Determination of cyclophosphamide and its metabolites in human plasma by high-performance liquid chromatography–mass spectrometry

A sensitive HPLC–MS method was developed for the simultaneous determination of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide (aldocyclophosphamide), 4-ketocyclophosphamide, caboxyphosphamide and 3-dechloroethylifosfamide in human plasma. 4-Hydroxycyclophosphamide was converted with methylhydroxylamine to the stable methyloxime form. We used a solid-phase extraction with C₁₈ cartridges followed by HPLC–MS with the single mass spectrometer SSQ 7000 of Finnigan. The limits of detection were 15 ng/ml for cyclophosphamide, 3-dechloroethylifosfamide and ketocyclophosphamide in each case and 30 ng/ml for carboxyphosphamide and 4-hydroxycyclophosphamide, respectively. First results of pharmacokinetics are shown.     

Effects of phosphoramide mustard and acrolein, cytotoxic metabolites of cyclophosphamide, on mouse limb development in vitro

Phosphoramide mustard and acrolein are toxic and reactive metabolites of the widely used anticancer drug and known teratogen cyclophosphamide. To study the mechanism(s) involved and to determine which of the active metabolites of cyclophosphamide is responsible for the production of limb malformations, the effects of exposure of cultured limb buds to phosphoramide mustard and acrolein were investigated. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to either phosphoramide mustard or acrolein (10 or 50 micrograms/ml) for the first 20 hours of the culture period. Exposure to phosphoramide mustard produced limb reduction malformations in both the fore- and hindlimbs; total limb bone area was greatly reduced, while the relative contribution of the paw to this area in forelimbs was increased. There was a fourfold reduction in both DNA and RNA; protein content was reduced only by one-half. Alkaline phosphatase activity was significantly decreased in fore- and hindlimbs exposed to phosphoramide mustard, whereas creatine phosphokinase activity was only reduced in hindlimbs in the limbs exposed to the higher concentration of phosphoramide mustard. Exposure to acrolein also produced malformed limbs with a mangled appearance; however, total limb bone area and the relative contribution of the long bones versus paw structures were not altered. Acrolein exposure had little effect on growth parameters such as DNA (decreased only in hindlimbs exposed to 50 micrograms/ml), RNA (increased in hindlimbs exposed to 50 micrograms/ml), or protein content. Alkaline phosphatase and creatine phosphokinase activities were not altered in acrolein-exposed fore- or hindlimbs. Thus, phosphoramide mustard and acrolein have dramatically different effects on developing limbs in vitro; this observation may indicate that they have different targets and/or mechanisms of action as teratogens in the limb. The effects of phosphoramide mustard are very similar to those of "activated" cyclophosphamide (4-hydroperoxycyclophosphamide).     

Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography-mass spectrometry

A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC-MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is approximately 20 fmol. Plasma CEPM concentration is determined using LC-MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.     

Influence of dietary carrot on cytostatic drug activity of cyclophosphamide and its main directly acting metabolite: induction of sister-chromatid exchanges in normal human lymphocytes, Chinese hamster ovary cells, and their DNA repair-deficient cell lines

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic anaemia (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5-2-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells (P less than 0.01), and to a lesser extent in human lymphocytes (p less than 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.     

Synthesis and cytotoxic activity of a glucuronylated prodrug of nornitrogen mustard

A new glucuronylated prodrug of nornitrogen mustard, incorporating the same spacer group as the doxorubicin prodrug HMR 1826, has been prepared. Upon exposure to E. coli beta-glucuronidase, fast hydrolysis occurs but a lower cytotoxicity against LoVo cancer cells is observed compared to the nornitrogen mustard alone. This is explained by cyclization of the intermediate carbamic acid to the inactive chloroethyl oxazolidinone.     

