Unstirred Water Layers and the Kinetics of Organic Cation Transport

Shibayama, Takahiro; Morales, Mark; Zhang, Xiao­hong; Martinez-Guerrero, Lucy Jazmin; Berteloot, A.; Secomb, Timothy W.; Wright, Stephen H.

doi:10.1007/s11095-015-1675-5

Cited by 18 publications

(12 citation statements)

References 58 publications

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“…The bias that an UWL introduces into measured values of K tapp is directly proportional to the J max of the transport process (Winne, 1973). We recently showed that simply changing the number of transporters expressed in the plasma membrane, thereby changing J max , can result in 5-to 10-fold changes in the measured K tapp values for OCT2-mediated transport of TEA and MPP (Shibayama et al, 2015). At steady state, the influence of UWLs and J max on an experimentally determined K t value is given by:…”

Section: Discussionmentioning

confidence: 99%

Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover

Severance

Sandoval

Wright

2017

J Pharmacol Exp Ther

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Organic cation (OC) transporter 2 (OCT2) mediates the first step in the renal secretion of many cationic drugs: basolateral uptake from blood into proximal tubule cells. The impact of this process on the pharmacokinetics of drug clearance as estimated using a physiologically-based pharmacokinetic approach relies on an accurate understanding of the kinetics of transport because the ratio of the maximal rate of transport to the Michaelis constant (i.e., J/ K) provides an estimate of the intrinsic clearance (Cl) used in in vitro-in vivo extrapolation of experimentally determined transport data. Although the multispecificity of renal OC secretion, including that of the OCT2 transporter, is widely acknowledged, the possible relationship between relative affinity of the transporter for its diverse substrates and the maximal rates of their transport has received little attention. In this study, we determined the J and apparent Michaelis constant (K) values for six structurally distinct OCT2 substrates and found a strong correlation between J and K; high-affinity substrates [K values <50 M, including 1-methyl-4-phenylpyridinium, or 1-methyl-4-phenylpyridinium (MPP), and cimetidine] displayed systematically lower J values (<50 pmol cm min) than did low-affinity substrates (K >200 M, including choline and metformin). Similarly, preloading OCT2-expressing cells with low-affinity substrates resulted in systematically larger-stimulated rates of MPP uptake than did preloading with high-affinity substrates. The data are quantitatively consistent with the hypothesis that dissociation of bound substrate from the transporter is rate limiting in establishing maximal rates of OCT2-mediated transport. This systematic relationship may provide a means to estimate Cl for drugs for which transport data are lacking.

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Section: Discussionmentioning

confidence: 99%

Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover

Severance

Sandoval

Wright

2017

J Pharmacol Exp Ther

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“…Although the need to control the UWL phenomenon in vitro has been recognized for absorption prediction, for the prediction of metabolic clearance this has been much less so. The depth of a UWL surrounding a membrane is greatest in an unstirred system and may be reduced by shaking or agitation (Williams et al, 1990;Avdeef et al, 2004;Shibayama et al, 2015). However, a widely held assumption persists that vigorous shaking has a detrimental effect on the structural integrity of isolated hepatocytes (Berry et al, 1991;Seglen, 2013), and consequently clearance assays are often performed under static or inadequately agitated conditions.…”

Section: Introductionmentioning

confidence: 99%

Importance of the Unstirred Water Layer and Hepatocyte Membrane Integrity In Vitro for Quantification of Intrinsic Metabolic Clearance

2017

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Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for the measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, the lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed nonviable by trypan blue staining showed undiminished metabolic activity for probe cytochrome P450 (P450) substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilization of the plasma membrane using saponin showed either full or minimal P450 activity, depending on the presence or absence of 1 mM NADPH, respectively. The shaking of incubations facilitated P450 metabolic rates up to 5-fold greater than static incubation, depending on intrinsic clearance, indicating the critical influence of the unstirred water layer (UWL). Permeabilization allowed static incubation metabolic rates to approach those of shaking for intact cells, indicating an artificially induced breakdown of the UWL. Permeabilization combined with shaking allowed an increased metabolic rate for saquinavir, resolving the membrane permeability limitation for this drug. These findings advance the interpretation of the rate-limiting processes involved in intrinsic clearance measurements and could be critical for successful in vitro prediction.

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“…It has been claimed recently that the unstirred water layer (UWL) should be accounted for when measuring transport kinetics using cultured cell systems. Otherwise, the apparent affinity of the substrate to the transporters may be underestimated (Shibayama et al, 2015). In our model, since the passive permeability was fitted when P-gp and the basolateral uptake transporter were fully inhibited by GF120918 (Tran et al, 2005), any effect of UWL will be included in the passive permeability coefficient.…”

Section: Discussionmentioning

confidence: 99%

Extrapolation of Elementary Rate Constants of P-glycoprotein–Mediated Transport from MDCKII-hMDR1-NKI to Caco-2 Cells

2016

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The best parameters for incorporation into mechanistic physiologically based pharmacokinetic models for transporters are system-independent kinetic parameters and active (not total) transporter levels. Previously, we determined the elementary rate constants for P-glycoprotein (P-gp)-mediated transport (on- and off-rate constants from membrane to P-gp binding pocket and efflux rate constant into the apical chamber) using the structural mass action kinetic model in confluent MDCKII-hMDR1-NKI cell monolayers. In the present work, we extended the kinetic analysis to Caco-2 cells for the first time and showed that the elementary rate constants are very similar compared with MDCKII-hMDR1-NKI cells, suggesting they primarily depend on the interaction of the compound with P-gp and are therefore mostly independent of the in vitro system used. The level of efflux active (not total) P-gp is also fitted by our model. The estimated level of efflux active P-gp was 5.0 ± 1.4-fold lower in Caco-2 cells than in MDCKII-hMDR1-NKI cells. We also kinetically identified the involvement of a basolateral uptake transporter for both digoxin and loperamide in Caco-2 cells, as found previously in MDCKII-hMDR1-NKI cells, due to their low passive permeability. This demonstrates the value of our P-gp structural model as a diagnostic tool in detecting the importance of other transporters, which cannot be unambiguously done by the Michaelis-Menten approach. The system-independent elementary rate constants for P-gp obtained in vitro are more fundamental parameters than those obtained using Michaelis-Menten steady-state equations. This suggests they will be more robust mechanistic parameters for incorporation into physiologically based pharmacokinetic models for transporters.

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Unstirred Water Layers and the Kinetics of Organic Cation Transport

Cited by 18 publications

References 58 publications

Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover

Correlation between Apparent Substrate Affinity and OCT2 Transport Turnover

Importance of the Unstirred Water Layer and Hepatocyte Membrane Integrity In Vitro for Quantification of Intrinsic Metabolic Clearance

Extrapolation of Elementary Rate Constants of P-glycoprotein–Mediated Transport from MDCKII-hMDR1-NKI to Caco-2 Cells

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