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The objective was to characterize intact complexes of drugs with proteins, particularly human serum albumin and a variety of enzymes using mass spectrometry to calculate the number of bound drug molecules. Drugs studied: (a) Antineoplastic: tamoxifen (372 Da); doxorubicin (544 Da), methotrexate (454 Da), bleomycin (1416 Da), suramin (1429 Da) and the novel benzyl styryl sulfone analog ON 1910Na (474 Da); (b) Other Drugs: Digoxin (791 Da); fluphenazine (438 Da), and trifluoroperazin (408 Da). Proteins studied: albumin (66 kDa) and the enzymes: papain (21 kDa), elastase (25 kDa), trypsin (24 kDa) and carbonic anhydrase (29 kDa). Methodology: One µL aliquots of incubates (30 min, 37°C) or plasma sample from patients on Phase I clinical trials with ON 01910.Na were injected directly into the ion source of a triple quadrupole mass spectrometer. Positive electrospray or nanospray mass spectra were obtained and molecular masses of the intact drug-protein complexes were obtained from multiple charged ion patterns.

Results: (a) Complex formation between the drug and protein commenced immediately upon exposure. (b) Albumin-drug interactions yielded mixtures simultaneously containing complexes with a varying number of bound drug molecules, e.g., up to 21 molecules of tamoxifen per albumin molecule vs. only up to 8 molecules of digoxin per albumin; (c) The number of bound molecules for each drug was different for different enzymes, e.g., carbonic anhydrase attached 13 molecules of tamoxifen vs. 11 molecules of digoxin; (d) Drug binding followed an apparently discontinuous digital progression, e.g., 10, 11, 13, 17, 19, and 21 molecules of tamoxifen bound to albumin but not 12, 14, 15, 16, 18, or 20; (e) Similar unique phenomena occurred using several antineoplastic drugs as well as other drugs; and also using several different enzymes. We hypothesize that conformational changes caused by binding may account for the exclusion of certain binding complexes, and that conformational changes may influence protein behavior and enzymatic activity. (Supported by the T. J. Martell Foundation for Leukemia, Cancer and AIDS Research and by Onconova Therapeutics, Inc.)

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA