Chemicals and Biologic Materials.

Bupropion, cimetidine, dextromethorphan, diclofenac, digoxin, fexofenadine, fluvastatin, furosemide, gemfibrozil, ketoconazole, irinotecan, MK-571, mycophenolic acid, pravastatin, propranolol, quinidine, tolbutamide, and verapamil were purchased from Sigma-Aldrich (St. Louis, MO). Oseltamivir, repaglinide, SN-38, memantine, and zosuquidar were obtained from Toronto Research Chemicals (Toronto, Canada). Atorvastatin, benzydamine, clozapine, famotidine, rosuvastatin, simvastatin, and valsartan were purchased from ChemPacific (Baltimore, MD), Honeywell Research Chemicals (Charlotte, NC), Memory Pharmaceuticals (Montvale, NJ), Prestwick Chemical (Illkirch, France), Cayman, LKT Laboratories (St. Paul, MN), and Lucerna Chem AG (Luzern, Switzerland), respectively. Cyclosporine A, efavirenz, and talinolol were provided from Syntex (Palo Alto, CA). Clopidogrel, ethynylestradiol, omeprazole, oxazepam [as internal standard for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis], rosiglitazone, and midazolam were synthesized at F. Hoffmann-La Roche Ltd. (Basel, Switzerland). Acetonitrile and PBS were obtained from Biosolve Chimie (Dieuze, France) and Thermo Fisher Scientific (Waltham, MA), respectively. DMSO (Sigma-Aldrich) was used to prepare stock solutions of the test drugs, resulting in 0.1% (v/v) DMSO concentrations in the final incubation samples.

Human cryopreserved hepatocytes for hepatic uptake and metabolism assays were obtained from BioIVT (Westbury, NY). Viability of hepatocytes after reconstitution was at least 80% throughout the study. Ready-to-use Human HepatoPac cultures (long-term hepatocyte cocultures; a pool of 10 donors) and stromal mouse fibroblasts (lots/donors: 8305/YFA and 9177/ACR; pooled) with the plates for incubations, application medium, and maintenance medium were acquired from BioIVT.

Passive Permeability in LLC-PK1 Cells.

LLC-PK1 cells overexpressing human P-glycoprotein (encoded by the multidrug resistance 1 gene) were obtained from Dr. A. Schinkel, The Netherlands Cancer Institute (Amsterdam, The Netherlands) under license agreement and used for monolayer permeability measurements as previously described (Poirier et al., 2014). Cells were cultured in Medium-199 supplemented with 10% (v/v) fetal calf serum (FCS), penicillin-streptomycin (10,000 IU/µg per milliliter), and colchicine (150 ng/ml).

Briefly, compounds dosed at 1 µM in phenol red-free Medium-199 supplemented with 10% (v/v) FCS and penicillin-streptomycin (10,000 IU/µg per milliliter) were evaluated for permeability in the apical-to-basolateral and basolateral-to-apical directions using a liquid handling robot (Tecan, Männedorf, Switzerland). Samples were collected from triplicate wells of donor and receiver compartments after a 3.5-hour incubation in the presence or absence of P-glycoprotein (P-gp) inhibitor (zosuquidar, 1 µM). Drug concentrations were measured by high-performance LC-MS/MS. The extracellular marker lucifer yellow (10 µM) was also added to ensure monolayer integrity; values exceeding 25 nm/s were eliminated from analysis. The incubation was conducted in triplicates.

For the permeability data assessment, the following equation was used:(1)where apparent permeability (P_app), A, C0, and dQ/dt represent the mean, bidirectional apparent permeability in the presence of P-gp inhibitor; the filter surface area; the initial concentration of test substances; and the amount per time period (3.5 hours), respectively. The recovery was calculated according to the following equation:(2)where C_r is the concentration in the receiver compartment at the end of experiment, V_r is the volume of the receiver compartment, C_d is the concentration in the donor compartment at the end of experiment, C_d0 is the initial concentration in the donor compartment, and V_d is the volume of the donor compartment. The acceptable range for mass balance recovery was 70%–120%.

Hepatic Uptake in Human Plated Hepatocytes.

