Challenges and Opportunities for In Vitro-In Vivo Extrapolation of Aldehyde Oxidase-Mediated Clearance: Toward a Roadmap for Quantitative Translation.

Underestimation of aldehyde oxidase (AO)-mediated clearance by current in vitro assays leads to uncertainty in human dose projections, thereby reducing the likelihood of success in drug development. In the present study we first evaluated the current drug development practices for AO substrates. Next, the overall predictive performance of in vitro-in vivo extrapolation of unbound hepatic intrinsic clearance (CLint,u) and unbound hepatic intrinsic clearance by AO (CLint,u,AO) was assessed using a comprehensive literature database of in vitro (human cytosol/S9/hepatocytes) and in vivo (intravenous/oral) data collated for 22 AO substrates (total of 100 datapoints from multiple studies). Correction for unbound fraction in the incubation was done by experimental data or in silico predictions. The fraction metabolized by AO (fmAO) determined via in vitro/in vivo approaches was found to be highly variable. The geometric mean fold errors (gmfe) for scaled CLint,u (mL/min/kg) were 10.4 for human hepatocytes, 5.6 for human liver cytosols, and 5.0 for human liver S9, respectively. Application of these gmfe's as empirical scaling factors improved predictions (45%-57% within twofold of observed) compared with no correction (11%-27% within twofold), with the scaling factors qualified by leave-one-out cross-validation. A road map for quantitative translation was then proposed following a critical evaluation on the in vitro and clinical methodology to estimate in vivo fmAO In conclusion, the study provides the most robust system-specific empirical scaling factors to date as a pragmatic approach for the prediction of in vivo CLint,u,AO in the early stages of drug development. SIGNIFICANCE STATEMENT: Confidence remains low when predicting in vivo clearance of AO substrates using in vitro systems, leading to de-prioritization of AO substrates from the drug development pipeline to mitigate risk of unexpected and costly in vivo impact. The current study establishes a set of empirical scaling factors as a pragmatic tool to improve predictability of in vivo AO clearance. Developing clinical pharmacology strategies for AO substrates by utilizing mass balance/clinical drug-drug interaction data will help build confidence in fmAO.

Influence of permeability enhancers on the paracellular permeability of metformin hydrochloride and furosemide across Caco-2 cells.

Permeability enhancers can affect absorption of paracellularly transported drugs. This study aims to evaluate effects of permeability enhancers (chitosan, methyl-ß -cyclodextrin, sodium caprate, sodium lauryl sulfate, etc.) on the permeability of paracellularly absorbed furosemide and metformin hydrochloride. Methyl thiazole tetrazolium bromide test was carried out to determine the drug concentrations in permeability study. Trans-epithelial electrical resistance (TEER) values determined to assess the integrity of tight junctions. Permeability enhancers were applied at different concentrations alone, in dual/triple combinations. Permeability was determined using human colorectal adenocarcinoma (Caco-2) cells (TEER > 400 Ω·cm2). Permeability enhancers have no significant effect (<2-fold; p > 0.05) on the permeability of furosemide (1.80 × 10-5 ± 4.55 × 10-7 cm/s); however, metformin permeability (1.36 × 10-5 ± 1.25 × 10-6 cm/s) increased significantly (p < 0.05) with 0.3% and 0.5% (w/v) chitosan (2.0- and 2.7-fold, respectively), 1% methyl-ß -cyclodextrin (w/v) (3.5-fold), 10 and 20 µmol/L sodium caprate (2.2- and 2.8-fold, respectively), and 0.012% sodium lauryl sulfate (w/v) (1.9-fold). Furosemide permeability increased significantly (p < 0.05) with chitosan-sodium lauryl sulfate combination (1.7-fold), and all triple combinations (1.4- to 1.9-fold). Chitosan containing dual/triple combinations resulted in significant increase (p < 0.05) in metformin permeability (1.7 to 2.8-fold). All results indicated that absorption of furosemide and metformin can be improved by the combination of permeability enhancers. Therefore, it can be evaluated for the formulation of development strategies containing furosemide and metformin by the pharmaceutical industry.

Hepatic transporter-mediated pharmacokinetic drug-drug interactions: Recent studies and regulatory recommendations.

