In vitro and in vivo efficacy and toxicity comparison between ApoGossypol and Gossypol, Bcl-2 antagonists

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Altered expression of Bcl-2-family proteins plays central roles in apoptosis dysregulation in cancer and leukemia, promoting malignant cell expansion and contributing to chemoresistance. In this study, we compared the toxicity and efficacy in mice of natural product (+)Gossypol and its semi-synthetic derivative (+)ApoGossypol and the purified (+) and (-) enantiomers of this compound, All of these compounds were shown to bind anti-apoptotic Bcl-2-family proteins with similar affinities, based on competitive BH3 peptide displacement assays and NMR-based chemical shift mapping experiments. Comparisons of growth inhibitory and cytotoxic activity of (+)Gossypol and (+)ApoGossypol using the NCI panel of 60 tumor cell lines, as well as cultured lymphoma cell lines and primary leukemia cells suggested that Gossypol and ApoGossypol have overlapping but non-identical cytotoxic mechanisms. Daily oral dosing studies showed that mice tolerate doses of ApoGossypol 2-4-times higher than Gossypol. Hepatotoxicity and gastrointestinal toxicity represented the major adverse activities of Gossypol, with ApoGossypol far less toxic. Efficacy was tested in transgenic mice in which Bcl-2 is over-expressed in B-cells, resembling low-grade follicular lymphoma in humans. In vitro, Bcl-2-expressing B-cells from transgenic mice were more sensitive to cytotoxicity induced by ApoGossypol than Gossypol, with LD50 values of 3-5 µM and 7.5-10 µM, respectively. In vivo, using the maximum tolerated dose of Gossypol (60 µmoles/kg) for sequential daily dosing (daily x 5 days x 3 weeks), ApoGossypol displayed superior activity to Gossypol in terms of reducing splenomegaly and reducing B-cell counts in spleens of Bcl-2 transgenic mice. At higher doses (120 µmole/kg), at which ApoGossypol but not Gossypol is tolerated, ApoGossypol showed even greater in vivo activity, reducing spleen weight by 40 ± 14% (p<0.001) and splenic B-cell counts by 66 ± 4% (p<0.0001). Preliminary studies of the (-)ApoGossypol and (+)ApoGossypol enantiomers suggested similar in vivo activity compared to racemic ApoGossypol. Taken together, these studies indicate that ApoGossypol is superior to parent compound Gossypol with respect to toxicology and efficacy, suggesting that further development of this compound for cancer therapy is warranted.