Nor-nitrogen Mustard Research Articles

Detection of mutagenic activity in urine samples using a new concentration procedure

A study performed with cyclophosphamide (CP) and nor-nitrogen mustard (NNM), one of its main urinary metabolites, has shown that separation on Polygosil® C-18 resin is preferable to one on XAD-2 resin as a means of concentrating the mutagenic activity present in urine of rats given cytostatics such as CP. Mutagenic activity was detected, using Salmonella typhimurium tester strain TA1535. While NNM is irreversibly bound to XAD-2 resin, it can be recovered after elution with methanol on Polygosil C-18. The better efficacy of Polygosil® C-18 in concentrating CP and its metabolite(s) was confirmed with experiments with urine of rats treated with increasing doses of CP.

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.

DNA-binding products of nornitrogen mustard, a metabolite of cyclophosphamide

Nornitrogen mustard was shown to bind to the N-7 position of guanine in DNA and guanosine. The initial binding product was NOR-G. N-(2-chloroethyl)- N-[2-(7-guaninyl)ethyl]amine but it was converted with time to a hydroxylated derivative. NOR-OH-G and to a cross-link, G-NOR-G. The NOR-OH-G adduct was also seen in the reactions of aziridineethanol. The cross-link appeared to depurinate slower than the two other adducts so that it was the main adduct after 72 h of incubation. The rate of reaction was higher at pH 6.0 than at pH 7.4 but even at pH 7.4 adducts were formed. When isolated NOR-G was incubated it was shown to be converted exclusively to NOR-OH-G at pH 5 but at pH 7.4 the main product was a dimer. The half-life of NOR-OH-G formation at pH 7.4 and 37°C was 4 h. The mustard arms of NOR-G and NOR-OH-G had pKa values of about 7.5 and 10, respectively. This was probably related to the somewhat higher rate of imidazole ring-opening of NOR-OH-gua as compared to NOR-gua. The lability of the imidazole ring of the guanosine adducts of nornitrogen mustard is intermediate to those of phosphoramide mustard and nitrogen mustard.

Estramustine-Induced Mitotic Arrest in Two Human Prostatic Carcinoma Cell Lines DU 145 and PC-3

In growth proliferation experiments on two human prostatic carcinoma cell lines, DU 145 cells were found to be more sensitive to the cytotoxic effect of estramustine and nor-nitrogen mustard than PC-3 cells. Estramustine was, however, much more cytotoxic in both cell lines than nor-nitrogen mustard. Cytogenetic experiments revealed that estramustine produced a drastic increase of the mitotic index in both these cell lines. This increase could be accounted for by the arrest of cells in their first treatment-metaphase. The arrested metaphases exhibited all the characteristics commonly found for stathmokinetic agents such as colchicine and vinca-analogues. No mitotic arrest was found for nor-nitrogen mustard but chromosomal aberrations were found at toxic concentrations. Estradiol exhibited minimal toxicity and caused no mitotic arrest in these cell lines. The mitotic arrest induced by estramustine was found to be reversible on removal of the drug.

Cytotoxic properties of estramustine unrelated to alkylating and steroid constituents

Using cultured HeLa S3 cells an ID50 of 2.5 micrograms/ml was found after a twenty-four-hour incubation with estradiol-17 beta- 3N -bis-(2-chloroethyl) carbamate (estramustine). Similar ID90 values were found in two Walker 256 rat carcinoma cell lines which were either sensitive or resistant to nitrogen mustards. Alkaline elution methodology revealed the complete absence of DNA strand breaks or cross-links in cells receiving up to 10 micrograms/ml estramustine for twenty-four hours. Nuclear uptake was 1.34 per cent of the available drug, one third of which was hydrophobically associated with the protein/phospholipid components of the nuclear matrix. In the human prostatic cell lines DU145 and PC3 , estramustine caused a drastic dose-dependent increase in the mitotic index. This increase resulted from an arrest of cells in metaphase, with highly contracted disoriented chromosomes present. Rapid reverse of the arrest on removal of drug resulted in cell death. Neither nor-nitrogen mustard nor estradiol demonstrated antimitotic properties. The lack of macromolecular alkylation together with the observed antimitotic effects predict a mechanism of action for estramustine which is distinct from either of its constituent components.

The hydrolysis of estramustine phosphate; in vitro studies.

The hydrolysis of estramustine phosphate by enzymes of blood, liver, intestines and prostate of man, dog and rat was studied by using specific analytical methods for the parent compound and some of its metabolites. Estramustine phosphate was stable in blood and plasma of different species but rapidly dephosphorylated to estramustine by liver, intestinal and prostatic enzymes. The prostate was the most active tissue. The ester bond by which nornitrogen mustard is linked to estradiol-17-phosphate was slowly cleaved by enzymes from the liver and prostate of the rat, dog and man. The dog tissues were more effective than those of the rat and man. Intestinal enzymes also exhibited the ability to hydrolyze the carbamic ester, but to a greater extent in the rat than in the dog. It is concluded that, with regard to the metabolism of estramustine, the rat is more similar to man than is the dog. Possible clinical implications of the biotransformation of estramustine phosphate are discussed.

