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PubMed1978

Cross-linking of DNA in L1210 cells and nuclei treated with cyclophosphamide and phosphoramide mustard.

Phosphoramide mustard, formed from cyclophosphamide in vivo and in vitro, may be the active metabolite of this drug. We have found phosphoramide mustard to be at least 100 times more potent than cyclophosphamide in inhibiting growth of two strains of the L1210 lymphoma in culture. Phosphoramide mustard also produced enlargement of cells, an effect not seen with cyclophosphamide. Phosphoramide mustard significantly increased the amount of cross-linked DNA after incubation with intact LM4 cells or nuclei isolated from these cells. Cyclophosphamide had a similar effect only in the isolated nuclei. These findings strengthen the proposed role of phosphoramide mustard as the active metabolite of cyclophosphamide. The effect of cyclophosphamide on nuclei is unexplained.

Alkylation↗

PubMed1980

Alkylation of guanosine and deoxyguanosine by phosphoramide mustard.

Phosphoramide mustard, an active metabolite of cyclophosphamide, has been reacted separately with guanosine and deoxyguanosine in aqueous solution at pH 7.4. The major adduct which was formed in each case has been isolated by reverse-phase high-pressure liquid chromatography. The structure of the major adduct, as determined by a combination of ultraviolet and field desorption mass spectrometry, is that of phosphoramide mustard, one arm of which has reacted with guanosine or deoxyguanosine in position 7. These adducts are much less stable than was 7-methylguanosine, and they decompose with a half-life of 1.3 hr at 37 degrees and pH 7.4. This instability may contribute to the action of phosphoramide mustard at a molecular level.

Alkylation↗

PubMed1995

Comparison of the protonation of isophosphoramide mustard and phosphoramide mustard.

The alkylating agent isophosphoramide mustard (IPM) spontaneously forms a relatively stable aziridine derivative which can be directly observed using NMR spectroscopy. The protonations of IMP and its aziridine were probed using 1H, 31P, 15N, and 17O NMR spectroscopy. The positions of the 31P, 15N, and 17O resonances of IPM between pH 2 and 10 each exhibit a single monobasic titration curve with the same pKa of 4.31 +/- 0.02. On the basis of a comparison with other compounds and our earlier work with phosphoramide mustard, the NMR results for IPM indicate that protonation occurs at nitrogen and not oxygen. Over this same pH range, each of the 1H, 31P, and 15N resonances of IPM-aziridine also show a single monobasic titration with a pKa of 5.30 +/- 0.09. The magnitude of the change in chemical shifts suggests that the protonation of the IPM-aziridine occurs at the ring nitrogen. Theoretical gas-phase calculations of PM, IPM, and IPM-aziridine suggest O-protonation to be more likely; however, aqueous phase calculations predict the N-protonated forms to be most stable. Furthermore, for PM and IPM-aziridine, which contain nonequivalent nitrogens, the theoretical calculations and experimental data both agree as to which nitrogen undergoes protonation. These results suggest that the IMP-aziridine remains unprotonated under physiological conditions and may, in part, explain the lower alkylating activity of IPM as compared to PM.

Antineoplastic Agents↗

PubMed1993

Protonation of phosphoramide mustard and other phosphoramides.

The chemistry of the bifunctional alkylating agent phosphoramide mustard and model phosphoramides was probed by multinuclear NMR spectroscopy as a function of pH. Between pH 1 and 11, both the 31P and 15N resonances for phosphoramide mustard displayed a single monobasic titration curve with a pKa of 4.9. The protonation below pH 4.9 correlates with the loss in reactivity of the mustard. The 17O NMR spectrum of 17O-enriched phosphoramide mustard shows little change with pH. The data on the mustard was compared to 15N and 31P NMR data on 15N-enriched phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide. Contrary to the conclusions of previous studies, our combined 31P, 15N, and 17O NMR results are more consistent with N-protonation of phosphoramide mustard rather than an O-protonation. Theoretical calculations on the phosphoramidic acid, phosphorodiamidic acid, and phosphoric triamide show O-protonation to be more stable in the gas phase. For the latter two compounds, the calculations suggest that N-protonation may be the most stable protonated form in the aqueous phase. These findings influence our understanding of the structure-activity relationships of phosphoramide mustards.

Amides↗

PubMed1989

31P NMR studies of the kinetics of bisalkylation by isophosphoramide mustard: comparisons with phosphoramide mustard.

