Phase I and II enzyme induction and inhibition by secoisolariciresinol diglucoside and it's aglycone
Boyd, Erin Margaret Rose
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The flaxseed lignan, secoisolariciresinol diglucoside (SDG), and its aglycone, secoisolariciresinol (SECO), have demonstrated benefits in the treatment and/or prevention of cancer, diabetes and cardiovascular disease. In order for the lignans to be used therapeutically, the safety of administration alone and in conjunction with other drugs must be determined. The primary cause of drug interactions is induction and inhibition of cytochrome P450 (CYP) and phase II enzymes. A preliminary screen was conducted to assess the potential for SECO and SDG to cause CYP inhibition. A method was established to assess for CYP, glutathione-S-transferase (GST) and uridine diphosphate-glucuronosyltransferase (UGT) induction in rat primary hepatocytes by real-time reverse transcription-polymerase chain reaction (RT-PCR).Preliminary assessments of inhibition measured the metabolism of testosterone to 6β-, 16α- and 2α-hydroxytestosterone, which corresponds to CYP3A, 2B/2C11 and 2C11 enzyme activity in rat hepatic microsomes by a validated high performance liquid chromatography (HPLC) method. Irreversible inhibition studies found that SDG is not an inhibitor of these isoforms up to 1000 μM. Secoisolariciresinol caused reversible inhibition of 6β-hydroxytestosterone at all testosterone concentrations, with an IC50 (inhibitor concentration causing 50% inhibition of enzyme) between 400 and 800 μM. Over the range of SECO concentrations tested, 10 – 1600 μM, 6β-hydroxytestosterone formation was reduced to 95 – 29% of control levels at 50 μM testosterone.Secoisolariciresinol caused a concentration-dependent increase in 16α-hydroxytestosterone formation at 50 μM testosterone. At 10 μM SECO, there was 90% of control activity, but at 1600 μM metabolite formation was 172% of control. The formation of 2α-hydroxytestosterone was not affected at any testosterone or inhibitor concentration. Thus, SECO appears to be a CYP3A inhibitor and a CYP2B activator at testosterone KM levels. The mechanism of reversible inhibition could not be determined due to the possibility of non-Michaelis-Menten kinetics observed with CYP3A inhibition and CYP2B activation. The gold standard in vitro model to assess induction is primary hepatocytes. A method was established that allowed for the isolation and culture of these cells. Positive controls caused induction of CYP mRNA levels after 24 hours treatment, demonstrating the ability of enzyme induction in the test system. Primers for real-time RT-PCR were designed that amplified CYP1A1, 1A2, 2B1, 2C11, 2C13, 2D1, 2D2, 3A1 and 3A2, GSTA2, A5 and P1, and UGT1A1, 1A7, 1A8, 2B1 and 2B12 genes. A preliminary assessment of transcriptional upregulation of drug metabolizing enzymes by SECO and SDG can be assessed in isolated and cultured rat primary hepatocytes.
DegreeMaster of Science (M.Sc.)
SupervisorKrol, Ed S.; Alcorn, Jane
CommitteeJanz, David M.; Blakley, Barry R.
Copyright DateApril 2007