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.     

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     

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.     

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     

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.     

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.     

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.     

Alkylating agents

With the approval of mechlorethamine by the FDA in 1949 for the treatment of hematologic malignancies, alkylating agents are the oldest class of anticancer agents. Even though their clinical use is far beyond the use of new targeted therapies, they still occupy a major place in specific indications and sometimes represent the unique option for the treatment of refractory diseases. Here, we are reviewing the major classes of alkylating agents and their mechanism of action, with a particular emphasis for the new generations of alkylating agents. As for most of the chemotherapeutic agents used in the clinic, these compounds are derived from natural sources. With a complex but original mechanism of action, they represent new interesting alternatives for the clinicians, especially for tumors that are resistant to conventional DNA damaging agents. We also briefly describe the different strategies that have been or are currently developed to potentiate the use of classical alkylating agents, especially the inhibition of pathways that are involved in the repair of DNA lesions induced by these agents. In this line, the development of PARP inhibitors is a striking example of the recent regain of interest towards the "old" alkylating agents.     

Alkylating potency of nitrosated amino acids and peptides.

The alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, Tyr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, Tyr-Tyr, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present during the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artificial sweetener aspartame); only Met under these conditions had a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Met produces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the first-order reaction rate for nitrite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uracil mustard revisited.

BACKGROUND: A patient with diffuse large cell lymphoma who had a complete response lasting 35 years following a 3-day course of uracil mustard stimulated a recall review of patients treated with this oral alkylating agent. METHODS: Records of patients treated with uracil mustard between 1958 and 1970 were reviewed. A current histologic review according to the International Formulation was performed when possible. Total doses of uracil mustard were similar to those of mechlorethamine, although there were variations in the dose schedule. RESULTS: Employing criteria used over 25 years ago to evaluate patients' responses, the overall regression rate for 94 non-Hodgkin lymphoma patients was 69.2% (complete response [CR] 23.4%). Of 62 patients with Hodgkin disease, 69.4% responded (CR 9.7%). For 39 patients with chronic lymphatic leukemia, the combined complete and partial response rate was 74% (CR 7.7%). Thrombocytopenia was the primary toxicity. CONCLUSIONS: Uracil mustard is an unmarketed, inexpensive oral alkylating agent that has been effective in the treatment of patients with lymphoma, chronic lymphatic leukemia, and thrombocythemia. Perhaps it should be reevaluated.     

Alkylating benzamides with melanoma cytotoxicity

Radioiodinated N-(2-(diethylamino)ethyl)benzamides have recently been discovered as selective agents for melanotic melanoma and are used for scintigraphic imaging in nuclear medicine. Owing to the high binding capacity, benzamide derivatives conjugated with alkylating cytostatics were synthesized and tested for their potential for targeted drug delivery. Conjugates of chlorambucil with procainamide (1), diethylaminoethylamine (2) and 2-pyrrolidin-1-yl-ethylamine (3), as well as 4-(bis(2-chloroethyl)amino)- (6,7) and 4-(N,N-diethyltriazeno)-substituted (8-10) benzamides, were synthesized. Cell uptake studies with B16 melanoma cells revealed high uptake of radioiodinated 1 and 2, while radiolabelled chlorambucil was found to lack this characteristic. These results were confirmed by biodistribution studies in a mouse melanoma model. Viability measurements revealed that all chlorambucil-benzamide derivatives showed higher toxicity against B16 melanoma and SK-MEL-28 cells than did the parent chlorambucil itself, and that the triazene derivatives were more potent than dacarbazine, which is currently used as a standard cytostatic drug in melanoma therapy. Of all the compounds tested in this series, the triazenes 9 and 10 showed the most promising targeting effect. The toxicity of these compounds against hepatoma cells (MH3924A) and, to a lesser extent, against mouse fibroblast (NIH 3T3) and cervix carcinoma (HeLa) cells was also enhanced, but they were not as toxic as dacarbazine (HeLa). These findings support the concept of a selective, benzamide-mediated in vivo delivery of cytostatics in melanoma cells, leading to enhanced efficacy.     

Antitumor activity of tetraacetylglucosamine mustard.

1,3,4,6-Tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose is active against L1210 leukemia, giving over 100% increased life-span at optimal dose. Against P388 leukemia, it gives 200% increased life-span with long-term survivors. The compound is most active when given i.p., but shows some activity when given s.c. than p.o., and is more potent (therapeutic and toxic effect) than mechlorethamine on both a molar and a mg basis. Of importance, the schedule dependency for the administration of 1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose in L1210 leukemia differs from most alkylating agents in that it is best given by multiple daily injections rather than as a single large injection on Day 1. This characteristic can be attributed to the amino-glucose moiety.     