The cryopreserved primary cells (male and female; mixed) were reconstituted in a tube containing prewarmed InVitroGRO cryopreserved platable medium at 37°C (BioreclamationIVT). To each well of a collagen I–coated 24-well tissue culture plate (BD BIOCOAT; BD Biosciences, San Jose, CA), 0.5 ml of the cell suspension was added corresponding to approximately a cell density of 0.3 × 10⁶ cells per well. The plates were then incubated at 37°C and 5% CO₂, with saturating humidity for 3 and 4 hours to allow for cell adherence, the medium was removed, and the cells were washed once with 1 ml of prewarmed Hanks’ balanced salt solution (HBSS; Thermo Fisher Scientific), supplemented with HEPES (20 mM), pH 7.4. The experiment was initiated by aspirating the wash buffer and then adding 0.3 ml of a prewarmed serum-free HBSS including the test compound in the absence and presence of the inhibitor cocktail on active transporters: MK-571 at 100 µM, cyclosporine A at 20 µM, and quinidine at 100 µM [inhibiting organic anion transporting polypeptides (OATPs), P-gp, breast cancer resistance protein (BCRP), MRPs, bile salt export pump, Na⁺-taurocholate cotransporting polypeptide, organic anion transporter (OAT) 2, and organic cation transporter (OCT) 1]. The inhibitor concentrations were optimized to achieve complete inhibition effects on hepatic transporters without any cell toxicity (in-house information). After the designated incubation time optimized to derive the linear uptake profile, the assay was stopped by washing the cells three times with ice-cold HBSS buffer containing 0.2% (v/v) bovine serum albumin. Subsequently, 0.3 ml of acetonitrile/water (75/25, v/v) containing internal standard was added to deprotonate the cells. The aliquot was transferred into LC-MS/MS to measure concentrations of the test drug. Using an additional cell plate by lysing the cells in 1% (v/v) Triton X-100, an average protein content (milligrams per milliliterl) was determined by the Pierce BCA assay kit with bovine serum albumin used as standard (Thermo Fisher Scientific). The uptake assay was conducted in triplicates.

Drug permeability clearance across the plasma membrane (PSinf: milliliters per minute per milligram protein) was determined from the test compound amount inside the cells (normalized to the cell protein amount and incubation time) divided by the measured concentration in the incubation medium. Contribution of an active uptake (PSinf,act) component (%) to the total permeability clearance (PSinf) was calculated with the following equation:(3)

ECCS Classification of Marketed Drugs Based on an Experimentally Defined Permeability Threshold.

Using Roche in-house synthesized discovery compounds from the same chemical series as a development compound previously reported to show active hepatic uptake in clinical studies (Kratochwil et al., 2018), P_app (nanometers per second) in the apical to basal direction in LLC-PK1 cells was plotted against the respective hepatic active uptake component (%). These compounds all included a carboxylic acid moiety, indicating potential of being substrates of OATP, and had MW >400. This correlation was used to set a permeability threshold for ECCS-based differentiation between classes 1/2 and 3/4. For verification, ECCS classification for 36 marketed drugs was conducted, followed by IVIVE for CLint,h prediction. Known enzyme and transporter interactions for these drugs are provided in Table 1.

Metabolism by Suspended Human Hepatocytes.

Primary pooled cryopreserved hepatocytes (male and female; mixed) were reconstituted in prewarmed William’s E media containing 10% FCS, 0.05 mg/ml streptomycin and 50 U/ml penicillin, and 0.4 mM l-glutamine from Thermo Fisher Scientific and 0.01 mg/ml gentamicin, 0.048 mg/ml hydrocortisone, and 0.004 mg/ml insulin from Sigma-Aldrich, to a final suspension density of 1 × 10⁶ cells/ml.

The incubation was performed fully automatically with Evo Liquid Handling System (Tecan) equipped with a CO₂ incubator with an orbital shaker from LiCONiC instruments (Montabaur, Germany). After the addition of test compounds at 1 µM, 0.1% v/v DMSO (marketed: n = 36) to the wells (1 × 10⁵ cells/well, 100 µl incubation volume), the 96-well hepatocyte suspension culture plates were incubated at 900 rpm in a 5% CO₂ at 37°C. Samples were quenched by addition of acetonitrile (including an internal standard) to the incubation well at the designated time points up to 2 hours. The incubation was conducted in n = 1 or 2. For intrinsic clearance, n = 1 denotes a single intrinsic clearance value determined using seven different independent incubation wells with different incubation times.

The natural logarithm of the percentage initial drug concentration in the incubation samples was plotted against time, and a linear regression analysis was applied using GraphPad Prism version 7.04 for Windows (GraphPad Software, La Jolla, CA). The in vitro CL_int was calculated from the slope of the linear regression:(4)

A conversion factor of 1 mg protein per million hepatocytes as determined previously (Kratochwil et al., 2017) was used to convert intrinsic clearance per million cells into intrinsic clearance per milligram hepatocyte protein.

Metabolism by Human HepatoPac.

Incubations for the same set of the test compounds (1 µM, 0.1% v/v DMSO) as conducted in suspension assays were performed in 96-well plates containing either a coculture of adherent hepatocytes (mixed, n = 5 for male and n = 5 for female) with mouse fibroblast control cells or control cells alone (5% CO₂ atmosphere and 37°C). The incubation media in human HepatoPac was identical with that in suspended hepatocytes. At defined time points (2, 18, 26, 48, 72, and 96 hours), whole wells were quenched with ice-cold acetonitrile containing an internal standard. Samples were then centrifuged appropriately, and the supernatant was analyzed by LC-MS/MS. The incubation was conducted in n = 1 or 2. For intrinsic clearance, n = 1 denotes a single intrinsic clearance value determined using eight different independent incubation wells with different incubation times.

