Curcumin, a dietary polyphenol, has been shown to have several preventive and therapeutic benefits in epidemiological studies. The chemopreventive potential of curcumin is related to its anti-inflammatory activity and is largely mediated through inhibition of the transcription factor NF-kB. However, curcumin has rather poor oral bioavailability (<1%). Much of orally dosed curcumin undergoes glucuronidation, resulting in the formation of inactive glucuronides. Thus, it is not clear how dietary curcumin exhibits chemopreventive activity despite not being absorbed into the systemic circulation in its active form. We proposed a prodrug hypothesis to explain this ‘bioavailability paradox’ of curcumin. β-glucuronidase is an enzyme that hydrolyzes the glycosidic bond of glucuronides. Previous studies have shown that the expression of this enzyme is elevated under inflammatory conditions and overexpressed in necrotic regions of tumors. Increased β-glucuronidase activity in the tumor tissue in comparison to its relatively minimal activity in normal cells potentially explains the bioavailability paradox with curcumin. We hypothesized that curcumin glucuronide is an inflammation-responsive natural prodrug that gets converted back to curcumin ‘on-demand’ at the site of action. Our studies were aimed at determining specific activity of β-glucuronidase based on mammary tumor type, stage, and model. β-glucuronidase activity was determined in mammary tumor tissues with HER-2+ (BALB-neuT, TuBo) and triple negative (4T1, JC, MDA-MB-231) phenotypes. Activity assay results showed that the highest rate of conversion was in 4T1 tumors as compared to the other tumor types. Immunohistochemistry (IHC) studies on primary human breast tumor tissue samples showed β-glucuronidase expression levels to be highest in HER-2+ type breast cancer compared to triple negative and ER/PR+ types. Also determined from the microarray of human tissue samples, as well as from Western blotting and fluorescence imaging studies, was the strong correlation between enzyme expression levels and stage of cancer: normal and benign stages showed the lowest levels of β-glucuronidase while the invasive/metastatic stage showed the highest expression levels. Using a self-microemulsifying drug delivery system (SMEDDS) formulation that was developed to improve the oral absorption, we aimed next to investigate the chemopreventive efficacy of curcumin following oral administration. Daily oral dosing of curcumin for one month in the orthotopic models described above showed that those with higher β-glucuronidase specific activities (JC, MDA-MB-231, and 4T1) benefitted the most from curcumin in limiting tumor growth rate. Pharmacokinetic studies with oral dosing of curcumin SMEDDS showed elevated levels of the glucuronide metabolite in plasma as compared to negligible levels of curcumin while the trend was reversed in the tumor tissue, providing further support to the prodrug activation hypothesis. The pharmacokinetics of curcumin glucuronide following intravenous dosing also confirmed conversion of the glucuronide to the parent compound in the tumor tissue. Overall, the work presented in this thesis demonstrated the potential of oral curcumin for breast cancer chemoprevention based on the enzymatic prodrug activation hypothesis.
University of Minnesota Ph.D. dissertation. November 2015. Major: Pharmaceutics. Advisor: Jayanth Panyam. 1 computer file (PDF); xxi, 188 pages.
Role of ß-Glucuronidase in the Chemopreventive Efficacy of Oral Curcumin: A Prodrug Hypothesis.
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