A Monofunctional Derivative of Melphalan:  Preparation, DNA Alkylation Products, and Determination of the Specificity of Monoclonal Antibodies That Recognize Melphalan−DNA Adducts

Abstract

Bifunctional alkylating agents, such as those based on nitrogen mustard, form important parts of many anti-cancer chemotherapy protocols and are responsible for increased incidences of secondary tumors in successfully treated patients. These drugs generally form a majority of monofunctional DNA adducts, although the bifunctional adducts appear to be necessary for their powerful cytotoxic and antitumor effects. The relative importance of bifunctional as opposed to monofunctional adducts in the varied biological consequences of drug exposure has not been studied in detail, particularly in relation to the role and specificity of biochemical responses to therapy-related DNA damage. A simple method is described for the preparation of useful quantities of a pure monofunctional derivative of the nitrogen mustard-based drug melphalan. Monohydroxymelphalan was prepared by partial hydrolysis, purified by reversed phase chromatography, and characterized by MS, NMR, and HPLC. Contamination with melphalan was </=0.2%. The heat labile DNA base adducts formed by monohydroxymelphalan were shown to contain undetectable levels of cross-linked species. The ratio of adenine to guanine adducts was 0.62, similar to the equivalent ratio for melphalan. The sequence-dependent pattern of alkylation of purified DNA was indistinguishable from that of melphalan, but required a higher dose to achieve comparable extents of reaction. The specificities of two monoclonal antibodies that recognize melphalan-DNA adducts were investigated using DNA alkylated with [3H]monohydroxymelphalan. Adducts on this DNA showed similar immunoreactivities to adducts formed by melphalan. This shows clearly that neither antibody was specific for cross-linked adducts and that it is therefore possible to quantify adducts formed by both monohydroxymelphalan and melphalan with high sensitivities. The availability of monohydroxymelphalan in addition to melphalan, together with sensitive immunoassays for adducts on extracted DNA and in individual cells, constitutes a useful system for investigating cellular responses to the DNA modifications formed by a clinically relevant drug.

Repair of DNA interstrand crosslinks as a mechanism of clinical resistance to melphalan in multiple myeloma

“…However, no evidence of strand break formation was detected in any of the patients' unirradiated, melphalan-treated cells. Indeed, studies with mono-melphalan, which produces equivalent levels of monoalkylation to melphalan but is unable to form ICLs, 34 have not revealed any significant formation of single strand breaks under the conditions used in the Comet assay (data not shown). Drug-induced apoptosis after melphalan treatment is possible particularly following the 40 hours after incubation.…”

p53 Elevation in Relation to Levels and Cytotoxicity of Mono- and Bifunctional Melphalan-DNA Adducts

“…All cells were grown in HEPES-buffered RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with fetal calf serum [10% (v/v)], glutamine (300 mg/l), penicillin (50 U/ml), streptomycin (50 g/ml), and neomycin (100 g/ml) at 37°C in 5% CO 2 . Monohydroxymelphalan was prepared as described previously (Tilby et al, 1998). Solutions of melphalan (Sigma-Aldrich, St. Louis, MO) and monohydroxymelphalan were prepared in acidified ethanol (Tilby et al, 1993) immediately before use and then diluted into culture medium to give final ethanol concentrations of 1% (v/v) for controls and for all exposures to alkylating agents.…”

“…DNA extraction and competitive ELISA methods using monoclonal antibody MP5/73 were performed as described elsewhere (Tilby et al, 1987(Tilby et al, , 1991(Tilby et al, , 1993. This technique shows equal sensitivity for DNA adducts formed by melphalan and monohydroxymelphalan (Tilby et al, 1998); nevertheless, independent standards of DNA alkylated with the appropriate radioactively labeled alkylating agent were included in every assay.…”

“…2), that is free of contamination with the bifunctional compound (Tilby et al, 1998). The DNA adducts formed by monohydroxymelphalan were compared with those formed by melphalan and were shown to be identical in nature and DNA sequence-related distribution, except that, as predicted, cross-linked products (guanine-guanine and guanine-adenine) were not formed after the monohydroxymelphalan treatment (Tilby et al, 1998). We have also described a sensitive immunoassay for DNA adducts induced by melphalan (Tilby et al, 1987).…”

“…We have also described a sensitive immunoassay for DNA adducts induced by melphalan (Tilby et al, 1987). This was shown to be applicable to clinical specimens (Tilby et al, 1993) and, with equal sensitivity, to adducts formed by monohydroxymelphalan (Tilby et al, 1998). These tools permit a detailed comparison of the cellular effects of chemically equivalent mono-and bifunctional adducts formed by a drug that is representative of, and relevant to, a large number of chemically and mechanistically related drugs, including agents currently being developed for targeted therapies (Melton et al, 1996).…”