Comparative Assessment of Extrapolation Methods Based on the Conventional Free Drug Hypothesis and Plasma Protein-Mediated Hepatic Uptake Theory for the Hepatic Clearance Predictions of Two Drugs Extensively Bound to Both the Albumin And Alpha-1-Acid Glycoprotein.

Bteich and coworkers recently demonstrated in a companion manuscript (J Pharm Sci 109: https://doi.org/10.1016/j.xphs.2020.07.003) that a protein-mediated hepatic uptake have occurred in an isolated perfused rat liver (IPRL) model for two drugs (Perampanel; PER and Fluoxetine; FLU) that bind extensively to the albumin (ALB) and alpha-1-acid glycoprotein (AGP). However, to our knowledge, there is no quantitative model available to predict the impact of a plasma protein-mediated hepatic uptake on the extent of hepatic clearance (CLh) for a drug binding extensively to these two proteins. Therefore, the main objective was to predict the corresponding CLh, which is an extension of the companion manuscript. The method consisted of extrapolating the intrinsic clearance from the unbound fraction measured in the perfusate or the unbound fraction extrapolated to the surface of the hepatocyte membrane by adapting an existing model of protein-mediated hepatic uptake (i.e., the fup-adjusted model) to include a binding ratio between the ALB and AGP. This new approach showed a relevant improvement compared to the free drug hypothesis particularly for FLU that showed the highest degree of ALB-mediated uptake. Overall, this study is a first step towards the development of predictive methods of CLh by considering the binding to ALB and AGP.

Impact of Extensive Plasma Protein Binding on the In Situ Hepatic Uptake and Clearance of Perampanel and Fluoxetine in Sprague Dawley Rats.

The main objective was to investigate the effect of albumin (ALB) and/or alpha-1-acid glycoprotein (AGP) on the hepatic clearance (CLh) of the drugs that bind, extensively, to both proteins. Isolated perfused livers from male Sprague Dawley rats (IPRL) were performed for perampanel (PER) and fluoxetine (FLU), using physiological solutions in four scenarios (n = 3 rats/scenario/drug): 1) without plasma proteins (WO), 2) with bovine ALB (40 g/L), 3) with bovine AGP (1 g/L), and 4) with mixture of both proteins (MIX). PER is poorly to moderately metabolized (hepatic extraction = 0.2-0.7), while FLU is highly metabolized (hepatic extraction = 0.8-0.99). The metabolic kinetics were fitted to the Michaelis-Menten model. For the PER, the parameters were Vmax = 90, 16.4, 86.1 and 16.9 (nmol/min/g liver) and unbound Km = 17, 1.7, 38.3 and 1.4 (µM) for the scenarios WO, with ALB, with AGP and with MIX, respectively. As for FLU, the parameters were Vmax = 65.5, 18.5, 33.8 and 12.2 (nmol/min/g liver) and unbound Km = 1.5, 0.03, 0.14 and 0.0466.31 (µM) in all four scenarios, respectively. In conclusion, a protein-mediated hepatic uptake likely occurred only at low concentrations for both drugs (i.e., therapeutic concentrations) in the presence of plasma proteins (except for the scenario of PER with AGP).

An overview of albumin and alpha-1-acid glycoprotein main characteristics: highlighting the roles of amino acids in binding kinetics and molecular interactions.

Although Albumin (ALB) and alpha-1-acid glycoprotein (AGP) have distinctive structural and functional characteristics, they both play a key role in binding a large variety of endogenous and exogenous ligands. An extensive binding to these plasma proteins could have a potential impact on drugs disposition (e.g. bioavailability, distribution and clearance), on their innocuity and their efficacy. This review summarizes the common knowledge about the structural and molecular characteristics of both ALB and AGP in humans, and about the most involved amino acids in their high-affinity binding pockets. However, the variability in residues found in binding pockets, for the same species, allows each plasma protein to interact differently with the ligands. The protein-ligand interaction influences differently the disposition of drugs that bind to either of these plasma proteins. The content of this review is useful for the design of new drug entities with high-binding characteristics, in qualitative and quantitative modelling (e.g. in vitro-in vivo extrapolations, 3D molecular docking, interspecies extrapolations), and for other interdisciplinary research.

The potential protein-mediated hepatic uptake: discussion on the molecular interactions between albumin and the hepatocyte cell surface and their implications for the in vitro-to-in vivo extrapolations of hepatic clearance of drugs.

