A Comprehensive In Vivo and In Vitro Assessment of the Drug Interaction Potential of Red Ginseng.

Purpose: Red ginseng is one of the world's most popular herbal medicines; it exhibits a wide range of pharmacologic activities and is often co-ingested with other herbal and conventional medicines. This open-label, randomized, 3-period study investigated the in vivo herb-drug interaction potential for red ginseng extract with cytochrome P-450 (CYP) enzymes and organic anion-transporting polypeptide (OATP) 1B1. METHODS: Fifteen healthy male volunteers (22-28 years; 57.1-80.8 kg) were administered a single dose of cocktail probe substrates (caffeine 100 mg, losartan 50 mg, omeprazole 20 mg, dextromethorphan 30 mg, midazolam 2 mg, and pitavastatin 2 mg) and single or multiple doses of red ginseng extract for 15 days. FINDINGS: The pharmacokinetic profiles of the probe substrates and metabolites after single- or multiple-dose administration of red ginseng extracts were comparable to the corresponding profiles of the control group. The geometric mean ratio of AUC0-t and 90% CIs for the probe substrate drugs between the control and multiple doses of red ginseng for 15 days were within 0.8 to 1.25 (CYP2C9, CYP3A4, and OATP1B1 probe substrates) or slightly higher (CYP1A2, CYP2C19, and CYP2D6 probe substrates). Additional assessments of the in vitro drug interaction potential of red ginseng extracts and the ginsenoside Rb1 on drug-metabolizing enzymes and transporters using human liver microsomes, cryopreserved human hepatocytes, and transporter-overexpressed cells were negative. IMPLICATIONS: Red ginseng poses minimal risks for clinically relevant CYP- or OATP-mediated drug interactions and is well tolerated. Clinical Research Information Service registry no.

Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human liver drug transporters.

17alpha-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-K(m) (Michaelis constant) model (K(m1) = 220 nM; K(m2) = 15.5 microM). Uptake was inhibited (approximately 90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na(+)/taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two K(m) values (87 nM and 141 microM), whereas OATP2B1- and NTCP-mediated uptake into HEK-293 cells conformed to single K(m) kinetics (10.7 and 2.6 microM, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression P-glycoprotein. Only vesicles that contained BCRP exhibited ATP-dependent uptake of EE2-Sul (K(m1) = 2.9 and K(m2) = 307 microM). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.

Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human kidney drug transporters.

17alpha-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-K(m) (Michaelis constant) kinetics (K(m) = 21.1 microM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-K(m) model (K(m1) = 1.6 microM; K(m2) = 195 microM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.

Current industrial practices of assessing permeability and P-glycoprotein interaction.

Combination of the in vitro models that are high throughput but less predictive and the in vivo models that are low throughput but more predictive is used effectively to evaluate the intestinal permeability and transport characteristics of a large number of drug candidates during lead selection and lead optimization processes. Parallel artificial membrane permeability assay and Caco-2 cells are the most frequently used in vitro models to assess intestinal permeability. The popularity of these models stems from their potential for high throughput, cost effectiveness, and adequate predictability of absorption potential in humans. However, several caveats associated with these models (eg, poor predictability for transporter-mediated and paracellularly absorbed compounds, significant nonspecific binding to cells/devices leading to poor recovery, variability associated with experimental factors) need to be considered carefully to realize their full potential. P-glycoprotein, among other pharmaceutically relevant transporters, has been well demonstrated to be the major determinant of drug disposition. The review article presents an objective analysis of the permeability and transporter models currently being used in the pharmaceutical industry and could help guide the discovery scientists in implementing these models in an optimal fashion.