Phosphoramide Mustard Research Articles

High-dose alkylating agent therapy: a review of clinical experiences.

Cancer Drug DeliveryVol. 1, No. 3 High-Dose Alkylating Agent Therapy: A Review of Clinical ExperiencesM.A. CORNBLEET, R.C.F. LEONARD, and J.F. SMYTHM.A. CORNBLEETSearch for more papers by this author, R.C.F. LEONARDSearch for more papers by this author, and J.F. SMYTHSearch for more papers by this authorPublished Online:2 Apr 2009https://doi.org/10.1089/cdd.1984.1.227AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byAlkylating Agents3 November 2010Application of hematopoietic progenitor assays for the estimation of hematotoxicity in ratsJournal of Pharmacological and Toxicological Methods, Vol. 31, No. 2A phase I trial of continuous-infusion cyclophosphamide in refractory cancer patientsCancer Chemotherapy and Pharmacology, Vol. 29, No. 1Delivery of vital drugs to the brain for the treatment of brain tumorsJournal of Controlled Release, Vol. 11, No. 1-3Physicochemical and pharmacokinetic parameters of seven lipophilic chlorambucil esters designed for brain penetrationCancer Chemotherapy and Pharmacology, Vol. 25, No. 5Brain and plasma pharmacokinetics and anticancer activities of cyclophosphamide and phosphoramide mustard in the ratCancer Chemotherapy and Pharmacology, Vol. 27, No. 1Drug Delivery to the Brain by Blood-Brain Barrier Circumvention and Drug ModificationOptimizing drug delivery to brain tumorsCancer Treatment Reviews, Vol. 14, No. 1Melphalan concentration dependent plasma protein binding in healthy humans and ratsEuropean Journal of Clinical Pharmacology, Vol. 32, No. 2 Volume 1Issue 3Jan 1984 To cite this article:M.A. CORNBLEET, R.C.F. LEONARD, and J.F. SMYTH.High-Dose Alkylating Agent Therapy: A Review of Clinical Experiences.Cancer Drug Delivery.Jan 1984.227-238.http://doi.org/10.1089/cdd.1984.1.227Published in Volume: 1 Issue 3: April 2, 2009PDF download

Relative mutagenicity and teratogenicity of cyclophosphamide and two of its structural analogs

In this report, cyclophosphamide was compared to two of its structural analogs, 5,5-dimethylcyclophosphamide and diethylcyclophosphamide, with respect to mutagenic and teratogenic activities. Mutagenicity was assessed using Salmonella typhimurium TA 1535; teratogenicity was assessed in Sprague-Dawley rats on day 20 of gestation after intra-amniotic drug administration on day 13. After metabolic activation, cyclophosphamide caused base substitution mutations in S. typhimurium TA 1535 and major structural defects in both intra-amniotically injected and contralateral uninjected fetuses. 5,5-Dimethylcyclophosphamide was neither mutagenic nor teratogenic. Diethylcyclophosphamide was not mutagenic but was teratogenic. However, diethylcyclophosphamide was less potent as a teratogen than cyclophosphamide and, unlike cyclophosphamide, caused malformations only in the intra-amniotically injected fetuses. Diethylcyclophosphamide does liberate acrolein after metabolic activation. If acrolein is responsible for the teratogenic effects of diethylcyclophosphamide, the other major cytotoxic metabolite of cyclophosphamide, phosphoramide mustard, may account for the difference in teratogenic potency between cyclophosphamide and diethylcyclophosphamide. These results would suggest that acrolein, although apparently not mutagenic, mediates the teratogenicity of diethylcyclophosphamide and a significant proportion of the teratogenicity of cyclophosphamide.

O-imino esters of N,N-bis(2-chloroethyl)phosphorodiamidic acid. Synthesis, X-ray structure determination, and anticancer evaluation.

Nine representatives of the title series of compounds [(ClCH2CH2)2NP(O)(NH2)ON = CRR'] were synthesized as potential anticancer prodrugs, based on the possibility of enzymatic reduction of the N-O bond to release the known cytotoxic agent phosphoramide mustard [1, (ClCH2CH2)2NP(O)(NH2)OH]. The dimethyl derivative (2, R = R' = CH3) exhibited a statistically significant, albeit low, level of anti-L1210 activity in mice. Derivative 2, which was shown by 31P NMR measurements to be very stable toward hydrolysis at 37 degrees C over a pH range of 5.7-7.4 (T1/2 congruent to 7-8 weeks), gave colorimetrically detectable amounts of alkylating material upon incubation with mouse liver slices: approximately 3-5% conversion after 20 min at 37 degrees C. A single-crystal X-ray study of 2 revealed an unusual hydrogen-bonded "ladder" and a very similar steric relationship for the NCH2CH2Cl and ON = CCH3 moieties.

