Identification and reactivity of the major metabolite (beta-1-glucuronide) of the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in humans.

1. The novel anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) is extensively metabolized by glucuronidation and 6-methylhydroxylation, resulting in DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA). 2. The major human urinary metabolite of DMXAA was isolated and purified by a solid-phase extraction (SPE) method. The isolated metabolite was hydrolysed to free DMXAA by strong base, and by beta-glucuronidase. Liquid chromatography-mass spectrometry (LC-MS) and spectral data indicated the presence of a molecular ion [M + 1]+ at m/z 459, which was consistent with the molecular weight of protonated DMXAA-G. 3. The glucuronide was unstable in buffer at physiological pH, plasma and blood with species variability in half-life. Hydrolysis and intramolecular migration were major degradation pathways. 4. In vitro and in vivo formation of DMXAA-protein adducts was observed. The formation of DMXAA-protein adducts in cancer patients receiving DMXAA was significantly correlated with plasma DMXAA-G concentration and maximum plasma DMXAA concentration. 5. At least five metabolites of DMXAA were observed in patient urine, with up to 60% of the total dose excreted as DMXAA-G, 5.5% as 6-OH-MXAA and 4.5% as the glucuronide of 6-OH-MXAA. 6. These data suggest that the major metabolite in patients' urine is DMXAA beta-1-glucuronide, which may undergo hydrolysis, molecular rearrangement and covalent binding to plasma protein. The reactive properties of DMXAA-G may have important implications for the pharmacokinetics, pharmacodynamics and toxicity of DMXAA.