Embryotoxicity of phenyl ketone analogs of cyclophosphamide

Phenylketophosphamide and phenylketoisophosphamide are preactivated acyclic ketone analogs of cyclophosphamide and isophosphamide with antitumor activity. These compounds undergo an elimination reaction to yield phosphoramide or isophosphoramide mustard and phenyl vinyl ketone. In this study, the embryotoxicity of phenylketophosphamide, phenylketoisophosphamide, and phenyl vinyl ketone were determined. Embryotoxicity was assessed in vitro in whole rat embryos cultured on day 10.5 of gestation in the absence and presence of an activating system derived from maternal liver. Both phenylketophosphamide and phenylketoisophosphamide were embryotoxic in the absence of metabolic activation. Moreover, there was no enhancement of this embryotoxicity in the presence of an activating system. A 10-microM concentration of phenylketophosphamide produced 100% malformed embryos, while this concentration of phenylketoisophosphamide was not teratogenic. At 25 microM phenylketoisophosphamide, all the surviving exposed embryos were malformed. Phenylketophosphamide was embryolethal to more than 50% of the exposed embryos at a concentration of 50 microM. In contrast, a concentration of phenylketoisophosphamide of 100 microM was required to produce significant embryolethality. Phenyl vinyl ketone was not embryotoxic at any of the concentrations tested. The major malformation observed, a hypoplastic prosencephalon, and the growth retardation effects were not only similar for phenylketophosphamide and phenylketoisophosphamide, but also similar to those previously reported for "activated" cyclophosphamide. Unlike the results with cyclophosphamide, where both phosphoramide mustard and the aldehydic metabolite of cyclophosphamide, acrolein, are toxic, the embryotoxic effects of phenylketophosphamide and phenylketoisophosphamide are mediated only by the mustard metabolite.     

Modulation of the control of mutagenic metabolites derived from cyclophosphamide and ifosfamide by stimulation of protein kinase A

The phosphorylation of the 2 major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in intact hepatocytes by the action of the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Under these conditions cyclophosphamide and ifosfamide (which are known to be activated by cytochrome P450 IIB1) were investigated for mutagenicity in Salmonella typhimurium TA1535 and TA100 and for cytotoxicity in TA1535. Cyclophosphamide and ifosfamide were transformed to mutagenic and cytotoxic metabolites by the hepatocytes. The activation of both drugs to mutagens was markedly reduced after pretreatment of the hepatocytes with the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Cyclophosphamide and ifosfamide activation were reduced to 51% and 38% of unstimulated controls respectively, when hepatocytes were incubated for 1 h with N6,O2'-dibutyryl-cAMP in the presence of the phosphodiesterase inhibitor theophylline, and Salmonella typhimurium TA1535 was used. A marked reduction in mutagenicity of cyclophosphamide (35% compared with unstimulated controls) was also observed under different experimental conditions, namely after pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP for 1.5 h without theophylline and using Salmonella typhimurium TA100 as target strain. Continued presence of the cytochrome P450 IIB1 and P450 IIB2 inducer phenobarbital in the stimulation medium increased the mutagenicity of cyclophosphamide and led to an even more marked reduction of mutagenicity by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. In order to investigate whether the observed changes were metabolism-related, the ifosfamide metabolite ifosfamide mustard which does not require metabolic activation by cytochrome P450 was studied under the same conditions. Its mutagenicity was indistinguishable after incubation with N6,O2'-dibutyryl-cAMP-treated or with unstimulated hepatocytes. Also the metabolic formation of cytotoxic metabolites from cyclophosphamide and ifosfamide but not that of ifosfamide mustard was markedly decreased by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. Thus the stimulation of protein kinase A in intact cells has important consequences for the control of genotoxic and cytotoxic metabolites and represents a fast and short-term regulation of it.     

Mutagenic activities of cyclophosphamide (NSC-26271) and its main metabolites in Salmonella typhimurium, human peripheral lymphocytes and Chinese hamster ovary cells

Cyclophosphamide (CPA) and its main metabolites were analyzed with respect to their mutagenic activities in Salmonella, human peripheral lymphocytes (PL), and Chinese hamster ovary (CHO) cells. In Salmonella, the compounds were activated with S9 mix from rat livers, which were unstimulated or stimulated with Aroclor 1254 or phenobarbital. For the enzyme inducers the following order of efficiency was found for all test compounds except carboxyphosphamide: phenobarbital greater than Aroclor 1254 greater than non-induced. The most potent mutagens in all 3 test systems were 4-OH-CPA, PAM and nor-HN2. S9 mix transforms 4-OH-CPA to strong mutagenic compounds in the Salmonella assay. All metabolites tested in the Salmonella assay were activated by S9 mix to higher mutagenic potential.     

