Intracellular uptake of agents that block the hERG channel can confound the assessment of QT interval prolongation and arrhythmic risk.

BACKGROUND: Oliceridine is a biased ligand at the µ-opioid receptor recently approved for the treatment of acute pain. In a thorough QT study, corrected QT (QTc) prolongation displayed peaks at 2.5 and 60 minutes after a supratherapeutic dose. The mean plasma concentration peaked at 5 minutes, declining rapidly thereafter. OBJECTIVE: The purpose of this study was to examine the basis for the delayed effect of oliceridine to prolong the QTc interval. METHODS: Repolarization parameters and tissue accumulation of oliceridine were evaluated in rabbit left ventricular wedge preparations over a period of 5 hours. The effects of oliceridine on ion channel currents were evaluated in human embryonic kidney and Chinese hamster ovary cells. Quinidine was used as a control. RESULTS: Oliceridine and quinidine produced a progressive prolongation of the QTc interval and action potential duration over a period of 5 hours, paralleling slow progressive tissue uptake of the drugs. Oliceridine caused modest prolongation of these parameters, whereas quinidine produced a prominent prolongation of action potential duration and QTc interval as well as development of early afterdepolarization (after 2 hours), resulting in a high torsades de pointes score. The 50% inhibitory concentration values for the oliceridine inhibition of the rapidly activating delayed rectifier current (human ether a-go-go current) and late sodium channel current were 2.2 and 3.45 µM when assessed after traditional acute exposure but much lower after 3 hours of drug exposure. CONCLUSION: Our findings suggest that a gradual increase of intracellular access of drugs to the hERG channels as a result of their intracellular uptake and accumulation can significantly delay effects on repolarization, thus confounding the assessment of QT interval prolongation and arrhythmic risk when studied acutely. The multi-ion channel effects of oliceridine, late sodium channel current inhibition in particular, point to a low risk of devloping torsades de pointes.

An ECV304 monoculture model for permeability assessment of blood-brain barrier.

OBJECTIVE: ECV304/C6 co-culture model is a widely used tool for BBB studies. However, cell source may influence the establishment of co-culture model and some C6 cells could damage the barrier integrity. Here, we established an ECV304 monoculture model and evaluated it in the respect of tightness, tight junction proteins and discriminative brain penetration. METHODS: The tightness of ECV304 cell layers was evaluated by the measurement of permeability to hydrophilic marker Lucifer yellow. Immunofluorescence method was explored to detect the expression of tight junction proteins occludin, claudin-5 and ZO-1 in ECV304 cells. The discriminative brain penetration of the model was assessed by a permeability testing of compounds with different penetration rates, including digoxin, quinidine, and propranolol. RESULTS: The ECV304 monolayers developed low permeability to Lucifer yellow (permeability coefficient: 0.31 ± 0.02 × 10-3 cm/min) and exhibited positive immunostaining of occludin, claudin-5 and ZO-1. The permeability coefficients of high permeable quinidine and propranolol across ECV304 cell layers were higher than that of low permeable digoxin by 3.6 and 2.8-fold, respectively. CONCLUSIONS: The ECV304 monoculture model developed tight paracellular barrier and discriminated between compounds with different permeability, indicating it as a potential in vitro model for BBB permeability assessment.

A simplified protocol employing elacridar in rodents: a screening model in drug discovery to assess P-gp mediated efflux at the blood brain barrier.

In the present study we have developed a simple, time, and cost effective in vivo rodent protocol to screen the susceptibility of a test compound for P-glycoprotein (P-gp) mediated efflux at the blood brain barrier (BBB) during early drug discovery. We used known P-gp substrates as test compounds (quinidine, digoxin, and talinolol) and elacridar (GF120918) as a chemical inhibitor to establish the model. The studies were carried out in both mice and rats. Elacridar was dosed intravenously at 5 mg/kg, 0.5 h prior to probe substrate administration. Plasma and brain samples were collected and analyzed using UPLC-MS/MS. In the presence of elacridar, the ratio of brain to plasma area under the curve (B/P) in mouse increased 2, 4, and 38-fold, respectively, for talinolol, digoxin, and quinidine; whereas in rat, a 70-fold increase was observed for quinidine. Atenolol, a non P-gp substrate, exhibited poor brain penetration in the presence or absence of elacridar in both species (B/P ratio ~ 0.1). Elacridar had no significant effect on the systemic clearance of digoxin or quinidine; however, a trend towards increasing volume of distribution and half life was observed. Our results support the utility of elacridar in evaluation of the influence of P-gp mediated efflux on drug distribution to the brain. Our protocol employing a single intravenous dose of elacridar and test compound provides a cost effective alternative to expensive P-gp knockout mice models during early drug discovery.

Derivation of occupational exposure limits based on target blood concentrations in humans.

