Comparison of Methods for Estimating Unbound Intracellular-to-Medium Concentration Ratios in Rat and Human Hepatocytes Using Statins

Abstract

It is essential to estimate concentrations of unbound drugs inside the hepatocytes to predict hepatic clearance, efficacy, and toxicity of the drugs. The present study was undertaken to compare predictability of the unbound hepatocyte-to-medium concentration ratios (K) by two methods based on the steady-state cell-to-medium total concentration ratios at 37°C and on ice (K) and based on their initial uptake rates (K). Poorly metabolized statins were used as test drugs because of their concentrative uptake via organic anion-transporting polypeptides. K values of these statins provided less interexperimental variation than the K values, because only data at longer time are required for K K values for pitavastatin, rosuvastatin, and pravastatin were 1.2- to 5.1-fold K in rat hepatocytes; K values in human hepatocytes also tended to be larger than corresponding K To explain these discrepancies, theoretical values of K and K were compared with true K (K), considering the inside-negative membrane potential and ionization of the drugs in hepatocytes and medium. Membrane potentials were approximately -30 mV in human hepatocytes at 37°C and almost abolished on ice. Theoretical equations considering the membrane potentials indicate that K values for the statins are 0.85- to 1.2-fold K, whereas K values are 2.2- to 3.1-fold K, depending on the ratio of the passive permeability of the ionized to nonionized forms. In conclusion, K values of anions are similar to K when the inside-negative membrane potential is considered. This suggests that K is preferable for estimating the concentration of unbound drugs inside the hepatocytes.

Physiologically‐Based Pharmacokinetic Modeling Analysis for Quantitative Prediction of Renal Transporter–Mediated Interactions Between Metformin and Cimetidine

“…Metformin (pKa of 12.3) exists mainly ionized at physiological pHs (thus, f o,union = f i,union = 0 and f o,ion = f i,ion = 1), and γ r and γ urine were calculated using the Nernst equation . Passive diffusion clearances of each compartment in the kidney were calculated using permeability measured in a parallel artificial membrane permeability assay system, surface area, γ r , and γ urine ( Table ) …”

“…To calculate γ h , γ r , and γ urine of cimetidine using Eq. , λ was set to be 0.1 as described previously …”

“…where CL int,sec is the intrinsic urinary secretion clearance; PS r,inf and PS r,eff are the intrinsic renal clearance via influx into and efflux out of renal cells, respectively; PS urine is the intrinsic efflux clearance from renal cells to the urinary lumen; PS r,dif,inf and PS r,dif,eff are the intrinsic passive clearance via influx into and efflux out of renal cells, respectively; PS urine,dif,inf and PS urine,dif,eff are the intrinsic passive clearance via influx into cells from the urinary lumen and efflux out of cells to the urinary lumen; R MATE/dif is the ratio of the intrinsic clearance of MATEs to the intrinsic passive diffusion clearance via efflux out of cells to the urinary lumen; γ r and γ urine are the passive influx-to-efflux ratio on the basolateral and luminal sides, respectively; λ is the ratio of passive diffusion for the ionized form to that for the unionized form; f o,union and f o,ion are the extracellular fractions of the unionized and ionized forms, respectively; f i,union and f i,ion are the intracellular fractions of the unionized and ionized forms, respectively; β kidney is a hybrid parameter reflective of the major rate-limiting steps of C Lint,sec Metformin (pKa of 12.3) exists mainly ionized at physiological pHs (thus, f o,union = f i,union = 0 and f o,ion = f i,ion = 1), and γ r and γ urine were calculated using the Nernst equation. 29 Passive diffusion clearances of each compartment in the kidney were calculated using permeability measured in a parallel artificial membrane permeability assay system, 30 surface area, γ r , and γ urine (Table S3). 29,31 Optimization of model parameters.…”

“…29 Passive diffusion clearances of each compartment in the kidney were calculated using permeability measured in a parallel artificial membrane permeability assay system, 30 surface area, γ r , and γ urine (Table S3). 29,31 Optimization of model parameters. Other unknown parameters (absorption rate constant, k a ; transit rate constant from the transit compartment to the intestinal compartment, k trans ; and R MATE/dif ) were optimized by fitting to the two clinical data sets obtained after oral administration of metformin at 1,500 mg (time profiles of plasma and blood concentrations and urinary excretion) 6 or 250 mg (DDI data between metformin and cimetidine).…”

Utilizing in vitro transporter data in IVIVE-PBPK: an overview

“…Another critical factor whether intracellular concentrations that drive efflux transport are accounted for [21]. Drug concentrations within the donor and acceptor compartments are dynamic; thus, the assumption that unbound drug concentrations on either side of a membrane are in thermodynamic equilibrium [22,23] is not applicable for drugs that are poorly permeable, actively transported, or extensively metabolized.…”