Stability Studies of Rooperol and Analogues by In Vitro Metabolism With HPLC/MS Detection




Bohanon, Amanda

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African Potato, Hypoxis hemerocallidea, has a long history of use by the indigenous people of South Africa to treat cancer and a variety of other diseases. Extracts from the corm contain the norligan bisglycoside hypoxoside, which is hydrolyzed in the body to yield the anticancer agent rooperol (1,5-bis(3’,4’-dihydroxyphenyl)pent-1-en-4- yne). Studies have shown that rooperol selectively inhibits the growth of several cancer cell lines. Phase I clinical trials of rooperol in lung cancer patients suggested promising activity with no dose-limiting toxicity. However, rooperol is rapidly converted to biologically inactive sulfate and glucuronide metabolites. Thus, a relatively small amount of the drug reaches the tumor site. Bioisosteric analogues of rooperol have been synthesized with the aim of increasing metabolic stability while preserving the anticancer properties of the parent drug. The goal of this research is to investigate the metabolic stability of rooperol and analogues. As part of this study, we employ an in vitro metabolism assay with porcine liver microsomes. Microsomes are supplemented with the cofactor UDP-glucuronic acid and the pore-forming peptide alamethicin. The suitability of this assay to characterize the Phase II metabolism of phenolic compounds was established with the plant phytochemical 3-hydroxytyrosol. The time-dependent metabolism of this compound was determined by HPLC assay, and the formation of the glucuronide Phase II metabolite was confirmed by HPLC/MS. The time course for disappearance of 3-hydroxytyrosol followed first-order kinetics with an apparent half-life of around 61 minutes. We have recently employed this assay to quantify the metabolic lability of rooperol. The disappearance of rooperol monitored by HPLC revealed an exceptionally short half-life of about 3 minutes. The identities of monoglucuronide and diglucuronide metabolites of rooperol were verified by HPLC/MS. The extreme metabolic lability of rooperol demonstrates the need to identify more metabolically stable analogues. We will discuss the integration of this in vitro metabolism assay in a work- flow designed to identify rooperol analogues exhibiting increased metabolic stability while retaining the cytotoxic activity of rooperol.



Rooperol, Hydroxytyrosol, Glucuronidation, In vitro, Metabolism, HPLC/MS


Bohanon, A. (2019). <i>Stability studies of rooperol and analogues by in vitro metabolism with HPLC/MS detection</i> (Unpublished thesis). Texas State University, San Marcos, Texas.


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