlorambucil-tertiary butyl ester, a 7-fold greater concentration integral was achieved by chlorambucil-tertiary butyl ester in brain at a 5-fold lower plasma concentration integral. Chlorambucil-tertiary butyl ester may be of value in the treatment of brain-sequestered tumors.     

Accelerated decomposition of 4-hydroxycyclophosphamide by human serum albumin

Cyclophosphamide, a widely used anticancer agent, requires initial metabolic activation to 4-hydroxycyclophosphamide (4-OHCP) to elicit its activity. The rate of decomposition of cis-4-OHCP was much faster in plasma than in buffer at pH 7.4. This plasma activity was not affected by treatment with acid (pH 1.3) or heat (60 degrees C for 30 min). The activity was retained in the macromolecular fraction (greater than 10,000) but not in the filtrate. Serum albumin was identified as the catalyst for the elimination step that generates phosphoramide mustard from aldophosphamide; albumin had no effect on the rate of ring opening of cis-4-OHCP to aldophosphamide. This catalytic activity was dependent on serum albumin concentration and independent of pH over the range of 6.5 to 7.5, in contrast to the buffer-catalyzed reaction. The catalytic rate constants kcat (pH 7.4, 37 degrees C) for phosphate buffer, human serum albumin, and bovine serum albumin were 1.13, 285, and 83 M-1 min-1, respectively. Pretreatment of cis-4-OHCP with serum albumin resulted in a time-dependent decrease in cytotoxic activity against L1210 tumor cells in vitro. These data suggest that the albumin-catalyzed reaction of cis-4-OHCP in plasma represents an important pathway for the transformation of cyclophosphamide metabolites and further emphasize the importance of considering phosphoramide mustard generated extracellularly versus intracellularly and the respective contributions of extracellular and intracellular phosphoramide mustard to cyclophosphamide cytotoxicity in vivo.     

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.     

Primary gene‐engineered neural stem/progenitor cells demonstrate tumor‐selective migration and antitumor effects in glioma

The prognosis of patients with glioblastoma multiforme (GBM) is generally poor after surgical tumor resection. With the aim of developing new adjuvant therapeutic strategies, we have investigated primary neural stem/progenitor cells (NSPC) in co‐cultures with glioma cells, and in a model of gene therapy on aggressively growing malignant glioma. NSPC exhibited tropism towards medium conditioned by glioma cells, and in adherent low‐cell density co‐culture, were attracted to, and fused with, tumor cells. Similarly, within 24–48 hr of co‐culture in suspension, NSPC‐tumor hybrids were observed, representing 2–3% of the total cell population. NSPC were then coinjected into mouse brain with GBM cells, employing NSPC expressing cyclophosphamide (CPA)‐activating enzyme cytochrome p450 2B6 (CYP2B6), which catalyzes CPA prodrug transformation into membrane diffusible DNA‐alkylating metabolites. Upon CPA administration, NSPC containing CYP2B6 elicited substantial impairment of tumor growth. When implanted intracerebrally at a distant site from the tumor, gene‐engineered NSPC specifically targeted GBM grafts, after traveling through brain parenchyma, and hindered tumor growth through local activation of CPA. Directed migration of primary NSPC corresponded closely with intracerebral and tumoral pattern of expression of vascular endothelial growth factor, which is a motility factor for NSPC. Overall, these findings indicate that therapeutic gene delivery mediated by primary NSPC is a potentially valid strategy for treatment of high‐grade gliomas.     

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.     

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.     

Effects of hypoxia and limited diffusion in tumor cell microenvironment on bystander effect of P450 prodrug therapy

Cytochrome P450 (CYP) enzyme 2B1 metabolizes the anticancer prodrug cyclophosphamide (CPA) to 4-hydroxy-CPA, which decomposes to the cytotoxic metabolites acrolein and phosphoramide mustard. We have evaluated the bystander cytotoxicity of CPA in combination with CYP2B1 gene-directed enzyme prodrug therapy using a cell culture-based agarose overlay technique. This method mimics the tumor microenvironment by limiting the diffusion of metabolites and by reducing the oxygen concentration to levels similar to those found in solid tumors. Under these conditions, the CYP activity of CYP2B1-expressing tumor cells was decreased by 80% compared to standard aerobic conditions. Despite this decrease in metabolic activity, a potent bystander effect was observed, resulting in up to 90% killing by CPA of a tumor cell population comprised of only approximately 20% CYP-expressing tumor cells. Similarly, transient transfection of a small fraction ( approximately 14%) of a human hepatoma Huh7 cell population with a CYP2B1 expression plasmid followed by short-term treatment with CPA (5 h) led to an eradication of 95% of the cells. No such bystander effect was observed without the agarose overlay. These findings suggest that the agarose overlay technique is very useful as an in vitro test system for investigation of the bystander effect of CYP/CPA and other enzyme/prodrug combinations under conditions that mimic the hypoxic conditions present in solid tumors in vivo.     

Antitumor activity of halogen analogs of phosphoramide,isophosphoramide, and triphosphoramide mustards,the cytotoxic metabolites of cyclophosphamide,ifosfamide, and trofosfamide

A series of halogen analogs of phosphoramide mustard, isophosphoramide mustard, and triphosphoramide mustard, the cytotoxic metabolites of the antitumor drugs cyclophosphamide, ifosfamide, and trofosfamide, respectively, was evaluated in vitro against human tumor cell lines and in vivo against experimental tumors to investigate the effect of replacement of chlorine with bromine or fluorine on the antitumor activity of the parent phosphoramide mustards. In the experimental tumors L1210 leukemia, B16 melanoma, mammary adenocarcinoma 16/C, and ovarian sarcoma M5076, the antitumor activity of the analogs was observed to be generally comparable with that of the parent mustards when chlorine was replaced by bromine but uniformly lower when chlorine was replaced by fluorine. Furthermore, the monobromo analog of isophosphoramide mustard displayed equal or somewhat greater activity in comparison with cyclophosphamide when evaluated against subcutaneously implanted L1210 leukemia with intraperitoneal drug treatment and against mammary adenocarcinoma 16/C.This work was financially supported by NIH, NCI grant PO1 CA34200     

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.     

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.     

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