2-Chloroacetaldehyde: a metabolite of cyclophosphamide in the rat

Chloroacetaldehyde has been shown to be a urinary metabolite of cyclophosphamide in the rat. It was identified in the urine of rats given a mixture of [ring C-4, 14C]-cyclophosphamide and [chloroethyl-3H]-cyclophosphamide as its 2,4-dinitrophenyl-hydrazone derivative by its 3H label and by its co-chromatography with chemically synthesised chloroacetaldehyde. The reaction of chloroacetaldehyde with another urinary metabolite of cyclophosphamide is discussed and a nornitrogen mustard adduct postulated. The toxicity of chloroacetaldehyde is also discussed particularly in relation to the urinary bladder.

Synthesis, Hydrolytic Reactivity, and Anticancer Evaluation of N- and O-Triorganosilylated Compounds as New Types of Potential Prodrugs

N- and O-Triorganosilylated compounds related to various anticancer agents were synthesized for evaluation as potential anticancer prodrugs. 1H-NMR and UV kinetic measurements of hydrolytic desily-lation were used to correlate relative rates of structural unmasking with steric bulk about the silicon reaction center. The tert-butyldimethylsilyl ester of chlorambucil and a number of O-triorganosilylated carbamate derivatives of nor-nitrogen mustard showed significant activity against P-388 lymphocytic leukemia in mice.

Growth and Cell Survival Following Treatment with Estramustine, Nor-Nitrogen Mustard, Estradiol and Testosterone of a Human Prostatic Cancer Cell Line (DU 145)

Estramustine at concentrations ranging from 3–40 × 10−6m inhibited the cell growth and clonogenic survival of a human prostatic carcinoma cell line (DU 145). This cell line was found to be unresponsive to estradiol and testosterone at concentrations ranging from 10−9m to 5 × 10−5m. Metabolism studies with estramustine showed that only a few per cent of the ester linkage was cleaved during the exposure period. This small amount of metabolism could possibly lead to the release of nor-nitrogen mustard, which was however found not to be as inhibitory as estramustine in this cell line. The results indicate that estramustine per se causes the cell death of hormone unresponsive human prostatic carcinoma cells in cell culture.

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.

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.

The pharmacokinetics of cyclophosphamide, phosphoramide mustard and nor-nitrogen mustard studied by gas chromatography in patients receiving cyclophosphamide therapy.

Abstract 1 Simple, accurate and specific gas-chromatographic methods for the estimation of derivatized phosphoramide and non-nitrogen mustards utilizing alkali-flame ionization detection are described. 2 The pharmacokinetics in plasma of cyclophosphamide, phosphoramide mustard, nor-nitrogen mustard and nitrobenzyl pyridine alkylating activity were investigated following administration of cyclophosphamide by intravenous and oral routes to patients with malignant disease 3 The mean T½ for cyclophosphamide was 8.88 h (s.d. 1.25 h) and the apparent volume of distribution (Vdβ) was 0.74 l kg-1 (s.d. 0.16 l kg-1). 4 The decline in plasma concentration of phosphoramide mustard was biphasic, the longer T½ being 8.68 h (s.d. 2.50 h). This was not significantly different from that of cyclophosphamide. This could indicate that the true biological T½ for phosphoramide mustard is identical with or shorter than that of cyclophosphamide. 5 The plasma concentrations of phosphoramide mustard following cyclophosphamide doses of known therapeutic efficacy are probably insufficient to produce important cytotoxic effects. This suggests that if phosphoramide mustard is the major alkylating metabolite derived from cyclophosphamide, it is transported in the blood in precursor form. 6 The mean plasma T½ of nor-nitrogen mustard was 3.31 h (s.d. 1.60 h) which was significantly different from that of cyclophosphamide. 7 The mean plasma T½ of the nitrobenzylpyridine alkylating activity was 9.81 h (s.d. 4.18 h) and did not significantly differ from that of cyclophosphamide. Although the area under the plasma alkylating activity concentration, time curve is related to the T½ of cyclophosphamide, the alkylating activity does not reflect the concentrations of the two plasma metabolites measured.

