The BA submodel in DILIsym comprises several components. It includes (i) the synthesis and metabolism of BAs in hepatocytes, (ii) the basolateral and canalicular active transport of BAs, (iii) the release of BAs from the gallbladder in humans, (iv) the synthesis of secondary BAs and deconjugation of BAs in the gut, (v) the recirculation of BAs from the gut and subsequent active uptake by the liver, and (vi) the regulatory effects of BAs on transporter expression and BA synthesis. The model contains representations of LCA and CDCA and its conjugates, the BA species most frequently linked to toxicity in in vitro experiments,20 (link),21 (link),33 (link),34 (link) and a “bulk” BA representation that contains the other BAs. A more detailed description of the BA homeostasis model and its parameterization is presented in the Supplementary Materials online.
To represent BA dynamics in humans, the model was optimized to the known profile of BAs in serum published by Trottier et al.,35 (link) as well as to overall concentrations of BAs in the liver measured by Setchell36 (link) and García-Cañaveras et al.37 (link) There were 47 system variables that were either unknown or expected to vary within the human population that were fit to the BA profile; a list of the variables used in the optimization is provided in theSupplementary Materials online. The optimization of the model based on the published BA profile was performed in a manner similar to the SimPops method outlined in previous publications, wherein a genetic algorithm was used to generate values of variable parameters that lead to model outcomes (in this case, BA profiles) that are within the range of experimental data.27 (link),28 (link) Using this optimization method allowed the selection of a population of 2,400 individuals with reasonable baseline BA concentrations for the large human sample population. A smaller, 10-parameter, 331-individual SimPops was also constructed for the purpose of model validation. While the large population was given artificially wide parameter ranges for the purpose of the sensitivity analysis, the small human sample population was constructed with more constrained parameter ranges for the purpose of approximating a plausible population of humans. For example, the transporter Vmax ranges included four orders of magnitude in the large population, but were constrained to ranges suggested by transporter expression profiles from Meier et al.25 (link) in the small population.
A smaller SimPops was constructed in rats for the validation against the experimental data. A population of 191 rats using a more limited 11 variable set (Supplementary Table S4 online) was generated. This population was intended to represent rats with both plausible serum BA concentrations and plausible values for the parameters that were varied. The baseline rat model was optimized to the BA profile from the control rats in the present experiment, and the SimPops was constructed around this baseline.
Details on the simulations performed for the model validation and for the model exploration can be found in theSupplementary Materials online. Multivariate analysis on the population sample was performed using JMP 9 from SAS (Cary, NC).
Multiple-dose glibenclamide study in rats For the BA-profiling experiments, 16 male 8- to 9-week-old CD-1 rats from Charles River Laboratories (Raleigh, NC) weighing between 200 and 300 g were randomized into four groups of four animals each. These groups were administered daily doses of either the vehicle control (0.5% hydroxypropylmethylcellulose /0.1% Tween 80 in water) or glibenclamide (Sigma-Aldrich, St Louis, MO) in vehicle via oral gavage for 7 days at three dose levels (300, 750, and 1,500 mg/kg/day). Details on the treatment of the rats are available in the Supplementary materials online.
Rats underwent a viability check twice per day, and detailed clinical observations were taken at least twice over the course of the study. Blood was drawn for serum BA profiles at 1, 3, 6, and 24 h after dosing on day 1 and day 7, and glibenclamide concentrations were measured for toxicokinetic analysis using the same blood samples from day 7. The animals were euthanized under isoflourane anesthesia on day 8 and underwent necropsy. Clinical and anatomic pathology data were collected from the animals, and these results are reported in theSupplementary Materials online. The serum BA concentrations from day 1 were used for comparison to the simulation results.
BA profiling in serum was performed using liquid chromatography–tandem mass spectrometry analysis at GlaxoSmithKline (Ware, UK). BAs in liver tissue 24 h after the final dose also were profiled; results from this analysis, as well as the liquid chromatography–tandem mass spectrometry analytical method, are presented in theSupplemental Materials online. This study was conducted in accordance with the GlaxoSmithKline Policy on the Care, Welfare and Treatment of Laboratory Animals and was reviewed by the Institutional Animal Care and Use Committee.
For this experiment, doses of glibenclamide were administered every 24 h for 7 days. Systemic exposure data were compared to simulation results for day 7; day 1 BA data were compared to simulated BA concentrations on day 1.
