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.     

Validation of a novel procedure for quantification of the formation of phosphoramide mustard by individuals treated with cyclophosphamide

Purpose

Use of the patient’s body surface area (mg m^?2) as a basis for dosing does not take individual variation in metabolic capacity and rate of clearance into account. Here, we evaluated a novel approach for individual monitoring of short-lived cytotoxic agents formed from cytostatic drugs such as cyclophosphamide (CP).

Methods

The accumulated blood dose of the cytotoxic active agent phosphoramide mustard (PAM) formed from CP was measured as a reaction product with hemoglobin (Hb adduct). This adduct, N-[2-(2-oxazolidonyl)ethyl]-valyl Hb (OzVal-Hb), was detached from Hb with the adduct FIRE procedure?, and the formed analyte was quantified using LC-MS/MS. This dose biomarker for PAM and the analytical procedure was evaluated in accordance with the guidelines on bioanalytical method validation formulated by the European Medicine Agency. The evaluated method was applied to quantify blood dose levels of PAM in female breast cancer patients (n = 12) before and after three cycles of polychemotherapy regimes containing CP.

Results

OzVal-Hb, a specific and stable biomarker, could be measured with great sensitivity (lower limit of quantification = 33 pmol g^?1 Hb), high accuracy (within ±20 %) and good repeatability (CV < 20 %). The inter-individual variability in the blood level of this adduct in women with breast cancer (n = 12) who received three doses of CP in combination with one or two other cytostatic drugs was 250 % following the first dose and approximately 150 % after each subsequent dose.

Conclusions

Measurement of the biomarker OzVal-Hb can be used to quantify the short-lived cytotoxic agent PAM in a single blood sample drawn several days after therapy. This procedure may aid in individualizing doses of CP, thereby improving efficacy while both reducing the risk of and increasing the predictability of side-effects.     

Brain and plasma pharmacokinetics and anticancer activities of cyclophosphamide and phosphoramide mustard in the rat

Summary By a sensitive and quantitative fluorometric assay, brain and plasma time-dependent concentration profiles were generated for phosphoramide mustard (PM) and active alkylating metabolites derived from cyclophosphamide (CPA) administration to rats. Whereas PM rapidly disappeared from plasma, with a monophasic half-life of 15.1 min, equimolar administration of CPA generated active metabolites in plasma that disappeared monoexponentially, with a composite half-life of 63 min. As a consequence, the time-dependent concentration integral of active alkylating metabolites derived from CPA administration, calculated between 5 min and infinity, was 3-fold that of PM. Pharmacokinetic parameters were calculated for each compound. The brain/plasma concentration-integral ratios of PM and active alkylating metabolites derived from CPA were 0.18 and 0.20, respectively. The cerebrovascular permeability-surface area product of PM was 7.5×10^–5s^–1, which is similar to that of other watersoluble anticancer agents that are restricted from entering the brain. The activities of a range of daily doses of PM and CPA were assessed against subcutaneous and intracerebral implants of Walker 256 carcinosarcoma tumor in rats. Inhibition of subcutaneous tumor growth by 50% was caused by CPA and PM doses of 6.6 and 12.0 mg/kg (daily for 5 consecutive days, starting 36 h after tumor implantation), respectively. However, administration of daily doses of up to 40 mg/kg did not significantly increase the survival of animals with intracerebral tumor implants. These studies indicate that active metabolites of CPA are restricted from entering the brain and that only subtherapeutic concentrations are achieved in brain tissue after systemic administration of CPA or PM.Abbreviations CPA cyclophosphamide - PM phosphoramide mustard - 4-HC 4-hydroxycyclophosphamide - AP aldophosphamide - PA cerebrovascular permeability-surface area product     

Intracavitary chemotherapy with activated cyclophosphamides and simultaneous systemic detoxification with protector thiols in Sarcoma 180 ascites tumor