Biotransformation of mafosfamide in P388 mice leukemia cells: intracellular 31P-NMR studies

The intracellular transformation of cis-mafosfamide has been studied in P388 mice leukemia cells using 31P-NMR spectroscopy. For this purpose the cells were entrapped in low-gelling-temperature agarose threads. Internal pH of the cells, determined from the position of the intracellular inorganic phosphate, was 7.2. The cell membrane was permeable to 4-hydroxycyclophosphamide and aldophosphamide and less permeable to phosphoramide mustard. 4-Ketocyclophosphamide and carboxyphosphamide signals were not detectable in cells either sensitive or resistant to oxazaphosphorine treatment.     

Gas chromatographic-mass spectrometric assay for N-2-chloroethylaziridine, a volatile cytotoxic metabolite of cyclophosphamide, in rat plasma

A sensitive and specific method for the quantitative analysis of N-2-chloroethylaziridine (CEA), a volatile cytotoxic metabolite of cyclophosphamide, has been developed using gas chromatography-mass spectrometry and stable isotope dilution techniques. The high volatility problem of CEA during isolation procedure was overcome by the combined use of a deuterium-labeled analog as the internal standard and a Snyder column-concentrator assembly. The assay was found to be linear from 16.7 to 2667 ng/ml in rat plasma with a routine detection limit of 5 ng/ml. The within-run precision at 33, 333 and 1333 ng/ml (n=6) was found to be 4.8, 4.9, and 6.1%, respectively. The between-run precision was 6.4% (n=6). The dichloromethane extraction recoveries at 33, 333, and 1333 ng/ml were found to be 101, 98, and 91%, respectively (all at n=6). However, the overall recovery through extraction and evaporation was only 18.3, 15.2, and 27.7% at 33, 333, and 1333 ng/ml levels, respectively. The analytical method was used to evaluate the generation of CEA from its precursors in sodium phosphate buffer, in cell culture media, and the degradation of CEA in these media. In pH 7.4, 0.067 M sodium phosphate buffer at 37°C, both phosphoramide mustard (PM) and nornitrogen mustard (NNM) were degraded in an apparent first-order fashion with half-lives of 24.8 and 14.5 min, respectively. The generated CEA was rather stable in this buffer and degraded with a half-life of 20 h. It was found that 32% PM and 91% NNM were converted to CEA in pH 7.4, 0.067 M sodium phosphate buffer at 37°C, respectively, and 41% PM was transformed into CEA in RPMI 1640 tissue culture media containing 10% FBS at 37°C. The generated CEA was very stabble in the culture media with a degradation half-life of 265 h.     

Denaturation of cytochrome P-450 by cyclophosphamide metabolites

Cyclophosphamide (CP) metabolites, acrolein and 4-hydroxy-CP, were found to denature rat liver microsomal cytochrome P-450, whereas another metabolite, phosphoramide mustard, CP $\text{per}\text{se}$ or its analog Ifosfamide had no effect. The denaturation produced by CP metabolites could be blocked by cysteine, suggesting an interaction between CP metabolite(s) and sulfhydryl groups in cysteine and probably in cytochrome P-450. These studies might explain the biochemical basis of the specific depression of various microsomal mixed function oxygenase activities produced by high doses of CP.     

The alkylation of hemoglobin S by nitrogen mustard. High resolution proton nuclear magnetic resonance studies.

Sickle cell hemoglobin (Hb S) treated with nitrogen mustard (bis(beta-chloroethyl)methylamine hydrochloride) gives two reaction products, one labile and one stable. After dialysis against buffer solution, the remaining stable product is found to inhibit the polymerization of deoxyhemoglobin S. High resolution proton nuclear magnetic resonance has been used to study the structure and function of this stable product and to investigate the nature of the binding sites of nitrogen mustard to the hemoglobin molecule. The NMR results suggest that the nitrogen mustard treatment of Hb S does not alter the heme environment or the subunit interfaces of the hemoglobin molecule. Moreover, the NMR spectra have also shown that the nitrogen mustard reacts with the beta2 histidines of the hemoglobin molecule and have suggested that several other surface amino acid residues of the hemoglobin molecule are also affected by the nitrogen mustard alkylation. These NMR findings are in good agreement with the data obtained from biochemical studies of nitrogen mustard-treated Hb S. The NMR spectra also indicate that nornitrogen mustard (which is also effective in inhibiting sickling) binds with the hemoglobin molecule in a manner identical with nitrogen mustard. Sulfur mustard, on the other hand, produces no observable changes in the aromatic proton resonances, which is consistent with the fact that it does not inhibit the polymerization of deoxy-Hb S.     

