Strong and Selective Inhibitory Effects of the Biflavonoid Selamariscina A against CYP2C8 and CYP2C9 Enzyme Activities in Human Liver Microsomes.

Like flavonoids, biflavonoids, dimeric flavonoids, and polyphenolic plant secondary metabolites have antioxidant, antibacterial, antiviral, anti-inflammatory, and anti-cancer properties. However, there is limited data on their effects on cytochrome P450 (P450) and uridine 5'-diphosphoglucuronosyl transferase (UGT) enzyme activities. In this study we evaluate the inhibitory potential of five biflavonoids against nine P450 activities (P450s1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A) in human liver microsomes (HLMs) using cocktail incubation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The most strongly inhibited P450 activity was CYP2C8-mediated amodiaquine N-dealkylation with IC50 ranges of 0.019~0.123 µM. In addition, the biflavonoids-selamariscina A, amentoflavone, robustaflavone, cupressuflavone, and taiwaniaflavone-noncompetitively inhibited CYP2C8 activity with respective Ki values of 0.018, 0.083, 0.084, 0.103, and 0.142 µM. As selamariscina A showed the strongest effects, we then evaluated it against six UGT isoforms, where it showed weaker inhibition (UGTs1A1, 1A3, 1A4, 1A6, 1A9, and 2B7, IC50 1.7 µM). Returning to the P450 activities, selamariscina A inhibited CYP2C9-mediated diclofenac hydroxylation and tolbutamide hydroxylation with respective Ki values of 0.032 and 0.065 µM in a competitive and noncompetitive manner. However, it only weakly inhibited CYP1A2, CYP2B6, and CYP3A with respective Ki values of 3.1, 7.9, and 4.5 µM. We conclude that selamariscina A has selective and strong inhibitory effects on the CYP2C8 and CYP2C9 isoforms. This information might be useful in predicting herb-drug interaction potential between biflavonoids and co-administered drugs mainly metabolized by CYP2C8 and CYP2C9. In addition, selamariscina A might be used as a strong CYP2C8 and CYP2C9 inhibitor in P450 reaction-phenotyping studies to identify drug-metabolizing enzymes responsible for the metabolism of new chemicals.

The inhibitory potential of Broussochalcone A for the human cytochrome P450 2J2 isoform and its anti-cancer effects via FOXO3 activation.

BACKGROUND: Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone compound isolated from the cortex of Broussonetia papyrifera (L.) Ventenat, exhibits several biological activities including potent anti-oxidant, antiplatelet, and cytotoxic effects. PURPOSE: The purpose of this study is to elucidate the inhibitory effect of BCA against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. STUDY DESIGN: The inhibitory effect of BCA on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its anti-cancer effect against human hepatoma HepG2 cells was also evaluated. METHODS: Two representative CYP2J2-specific probe substrates, astemizole and ebastine, were incubated in HLMs with BCA. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. To investigate the binding model between BCA and CYP2J2, we carried out structure-based docking simulations by using software and scripts written in-house. RESULTS: BCA inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities in a concentration dependent manner with Ki values of 2.3 and 3.7 µM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner with activation of apoptosis related proteins. CONCLUSION: Overall, this was the first report of the inhibitory effects of BCA on CYP2J2 in HLMs. The present data suggest that BCA is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities.

Acetylshikonin is a novel non-selective cytochrome P450 inhibitor.

Acetylshikonin is a biologically active compound with anti-cancer and anti-inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma. An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC50 values of 1.4-4.0 µ m. Pre-incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC50 values of 2.5, 3.6 and 0.5 µ m, respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF-525A.

Inhibitory Effect of Selaginellins from Selaginella tamariscina (Beauv.) Spring against Cytochrome P450 and Uridine 5'-Diphosphoglucuronosyltransferase Isoforms on Human Liver Microsomes.

Selaginella tamariscina (Beauv.) has been used for traditional herbal medicine for treatment of cancer, hepatitis, and diabetes in the Orient. Numerous bioactive compounds including alkaloids, flavonoids, lignans, and selaginellins have been identified in this medicinal plant. Among them, selaginellins having a quinone methide unit and an alkylphenol moiety have been known to possess anticancer, antidiabetic, and neuroprotective activity. Although there have been studies on the biological activities of selaginellins, their modulatory potential of cytochrome P450 (P450) and uridine 5'-diphosphoglucuronosyltransferase (UGT) activities have not been previously evaluated. In this study, we investigated the drug interaction potential of two selaginellins on ten P450 isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and 3A) and six UGT isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) using human liver microsomes and liquid chromatography-tandem mass spectrometry. Selaginellin and selaginellin M had high inhibitory potential for CYP2C8-mediated amodiaquine O-demethylation with IC50 values of 0.5 and 0.9 µM, respectively. Selaginellin and selaginellin M also showed medium inhibitory potential against CYP2C9, CYP2J2, UGT1A1, and UGT1A3 (1 µM < IC50 < 5 µM). These two selaginellins had low inhibitory potential against CYP1A2, CYP2A6, CYP2E1, and UGT1A6 (IC50 > 25 µM). This information might be helpful to predict possible drug interaction potential of between selaginellins and co-administered drugs.