Transporter-mediated drug-drug interactions are one of the major mechanisms in pharmacokinetic-based drug interactions and correspondingly affecting drugs' safety and efficacy. Regulatory bodies underlined the importance of the evaluation of transporter-mediated interactions as a part of the drug development process. The liver is responsible for the elimination of a wide range of endogenous and exogenous compounds via metabolism and biliary excretion. Therefore, hepatic uptake transporters, expressed on the sinusoidal membranes of hepatocytes, and efflux transporters mediating the transport from hepatocytes to the bile are determinant factors for pharmacokinetics of drugs, and hence, drug-drug interactions. In parallel with the growing research interest in this area, regulatory guidances have been updated with detailed assay models and criteria. According to well-established preclinical results, observed or expected hepatic transporter-mediated drug-drug interactions can be taken into account for clinical studies. In this paper, various methods including in vitro, in situ, in vivo, in silico approaches, and combinational concepts and several clinical studies on the assessment of transporter-mediated drug-drug interactions were reviewed. Informative and effective evaluation by preclinical tools together with the integration of pharmacokinetic modeling and simulation can reduce unexpected clinical outcomes and enhance the success rate in drug development.

Development and characterization of metformin hydrochloride- and glyburide-containing orally disintegrating tablets.

Diabetes is characterized by chronic hyperglycemia. Although metformin hydrochloride (MHCl)- and glyburide (GLB)-containing conventional tablets are available in the market and used to treat diabetes, orally disintegrating tablets (ODTs) containing the combination of these drugs are not commercially available. Therefore, the aim of this study was to prepare ODTs containing MHCl and GLB by direct-compression (DC-ODTs) and freeze-drying (FD-ODTs) methods. Physical properties of the powder mixture of DC-ODT formulation were determined (Angle of repose: 37.18 ± 1.27°; compressibility index: 20.31 ± 1.06%; Hausner ratio: 1.25 ± 0.03). Its moisture content was 0.3 ± 0.09%. The hardness values and the disintegration times for DC-ODTs and FD-ODTs were 221.60 ± 40.82 and 66.54 ± 2.68 N, and 80 and 30 s, respectively. Friability values were 0.24% for DC-ODTs and 0.38% for FD-ODTs. In uniformity-of-mass for single-dose-preparations test, the average weight was 684.38 ± 1.97 mg for DC-ODTs and 342.93 ± 2.4 mg for FD-ODTs, with less than 5% deviation for all 20 tablets. Water-absorption ratio for DC-ODTs was 1.30 ± 0.05. More than 90% of MHCl and GLB were dissolved within 5 min in both DC-ODTs and FD-ODTs. Although Caco-2 permeability of MHCl was influenced by the ODTs, GLB permeability was not. These results indicated that MHCl- and GLB-containing ODTs may be used as promising formulations for the treatment of diabetes.

An Isolated Perfused Rat Liver Model: Simultaneous LC-MS Quantification of Pitavastatin, Coproporphyrin I, and Coproporphyrin III Levels in the Rat Liver and Bile.

The isolated perfused rat liver (IPRL) model provides a mechanistic understanding of the organic-anion-transporting polypeptide (OATP/Oatp)-mediated pharmacokinetics in the preclinical evaluation, which often requires the use of control substrates (i.e., pitavastatin) and monitoring endogenous biomarkers (coproporphyrin I and III). This study aimed to develop and validate an LC-MS method allowing the simultaneous quantification of pitavastatin, coproporphyrin I (CPI), and coproporphyrin III (CPIII) in rat liver perfusion matrices (perfusate, liver homogenate, bile). The analysis was performed on a C18 column at 60 °C with 20 µL of sample injection. The mobile phases consisted of water with 0.1% formic acid and acetonitrile with 0.1% formic acid with a gradient flow of 0.5 mL/min. The assay was validated according to the ICH M10 Bioanalytical Method Validation Guideline (2022) for selectivity, calibration curve and range, matrix effect, carryover, accuracy, precision, and reinjection reproducibility. The method allowing the simultaneous quantification of pitavastatin, CPI, and CPIII was selective without having carryover and matrix effects. The linear calibration curves were obtained within various calibration ranges for three analytes in different matrices. Accuracy and precision values fulfilled the required limits. After 60 min perfusion with pitavastatin (1 µM), the cumulative amounts of pitavastatin in the liver and bile were 5.770 ± 1.504 and 0.852 ± 0.430 nmol/g liver, respectively. CPIII was a more dominant marker than CPI in both liver (0.028 ± 0.017 vs 0.013 ± 0.008 nmol/g liver) and bile (0.016 ± 0.011 vs 0.009 ± 0.007 nmol/g liver). The novel and validated bioanalytical method can be applied in further IPRL preparations investigating Oatp-mediated pharmacokinetics and DDIs.