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs.

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.

ChemInform Abstract: SYNTHESIS AND STUDY OF A SPIN‐LABELED CYCLOPHOSPHAMIDE ANALOG 3‐(1‐OXY‐2,2,6,6‐TETRAMETHYL‐4‐PIPERIDINYL)CYCLOPHOSPHAMIDE

AbstractDurch Addition des Aminopiperidins (I) an Acrylsäureethylester (II) erhält man den Aminocarbonsäureester (III), der über (IV) das im Titel genannte Cyclophosphamid (V) ergibt.

31P NMR kinetic studies of the intra- and intermolecular alkylation chemistry of phosphoramide mustard and cognate N-phosphorylated derivatives of N,N-bis(2-chloroethyl)amine.

31P NMR kinetic studies of the intra- and intermolecular alkylation chemistry of phosphoramide mustard and cognate N-phosphorylated derivatives of N,N-bis(2-chloroethyl)amine.

Synthesis and study of a spin-labeled cyclophosphamide analogue, 3-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide.

3-(1-Oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (7) was isolated in 36% yield following H2O2-Na2WO4 oxidation of 3-(2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (6), which was synthesized in three steps (25% yield) starting with 4-amino-2,2,6,6-tetramethylpiperidine. Binding of 7 to mouse liver microsomes was investigated by optical and electron spin resonance spectroscopy. Compared with the mouse liver microsomal metabolism of 1, separate incubations of 6 and an ca. 1:1 mixture of 1 and 6 gave approximately 90 and 60% less acrolein, respectively. A spin-labeled metabolite of 7, viz., N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)phosphoramide mustard (9), was synthesized and its intramolecular O-alkylation at pH 7.4, 37 degrees C, was studied by 31P NMR spectroscopy. Compounds 7 and 9 were inactive in screening tests against L1210 lymphoid leukemia in mice.

Mutagenic, teratogenic and pharmacokinetic properties of cyclophosphamide and some of its deuterated derivatives

To elucidate which metabolic pathway leads to the ultimate mutagenic and teratogenic metabolite of cyclophosphamide (CPA), the mutagenicity in vitro as well as the teratogenicity in vivo and the pharmacokinetics of CPA and several deuterated analogs (5,5-d 2-CPA, 4,4-d 2-CPA and 4,4–6,6-d 4-CPA) were compared. 5,5-d 2-CPA was less mutagenic (isotope effects between 1.7 and 12.3 were found for sister-chromatid exchanges and structural chromosomal aberrations in CHO cells and in the Ames test) and less teratogenic (deuterium isotope effect between and 3) than CPA and the other deuterated analogs. Because the concentrations of 5,5-d 2-CPA in pregnant mice and their embryos were equal to or even exceeded those of CPA and the other deuterated analogs, a particular metabolic pathway involving a fission of the C-D or C-H bond on the C-5 rather than the parent drug must be responsible for the isotope effect observed. It had previously been shown (Cox et al., 1976) that the 5,5-d 2-CPA has a decreased anti-tumor activity in vivo (isotope effect 7–13) which was accompanied by a decreased formation of phosphoramide mustard and acrolein in vitro (isotope effect 5.3). Our results suggest that phosphoramide mustard is an important proximate metabolite of CPA with regard to the mutagenicity and teratogenicity of the drug. These results show for the first time that labeling of drugs with stable isotopes offers a promising approach to the study of the mechanism of the mutagenic and teratogenic actions of drugs.

O-Methylhydroxylamine as a New Trapping Reagent for Quantitative Studies of 4-Hydroxycyclophosphamide and Aldophosphamide

31P- and 1H-NMR spectroscopy were used to demonstrate that the primary metabolites of the anticancer drug cyclophosphamide (4-hydroxycyclophosphamide and its acyclic tautomer, aldophosphamide) are quantitatively converted by O-methylhydroxylamine, at pH 7.4 and 37o, into the E and Z isomers of aldophosphamide O-methyl oxime. These trapping products are readily extracted from aqueous media with either chloroform or ethyl acetate, are stable at pH 6-8 toward oxime hydrolysis and elimination of phosphoramide mustard (a secondary metabolite of cyclophosphamide), and showed no evidence for transox-imination with either ketone or aldehyde acceptors. All of these features support the use of aldophosphamide O-methyl oxime in quantitative studies related to cyclophosphamide metabolism.

