Mass spectrometry of intact complexes of benzyl styryl sulfone ON1910Na with albumin using direct serum injection

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The benzyl styryl sulfone analog (ON1910Na, 473.5 Da) is currently in Phase I clinical trials. The objective was to characterize the intact complexes formed between drug and serum albumin in treated mice and in patients. Molecular masses indicated the number of drug molecules in the complex at various dose levels and sampling times. Serum and plasma were obtained from treated mice with various solid tumors and patients. One μL aliquots were injected directly into the mass spectrometer. Positive electrospray mass spectra were obtained with a triple quadrupole instrument. Molecular masses of the intact drug-albumin complexes were obtained from multiple charged ion patterns, based on the interrupted incremental augmentation of albumin masses, using conventional transformation software. Unbound 1910Na was quantified by selected reaction monitoring (m/z 474 to 216 transition). Complex formation between the drug and albumin commenced immediately upon exposure. The smooth lines of the bell-shaped multiply charged ion patterns of serum albumin became ragged. The molecular masses of the intact complexes and the number of bound drug molecules could be determined from the new peaks. Masses were determined to 0.1%. Data from mouse and human samples were comparable but not the same. Up to 21 drug molecules were bound per albumin molecule depending upon drug dose, quantity of unbound drug, and time of sampling. The number of drug molecules added were discontinuous, suggesting conformational changes permitting certain numbers of drug molecules to bind but excluding others. The technique developed demonstrates that free drug concentration and drug-albumin binding data can be measured directly in untreated serum or plasma, thus avoiding iatrogenic artifacts. (Supported by the T. J. Martell Foundation for Leukemia, Cancer and AIDS Research and by Onconova Therapeutics, Inc.).