Synthesis and first in vitro cytotoxicity studies of bis(2-chloroethyl) amino group containing polymers. Pharmacologically active polymers: 22

Monomers containing the cytotoxic bis(2-chloroethyl)amino group (nor-nitrogen mustard), linked in deactivated form via urethane and O-acylated hydroxamic acid bonds to polymerizable methacrylic acid derivatives, have been prepared. Besides the homopolymerization, these monomers were copolymerized with hydrophilic comonomers to obtain water-soluble polymers. The linkages used are expected to undergo intracellular enzymatic or hydrolytic cleavage, releasing the cytotoxic bis(2-chloroethyl)amino moiety from the polymeric carrier. Preliminary in vitro cytotoxicity data for the polymers against three rat and mouse experimental tumour lines (Walker 256 carcinosarcoma, L1210 murine leukaemia and ADJ/PC6A plasma cell tumour) are reported. An approximately 10 2 fold difference in cytotoxic potency was found, depending on the type of the cleavable spacer group.

Cytogenetic toxicity of cyclophosphamide and its metabolites in vitro.

The effects of cyclophosphamide and three of its known metabolites (nitrogen mustard, acrolein, and nor-nitrogen mustard on chromosome breakage and sister chromatid exchange (SCE) were analyzed in vitro with and without the presence of a metabolic activating system (liver S9). We confirmed that cyclophosphamide induced chromosome breakage and SCE only in the presence of S9. After metabolism, cyclophosphamde was more active than the other agents in inducing SCE. Thus, the agent(s) directly responsible for this induction of a high SCE rate was not analyzed in this study. On the other hand, acrolein was most toxic to cell proliferation and most active in inducing chromosome breakage. The cytogenetic toxicity of these agents in comparison with their mutagenic and therapeutic activities is discussed.

Mutagenic activity of major mammalian metabolites of cyclophosphamide toward several genes ofEscherichia coli

Representative intermediates of the major mammalian metabolic pathway of cyclophosphamide, a drug that is not mutagenic as such unless it is metabolically activated, were assayed for their direct mutagenic activity toward a bacterial indicator strain, Escherichia coli 343/113. The compounds tested were 4-hydroperoxycyclophosphamide and the two urinary metabolites, carboxyphosphamide and 4-ketocyclophosphamide. Further, the degradation products of 4-hydroxycyclophosphamide, phosphoramide mustard, acrolein, and nornitrogen mustard, were also tested. The mutagenicity test systems were those used previously to demonstrate the liver enzyme-mediated mutagenic activity of cyclophosphamide in E. coli 343/113: stationary cell suspensions were treated for 180 min at 37 degrees C with different concentrations of the compound under test; the induction of forward mutations from 5-methyltryptophan sensitivity to resistance (MTR) and from galRs18 to gal+ as well as back mutations from arg56 to arg+ was measured by plating aliquots of the treated bacterial population on different selective mutation media. Except for acrolein, all cyclophosphamide metabolites tested are directly mutagenic toward E. coli 343/113. With all substances the highest induced mutation frequency is that of arg+ mutations, followed by gal+ and MTR mutations; this indicates that mostly base-pair substitution type mutations are induced. The mutagenic potential, however, differs greatly between compounds at concentrations between 0.1 and 20mM. The results show that the first step in the mammalian biodegradation of cyclophosphamide gives rise to compounds that are directly mutagenic, and that this mutagenicity is retained and even enhanced through all further metabolic steps to produce the compound of highest mutagenicity, nornitrogen mustard.

Review of the Metabolism and Mode of Action of Cyclophosphamide

A review of recent studies of the pharmacology of cyclophosphamide is presented. Particular attention is paid to the use of mass spectrometry in the identification of the active compound phosphoramide mustard as a circulating human metabolite of cyclophosphamide, the development of an assay method for cyclophosphamide, phosphoramide mustard, and nornitrogen mustard, and a demonstration that alkylation by phosphoramide mustard proceeds through a symmetrical aziridinium ion.

Evaluation of extracorporeal alkylation of red cells as a potential treatment for sickle cell anemia

Nitrogen mustard and nor-nitrogen mustard inhibit sickling, but the concentrations required would be associated with unacceptable toxicity if these agents were administered to patients. Red cells could be treated extracorporeally and infused back into donors, if the alkylating agent could be removed or inactivated, if the treatment per se did not significantly shorten red cell survival, and if viable alkylated lymphocytes could be eliminated from the treated blood. To estimate whether these conditions could be met in a clinical trial, red cells from four dogs were alkylated at 6-wk intervals. No toxic reactions were observed, although not all nor-nitrogen mustard was removed by the washing procedure. Red cell survival was shortened to about half that of control cells, using concentrations of alkylating agent which reduce sickling by 50%. Lymphocytes from treated blood could still exclude trypan blue, but could not be shown to circulate after reinfusion into donor dogs. If alkylating agents are used to treat patients' cells, inhibition of sickling may outweigh the shortening of red cell life span induced by the treatment; blood should probably be irradiated before infusion to avoid administration of alkylated and potentially mutated, but viable, lymphocytes.