Toxicity, interstrand cross-links and DNA fragmentation induced by ‘activated’ cyclophosphamide in yeast: Comparative studies on 4-hydroperoxy-cyclophosphamide, its monofunctional analogon, acrolein, phosphoramide mustard, and nor-nitrogen mustard

Activated cyclophosphamide (CP) is known to achieve its cytotoxic and alkylating capacity upon spontaneous hydrolytic breakdown of the oxazaphosphorine ring structure. Treatment of yeast cells with the chemically activated form of CP (4-hydroperoxy-CP, 4-OOH-CP) and with several potentially toxic cleavage products revealed that cytotoxicity is closely linked to the formation of DNA interstrand cross-links and to DNA fragmentation. While this holds true for 4-OOH-CP and its bifunctional alkylating breakdown products, phosphoramide mustard (PM) and nor-nitrogen mustard (NNM), equimolar concentrations of acrolein and the monofunctional analogon of activated CP were inactive. NNM, the ultimate cleavage product within the successive degradation of the oxazaphosphorine structure was five times more toxic than 4-OOH-CP, whereas the cytotoxic action of PM was only slightly enhanced. The high cytotoxicity of NNM was matched by its ability to induce DNA interstrand cross-links: at concentrations and treatment times producing equal cell killing, 4-OOH-CP and NNM produced the same extent of cross-linking and DNA fragmentation. Biochemical potency of NNM is in contrast to data found with the NBP colorimetric assay which suggest that NNM loses its alkylating activity at neutral pH. 4-OOH-CP and PM are much more stable than predicted from half-life measurements performed via the NBP colorimetric assay: they retain a considerable fraction of their cytotoxic and cross-linking activity in spite of a 12-h preincubation at pH 7 and 36°C.

Metabolic activation of chemical carcinogens.

Ideally the end result may differ, but there are similarities between the metabolic pathways of chemical carcinogens and drugs, for both are xenobiotics or foreign substances. Furthermore, certain drugs may increase the risk of a possible carcinogenic effect after long-term administration. In these cases judicious consideration of the risk versus the benefit to the patient is necessary. Drugs more likely to bear this hazard are those used to treat neoplastic or autoimmune diseases, situations where the benefit outweighs future risks. An early example of this type drag was chlornaphazine which eventually led to bladder tumors in the patients, understandable because of its 2-naphthylamine skeleton [1]. Chlornaphazine and other alkylating compounds represent primary or reactive carcinogens which can interact with cellular constituents as such (fig. 1) [2]. Despite the presence of alkylating groups in the widely used cyclophosphamide, this drug apparently does not react as such. Metabolism through 4-hydroxycyclophosphamide and aldophosphamide affords phosphoramide mustard [3, 4] which can form adducts with nucleosides [5]. Thus the cyclophosphamide metabolites act as transport forms of the ultimate cytotoxic metabolite [4].KeywordsPolycyclic Aromatic HydrocarbonAromatic AmineCarcinogenic EffectChemical CarcinogenCellular MacromoleculeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

5-Fluoro- and 5-chlorocyclophosphamide: synthesis, metabolism, and antitumor activity of the cis and trans isomers.

In seeking analogues of cyclophosphamide (1) having improved antitumor activity by virtue of accelerated formation of the cytotoxic metabolite phosphoramide mustard, cis and trans isomers of 5-fluoro- and 5-chlorocyclophosphamide (9, 10, 11 and 12, respectively) were synthesized by condensation of the appropriate 3-amino-2-halopropan-1-ol (13 or 26) with N,N-bis(2-chloroethyl)phosphoramidic dichloride (14). The metabolism of the halocyclophosphamides by rat liver microsomes was stereoselective; the cis isomers (9 and 11) were poorly metabolized, whereas the trans isomers (10 and 12) were metabolized with efficiency comparable to that of cyclophosphamide. However, there was no evidence that the yield of phosphoramide mustard produced by the trans analogues were significantly greater than that from cyclophosphamide following microsomal 4-hydroxylation. Hence, the halogen substituents did not accelerate beta-elimination of acrolein from the acyclic aldehydo tautomers. As expected, the poorly metabolized cis-5-fluoride (9) had little activity against the ADJ/PC6 tumor in mice. However, the cis-5-chloride (11) was as active as the trans isomer (12) and each had approximately half the therapeutic index of 1. The trans-5-fluoride (10) was much less active, having an ED90 value some 16-fold that of 1.

