Analysis of bioconversion from apogossypol hexaacetate to apogossypol, a novel proapoptotic compound
Authors: Lori Coward, Corenna Kerstner-Wood, Pat Noker, Greg Gorman, Barbara Becattini, Maurizio Pellecchia, John Reed, Lee Jia
Journal: Cancer Research (2005)

2259   Overexpression of anti-apoptotic Bcl-2-family proteins Bcl-2 or Bcl-X L  occurs during apoptosis dysregulation in many malignancies, resulting in resistance to cytotoxic anticancer drugs. Recent studies showed that Bcl-2 and Bcl-X L  are targets of (±)Gossypol (Kitada, et al.  J Med Chem  46: 4258, 2003), which has then tested for treatment of metastatic adrenal cancer, glioblastoma and breast cancer with low but measurable responses. (-)Gossypol was also developed and showed improved efficacy and pharmacokinetic properties to some extent (Jia, et al. 2003 AACR-NCI-EORTC). It seems that the presence of two highly reactive aldehyde groups in Gossypol may be a limitation to its therapeutic successes. By using a combination of molecular modeling and structural biology techniques, Apogossypol (AG), which lacks the aldehydic groups and retains pro-apoptototic activity, has recently been synthesized and shown to bind to and inhibit the Bcl-2-family proteins, Bcl-2 and Bcl-X L , inducing apoptosis of the tumor cell lines (Becattini  et. al .,  Chem Biol.  11:389-9, 2004). However, due to the inherent instability of AG in the solid form, the compound has been synthesized as a hexaacetate prodrug form, a very stable compound both in solution and in solid form. Apogossypol hexaacetate (AGA) lacks apoptotic activity because the acetate groups hinder the binding of the compound to the Bcl-2 proteins. Therefore, the acetates must be cleaved for the molecule to exhibit activity  in vivo . In preparation for preclinical studies, a method was developed and validated for the analysis of AG in mouse plasma from mice dosed with AGA. The methodology involves protein precipitation of plasma samples followed by LC/MS/MS analysis. The validated concentration range extends from 10-2,000 ng/mL using 100 μL of mouse plasma, with extraction recoveries averaging 90%. The quantitation is based on internal standardization using apigenin as the internal standard. This method was used to determine the short-term stability of AG in mouse plasma stored at room temperature, on ice, and at 37 degrees C. AG was found to be stable for at least 1 hour at room temperature and 3 hours when stored on ice, however, it appeared degraded in plasma in less than 30 minutes at 37 °C. The stability of AG at 37 °C was increased by the addition 1% ascorbic acid to plasma. Incubation of AGA at 0.2, 1 and 5 μg/ml with mouse plasma for 30 min at 37 °C resulted in essentially complete conversion of AGA in AG (>100%). The conversion appears to be temperature-dependent because lowering temperatures delayed the conversion rate. The addition of 1% ascorbic acid to the incubation also delayed the conversion rate. The bioconversion was also demonstrated in mice after administration of AGA. These results suggest that AGA will be a useful prodrug form of apogossypol and may have considerable theoretical and practical significance (Supported by NCI Contract No. N01-CM-07110).