Activated cyclophosphamides such as 4-sulfoethylthiocyclophosphamide (mafosfamide) are suitable for a local intracavitary chemotherapy, whereas cyclophosphamide requires a metabolic activation. Mafosfamide administered i.p. in mice was less toxic (50% lethal dose, 640 mg/kg) than its i.v. application (50% lethal dose, 480 mg/kg). A further remarkable reduction of toxicity with an increase of the 50% lethal dose of mafosfamide to 1500 mg/kg was obtained by the simultaneous i.v. application of the protector thiol cysteine (mafosfamide:cysteine ratio, 1:5 on molar weight basis). In comparison with the i.v. injection of mafosfamide, the local i.p. application resulted in a 20 times higher concentration versus time product of peritoneal drug levels. The molar ratio of sulfhydryl groups to activated cyclophosphamide (resorbed from the peritoneal cavity) remained high in blood. Therapy studies on Sarcoma 180 ascites tumor of mice revealed that the coadministration of cysteine i.v. in mafosfamide i.p. treatment is superior to mafosfamide i.p. application alone. On the contrary, the simultaneous i.p. application of cysteine is accompanied by a loss of antitumor efficacy. The regimen of local i.p. chemotherapy with activated cyclophosphamide and simultaneous systemic detoxification by an appropriate thiol allows the reduction of the systemic toxicity of treatment without influence on the cancerotoxic activity at the site of local injection and the exposing of the intraabdominal tumor to a much higher concentration of the cytostatic agent.     

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

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

A 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     

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.     

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

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

The effect of treatment with L-phenylalanine mustard and L-phenylalanine mustard antibody on the survival time of mice with intracerebral ependymoblastomas

Previous work has shown a decreased rate of growth of mouse ependymoblastomas treated with an antigen which fixes to the cell wall, followed by an antibody to the tumor cell marker. The present study was designed to compare the survival time of mice with intracerebrally implanted ependymoblastomas that were treated with the above method with the survival time of controls. Eight groups of C₃ H male mice were subjected to intracerebral implantation of ependymoblastoma. Five days following implantation, each group was subdivided into four subgroups, and the subgroups were treated with intravenous normal saline, intravenous antibody, intracarotid L–phenylalanine mustard and intracarotid L–phenylalanine mustard followed by intravenous antibody, respectively. The treated mice showed statistically significant increase of survival time, as compared with the controls. It is possible that by changing some of the treatment parameters, more long-term results can be achieved.     

Chlorophyllin protects cells from the cytostatic and cytotoxic effects of quinacrine mustard but not of nitrogen mustard

Chlorophyllin (CHL), the sodium and copper salt of chlorophyll, is capable of inhibiting the mutagenic activity of many chemical compounds. Several mechanisms have been advanced to explain the antimutagenic activity of CHL, including its antioxidant properties and its ability to form complexes with mutagens. The present study was designed to reveal whether the heterocyclic aromatic nature of a potential mutagen is essential to its sensitivity to CHL. Toward this end, the inhibitory effect of CHL on two compounds of similar chemical reactivity (mustards), that either embodied an aromatic structure (quinacrine mustard; QM) or did not (nitrogen mustard; NM), were compared. Human leukemic HL-60 and breast carcinoma MCF-7 cells were treated with QM or NM in the absence or presence of various concentrations of CHL. Both QM and NM when administered for 1-2 h at micromolar concentrations exerted similar effects; both arrested cells in G2 phase of the cell cycle, induced apoptosis and reduced the clonogenicity of MCF-7 cells. The simultaneous addition of 0.22 M CHL to cultures receiving QM virtually abolished the QM-induced inhibition of cell growth and clonogenicity. In contrast, CHL had no effect on reducing the cytostatic or cytotoxic activity of NM. CHL alone, at a concentration of 0.22 M, had minimal effect on growth of HL-60 cells slightly perturbing their progression through G2. The results are consistent with the model that explains the inhibition of the activity of mutagens or antitumor drugs with aromatic structures by CHL as mediated by its ability to sequester these molecules within heterologous mutagen:CHL complexes that are maintained by stacking interactions. Therefore, excess of chlorophyll in the diet, by sequestering aromatic mutagens (or antitumor drugs with a heterocyclic structure, if taken orally), may inhibit their accessibility to cells, thereby reducing their activity.     

Interactions of the carcinogen 4-nitroquinoline 1-oxide with the non-protein thiols of mammalian cells.

