Determination of Intracellular Unbound Concentrations and Subcellular Localization of Drugs in Rat Sandwich-Cultured Hepatocytes Compared with Liver Tissue

Pfeifer, Nathan D.; Harris, Kevin B; Yan, Grace; Brouwer, Kim L. R.

doi:10.1124/dmd.113.052134

Cited by 35 publications

(61 citation statements)

References 51 publications

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“…It is worth noting that quantification of intracellular concentration of a drug is, in general, a difficult task. It is well known that nonspecific binding is present in many hepatocyte incubations (Witherow and Houston, 1999;Austin et al, 2005;Lu et al, 2006;Chu et al, 2013;Pfeifer et al, 2013). In addition, the presence of Matrigel in the SCH may hinder the total removal of extracellular TP, leading to an overestimation of intracellular concentrations.…”

Section: Groupsmentioning

confidence: 99%

Assessment of the Roles of P-glycoprotein and Cytochrome P450 in Triptolide-induced Liver Toxicity in Sandwich-Cultured Rat Hepatocyte Model

et al. 2013

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Triptolide (TP), a main bioactive component of Tripterygium wilfordii Hook F., is a promising agent for treatment of autoimmune diseases. However, a high incidence of dose-limiting hepatotoxicity was observed in the clinic. Sandwich-cultured rat hepatocyte model was used in this study to identify the involvement of P-glycoprotein (P-gp) in TP disposition and to evaluate TP-induced hepatotoxicity after modulation of P-gp by the known inhibitors, ritonavir and tariquidar, and known inducers, phenobarbital, quercetin, and H 2 O 2 . Our data showed that biliary clearance of TP reduced 73.7% and 84.2% upon treatment of ritonavir (25 mM) and tariquidar (5 mM), respectively. In contrast, increases of 346%, 280%, and 273% in biliary clearance of TP were observed with treatment of phenobarbital (1.0 mM), quercetin (20 mM), and H 2 O 2 (0.5 mM), respectively. The TP-induced hepatotoxicity increased by twofold when CYP activity was blocked by 1-aminobenzotriazole, suggesting that CYP and P-gp may both contribute to the detoxification of TP in the SCRH model. In addition, hepatotoxicity and the expression of apoptosis proteins Bax and Bcl-2 were correlated qualitatively with the TP exposure duration and its intracellular concentration, which, in turn, can be modulated by P-gp inhibitors or inducers. Our results for the first time demonstrated that in addition to CYP-mediated metabolism, P-gp also plays an important role in the disposition of TP and TP-induced hepatotoxicity. Thus, the modulation of canalicular P-gp has a potential to cause drug-drug interaction between TP and the coadministered P-gp inhibitors or inducers in the clinic.

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Section: Groupsmentioning

confidence: 99%

Assessment of the Roles of P-glycoprotein and Cytochrome P450 in Triptolide-induced Liver Toxicity in Sandwich-Cultured Rat Hepatocyte Model

et al. 2013

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“…However, accurate model predictions of the experimental C out , total liver concentrations, and cumulative biliary ATV amounts increase confidence in the prediction of unbound intracellular concentrations in this study. Organelle binding of drugs to calculate unbound intracellular concentrations has been reported for ritonavir, rosuvastatin (Pfeifer et al, 2013), and metformin (Chien et al, 2016). The complex interplay between uptake, metabolism, and efflux and its effect on unbound intracellular concentrations can be addressed with a number of approaches (e.g., sandwich cultured hepatocytes) (Pfeifer et al, 2013).…”

Section: Intracellular Unbound Atorvastatin Concentrationsmentioning

confidence: 99%

“…Organelle binding of drugs to calculate unbound intracellular concentrations has been reported for ritonavir, rosuvastatin (Pfeifer et al, 2013), and metformin (Chien et al, 2016). The complex interplay between uptake, metabolism, and efflux and its effect on unbound intracellular concentrations can be addressed with a number of approaches (e.g., sandwich cultured hepatocytes) (Pfeifer et al, 2013). Our approach uses a simple compartmental model parameterized with complex experimental data from the liver perfusion studies.…”

Section: Intracellular Unbound Atorvastatin Concentrationsmentioning

confidence: 99%

“…Current methods to indirectly determine intracellular free drug concentrations include microdialysis, fluorescence imaging, tomography imaging, capillary electrophoresis, equilibrium dialysis of tissues, microscopic imaging and particle induced photon emission (Chu et al, 2013), secondary ion MS (Dollery, 2013), and differential centrifugation in sandwich-cultured hepatocytes (Pfeifer et al, 2013). Most of these techniques have practical or financial limitations.…”

Section: Introductionmentioning

confidence: 99%

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Intracellular Unbound Atorvastatin Concentrations in the Presence of Metabolism and Transport