Evaluation of Extracorporeal Alkylation of Red Cells as a Potential Treatment for Sickle Cell Anemia

Nitrogen mustard and nor-nitrogen mustard inhibit sickling, but the concentrations required would be associated with unacceptable toxicity if these agents were administered to patients. Red cells could be treated extracorporeally and infused back into donors, if the alkylating agent could be removed or inactivated, if the treatment per se did not significantly shorten red cell survival, and if viable alkylated lymphocytes could be eliminated from the treated blood. To estimate whether these conditions could be met in a clinical trial, red cells from four dogs were alkylated at 6-wk intervals. No toxic reactions were observed, although not all nor-nitrogen mustard was removed by the washing procedure. Red cell survival was shortened to about half that of control cells, using concentrations of alkylating agent which reduce sickling by 50%. Lymphocytes from treated blood could still exclude trypan blue, but could not be shown to circulate after reinfusion into donor dogs. If alkylating agents are used to treat patients' cells, inhibition of sickling may outweigh the shortening of red cell life span induced by the treatment; blood should probably be irradiated before infusion to avoid administration of alkylated and potentially mutated, but viable, lymphocytes.

Metabolic effects of antisickling amounts of nitrogen and nor-nitrogen mustard on rabbit and human erythrocytes

Nitrogen mustard (NH2) and Nor-nitrogen mustard (Nor-HN2) both inhibit the polymerization of deoxyhemoglobin S in solution and in intact erythrocytes. Metabolic studies were undertaken to determine the feasability of an extracorporeal treatment with these or related agents. Glucose utilization, hexose monophosphate shunt activity, methemoglobin reduction, and incubation with acetylphenylhydrazine for Heinz body formation were performed, as well as specific assays for hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glutathione reductase, ATP, reduced glutathione (GSH), and survival of autologous mustard-treated cells in rabbits. HN2 was found to enter red cells rapidly and bind to intracellular contents. Metabolic studies revealed no significant inhibition or alteration of function by Nor-HN2 at 10 mg/ml of whole blood. Rabbit red cell survival was also normal. HN2, however, inhibited glutathione reductase and blocked the free sulfhydryl group of GSH by forming serveral addition products of alkylated GSH. Heinz body test with acetylphenylhydrazine became positive in HN2-treated cells, and rabbit red cell survival was shortened considerably in the concentration range used to inhibit sickling. Ascorbic acid stimulation of the hexose shunt pathway was inhibited by HN2, but methylene blue stimulation remained unaffected. 14-C-HN2 remains bound to red cells in vivo, and the disappearance of radioactivity is similar to that found with 14-C-DFP (disopropylfluorophosphate). Oxygen affinity of both HN2 and Nor-HN2 treated human red cells remains virtually the same as that found in control samples. It is concluded that Nor-HN2 may be a suitable agent for an extracorporeal therapy, and that each mustard needs to be evaluated individually for its antisickling effects and its suitability for extracorporeal use.

Metabolic Effects of Antisickling Amounts of Nitrogen and Nor-Nitrogen Mustard on Rabbit and Human Erythrocytes

Nitrogen mustard (NH2) and Nor-nitrogen mustard (Nor-HN2) both inhibit the polymerization of deoxyhemoglobin S in solution and in intact erythrocytes. Metabolic studies were undertaken to determine the feasability of an extracorporeal treatment with these or related agents. Glucose utilization, hexose monophosphate shunt activity, methemoglobin reduction, and incubation with acetylphenylhydrazine for Heinz body formation were performed, as well as specific assays for hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glutathione reductase, ATP, reduced glutathione (GSH), and survival of autologous mustard-treated cells in rabbits. HN2 was found to enter red cells rapidly and bind to intracellular contents. Metabolic studies revealed no significant inhibition or alteration of function by Nor-HN2 at 10 mg/ml of whole blood. Rabbit red cell survival was also normal. HN2, however, inhibited glutathione reductase and blocked the free sulfhydryl group of GSH by forming serveral addition products of alkylated GSH. Heinz body test with acetylphenylhydrazine became positive in HN2-treated cells, and rabbit red cell survival was shortened considerably in the concentration range used to inhibit sickling. Ascorbic acid stimulation of the hexose shunt pathway was inhibited by HN2, but methylene blue stimulation remained unaffected. 14-C-HN2 remains bound to red cells in vivo, and the disappearance of radioactivity is similar to that found with 14-C-DFP (disopropylfluorophosphate). Oxygen affinity of both HN2 and Nor-HN2 treated human red cells remains virtually the same as that found in control samples. It is concluded that Nor-HN2 may be a suitable agent for an extracorporeal therapy, and that each mustard needs to be evaluated individually for its antisickling effects and its suitability for extracorporeal use.