Influence of metabolic factors on the mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster

This paper describes the influence of changes in metabolic activity on the in-vivo mutagenic effectiveness of cyclophosphamide in Drosophila melanogaster. A dose-dependent increase in mutagenicity was observed until a plateau value is reached which was increased only slightly after enzyme induction with Aroclor 1254, whereas induction with phenobarbital resulted in a decrease, especially when cyclophosphamide was applied by injection. Treatment of the adult males with inhibitors of the monoamine oxidase (MAO, EC 1.4.3.4), such as iproniazid (Ipr), benzimidazole or tryptamine, led to a marked increase of the mutagenic effectiveness of cyclophosphamide especially in spermatocytes. This indicates the importance of metabolic de-activation processes for the limited mutagenicity of cyclophosphamide in Drosophila. The principal active metabolite of cyclophosphamide, phosphoramide mustard, is extensively de-activated by enzymes that can be inhibited by 1-phenylimidazole (PhI), presumably cytochrome P-450 (EC 1.14.14.1), but not by those blocked by MAO inhibitors. Inhibition of the FAD-containing dimethylaniline monooxygenase (FDMAM, EC 1.14.13.8) by N,N-dimethylbenzylamine (N,N-DMB) resulted in some increase in cyclophosphamide mutagenicity only in spermatids. The marginal mutagenicity of cyclophosphamide in Drosophila larvae could not be increased either by cytochrome P-450 induction with phenobarbital or by MAO inhibition with Ipr. In contrast to the failure of cyclophosphamide to induce rod-chromosome loss, a considerable activity was found when a ring-shaped chromosome was used. Similar to the sex-linked recessive lethal (SLRL) test, ring-X loss frequency could be enhanced by simultaneous treatment with MAO inhibitors. The observed ring-X loss frequency declined when males treated with cyclophosphamide were mated to DNA-repair deficient mei-9L1 females. Cyclophosphamide produces chromosome breaks, detected as 2-3 translocations, in Drosophila spermatocytes, the stage in spermatogenesis that is also the most sensitive to the induction of SLRL mutations.     

Comparison of sister-chromatid exchange frequencies in mouse T- and B-lymphocytes after exposure to 4-hydroxycyclophosphamide or phosphoramide mustard

The present study was designed to investigate the genotoxicity of 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), both reactive metabolites of cyclophosphamide (CP), for possible differences in SCE-inducing activity in mouse T- and B-lymphocytes. Mouse peripheral blood lymphocytes were isolated and stimulated to divide with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (a polyclonal B-cell activator). Significant concentration-dependent increases in SCE frequencies were observed for both 4-OHCP and PAM with both mitogens, with 4-OHCP being almost twice as potent as PAM. There was no difference in SCE response between T- and B-lymphocytes after exposure to either PAM or 4-OHCP. These data do not support the idea that the difference in SCE response in T- and B-lymphocytes by CP in vivo is due to differential responses to either of the proposed putative metabolites of CP.     

Inhibition of NADPH-cytochrome P450 reductase by cyclophosphamide and its metabolites

Cyclophosphamide (CP) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome-P450 reductase and microsomal mixed function oxidase (MFO) activities. $\text{In vitro}$ CP, its metabolites (acrolein, phosphoramide mustard, 4-keto CP and nor-nitrogen mustard) and Ifosfamide, which is an analog of CP, were tested for their effects on the reductase activity. Only acrolein produced a significant loss of the reductase (66%). This loss of activity could be prevented by the presence of cysteine in the incubation mixture. Acrolein also produced a dose dependent loss of the activity when incubated with the purified reductase. These data suggest that CP-induced loss of the reductase results from interaction between CP metabolite acrolein and critical sulfhydryl groups in the reductase.     