An approach for deriving occupational exposure limits (OEL) for pharmaceutical compounds is the application of safety factors to the most appropriate pre-clinical toxicity endpoint or the lowest therapeutic dose (LTD) in humans. Use of this methodology can be limited when there are inadequate pre-clinical toxicity data or lack of a well-defined therapeutic dose, and does not include pharmacokinetic considerations. Although some methods have been developed that incorporate pharmacokinetics, these methods do not take into consideration variability in response. The purpose of this study was to investigate how application of compartmental pharmacokinetic modeling could be used to assist in the derivation of OELs based on target blood concentrations in humans. Quinidine was used as the sample compound for the development of this methodology though the intent was not to set an OEL for quinidine but rather to develop an alternative approach for the determination of OELs. The parameters for the model include body weight, breathing rate, and chemical-specific pharmacokinetic constants in humans, data typically available for pharmaceutical agents prior to large scale manufacturing. The model is used to simulate exposure concentrations that would result in levels below those that may result in any undesirable pharmacological effect, taking into account the variability in parameters through incorporation of Monte Carlo sampling. Application of this methodology may decrease some uncertainty that is inherent in default approaches by eliminating the use of safety factors and extrapolation from animals to humans. This methodology provides a biologically based approach by taking into consideration the pharmacokinetics in humans and reported therapeutic or toxic blood concentrations to guide in the selection of the internal dose-metric.

Evaluation of a Cmin and a normalized Cmin method for the confirmation of steady-state in bioequivalence studies.

PURPOSE: Two methods to confirm attainment of steady-state conditions in multiple-dose bioequivalence studies are described and evaluated: (1) the Cmin method and (2) the Area Below the Cmin plasma-concentration-versus-time-curve method (ABCM method). METHODS: Cmin Method-After repetitive drug administration to presumed steady-state, successive trough, or Cmin, values are evaluated to determine if they are equal. ABCM Method-The ABCM of successive doses from dose two to presumed steady-state [ABCM(ss)] are divided by the ABCM for the first dose, ABCM(t), to give ABCM(ss)/ ABCM(t)=R, which describes the increase in ABCM(n) with successive doses. The quantity, R, is then divided by an accumulation ratio to render the value independent of intra-subject clearance differences. Monte Carlo simulations were done to test the effects of data error and slow-clearing subpopulations on the method's performance. Data from multiple-dose bioequivalence studies were evaluated using confidence intervals for both methods to determine how well each predicted steady-state for immediate-release and controlled-release drug products. RESULTS/CONCLUSIONS: The Cmin method more accurately predicted the attainment of steady-state conditions for immediate-release formulations compared to the ABCM method. Conversely, the ABCM procedure more accurately predicted the attainment of steady-state conditions for controlled-release formulations compared to the Cmin method. The simulation results were further supported by the experimental data.

Comparison of single and multiple dose pharmacokinetics using clinical bioequivalence data and Monte Carlo simulations.

The purpose of this study was to evaluate the relative performance and usefulness of single dose (SD) and multiple dose (MD) regimens for bioequivalence (BE) determination. Drugs such as indomethacin, procainamide, erythromycin, quinidine, nifedipine were tested for BE under SD and MD dose regimens. Drugs characterized by low accumulation indices (AI) showed virtually no change in the 90% confidence interval (CI) of AUC and CMAX upon multiple dosing. On the other hand, drugs with higher AI appeared to have smaller CI at steady-state. For example, the CI range of AUC and CMAX of quinidine (AI of 1.54) decreased from 26 to 12 and from 22 to 12, respectively, upon multiple dosing. A Monte Carlo simulation study of SD and MD bioequivalence trials was performed. The probability of failing the bioequivalence test was evaluated for several situations defined by different levels of variability and correlation in ka constants, presence or absence of inter- and/or intra-individual variability in clearance (CL) and volume of distribution (V), and different degrees of accumulation. All the possible combinations of these factors were tested with SD and MD study designs. All simulations used 1000 data sets with 30 subjects in each data set for a total of 144 unique designs (total of 144,000 simulations of bioequivalence trials). Upon multiple dosing, narrowing of CI ranges was observed for drugs simulated to have high AI high variability and a large difference in absorption constants (ka) between test and reference formulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Population pharmacokinetics of quinidine.

1. Population pharmacokinetic parameters of quinidine were determined based on 260 serum drug concentration measurements in 60 patients treated for arrhythmias with quinidine sulphate or quinidine bisulphate (Kinidin duriles) orally. 2. Quinidine kinetics were best described by a two compartment model with zero order absorption from the gastrointestinal tract. The pharmacokinetics are influenced by severe heart or liver failure and renal function impairment. No effect was found for mild or moderate heart failure, for age, for body weight or for coadministration of nifedipine. 3. Population pharmacokinetic parameters of quinidine (assuming 100% bioavailability of oral quinidine sulphate) were: nonrenal clearance for patients without severe heart and liver failure 12.6 l h-1, reduction in patients with severe heart or liver failure to 6.8 l h-1, renal clearance (l h-1) related to creatinine clearance (ml min-1), proportionality constant 0.0566, volume of distribution of the central compartment 161 l, maximum serum drug concentration 1.4 h after administration of quinidine sulphate and 6.0 h after administration of quinidine bisulphate. 4. The results were validated by predicting the serum drug concentration in a separate group of 30 patients. The model reliably predicted both the population average and the variability of the serum concentration of quinidine. 5. Using Monte Carlo computer simulations, an a priori dosing regimen was derived that should maximize the proportion of patients having quinidine serum concentrations within the recommended range (2-5 mg l-1): initial dose of 600 mg quinidine sulphate in all patients, 3 h later first maintenance dose of quinidine bisulphate.(ABSTRACT TRUNCATED AT 250 WORDS)