Identification of three alkylated nucleotide adducts from the reaction of guanosine 5'-monophosphate with phosphoramide mustard

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTIdentification of three alkylated nucleotide adducts from the reaction of guanosine 5'-monophosphate with phosphoramide mustardVanessa T. Vu, Catherine C. Fenselau, and O. Michael ColvinCite this: J. Am. Chem. Soc. 1981, 103, 24, 7362–7364Publication Date (Print):December 1, 1981Publication History Published online1 May 2002Published inissue 1 December 1981https://doi.org/10.1021/ja00414a067RIGHTS & PERMISSIONSArticle Views156Altmetric-Citations57LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (414 KB) Get e-Alerts Get e-Alerts

Irreparable DNA cross-links and mammalian cell lethality with bifunctional alkylating agents

The appearance and removal of DNA cross-links in C3H10T1/2 cells has been investigated with closely related bifunctional alkylating agents having widely different kinetics of alkylating activity. Concentrations of nitrogen mustard, phosphoramide mustard and melphalan demonstrating equivalent levels of cell lethality indicate a correlation between the appearance of residual DNA cross-links, transformation and the loss of colony formation ability. Residual DNA cross-links appear to remain after repair is nearly complete by 24 h and consist of both the interstrand DNA and DNA-protein varieties. These adducts occur in the general population of cells despite the fact that these cells retain their DNA cross-link repair capabilities. Less than 100 interstrand DNA cross-links and a 10-fold greater number of DNA-protein cross-links are estimated to remain in the DNA after a 66% lethal dose of these compounds. Due to the obvious complications involving DNA replication in the presence of such lesions, residual DNA cross-links may possibly be associated with lethality in these cells. Current evidence demonstrating the formation of additional sites of alkylation such as irreparable phosphotriester adducts by several alkylating agents may suggest a role for a specific type of DNA damage in this process.

Chemosensitivity of human neoplasms with in vitro clone formation. Experience at the University of Southern California - Los Angeles County Medical Center.

We analyze experience with 600 specimens for in vitro chemosensitivity assessment of human neoplasms utilizing a soft agar colony-forming technique. Good test reproducibility is demonstrated. Disaggregation with collagenase enhances yield and does not alter chemosensitivity profiles. Therapeutic exposure to chemotherapy prior to biopsy reduces in vitro sensitivity to the specific agents used in vitro. The cyclophosphamide derivatives 4-hydroperoxycyclo phosphamide (4-HC) and phosphoramide mustard are active in vitro, and produce comparable rank order sensitivities among tested tumors. There is marked reduction of in vitro 4-HC sensitivity in patients with prior therapeutic cyclophosphamide exposure, supporting the use of this derivative in test systems. Rank order of test results among specimens is compared at 0.1 microgram and 10 microgram drug/ml. Substantial differences in rank order at these two dose levels are demonstrated, indicating that the in vitro test dose selected is an important variable.

Teratogenicity of cyclophosphamide metabolites: Phosphoramide mustard, acrolein, and 4-ketocyclophosphamide in rat embryos cultured in vitro

Phosphoramide mustard, acrolein, and 4-ketocyclosphosphamide, known stable metabolic products of bioactivated cyclophosphamide, were tested for their teratogenicity against rat embryos grown in vitro from Day 10 to Day 11 of gestation. Results indicate that phosphoramide mustard is the teratogenic metabolite of cyclophosphamide, since the effects of phosphoramide mustard exactly parallel those of bioactivated cyclophosphamide. These effects are reductions in total embryo protein content, crown-rump length and number of somites, characteristic malformations, and pattern of cell necrosis. Acrolein at a dose equimolar to a dose of bioactivated cyclophosphamide which produced malformations in 100% of treated embryos had no effect on any of the parameters measured. An equimolar dose of 4-ketocyclophosphamide had no effect on total embryo protein content or crown-rump length and number of somites, but consistently produced some malformed embryos. The kinds of malformations observed, however, are not seen in embryos treated with phospharamide mustard or bioactivated cyclophosphamide.

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. 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.

Investigation on cyclophosphamide treatment during the preimplantation period. II. In vitro studies on the effects of cyclophosphamide and its metabolites 4-OH-cyclophosphamide, phosphoramide mustard, and acrolein on blastulation of four-cell and eight-cell mouse embryos and on their subsequent development during implantation.

