Almost of drugs in the market
Almost 75% of drugs in the market undergo deactivation though oxidation, reduction or hydrolysis by cytochrome P450 (CYP) enzymes, and then, either directly or by facilitated excretion from the body (Guengerich, 2008). The CYP3A subfamily is of particular interest because of its broad substrate specificity (van Waterschoot and Schinkel, 2011), and as it is notable that roughly 50% of drugs currently used are metabolized through CYP3A (Guengerich, 2008). In fact, induction or inhibition of the CYP3A by different drugs and/or chemicals may lead to treatment failure, or result in direct hepatic injury and DDI-mediated toxicity (Sun et al., 2015). Therefore, the U.S. Food and Drug Administration (FDA) have recommended that in vitro CYP-associated metabolic studies should be carried out to predict the potential DDI. Recently, we have reported that CuE affects the pharmacokinetics and pharmacodynamics of warfarin via the inhibition of CYP2C activity in rats (Ding et al., 2015). However, the studies on the CYP-mediated DDI of CuE are still limited. In particular, the effects of CuE on human CYP3A remain unknown.
P-glycoprotein (P-gp; MDR1; ABCB1) exists not only in the apical (luminal) membrane of many normal tissues but also in tumor cells, and acts as an efflux pump to xenobiotics, including drugs (Zhou, 2008). Activity of P-gp in the intestinal epithelium can restrict the uptake of substrates from the gut cavity, decreasing the splenic volume calculator of drugs, whereas in liver and kidney, it facilitates the excretion of substrates into bile and urine, respectively (van Waterschoot and Schinkel, 2011). In addition, P-gp is also expressed in the barrier tissues of the body, such as placenta, brain, and testis, to prevent the invasion of harmful substances and protect these organs. In contrast, P-gp expressed in cancer cells is thought to be the most prevalent transporter that effluxes the drug out of the cell, leading the generation of multiple drug resistance (MDR) phenomenon (Eichhorn and Efferth, 2012). Since CYP3A and P-gp share an extensive overlap between their substrates, and work synergistically in body system to limit the systemic exposure to many orally ingested drugs (van Waterschoot et al., 2009), modulation on CYP3A and P-gp activities may dramatically affect drug bioavailability and safety. Therefore, the effects of CuE on P-gp will affect the absorption and excretion of drugs, and then influence the bioavailability and drug safety. In particular, the inhibition of CYP3A and P-gp simultaneously may result in a severe toxicity of certain drugs, especially drugs with narrow therapy window (van Waterschoot et al., 2009, Zhuang et al., 2013).
The aim of this study was to comprehensively assess CuE related hepatotoxicity and potential drug-drug interactions involving CYP3A and P-gp. To evaluate the hepatotoxicity of CuE, we employed HepG2 cell model to predict the toxicity by MTS, sulforhodamine B (SRB), neutral red uptake (NRU) and apoptosis assays. To investigate the effects of CuE on CYP3A and P-gp, human and rat liver microsomes incubation system, Caco-2 transport model and 3D organoid model were used in vitro. In in vivo studies, we investigated the effects of CuE on the oral pharmacokinetics of indinavir, a substrate of both CYP3A and P-gp, in rats.
Materials and methods
Discussion CuE showed to possess a number of biological activities, but its hepatotoxicity remains unclear. Moreover, the widespread use of CuE-containing medicines has led to increasing concerns on potential drug-drug interactions through metabolic enzyme pathways. In this study, we firstly employed HepG2 cell model to investigate the hepatotoxicity of CuE. Then we assessed the effects of CuE on rat CYP3A and P-gp expression and activities both in vitro and in vivo. The present study used three cytotoxic assays including MTS, SRB and NRU to avoid underestimation or overestimation of the cytotoxicity of CuE on HepG2 cells. The results obtained from these assays presented well correlation one each other. At the same time, CuE showed much lower IC50 values than troglitazone (positive control) in three assays, suggesting the strong cytotoxicity on HepG2 cells. The mechanisms of toxic action may include direct cytotoxicity and inhibition of cell proliferation. Our present findings demonstrated that CuE presents direct cytotoxicity on HepG2 cells, although it also induces apoptosis. Previous studies evaluated the liver protective activity of cucurbitacins using HepG2 cells (Bartalis and Halaweish, 2011). For example, cucurbitacins B, D and E show a high protection (74%−83%), but they have low toxic-to-active dose, which means that most cucurbitacins present narrow dosage window from protective dose to toxic dose (Bartalis and Halaweish, 2011). Moreover, the IC50 value of CuE on HepG2 by MTT was reported at 15.25µM (Bartalis and Halaweish, 2011), which is close to our data (15.98 ± 4.33µM) by MTS. Furthermore, the EC50 value of protection function on HepG2 was only 3.20µM (Bartalis and Halaweish, 2011), showing a narrow protective concentration scope. Hence, it is worth to perform further studies to identify its mechanism of hepatotoxicity in humans.