Short-term glibenclamide studies in rats For the short-term studies, male Han Wistar rats (substrain AlpkHsdBrlHan:WIST; AstraZeneca, Macclesfield, UK) of 10–12-week age (300–400 g) were used. Details on the treatment of the rats used in this study are available in the Supplemental Materials online. All animals were treated in accordance with approved UK Home Office license requirements.
Two experiments were performed in which total plasma BA concentrations and glibenclamide plasma concentrations were determined. Glibenclamide (Sigma-Aldrich) was formulated as a solution or suspension in hydroxypropyl-β-cyclodextrin (Acros Organics, distributed by Fisher Scientific, Loughborough, UK) in aqueous 0.2 mol/l Na2CO3/NaHCO3 buffer (pH 10). In the first experiment (29 animals), four groups of five animals received a single dose via oral gavage of 50, 250, and 500 mg/kg glibenclamide or 10% (w/v) hydroxypropyl-β-cyclodextrin vehicle alone, and blood samples were taken at 1, 6, and 24 h after dosing; an additional three animals per glibenclamide-treated group received a single dose of 50, 250, and 500 mg/kg glibenclamide, and blood samples were taken at 1, 3, 6, 12, and 24 h after dosing. In the second experiment (20 animals), two groups of five animals each received two oral doses of 250 and 500 mg/kg glibenclamide in 20% (w/v) hydroxypropyl-β-cyclodextrin vehicle at 0 and 4 h, and blood samples were taken predose and at 0.5, 1, 2, and 4 h after dosing; blood samples from an additional five animals per glibenclamide-treated group were taken predose and 1 h after dosing to increase the dataset for this time point. Details on the blood-sampling procedure are located in theSupplementary Materials online. Animals were dosed 2 h into the light cycle.
To represent BA dynamics in humans, the model was optimized to the known profile of BAs in serum published by Trottier et al.,35 (link) as well as to overall concentrations of BAs in the liver measured by Setchell36 (link) and García-Cañaveras et al.37 (link) There were 47 system variables that were either unknown or expected to vary within the human population that were fit to the BA profile; a list of the variables used in the optimization is provided in the
A smaller SimPops was constructed in rats for the validation against the experimental data. A population of 191 rats using a more limited 11 variable set (
Details on the simulations performed for the model validation and for the model exploration can be found in the
Rats underwent a viability check twice per day, and detailed clinical observations were taken at least twice over the course of the study. Blood was drawn for serum BA profiles at 1, 3, 6, and 24 h after dosing on day 1 and day 7, and glibenclamide concentrations were measured for toxicokinetic analysis using the same blood samples from day 7. The animals were euthanized under isoflourane anesthesia on day 8 and underwent necropsy. Clinical and anatomic pathology data were collected from the animals, and these results are reported in the
BA profiling in serum was performed using liquid chromatography–tandem mass spectrometry analysis at GlaxoSmithKline (Ware, UK). BAs in liver tissue 24 h after the final dose also were profiled; results from this analysis, as well as the liquid chromatography–tandem mass spectrometry analytical method, are presented in the
For this experiment, doses of glibenclamide were administered every 24 h for 7 days. Systemic exposure data were compared to simulation results for day 7; day 1 BA data were compared to simulated BA concentrations on day 1.
Two experiments were performed in which total plasma BA concentrations and glibenclamide plasma concentrations were determined. Glibenclamide (Sigma-Aldrich) was formulated as a solution or suspension in hydroxypropyl-β-cyclodextrin (Acros Organics, distributed by Fisher Scientific, Loughborough, UK) in aqueous 0.2 mol/l Na2CO3/NaHCO3 buffer (pH 10). In the first experiment (29 animals), four groups of five animals received a single dose via oral gavage of 50, 250, and 500 mg/kg glibenclamide or 10% (w/v) hydroxypropyl-β-cyclodextrin vehicle alone, and blood samples were taken at 1, 6, and 24 h after dosing; an additional three animals per glibenclamide-treated group received a single dose of 50, 250, and 500 mg/kg glibenclamide, and blood samples were taken at 1, 3, 6, 12, and 24 h after dosing. In the second experiment (20 animals), two groups of five animals each received two oral doses of 250 and 500 mg/kg glibenclamide in 20% (w/v) hydroxypropyl-β-cyclodextrin vehicle at 0 and 4 h, and blood samples were taken predose and at 0.5, 1, 2, and 4 h after dosing; blood samples from an additional five animals per glibenclamide-treated group were taken predose and 1 h after dosing to increase the dataset for this time point. Details on the blood-sampling procedure are located in the