Preimplantation mouse embryos were cultured for 48 hours from the four-cell and eight-cell stage to the blastocyst stage in the presence of cyclophosphamide (CPA) or one of its metabolites-4-hydroperoxy-CPA (4-HP-CPA), phosphoramide mustard (PAM), and acrolein (Acr)--to identify the metabolite which is embryotoxic after CPA treatment of pregnant mice during the preimplantation period. The dose-response relations for the inhibition of blastulation revealed identical inhibition curves for PAM and 4-HP-CPA (in solution 4-HP-CPA immediately decomposes to 4-hydroxy-CPA (4-OH-CPA)). These two metabolites are inhibiting blastulation in vitro at concentrations that are 10,000 times lower than CPA and 100 times lower than acrolein. When blastocysts which had developed in the presence of CPA and its metabolites in vitro were subsequently cultured in inhibitor-free medium NCTC-109, the same dose-response relationship pattern was obtained. Since 4-OH-CPA decomposes into acrolein and PAM in vivo and in vitro and since PAM and 4-OH-CPA exhibit identical embryotoxicity towards preimplantation embryos in vitro, PAM probably also is an active embryotoxic CPA metabolite in vivo before implantation. This result is discussed in relation to the importance of alkylating CPA metabolites in cancer treatment and in teratological studies during organogenesis.

Sister-chromatid exchange analyses in rodent maternal, embryonic and extra-embryonic tissues: transplacental and direct mutagen exposures.

Sister-chromatid exchange (SCE) analyses were conducted in maternal, embryonic and extraembryonic tissues of pregnant rats and mice. The various tissues were substituted in vivo with 5-bromodeoxyuridine (BrdU) by implantation of a BrdU tablet in pregnant animals at mid-gestation. Following maternal exposure to 5-20 mg/kg cyclophosphamide, embryonic liver cells demonstrated dose-dependent SCE increases up to 10-fold that of control. Rat embryos revealed little intralitter variability for this transplacental effect. Maternal marrow and yolk sac cells examined in the rat also underwent significant increases in SCE, although to different extents. While marrow SCE frequencies were similar to those of embryo liver, yolk sac SCE frequencies were generally much lower. SCE analyses were also conducted in rat yolk sac cells substituted in vivo with BrdU and subsequently explanted to whole-embryo culture. In vitro exposure to cyclophosphamide at concentrations up to 100 microgram/ml had no SCE-inducing effect. However, similar exposures to phosphoramide mustard, a presumed metabolite of cyclophosphamide, caused dose-dependent increases in SCE up to 8-fold higher than control at 2 microgram/ml. Thus, cyclophosphamide appears to require maternal metabolic activation in order to cause an increased SCE frequency in yolk sac cells. The system described permits versatile SCE analyses which can help to define relative maternal and embryo tissue-specific sensitivities to chemical-induced genetic damage.

Effect of renal insufficiency on the pharmacokinetics of cyclophosphamide and some of its metabolites.

Cyclophosphamide pharmacokinetics were studied in seven patients with moderate to severe renal insufficiency (creatinine clearances 0-51 ml . min-1), and compared with a matched control group of patients with normal renal function. The mean half-life of cyclophosphamide following intravenous administration in the normal group was 8.21 +/- 2.33 (SD) h whilst that in renal failure was 10.15 +/- 1.80 h: these were significantly different. The total body clearance in the normal control group was 58.6 +/- 10.9 ml . kg-1h-1 which was significantly larger than in renal failure where it was 48.8 +/- 10.9 ml . kg-1h-1. Vd beta, Vdss and Vc were not significantly different between the two groups. A linear relationship exists between beta, the first order disposition rate constant and endogenous creatinine clearance since this drug shows a relatively small degree of compartmentalisation. The plasma half-life of phosphoramide mustard, a cytotoxic metabolite of cyclophosphamide, shows a parallel and significant increase in renal failure with the parent compound. The t1/2 in normal patients was 8.33 +/- 2.0 h, whilst in the renal failure group it was 13.37 +/- 4.23 h. Total alkylating activity as measured by the nitrobenzyl-pyridine reaction showed a significant increase in renal failure. This data suggests that in pharmacokinetic terms it may not be necessary to alter the dose of cyclophosphamide until there is severe renal impairment. Further studies correlating the efficacy and toxicity of the drug with its pharmacokinetics in renal failure are necessary.