Comparison of the effects of 1,2-dimethylhydrazine and cyclophosphamide on micronucleus incidence in bone marrow and colon

An in vivo micronucleus assay has been developed that utilizes colonic epithelial cells. The genotoxic effects of 1,2-dimethylhydrazine (54-07-3), a colon carcinogen, and of the nitrogen mustard, cyclophosphamide (50-18-0), on the bone-marrow polychromatic erythrocytes and on colonic epithelium from mice were compared using micronucleus induction in each organ as the end point. In the bone marrow, cyclophosphamide was a potent inducer of micronuclei, while 1,2-dimethylhydrazine administration had little effect on the micronucleus incidence. In the colon, 1,2-diemthylhydrazine was an effective inducer of micronuclei. Thus, the colonic micronucleus assay appears to be a potentially useful test for the detection of colon carcinogens.     

Studies on the mechanism of action of diallyl sulfide, an inhibitor of the genotoxic effects of cyclophosphamide

Diallyl sulfide, a component of garlic oil, has been previously shown to inhibit induction of nuclear aberrations in the colons of mice treated with 1,2-dimethylhydrazine, a colon carcinogen. The ability of this agent to block cyclophosphamide-induced nuclear aberrations in urinary bladder and hair follicles was tested. [14C]Cyclophosphamide was injected and urine was collected to determine the disposition of metabolites of cyclophosphamide in mice treated with diallyl sulfide. This agent was capable of blocking nuclear aberration induction by cyclophosphamide in bladder and hair follicles. The effect was not mediated through inhibition of mitosis as determined by [3H]thymidine autoradiography in hair follicles. Diallyl sulfide pretreatment decreased the amount of radioactivity excreted in the urine in the first 24 h following cyclophosphamide treatment and blocked the appearance of acrolein, a cytotoxic metabolite of cyclophosphamide, in the urine over this time period. These results suggest that diallyl sulfide acts by conjugating the toxic metabolites of cyclophosphamide, thereby limiting their systemic circulation and diverting their route of excretion from the urine.     

The induction of sister chromatid exchanges by cyclophosphamide in the presence of differently induced microsomal fractions of rat liver.

The induction of sister chromatid exchanges can be monitored by a test that incorporates the factor of metabolic activation in a simple manner. The results with cyclophosphamide show that in this test the induction of the metabolizing enzymes of the rat liver homogenates used is very important. 3-Methylcholanthrene induces little if any extra conversion of cyclophosphamide to SCE-inducing metabolites, compared with no induction. Aroclor 1254 and phenobarbital however, were very good inducers. The difference found between the liver homogenates concerning SCE induction corresponded with the differences in cyclophosphamide metabolism, which was measured as the decrease in NADPH induced by cyclophosphamide.     

Comparison of the cytogenetic effects of cyclophosphamide on monkey lymphocytes in vivo and in vitro

The comparative in vivo and in vitro study of chromosomal aberrations and SCE induced by cyclophosphamide (CP) in macaca rhesus lymphocytes was performed. The dose of mutagenic exposure for quantitative estimation of effects was determined as a product of concentration of alkylating CP metabolites on the exposure time. The mutagenic effect caused by the same doses of CP (CP metabolites) appeared similar in vivo and in vitro. This suggests that the results obtained in adequate in vitro mutagen-testing experiments may be quantitatively extrapolated for the in vivo conditions.     

Chronobiologic fluctuation of cyclophosphamide induced urinary bladder damage in mice

Cyclophosphamide is the most widely used alkylating agent in clinical medicine. The usefulness of this drug is often limited by its propensity to produce hemorrhagic cystitis. To be active cyclophosphamide must be metabolized by the mixed function oxidase system. It has been previously demonstrated that the oncolytic activity and host lethality of cyclophosphamide are dependent upon circadian fluctuations. When cyclophosphamide is administered i.p. to male mice there is a dose dependent increase in urinary bladder weight. Histopathologic examination of these bladders revealed hemorrhage, edema, inflammation and stretching of the epithelial lining. When administered i.p. at 4-h intervals throughout a 24-h time period, cyclophosphamide produced maximum bladder damage when administered at 0500 and 1700 and little or no damage to the bladder when administered at 0100 or 1300. These studies suggest that cyclophosphamide induced cystitis, a toxicity resulting from the metabolic production of acrolein, may also be dependent upon chronobiologic fluctuations.