The carcinogen 4-nitroquinoline 1-oxide (4-NQO) was found to rapidly deplete non-protein thiols (NPSH) from Ehrlich ascites tumor cells and V79 Chinese hamster fibroblasts. The effects of NPSH on 4-NQO metabolism were studied by measuring 4-hydroxyaminoquinoline 1-oxide formation, CN- -insensitive oxygen consumption, and reduction of ferricytochromes c + c1 in normal cells and in cells pretreated with the thiol reagent N-ethylmaleimide. Removal of thiols before treatment with 4-NQO resulted in increased production of 4-hydroxyaminoquinoline 1-oxide and increased production of nitro radicals. The NPSH thus appeared to play a significant role in 4-NQO detoxification. Glutathione, when present in culture medium during 4-NQO treatment, protected V79 cells from 4-NQO toxicity. Several mechanisms for reaction of 4-NQO with intracellular NPSH were indicated. Both V79 and Ehrlich cells contained appreciable amounts of glutathione S-transferase (EC 2.5.1.18), which catalyzes the nucleophilic substitution of the nitro group of 4-NQO with thiols. Greater thiol loss under oxic than under hypoxic conditions suggested oxidation by superoxide, peroxide, or hydroxyl radical formed in the course of 4-NQO reduction. In addition, reaction of thiols with nitro radicals or with nitrosoquinoline 1-oxide was indicated by the inhibitory effect of glutathione on oxygen consumption in solutions of 4-NQO and sodium ascorbate.     

Formation of O6-ethylthioethylguanine in DNA by reaction with the sulfur mustard, chloroethyl sulfide, and its apparent lack of repair by O6-alkylguanine-DNA alkyltransferase

The reactions of the monofunctional sulfur mustard, chloro-ethylethyl sulfide, with calf thymus DNA have been studied in vitro.In addition to extensive alkylation of the 7 position of guanineand the 3 position of adenine, - 0.1% of the total alkylationis on the O⁶ position of guanine. Since chloroethyl ethyl sulfideis a model for S-adenosylmethionine and for the reactive intermediatesgenerated from ghitathione by certain environmental agents,this result establishes a route by which these compounds couldbe mutagenic. A DNA substrate modified for repair studies hasbeen produced by incubating calf thymus DNA with [¹⁴C]chloroethyl-labelledchloroethyl ethyl sulfide. This DNA contains 6 nmol/mg of alkylatedproducts including 6 pmol/mg O⁶-ethylthioethylguanine. Incubationof this substrate with up to a 20-fold excess of mammalian (O⁶-alkylguanine-DNAalkyltransferase has indicated that O⁶-ethylthioethylguanineis either not a substrate or a poor one for this DNA repairsystem.     

芥菜籽对化学诱导小鼠大肠肿瘤形成过程中体内抗氧化状态的影响

  目的  通过观察小鼠体内自由基水平和抗氧化酶活性的影响, 探讨芥菜籽对氧化偶氮甲烷(azoxymethane, AOM)诱导的小鼠大肠肿瘤的预防作用机制。  方法  60只昆明品系小鼠随机分为AOM模型组、AOM+5%MS、AOM+10%MS干预组和正常对照组。观察记录各组小鼠肿瘤发生率; 检测小鼠血清中抗氧化酶(SOD、CAT、GSH-PX)的活力和脂质过氧化产物丙二醛(MDA)的水平。  结果  正常对照组小鼠无肿瘤发生。AOM模型组、5%MS和10%MS干预组三组间肿瘤发生率有差异(χ2=6.607, P=0.048);AOM模型组小鼠平均肿瘤数为(2.20±1.21)个, 而5%MS和10%MS干预组小鼠平均肿瘤数分别为(1.07±1.10)个和(0.67±0.89)个, 与模型组相比有显著性差异(P < 0.05)。小鼠血清抗氧化酶SOD、CAT、GSH-PX的活力: AOM模型组明显低于正常对照组(P < 0.05);MS干预组与AOM模型组相比均有所提高(P < 0.05);其中10%MS干预组提高小鼠血清抗氧化酶活力的作用最明显, 与5%MS干预组相比有显著性差异(P < 0.05)。小鼠血清脂质过氧化产物(MDA)含量: AOM模型组明显高于正常对照组(P < 0.05);MS干预组与AOM模型组相比均有所下降(P < 0.05);其中10%MS干预组降低小鼠血清MDA的效果最明显, 与5%MS干预组相比有显著性差异(P < 0.05)。  结论  芥菜籽能明显降低AOM诱导的小鼠大肠癌发生率并减少小鼠的平均肿瘤数, 且随芥菜籽浓度的增加, 其预防作用增强。芥菜籽能明显增强体内抗氧化的能力, 提高体内自由基清除酶的活性, 其预防机制可能与其抗氧化作用有关, 通过减少自由基对细胞的损伤, 预防基因突变和肿瘤发生。