Introduction: In quantitative modeling, the resolving of underpredictions and overpredictions of hepatic clearance (CLh) makes a top priority for pharmacokinetic modelers. Clearly, the 'protein-mediated hepatic uptake' is a violation of 'the free drug hypothesis', but the lack of its consideration in CLh-predictive approaches may be one of the reasons to explain the discrepancies between predicted and observed values. Areas covered: We first review the two 'albumin-facilitated hepatic uptake' models that were recently challenged to improve the in vitro-to-in vivo extrapolation (IVIVE) of CLh by reducing the underprediction bias, particularly in the absence of albumin (ALB) in vitro compared to the presence of ALB in vivo. Second, we identify three types of interactions related to the ALB-bound drug moiety (i.e., ALB-lipids, ALB-proteins, and ALB-ligand allosteric interactions) that may be behind the 'ALB-mediated hepatic uptake' mechanism(s) for highly bound drugs. Main keywords used in our search are IVIVE; albumin; allostery; protein-mediated uptake; hepatic clearance; polarized hepatocytes. Expert opinion: Understanding the implication of these interactions and the enzyme/transporter interplay for each drug would help selecting the appropriate IVIVE model. Therefore, we have proposed a tree of decision for guidance. The next step is to improve the 'ALB-facilitated hepatic uptake' models to cover the remaining uncertainties.

Predictions of bisphenol A hepatic clearance in the isolated perfused rat liver (IPRL): impact of albumin binding and of co-administration with naproxen.

1. This study aimed (i) to characterise hepatic clearance (CL) of bisphenol A (BPA) and naproxen (NAP) administered alone or in binary mixtures to highlight the influence of a binding to albumin (ALB) using an isolated perfused rat liver (IPRL) system; and (ii) to compare results of prediction algorithms with measured clearance rates. 2. The IPRL system and liver microsomes were used to determine the metabolic constants of BPA and NAP either in the presence or absence of ALB. In this study, the IPRL was used as proxy for the in vivo situation. Accordingly, diverse in vitro-to-in vivo and in vivo-to-in vivo extrapolations (IVIVEs) were made to predict CL of BPA determined in situ/in vivo with ALB from metabolic data determined without ALB by using different binding correction methods (i.e., direct and conventional scaling as well as a novel scaling considering an ALB-facilitated uptake mechanism). 3. The addition of ALB significantly influenced the liver kinetics of BPA and NAP either administered alone or in binary mixtures, which was reflected in the Michaelis-Menten constants. Analysis of concomitant exposures of BPA and NAP gave a fully competitive inhibition. Furthermore, the IVIVE method based on the ALB-facilitated uptake mechanism provided the most accurate predictions of CLin vivo as compared with the other IVIVE methods when the impact of ALB is considered. 4. Our findings support the notion that high binding to ALB reduces the biotransformation of BPA and NAP when administered alone or in mixtures in the IPRL system. However, the free drug concentration in liver in vivo is probably higher than expected since the IVIVE method based on a potential ALB-facilitated uptake mechanism is the most robust prediction method. Overall, this study should improve the physiologically-based pharmacokinetic (PBPK) modelling of chemical-drug interactions.

Supplemental Analysis of the Prediction of Hepatic Clearance of Binary Mixtures of Bisphenol A and Naproxen Determined in an Isolated Perfused Rat Liver Model to Promote the Understanding of Potential Albumin-Facilitated Hepatic Uptake Mechanism.

The hepatic clearance (CL) of bisphenol A (BPA) in the isolated perfused rat liver (IPRL) model has been studied for the impact of albumin (ALB) binding and coadministration with naproxen (NAP) in a companion manuscript (Bounakta et al. Xenobiotica. 2017;3:1-13.). We reported that the extrapolations of hepatic CL of BPA, NAP, and the binary mixtures between 2 ALB concentrations did not follow the free drug hypothesis; however, potential ALB-facilitated hepatic uptake mechanism(s) were highly suspected. Therefore, the objective of the present study was to reanalyze the IPRL data to provide a deeper quantitative extrapolation of CL; however, the focus was made on the impact of ALB binding on the intrinsic clearance (CLint) versus unbound CLint instead of only the global hepatic CL to verify whether the concept of ALB-facilitated hepatic uptake still holds for these 2 additional parameters for binary mixtures. Firstly, the variations in CLint that were observed between the IPRL model using no ALB and ALB in the perfusates were compared to the corresponding variations in the unbound fraction measured in the perfusates (fup) according to the free drug hypothesis, or to the variations in the fup values adjusted for potential ALB-facilitated uptake mechanism (i.e., fup-adjusted). The parameter fup-adjusted showed a greater predictability compared to fup (average fold error ∼ 1 vs. 5.2), suggesting that both the free and bound drug moieties should be available for hepatic uptake. Secondly, the supplemental data analysis showed a greater decrease in unbound Km than in Vmax resulting in increased uptake CLint of the unbound drug (Vmax/unbound Km) with increased ALB concentration at a given substrate concentration, which is compatible with an ALB-facilitated hepatic uptake mechanism. Interestingly, the unbound CLint increased by a factor that corresponds to the bound drug moiety also assumed available for hepatic uptake. These additional findings corroborate the recent literature. Overall, this study showed the importance of quantifying any differential of ALB concentration (in vitro vs. in vivo or hypoalbuminemia in vivo vs. hyperalbuminemia in vivo) in the IPRL-based, in vitro-to-in vivo or in vivo-to-in vivo extrapolation-based or physiologically based pharmacokinetics-based CL prediction of chemical-drug interactions between xenobiotics significantly bound to ALB.