Introduction: The objective of this study was to provide solubility, rabbit intestinal permeability, Caco-2 permeability, and hepatocyte metabolism information in a blinded fashion for the performance evaluation of the physiological based predictive absorption model (IDEA ) using a set of eight diverse drug compounds. The compounds were derived from different therapeutic classes and were provided by Hoffman-LaRoche, Ltd. Trega has developed a physiological based absorption model (IDEA TM Predictive Model) which was trained using a database of 56 non-metabolized compounds and a total of 85 drug-dose combinations. The in vivo training data was obtained from clinical pharmacokinetic studies provided by a consortium of five pharmaceutical companies together with Trega Biosciences.
Experimental A. Solubility Testing: Simulated gastric fluid (pH 1.5, NaCl/HCl) Simulated intestinal fluid (pH 5.0, 6.5, 7.0 and 7.5; KH 2 PO 4 /NaOH) N = 3 determinations for each pH Incubate at 37 o C (rotation), 4 hours pH adjust, if necessary, and incubate additional 2 hours Filter aliquot (0.45 M) Analysis: HPLC/UV
B. Rabbit Intestinal Permeability: Directionality: A B and B A Male New Zealand white rabbits (2.5-3 kg) (overnight fasting) Intestinal Segments: duodenum, jejunum, ileum, and descending colon Ussing side-by-side diffusion chamber/ 37 °C/ gas lift mechanism using 1-2 mL/min 95% O 2 and 5% CO 2 Donor conc: 100 µM including 1% DMSO Apical and Basolateral: 1.5 mL pH 7.4 Ringers buffer Donor sampling: 100 µL at beginning and final Receiver sampling: 100 µL at 30, 50, 70, and 90 min N=3 chambers per compound Analysis: LC-MS/ HPLC-UV/ LSC Peff (cm/sec) = (dX/dt)/(A*Co*60), where X: mass transported, A: surface area, 0.636 cm 2 and Co: initial donor drug concentration
C. Caco-2 Permeability: Directionality: A B and B A Caco-2: 20-23 day/ Passage 30-40/Surface Area: 0.31 cm 2 Donor conc: 100 µM including 1% DMSO A: 300 µL pH 7.4 (or pH 6.5 Mes)/ B: 1200 µL pH 7.4 Ringers buffer Donor side sampling: 20 µL at beginning and final Receiver side sampling: 100 µL at 30, 50, 70, and 90 min Incubation at 50 oscillations per minute, 37 °C, 5%CO 2, 95% humidity N=4 wells per compound Analysis: LC-MS, HPLC-UV, or LSC Peff (cm/sec) = (dX/dt)/(A*Co*60), where X: mass transported, A: surface area and Co: initial donor drug concentration Monolayer integrity: Pre and post-test TEER measurements Transport markers: Atenolol, propranolol
D. Hepatocyte Metabolism: Objective: Determine the metabolic stability of each test compound in human cryopreserved hepatocytes. 96-well plate format Cell Density –pooled cryopreserved hepatocyte suspensions (N = 6 human donors) –1 x 10 5 cells/well Single substrate concentration (5.0 µM, 0.025% DMSO final) Incubate 37° C, 5% CO 2 Time Points: 0, 30, 60 and 240 min (N = 3/time point) Terminate reaction w/ addition of organic solvent Precipitate protein Analyze supernatant by LC/MS Report: % parent remaining
Hepatocyte Viability Evaluations: Cellular Viability –Trypan blue exclusion –Cell viability and yield –Initiation and termination of experiment Metabolic Viability –7-Ethoxycoumarin metabolic profile (75 µM) –Testosterone metabolic stability (5 µM) –Atenolol metabolic stability (5 µM) Negative Control –Test compound incubated w/o cells –Assessed at 60 and 240 minutes post-incubation
Results A. Solubility Profiles: High Solubility (>90 mg/mL): Compounds B, C and G Low Solubility (<1 mg/mL): Compounds A, D, H (except pH 5) and I
B. Rabbit Intestinal Permeability (A B, B A Profiles) Mean values (±SD) from N=3 replicates
Rabbit Intestinal Permeability: Results –A B Permeability Compounds B, D and H: >5 x 10 -6 cm/sec (high permeability) Compounds C and G: 1 - 5 x 10 -6 cm/sec (intermediate permeability) Compounds A, E, and I: <1 x 10 -6 cm/sec (low permeability) –Transport processes Passive transport (A B B A): Compounds D and H Facilitated transport –Influx transport (A B > B A): Compounds A, B, C –Efflux transport (A B< B A): Compounds E, G and I
C. Caco-2 Permeability Mean values (±SD) from N=4 replicates
Caco-2 Permeability: Results –TEER values within range of historical dataset (> 200 /cm 2 ) –Transport markers (N=12 separate experiments, A B transport) Atenolol: 1.08 (0.69 - 1.80) x 10 -6 cm/sec (low permeability) Propranolol: 28.53 (18.50 - 36.80) x 10 -6 cm/sec (high permeability) –pH 7.4 Apical/pH 7.4 Basolateral Protocol A B Permeability –Compounds B and D: >5 x 10 -6 cm/sec (high permeability) –pH 6.5 (Mes) Apical/pH 7.4 Basolateral Protocol A B Permeability –Compounds D, H, and I: >5 x 10 -6 cm/sec (high permeability)
Metabolic Stability: Results Positive Controls –Cellular and metabolic viability markers were consistent with historical data. Negative Controls –No evidence for non-enzymatic degradation of Roche compounds. Roche Validation Set –All compounds exhibited metabolic stability with the exception of: Roche D Roche G
Performance of Hepatocyte Assay: *7-EC: 7-ethoxy coumarin, 7-HC: 7-hydroxy coumarin, 7-HCG: 7- hydroxy coumarin glucuronide, and 7-HCS: 7- hydroxy coumarin sulfate. *Data represent the mean (±SD). N=23 determinations for viability, testosterone, 7-EC screening. N=8 determinations for atenolol screening.
Performance of the IDEA TM Absorption Model The IDEA absorption model predicted the human absorption of all 8 compounds within the specified criteria established by Hoffman- LaRoche, Ltd.
Comparison of observed FDp estimates (Hoffman-LaRoche Ltd) with FDp-predicted (IDEA TM Model)
Conclusion: The primary in vitro data inputs used for the model included rabbit intestinal permeability from various regions and solubility determined at various pH values. The compounds were very diverse in terms of their physical and chemical properties: MW: 200 - 552, solubility (22 ng/mL to >100 mg/mL), permeability (0.14 to 25 10 -6 cm/s), and pharmacokinetic properties. IDEA Predictive Model, a physiological based predictive absorption model, accurately predicted relevant absorption outcomes for a diverse blinded, external validation set of drugs.