31P nuclear magnetic resonance spectroscopy was used to measure the pKa (4.28 +/- 0.2) of isophosphoramide mustard (IPM) at 20 degrees C and to study the kinetics and products of the decomposition of IPM at a solution pH value of ca. 7.4 and at temperatures between 20 and 47 degrees C in the presence of nucleophilic trapping agents. At 37 degrees C, the half-life for the first alkylation was ca. 77 min and ca. 171 min for the second alkylation; these data may be compared with those for phosphoramide mustard (Engle, T.W.; Zon, G.; Egan, W.J. Med. Chem. 1982, 25, 1347), wherein the half-lives for the first and second alkylations are approximately the same (18 min). The rate of fragmentation of aldoifosfamide to IPM and acrolein was also studied by NMR spectroscopy (pH 7.0; 37 degrees C; 0.07 M phosphate); under the noted conditions, the half-life of aldoifosfamide was found to be ca. 60 min.

Alkylating Agents↗

PubMed1987

Kinetics of hydrolysis in vitro of nornitrogen mustard, a metabolite of phosphoramide mustard and cyclophosphamide.

A gas chromatographic (GC-ECD) method was developed for the determination of nornitrogen mustard (NOR) and its hydrolysis products. The method was based on derivatization by heptafluorobutyric anhydride. The structures of the derivatives of NOR were established by GC-MS. The method was used to characterize the rate of transformation of NOR and phosphoramide mustard (PAM), important metabolites of cyclophosphamide, into secondary products in vitro at 37 degrees C and pH 7.4. The rate of disappearance of NOR had a half-life of 20 min. The half-life of appearance of N-(2-chloroethyl)-N-(2-hydroxyethyl)amine (NOR-OH) was 19 min. While most NOR appeared to be converted to NOR-OH, the yield of N,N-bis(2-hydroxyethyl)amine (NOR-OH-OH) was a small fraction of the starting material. The disappearance of NOR, when PAM was used as a starting material, had a half-life of 19 min; in these experiments NOR-OH and NOR-OH-OH were relatively much more abundant compared to when NOR was used as the starting material.

Amides↗

PubMed1982

Vomiting induced by cyclophosphamide and phosphoramide mustard in cats.

Cyclophosphamide and phosphoramide mustard produce significant vomiting. Cyclophosphamide is metabolized to phosphoramide mustard, which may ultimately contribute to vomiting after cyclophosphamide administration. The role of the chemoreceptor trigger zone (CTZ) in vomiting caused by these agents is unknown. We studied the emetic syndromes produced by iv and intracerebroventricular cyclophosphamide and phosphoramide mustard in unanesthetized normal and CTZ-ablated cats. Iv cyclophosphamide produced vomiting unpredictably, with a mean latency of 54 +/- 9 mins (mean +/- SE) in cats that vomited. A dose-response relationship was found for phosphoramide mustard-induced emesis. A dose of 200 mg/kg was consistently effective, with a mean latency of 127 +/- 6 mins. Neither agent produced predictable emesis by the intracerebroventricular route of administration. One of four CTZ-ablated cats vomited after 300 mg/kg of cyclophosphamide. Since cyclophosphamide was an unpredictable emetic stimulus, it was not possible to further evaluate the effect of CTZ ablation on cyclophosphamide-induced vomiting. However, CTZ-ablated cats given 200 mg/kg of phosphoramide mustard vomited significantly less frequently (P = 0.05 by chi-square test) and with a longer latency than nonablated animals. A temporary, severe neurotoxic reaction was observed in cats receiving greater than or equal to 3400 mg/kg of cyclophosphamide, which may have had an inhibitory effect on emesis. Phosphoramide mustard was found to be a potent emetic stimulus in cats and may contribute to the emetic response following cyclophosphamide administration. Analysis of latency data suggests that in the cat other cyclophosphamide metabolites may also contribute to the emetic syndrome.

Animals↗

PubMed1991

Chemically stable, lipophilic prodrugs of phosphoramide mustard as potential anticancer agents.

Benzyl phosphoramide mustard (3), 2,4-difluorobenzyl phosphoramide mustard (4), and methyl phosphoramide mustard (5) were examined as lipophilic, chemically stable prodrugs of phosphoramide mustard (2). These phosphorodiamidic esters are designed to undergo biotransformation by hepatic microsomal enzymes to produce 2. The rate of formation of alkylating species, viz., 2, from these prodrugs and their in vitro cytotoxicity toward mouse embryo Balb/c 3T3 cells were comparable to or better than that of cyclophosphamide (1). Preliminary antitumor screening against L1210 leukemia in mice, however, suggests that these prodrugs are devoid of any significant antitumor activity in vivo.