Kulkarni¹,

Korzekwa²,

Nagar³

2016

Journal of Pharmacology and Experimental Therapeutics

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Accurate prediction of drug target activity and rational dosing regimen design require knowledge of drug concentrations at the target. It is important to understand the impact of processes such as membrane permeability, partitioning, and active transport on intracellular drug concentrations. The present study aimed to predict intracellular unbound atorvastatin concentrations and characterize the effect of enzyme-transporter interplay on these concentrations. Single-pass liver perfusion studies were conducted in rats using atorvastatin (ATV, 1 mM) alone at 4°C and at 37°C in presence of rifampin (RIF, 20 mM) and 1-aminobenzotriazole (ABT, 1 mM), separately and in combination. The unbound intracellular ATV concentration was predicted with a five-compartment explicit membrane model using the parameterized diffusional influx clearance, active basolateral uptake clearance, and metabolic clearance.Chemical inhibition of uptake and metabolism at 37°C proved to be better controls relative to studies at 4°C. The predicted unbound intracellular concentration at the end of the 50-minute perfusion in the 1ABT , 1ABT1RIF, and the ATV-only groups was 6.5 mM, 0.58 mM, and 5.14 mM, respectively. The predicted total liver concentrations and amount recovered in bile were within 0.94-1.3 fold of the observed value in all groups. The fold difference in total liver concentration did not always extrapolate to the fold difference in predicted unbound concentration across groups. Together, these results support the use of compartmental modeling to predict intracellular concentrations in dynamic organ-based systems. These predictions can provide insight into the role of uptake transporters and metabolizing enzymes in determining drug tissue concentrations.

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“…Some high-throughput methods have been used to measure cellular total and unbound inhibitor concentrations ([I] t,cell and [I] u,cell , respectively) (Mateus et al, 2013). However, the isolation of cytosol and measurement of cytosolic total and unbound inhibitor concentrations ([I] t,cyt and [I] u,cyt , respectively) add complexity (Pfeifer et al, 2013b). Thus, [I] t,cyt or [I] u,cyt has not been adopted routinely into the prediction of efflux transporterbased drug interactions.…”

Section: Introductionmentioning

confidence: 99%

Prediction of Altered Bile Acid Disposition Due to Inhibition of Multiple Transporters: An Integrated Approach Using Sandwich-Cultured Hepatocytes, Mechanistic Modeling, and Simulation

Guo

Yang

Brouwer

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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Transporter-mediated alterations in bile acid disposition may have significant toxicological implications. Current methods to predict interactions are limited by the interplay of multiple transporters, absence of protein in the experimental system, and inaccurate estimates of inhibitor concentrations. An integrated approach was developed to predict altered bile acid disposition due to inhibition of multiple transporters using the model bile acid taurocholate (TCA). TCA pharmacokinetic parameters were estimated by mechanistic modeling using sandwich-cultured human hepatocyte data with protein in the medium. Uptake, basolateral efflux, and biliary clearance estimates were 0.63, 0.034, and 0.074 mL/min/g liver, respectively. Cellular total TCA concentrations (C t,Cells ) were selected as the model output based on sensitivity analysis. Monte Carlo simulations of TCA C t,Cells in the presence of model inhibitors (telmisartan and bosentan) were performed using inhibition constants for TCA transporters and inhibitor concentrations, including cellular total inhibitor concentrations ([I] t,cell ) or unbound concentrations, and cytosolic total or unbound concentrations. For telmisartan, the model prediction was accurate with an average fold error (AFE) of 0.99-1.0 when unbound inhibitor concentration ([I] u ) was used; accuracy dropped when total inhibitor concentration ([I] t ) was used. For bosentan, AFE was 1.2-1.3 using either [I] u or [I] t . This difference was evaluated by sensitivity analysis of the cellular unbound fraction of inhibitor (f u,cell,inhibitor ), which revealed higher sensitivity of f u,cell,inhibitor for predicting TCA C t,Cells when inhibitors exhibited larger ([I] t,cell /IC 50 ) values. In conclusion, this study demonstrated the applicability of a framework to predict hepatocellular bile acid concentrations due to drug-mediated inhibition of transporters using mechanistic modeling and cytosolic or cellular unbound concentrations.

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Determination of Intracellular Unbound Concentrations and Subcellular Localization of Drugs in Rat Sandwich-Cultured Hepatocytes Compared with Liver Tissue

Cited by 35 publications

References 51 publications

Assessment of the Roles of P-glycoprotein and Cytochrome P450 in Triptolide-induced Liver Toxicity in Sandwich-Cultured Rat Hepatocyte Model

Assessment of the Roles of P-glycoprotein and Cytochrome P450 in Triptolide-induced Liver Toxicity in Sandwich-Cultured Rat Hepatocyte Model

Intracellular Unbound Atorvastatin Concentrations in the Presence of Metabolism and Transport

Prediction of Altered Bile Acid Disposition Due to Inhibition of Multiple Transporters: An Integrated Approach Using Sandwich-Cultured Hepatocytes, Mechanistic Modeling, and Simulation

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