Prediction of Biliary Efflux Clearance of Rosuvasatatin Using Efflux Transporter-Expressing Cell Lines and Quantitative Proteomics
Clearance refers to the capability by which substance is eliminated from body via metabolism or direct excretion[1]. The liver is the major metabolism and clearance organ in the body where drugs are removed from the blood circulation by metabolism and biliary excretion. For drugs which cannot passively transport, basolateral uptake is the first step in hepatic clearance of compounds from blood circulation which requires uptake transporters in the basolateral membrane, such as the sodium-dependent taurocholate cotransporting polypeptide (NTCP), the organic anion transporting polypeptides (OATPs), organic anion transporters (OATs), and organic cation transporters (OCTs)[2]. After the drug is taken up into the hepatocytes, the drug metabolized by the drug metabolizing enzymes, and parent compound or its metabolite(s) are back fluxed into the blood circulation by the efflux transporter such as multidrug resistance-associated protein (MRP) 3 and MRP4 expressed on the sinusoidal membrane or in bile canal by MRP2, breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), bile salt export pump (BSEP), and MATE1 expressed on the canalicular membrane [3,4,5]. Thus, uptake and efflux transporters can be very important in clearance of drugs.
Determination of the predominant clearance mechanism of drug candidates in human has increasingly gained importance during drug development since efficacy and safety are directly impacted by the pharmacokinetic behaviors of a drug[6]. However, predicting the hepatobiliary clearances, especially biliary efflux clearance, remains a challenge, because obtaining clinical bile samples is very difficult in practice, so determining in vivo biliary secretion in humans is highly dependent using in vitro models for predicting in vivo clearance, which is called in vitro-in vivo extrapolation (IVIVE)[7,8]. This method is a promising tool for prediction of the pharmacokinetics of clearance which cannot be easily observed in vivo. And it expands the predictive capabilities of PBPK modeling approaches for prediction of pharmacokinetics based on in vitro data to a wider range of compounds.
When it comes to scale up in vitro data to in vivo, there are three approaches have been used: empirical scaling factor, relative activity factor (RAF) and relative activity factor (REF)[9,10]. The empirical scaling factor is drug-dependent thus difficult to be generalized. The RAF can be used only when transporter-specific substances are available for use both in vitro and in vivo, which casts a limitation in some situations. In REF approach, intrinsic clearance of each transporter is calculated and is extrapolated based on relative protein expression in vivo compared to in vitro.
In general, several drug transporters expressed on the same membrane act to transport substrates in the same direction, with overlapping substrate specificities. In this study, our model drug is rosuvstatin (RSV) which can be transported by several transporters. Thus, membrane penetration of substrates is often achieved by multiple transporters. We consider it necessary to determine the contribution of each transporter to the hepatic transport of drugs because such information enables us to predict changes in hepatic intrinsic clearance when the expression of transporters are altered by genetic polymorphisms, disease, or drug-drug interactions. Therefore, we choose REF as our approach in IVIVE.
As for the in vitro model, sandwich-cultured hepatocytes (SCH) are thought to be a typical and effective in vitro model for transporter-mediated hepatobiliary clearance which is considered to maintain the cell polarity and membrane components [11]. It has many transporters and drug metabolizing enzymes which are working synergistically [12]. Therefore, it is difficult to figure out contribution of individual transporter due to absence of specific inhibitors for many transporters. Besides, the unknown expression transporters between the SCH and the liver and the change of transporter levels after culturing[13]. In addition, it is expensive, time-consuming and not suitable for high throughput analysis. Therefore, alternative approaches are needed.
We previously succeeded the prediction of the in vivo sinusoidal uptake clearance of rosuvastatin (RSV) in rats using transporter-overexpressing cell lines and the quantitative proteomics[14]. Therefore, the aim of the present study was to determine the in vivo biliary clearance of RSV can be predicted using the efflux transporter-overexpressing polarized cells. We selected RSV as a model substrate, because it is predominantly excreted into the bile [15,16]. In addition, BCRP and MRP2 (and probably P-gp) are involved in RSV efflux into the bile with unchanged drug form [17]. Thus, we use these efflux transporter-expressing cell lines determine intrinsic clearance of RSV by each transporter and targeted quantitative proteomics to quantified the abundance of transporter protein of in theses cell lines.
To sum up, our goal is to predict transporter-mediated hepatobiliary clearance of RSV using efflux transporter-expressing cell lines and quantitative proteomics. To achieve this goal, firstly, we should obtain efflux transporter (P-gp, BCRP, MRP2)-expressing cell lines. Then, calculate the clearance of transport of RSV of each individual efflux transporter and passive diffusion ratio using transwell transport assay. At the same time, determine the transporter expression using quantitative proteomics, then scale the in vitro efflux clearance to the in vivo using the protein expression data. In addition, we will confirm our predictions with data obtained from ongoing clinical study using positron emission tomography (PET) study to measure in vivo hepatobiliary clearance of RSV. Hopefully we can successfully predict hepatobiliary clearance of RSV thus providing a new approach to estimate hepatobiliary clearance. In this way, we only need to use in vitro model to predict the in vivo biliary clearance of drug, which will be more economical, time-saving.
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