Animals↗

PubMed1985

Kinetics of phosphoramide mustard hydrolysis in aqueous solution.

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.

Bromides↗

PubMed1989

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).

Acrolein↗

PubMed2004

1,3- vs 1,5-intramolecular alkylation reactions in isophosphoramide and phosphoramide mustards.

It is well-established that at pH 7.4, intramolecular 1,3-N-alkylation reactions in isophosphoramide mustard (IPM) and phosphoramide mustard (PM) produce electrophilic alkylating agents with aziridinyl moieties. To investigate the role of 1,5-intramolecular cyclizations in the chemistry of IPM and PM, the five-membered ring phospholidine products of these reactions were independently synthesized and characterized by (31)P NMR. In 0.33 M BisTris, pH 7.4, 37 degrees C, the intramolecular O-alkylation product of IPM [2-(2-chloroethylamino)-2-tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (11)] had a chemical shift of delta 33.0 and a half-life of 3.3 h. The O-alkylation product of PM [2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphospholidine-2-oxide (12)] displayed a chemical shift of delta 30.6 and a half-life of 26.9 h. For both IPM and PM, 1,5-N-alkylation provides the same product [1-(2-chloroethyl)-2-hydroxy-tetrahydro-2H-1,3,2-diazaphospholidine-2-oxide (13)]. Because of its instability, 13 was generated in situ and was not isolated; however, the chemical shift (delta 33.0) and reactivity (half-life 0.3 h at 25 degrees C) of the species attributed to 13 were consistent with the assigned structure. Resonances with (31)P NMR chemical shifts indicative of 11 or 12 did not appear in reaction solutions of IPM or PM. The compound assigned as 13 gave hydrolysis products that were not found in reaction solutions of IPM or PM. The collective data supported the conclusion that intramolecular 1,5-alkylations do not contribute to the chemistry of IPM or PM in aqueous solutions at pH 7.4, 37 degrees C. Conversely, 11 and 12 were found to be the major if not exclusive products formed in DMSO solutions of the respective cyclohexylammonium salts of IPM and PM. Both 11 and 12 were relatively noncytotoxic against a series of cell lines, but there were differences in mutagenicities. Chinese hamster ovary cells were exposed to 11 or 12 for one half-life of each compound; 11 was nonmutagenic up to 500 microM, while 12 (500 microM) was mutagenic with 246 mutant colonies/10(6) surviving cells.

Alkylation↗

PubMed1982

Conversion of 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein mediated by bifunctional catalysis.

The rates at which 4-hydroperoxycyclophosphamide and 4-hydroxycyclophosphamide are converted to phosphoramide mustard and acrolein were determined as a function of buffer composition, buffer concentration, and pH. Conversion of 4-hydroperoxycyclophosphamide to 4-hydroxycyclophosphamide in 0.5 M Tris buffer, pH 7.4, 37 degrees, was first-order (k = 0.016 min-1), but subsequent conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein under these conditions was negligible. Phosphoramide mustard and acrolein were readily generated from 4-hydroperoxycyclophosphamide or 4-hydroxycyclophosphamide when either of these agents was placed in phosphate buffer. Conversion of 4-hydroxycyclophosphamide to phosphoramide mustard and acrolein was first-order with respect to 4-hydroxycyclophosphamide (k = 0.126 min-1 in 0.5 M phosphate buffer, pH 8, 37 degrees) as well as first-order with respect to phosphate serving as a catalyst. The rate-determining step in the reaction was pH dependent only insofar as the hydrogen ion concentration governed the relative amounts of monobasic and dibasic phosphate present. Pseudo-first-order rate constants were 0.045 M-1 min-1 for monobasic phosphate and 0.256 M-1 min-1 for dibasic phosphate. The role of phosphate in this reaction was as that of a bifunctional catalyst. The reaction was not subject to specific or general, acid or base, catalysis. Other bifunctional catalysts such as glucose-6-phosphate and bicarbonate also catalyzed the reaction, albeit less efficiently. Aldophosphamide apparently exists only transiently; its presence could not be established by 31P nuclear magnetic resonance spectroscopy. We conclude that, in the reaction sequence 4-hydroxycyclophosphamide leads to aldophosphamide leads to phosphoramide mustard + acrolein, the conversion of 4-hydroxycyclophosphamide to aldophosphamide is rate limiting and is subject to bifunctional catalysis; this reaction can proceed efficiently only in the presence of a bifunctional catalyst. Assuming that the oncotoxic specificity of cyclophosphamide resides with 4-hydroxycyclophosphamide and that its cytotoxic effect at therapeutic doses is largely mediated by phosphoramide mustard released within cells, these observations offer the possibility that the intracellular concentration of bifunctional catalysts, whether in the form of inorganic phosphates, organic phosphates, enzymes, or other species, serve as important determinants with regard to the oncotoxic potential and specificity of cyclophosphamide. Similarly, the concentration of bifunctional catalysis in the urine as well as the pH of the urine may be important with regard to the potential of cyclophosphamide to induce, via acrolein, hemorrhagic cystitis.