Mechanism of resistance of Yoshida sarcoma to nitrogen mustard. 3. Mechanism of suppressed transport of nitrogen mustard

Mechanism of decreased transport of nitrogen mustard (HN₂) in HN₂-resistant Yoshida sarcoma cells was investigated. HN₂ uptake by Yoshida sarcoma cells proved to be temperature sensitive, saturable, affected by metabolic inhibitors, and also repressed competitively by choline chloride. In resistant cells, the uptake of choline was markedly depressed. Kinetics of choline transport in resistant cells was comparatively examined, and decreased V_max and increased K_m were observed in some resistant sublines. Accordingly, decrease in choline uptake may be due to decreased activity of choline transport-carrier, both qualitatively and quantitatively. Decreased HN₂ uptake by resistant cells seemed to be produced by the same mechanism.     

Comparative genotoxicity of nitrogen mustard and nor-nitrogen mustard

Nitrogen mustard and nor-nitrogen mustard were investigatedin Salmonella typhimurium and human lymphocytes for genotoxicity.Point mutations were assessed using the plate test, the conventionalspot test and a method in which the substances were not in directcontact with the microorganisms. Both compounds were active,but nornitrogen mustard was far more potent than nitrogen mustardin all bacterial systems. Cytogenetic experiments with humanlymphocytes revealed that both compounds induced a dose dependentincrease in chromosomal aberrations and sister chromatid exchanges,but that nitrogen mustard was 10 times more potent than nor-nitrogenmustard. Thus, the spectrum of activity for nor-nitrogen mustardand nitrogen mustard differ. Nor-nitrogen mustard was more effectivein inducing point mutation damage than nitrogen mustard anda reversal of potency was found for cytogenetic damage. Theseresults indicate that although these two substances induce thesame type of DNA lesions the amounts of the different DNA adductsvary.     

Mechanism of resistance of Yoshida sarcoma to nitrogen mustard. II. Further evidence for suppressed transport of nitrogen mustard

Sensitivity of Yoshida sarcoma lines resistant to nitrogen mustard (HN²) was not enhanced by pretreatment in vitro with SH-blocking agents such as 2,2′-dithiodi-pyridine. This fact seems to indicate that content of cellular sulfhydryl groups did not play a great role in acquisition of resistance to HN². No difference was observed between the sensitive and resistant lines in retention rate of HN² bound to DNA of the cells during incubation of the cells in an HN-free medium. These results suggested no considerable difference in the ability to repair DNA in both lines. Further evidence was added for suppressed transport through the cell membranes of the resistant lines. Damage to cell membranes was proved by vital staining when a concentration of SH-blocking agents in the pretreatment reached a certain level, and the binding rate of HN² to DNA of the resistant lines was recovered almost to the same level of the sensitive line by this pretreatment. However, the binding rate of the sensitive line was not changed by pretreatment with the blocking agents at any concentrations.     

A Comparative Analysis of Alkylating Agent and Epipodophyllotoxin-Related Leukemias

This review deals with the differences between leukemias—induced by alkylating agents as opposed to a “new form” of treatment related leukemia due to prior exposure to epipodophyllotoxins the latter having a short treatment—disease onset interval, absence of a MDS phase, a monocytic component and cytogenetic abnormalities involving the 11q23 band. The link between the existence of oncogenes or tumor suppressor genes located on the involved portion of chromosome 11 and the development of epipodophyllotoxin—related leukemia still needs to be examined. Alkylating agents—induced leukemias have a longer treatment—disease onset interval, have a prior myelodysplastic syndrome, and are most frequent myelo-blastic or myelomonocytic in nature. Karyotype analysis reveals partial or complete deletion of chromosomes no. 5 or 7. This form of leukemia is highly resistant to treatment in the majority of cases. Some of the possible molecular mechanisms of leukemogenesis are discussed.     

Alkylating agents and nucleoside analogues in the treatment of B cell chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world. The natural clinical course is highly variable and chemotherapy is usually not indicated in early and stable disease. Treatment is needed in the progressive form of this leukemia. Chlorambucil, with or without steroids, has been for many years the drug of choice in the treatment of CLL. More recently, treatment approaches have included nucleoside analogues, (NA) fludarabine (FAMP) and cladribine (2-CdA, 2-chlorodeoxyadenosine), which seem to be the treatment of choice for patients failing standard therapies. Their role as first line therapy is being investigated in randomized trials and the results have recently been published. These studies have shown a higher overall response and complete remission (CR) rate and longer response duration in patients treated initially with NA than with chlorambucil or cyclophosphamide-based combination regimens. In contrast, overall survival is similar in patients treated with NA and alkylating agents. However, the randomized trials were designed as crossover studies which may influence survival. Combined use of NA with other cytotoxic drugs, cytokines, monoclonal antibodies and other agents may increase the CR and prolong survival time. However, the results of randomized trials comparing combination treatment with NA alone are not yet available. In conclusion, alkylating agents still have an important place in the routine management of the majority of CLL patients. NA should be routinely used as second line treatment and possibly as first line therapy in younger patients, who are candidates for potentially curative treatment such as stem cell transplantation and/or monoclonal antibodies.