LKY-047: First Selective Inhibitor of Cytochrome P450 2J2.

Highly selective cytochrome P450 CYP2J2 (CYP2J2) inhibitors suitable for reaction phenotyping are currently not available. (7S)-(+)-(4-Nitro-phenyl)-acrylic acid, 8,8-dimethyl-2-oxo-6,7-dihydro-2H,8H-pyrano[3,2-g]chromen-7-yl-ester (LKY-047), a decursin derivative, was synthesized, and its inhibitor potencies toward CYP2J2 as well as other cytochrome P450 (P450) enzymes in human liver microsomes (HLM) were evaluated. LKY-047 was demonstrated to be a strong competitive inhibitor of CYP2J2-mediated astemizole O-demethylase and terfenadine hydroxylase activity, with Ki values of 0.96 and 2.61 µM, respectively. It also acted as an uncompetitive inhibitor of CYP2J2-mediated ebastine hydroxylation with a Ki value of 3.61 µM. Preincubation of LKY-047 with HLMs and NADPH did not alter inhibition potency, indicating that it is not a mechanism-based inhibitor. LKY-047 was found to be a selective CYP2J2 inhibitor with no inhibitory effect on other human P450s, such as CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A (IC50 > 50 µM). These in vitro data support the use of LKY-047 as a selective CYP2J2 inhibitor with potential application in the identification of P450 isoforms responsible for drug metabolism in reaction phenotyping assays.

Identification of acetylshikonin as the novel CYP2J2 inhibitor with anti-cancer activity in HepG2 cells.

BACKGROUND: Acetylshikonin is one of the biologically active compounds derived from the root of Lithospermum erythrorhizon, a medicinal plant with anti-cancer and anti-inflammation activity. Although there have been a few previous reports demonstrating that acetylshikonin exerts anti-cancer activity in vitro and in vivo, it is still not clear what is the exact molecular target protein of acetylshikonin in cancer cells. PURPOSE: The purpose of this study is to evaluate the inhibitory effect of acetylshikonin against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. STUDY DESIGN: The inhibitory effect of acetylshikonin on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its cytotoxicity against human hepatoma HepG2 cells was also evaluated. METHOD: Astemizole, a representative CYP2J2 probe substrate, was incubated in HLMs in the presence or absence of acetylshikonin. After incubation, the samples were analyzed by liquid chromatography and triple quadrupole mass spectrometry. The anti-cancer activity of acetylshikonin was evaluated on human hepatocellular carcinoma HepG2 cells. WST-1, cell counting, and colony formation assays were further adopted for the estimation of the growth rate of HepG2 cells treated with acetylshikonin. RESULTS: Acetylshikonin inhibited CYP2J2-mediated astemizole O-demethylation activity (Ki = 2.1µM) in a noncompetitive manner. The noncompetitive inhibitory effect of acetylshikonin on CYP2J2 enzyme was also demonstrated using this 3D structure, which showed different binding location of astemizole and acetylshikonin in CYP2J2 model. It showed cytotoxic effects against human hepatoma HepG2 cells (IC50 = 2µM). In addition, acetylshikonin treatment inhibited growth of human hepatocellular carcinoma HepG2 cells leading to apoptosis accompanied with p53, bax, and caspase3 activation as well as bcl2 down-regulation. CONCLUSION: Taken together, our present study elucidates acetylshikonin displays the inhibitory effects against CYP2J2 in HLMs and anti-cancer activity in human hepatocellular carcinoma HepG2 cells.

Screening of non-steroidal anti-inflammatory drugs for inhibitory effects on the activities of six UDP-glucuronosyltransferases (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) using LC-MS/MS.

Non-steroidal anti-inflammatory drugs (NSAIDs) are used widely to relieve pain and to decrease inflammation. Several clinical studies have reported that NSAIDs inhibit uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. Therefore, the study evaluated the inhibitory potential of 15 NSAIDs on the activities of six UGT isoforms (i.e. UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes (HLMs). Among the 15 NSAIDs tested here, mefenamic acid and diclofenac inhibited all UGTs tested in this study. Piroxicam and niflumic acid inhibited UGT1A9 activity (IC50 = 73.8 µm and 0.38 µm, respectively) and naproxen selectively inhibited UGT2B7 activity (IC50 = 53.1 µm), whereas it did not inhibit the other UGTs tested (IC50 > 200 µm). Diflunisal inhibited the UGT1A1 (IC50 = 33.0 µm) and UGT1A9 (IC50 = 19.4 µm). Acetaminophen, fenoprofen, ibuprofen, ketoprofen, meloxicam, phenylbutazone, salicylic acid and sulindac showed negligible inhibitory effects on the six UGTs (IC50 > 100 µm). These results suggest that some NSAIDs have the potential to inhibit UGTs in vitro.

Escherichia coli as reservoir for macrolide resistance genes.

The plasmid-borne mph(A) gene that confers resistance to azithromycin and has recently emerged in Shigella sonnei is present in multidrug- and non-multidrug-resistant Escherichia coli isolates from 4 continents. Further spread of mph(A) to Shigella and Salmonella spp. may be expected.