Acrolein↗

PubMed1984

Alkylation of guanosine by phosphoramide mustard, chloromethine hydrochloride and chlorambucil.

Guanosine was reacted in vitro with phosphoramide mustard, chloromethine hydrochloride, and chlorambucil. The products were isolated by HPLC and characterized by UV and fluorescence spectroscopy, and C-8 tritium exchange. The primary products were 7-alkylguanosines according to such evidence. Phosphoramide mustard had 1/10 of the apparent alkylation activity of two other mustards. The primary 7-alkylguanosines were unstable at pH 7.4 and 37 degrees; t1/2 were 3 min. for chloromethine hydrochloride, 2.7 hrs for chlorambucil and 3.0 hrs for phosphoramide mustard. Both dechlorination at the unbound arm of the mustard and imidazole ring opening og guanosine appeared to account for such instability.

Alkylation↗

PubMed1991

Phosphoramide mustard is responsible for the ovarian toxicity of cyclophosphamide.

Although cyclophosphamide (CPA) is an ovarian toxicant, the responsible metabolite(s) have not been identified. The purpose of these experiments was to determine if phosphoramide mustard or acrolein were the proximate toxicants produced by metabolic activation of CPA. To do this analogs of CPA known to generate either phosphoramide mustard or acrolein in vivo were assessed for their ability to produce ovarian toxicity as measured by differential follicle destruction, ovarian volume loss, and uterine weight loss and compared to the effects produced by CPA. Phosphoramide mustard cyclohexylamine salt (PMC) and trans-4-phenylcyclophosphamide (T4P), both of which generate phosphoramide mustard, and didechlorocyclophosphamide (DCPA) and allyl alcohol (AA) which generate acrolein were administered ip to female C57BL/6N mice, 10-12 weeks old, at doses equimolar to 0, 25, 75, 200, or 500 mg/kg of CPA. Three days later the animals were killed, their uterine weights measured and their ovaries removed, fixed, and serially sectioned. Only PMC and T4P produced ovarian toxicity. On an equimolar basis these compounds were over twice as potent as CPA. Both caused a significant reduction in uterine weight (to 50% of controls) at doses of 200 (PMC) and 150 mg/kg (T4P). PMC and T4P also caused a 50% reduction in ovarian volume at doses above 75 mg/kg. Primordial follicles were most sensitive; ED50s were 76.9, 25.3, and 19.3 mg/kg (0.276, 0.091, and 0.069 mmol/kg) for CPA, PMC, and T4P, respectively. Growing follicle numbers were also reduced by T4P and PMC, an effect not seen with CPA treatment. Finally, antral follicles were significantly reduced by all doses of PMC, and with T4P at doses greater than 75 mg/kg. The highest doses of PMC, T4P, and CPA all caused a reduction in antral follicle numbers to less than one percent of controls. Didechlorocyclophosphamide (DCPA) and allyl alcohol (AA), compounds that generate acrolein but not phosphoramide mustard in vivo, had no effect on any of the parameters measured even when injected directly into the ovary. This suggests that phosphoramide mustard is responsible for CPA ovarian toxicity. The greater potency of PMC and T4P compared to CPA is likely the result of these compounds bypassing important detoxification steps, therefore, more of the parent compound reaches the ovary as the toxic metabolite.

1-Propanol↗

PubMed1993

Cyclophosphamide modulates rat hepatic cytochrome P450 2C11 and steroid 5 alpha-reductase activity and messenger RNA levels through the combined action of acrolein and phosphoramide mustard.