Measured concentrations in the medium were plotted against incubation time (minutes) and a linear fit made to the data with emphasis upon the initial rate of compound disappearance. This calculation was performed for samples prepared from HepatoPac and fibroblast control cells (stromal cells). The slope of the fit was then used to calculate the apparent intrinsic clearance:(5)

The apparent intrinsic clearance in hepatocytes was corrected for fibroblast metabolism. Each well in HepatoPac plates has 75% surface area as fibroblasts and 25% surface area as hepatocytes (Khetani and Bhatia, 2008; Chan et al., 2019). The in vitro intrinsic clearance determined from the fibroblast lysate according to eq. 5 were multiplied by 0.75 and then subtracted from the respective value determined in HepatoPac plates, to exclude metabolic clearance by fibroblasts. Hence, the hepatocyte-specific intrinsic clearance (CLint,met) was calculated using eq. 6:(6)

LC-MS/MS Analysis.

Calibration samples for test compounds were prepared by spiking stock solution of the test substance into the blank culture medium used for the experiment (1%, v/v, DMSO), followed by quenching with 2-fold volumes of acetonitrile containing 0.1 µg/ml oxazepam as internal standard. All samples were 10-fold diluted in a mixture of acetonitrile and water (66:33, v/v). The high-performance liquid chromatography system consisted of 20AD Shimadzu pumps (Kyoyo, Japan) and an HTS CTC PAL autosampler (CTC Analytics AG; Zwingen, Switzerland). Sample solutions (1 µl) were injected into the analytical column heated to 60°C (Supelco Ascentis Express C18; 2 cm × 2.1 mm, particle size: 2.7 µm; Sigma-Aldrich). To elute the compounds, the following mobile phases were used: phase A, formic acid 0.5% in water:methanol = 95:5 (v/v); phase B, acetonitrile. A high pressure linear gradient from 0% to 95% B in 40 seconds was applied at a flow rate of 600 µl/min. Mass spectrometric detection with multiple reaction monitoring was operated in the positive ion mode with an API6500 mass spectrometer equipped with a TurboIonSpray source (IonSpray Voltage 5500 V; Sciex, Framingham, MA). Analyst 1.6.3 software (Sciex) was used for data processing using linear regression with 1/x² weighting on peak area ratio. The precision and accuracy of linear regression of the standard curve samples was between 80% and 120%.

Prediction of Human Hepatic Intrinsic Clearance from the In Vitro Assays.

Using the measured clearance values (CLint,met from hepatocyte suspension and HepatoPac), the intrinsic hepatic unbound clearance (CLint,h,u) was estimated according to eq. 7:(7)where fu(inc) is the unbound fraction in incubates. The fu(inc) values were predicted with the dilution method (Schuhmacher et al., 2000) based on the reported unbound fractions in plasma (fu,p), without conducting experimental measurements (Table 2). Correspondence of calculated and measured fu(inc) values was confirmed with a limited test compound set (midazolam, quinidine, diclofenac, tolbutamide, and dextromethorphan) for which measured fu(inc) values were previously available: 0.19, 1, 0.02, 0.35, and 1, respectively (Kratochwil et al., 2017). This provided validity of the use of the dilution method for fu(inc) prediction. For measured intrinsic uptake clearance (PSinf), fu(inc) was assumed as 1, since the assays were conducted in the absence of serum protein.

It was assumed that intrinsic process clearance values in the current in vitro assays were measured under linear conditions: substrate concentration (S) << Km.

For CLint,h,u prediction, the following physiologic scaling factors were applied: hepatocellularity, 120 (10⁶ cells = mg protein/g liver); liver weight, 22 (g liver/kgbw); and body weight, 70 (kgbw). The previous study results from the protein determination using human hepatocyte suspensions indicated that the 10⁶ cells was equivalent to 1 mg protein content (Kratochwil et al., 2017). The identical relationship between milligrams protein and cell numbers is assumed with HeatoPac, which the identical protein content per hepatocyte is applied for scaling the hepatocyte CLint,met after subtraction of the background turnover measured by the incubation with fibrobrasts as indicated in eq. 6. Otherwise, the scaling factors were generally taken from the system parameter database (Johnson et al., 2005).

Estimation of Human In Vivo Hepatic (Intrinsic) Clearance as Reference.

Pharmacokinetic parameters for reference compounds were collected from publications, clinical pharmacology review documents and drug prescribing labels. Hepatic plasma (organ) clearance CLh,p was calculated from total plasma clearance CLtot,p by subtraction of renal CLr,p and other pathway clearances CLothers,p:(8)

The fu,p and the blood-to-plasma concentration ratio (Rb) were used for the calculation of fu,b = fu,p/Rb, assuming Rb = 1 if no data were available (Table 2).

Hepatic organ blood clearance (CLh,b) was obtained as CLh,b = CLh,p/Rb. Finally, the in vivo intrinsic hepatic clearance was calculated with the well stirred model as follows:(9)where Qh is hepatic blood flow rate (23.2 ml/min per kilogram; Johnson et al., 2005). Key pharmacokinetic (PK) parameters used for evaluation of the current IVIVE performance for CLint,h,u prediction are summarized in Table 2. Derivations of the in vivo CLtot,p, CLr,p, and CLothers,p are summarized in Supplemental Table 1 with the literature source information.