Cyclophosphamide treatment of adult male rats leads to sustained decreases in several liver microsomal cytochrome P450 (CYP) activities, including CYP 2C11-catalyzed cyclophosphamide activation, via a process that is associated with a feminization of the overall pattern of liver enzyme expression (G. A. LeBlanc and D. J. Waxman, Cancer Res., 50:5720-5726, 1990). The present study compares the effects of cyclophosphamide and its isomeric analogue ifosphamide on the gender-dependent expression of hepatic CYP 2C11 and steroid 5 alpha-reductase in adult male rats and also examines the role of the cyclophosphamide metabolites acrolein and phosphoramide mustard in feminizing the expression of these liver enzymes. Ifosphamide (a) suppressed the male-specific CYP 2C11 mRNA and CYP 2C11-catalyzed liver microsomal testosterone 2 alpha-hydroxylation and cyclophosphamide and ifosphamide 4-hydroxylation and (b) elevated the female-dominant liver enzyme steroid 5 alpha-reductase and its mRNA 7-9 days after drug treatment, both occurring in a manner similar to that of cyclophosphamide, but requiring a 50% higher dose (180 mg/kg, single i.p. injection) to achieve these effects. This pattern of response could not be achieved by treatment of rats with acrolein or with cyclophosphamide analogues that decompose to acrolein without formation of phosphoramide mustard. In contrast, phosphoramide mustard treatment (100 mg/kg) did modulate microsomal CYP 2C11 and steroid 5 alpha-reductase activities. Treatment with a lower dose (50 mg/kg) of phosphoramide mustard or with the acrolein precursor 4-hydroperoxydechlorocyclophosphamide (200 mg/kg) alone did not affect liver enzyme expression, whereas the combination of these agents produced an overall pattern of response that was similar to that conferred by cyclophosphamide. These studies establish that ifosphamide is less potent than cyclophosphamide in modulating the pattern of cytochrome P450 and steroid 5 alpha-reductase expression and that phosphoramide mustard is responsible for the modulation of liver enzyme expression by cyclophosphamide, with acrolein potentiating the modulating activity of the mustard.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1987

Analysis of phosphoramide mustard by reversed-phase ion pair high pressure liquid chromatography.

The analysis of phosphoramide mustard by reversed-phase ion pair high pressure liquid chromatography using ultraviolet detection is described. The method will enable measurement of serum concentrations of phosphoramide mustard over a period of approximately 24 h after an i.v. dose of 60 mg/kg of cyclophosphamide and requires 0.5 ml of serum or plasma. The between-day precision of the assay at concentrations of 0.34, 2.76, and 6.90 mg/L gave coefficients of variation of 16, 10, and 9%, respectively. The percentage analytical recovery of phosphoramide mustard is 35%. Preliminary results providing pharmacokinetic data on two adults undergoing high-dose cyclophosphamide treatment prior to bone marrow transplant are presented.

Chromatography, High Pressure Liquid↗

PubMed1984

Plasma concentrations of 4-hydroxycyclophosphamide and phosphoramide mustard in patients repeatedly given high doses of cyclophosphamide in preparation for bone marrow transplantation.

Plasma half-life and area under the curve (AUC) values for cyclophosphamide were determined in patients given this agent iv at doses of 50-60 mg/kg/infusion. Apparent plasma half-life and AUC values for the metabolites 4-hydroxycyclophosphamide and phosphoramide mustard were also determined in some of these patients. Disappearance from the plasma of the parent compound as well as that of the metabolites was approximately first-order. Plasma half-life values for cyclophosphamide ranged from 45 to 480 mins; AUC values ranged from 10 to 188 mM X min. As expected, AUC values for cyclophosphamide increased approximately linearly with an increase in its plasma half-life. Apparent plasma half-life values for 4-hydroxycyclophosphamide and phosphoramide mustard increased approximately linearly with an increase in plasma half-life values for cyclophosphamide; the slopes of these relationships were 1.35 and 1.97, respectively, but did not quite extrapolate to zero. AUC values for 4-hydroxycyclophosphamide and phosphoramide mustard remained approximately constant at about 5 and 15 mM X min, respectively, over the relatively wide range of plasma half-life and AUC values obtained for cyclophosphamide. On the basis of these observations we suggest that (a) changes in the rate of cyclophosphamide hydroxylation, effected by whatever means, will not alter the systemic therapeutic and toxic responses to a given dose of cyclophosphamide, given that the cytotoxic effects of this agent are directly proportional to AUC values of 4-hydroxycyclophosphamide and/or phosphoramide mustard, and (b) in most cases, 4-hydroxycyclophosphamide, and not phosphoramide mustard, is likely to be the circulating metabolite of therapeutic importance in humans since the AUC values for phosphoramide mustard exceeded those for 4-hydroxycyclophosphamide by only a factor of 3 and tumor and bone marrow cells proliferating in culture are generally substantially (8-25-fold) more sensitive to 4-hydroxycyclophosphamide.

Adolescent↗

PubMed1989

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.

Acetylcysteine↗