*2.3. Bidirectional Permeability Assay*

Bidirectional permeability assays were performed as described previously [13,14]. Briefly, compounds were diluted in transport buffer (128.13 mM NaCl, 5.36 mM KCl, 1 mM MgSO4, 1.8 mM CaCl2, 4.17 mM NaHCO3, 1.19 mM Na2HPO4, 0.41 mM NaH2PO4, 15 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 20 mM glucose, pH 7.4) containing 0.25% bovine serum albumin to a final concentration of 10 µM and added to the apical or basal compartment. In indicated experiments, inhibitors were added to both compartments. Cells were incubated with the compounds for up to 2 h. Samples from the opposite compartment were taken at different timepoints. Compound concentrations in the samples were determined by HPLC-MS/MS (standard equipment: HPLC series 1000 or higher from Agilent, Santa Clara, CA, USA, and mass spectrometers API 4000 or higher from AB Sciex, Toronto, ON, Canada). Prior to bioanalysis samples were spiked with internal standard solution and diluted with acetonitrile (ACN) for protein precipitation. Measurement was operated in multiple reaction monitoring (MRM) mode. Quantification was performed using external calibration. Apparent

permeability coefficients in the apical to basal direction (*Papp,AB*) and in the basal to apical direction (*Papp,BA*) and efflux were calculated as follows

$$P\_{app,AB} = \frac{Q\_{AB}}{(\mathbb{C}\_0 \times s \times t)}$$

$$P\_{app,BA} = \frac{Q\_{BA}}{(\mathbb{C}\_0 \times s \times t)}$$

$$Efflux = \frac{P\_{app,BA}}{P\_{app,AB}}$$

where *Q* is the amount of compound recovered in the receiver compartment after the incubation time *t*, *C*<sup>0</sup> the initial compound concentration given to the donor compartment, and s the surface area of the Transwell inserts. Efflux ratio is calculated as the quotient of *Papp,BA* to *Papp,AB*. As quality controls, one reference P-gp substrate (apafant) and one low permeable compound (BI internal reference, *Papp* ≈ 3 × 10−<sup>7</sup> cm/s, no efflux) is included in every assay plate. In addition, Transepithelial electrical resistance (TEER) values are measured for each plate before the permeability assay and total recovery in donor and receiver compartments was determined for each compound. All these parameters (efflux of apafant, *Papp* values of the low permeable compound, TEER values, and total recovery) are used to ensure the quality of the assays.

#### *2.4. Measurement of DME Activities in EpiIntestinal and Caco-2*

For measurements of DME activities in EpiIntestinal microtissues and Caco-2 cells, both were cultured in 96-well Transwell inserts. Drugs (Table 1) were dissolved in the respective solvent at 200× concentration and diluted in a pre-warmed transport buffer. Diluted substrate solution was applied to the apical (100 µL) and basal (250 µL) compartment of the Transwell and incubated at 37 ◦C and 5% CO<sup>2</sup> and 60 rpm continuous shaking. DME activities were determined by monitoring metabolite formation in basal compartment over time (0, 0.5, 1, 2, 3 and 4 h) with LC-MS/MS. For LC-MS/MS, an HTS-xt PAL autosampler (CTC Analytics), LC 1290 infinity G4220A (Agilent Technologies), column oven (Agilent Technologies) and 6500 TripleQuad (AB Sciex) were used. Chromatographic separation of samples was performed on YMC Triart C18 (1.9 µm, 30 × 2 mm; YMC Europe, Dinslaken, Germany) LC analytical column. Quantification of all metabolites listed in Table 1 was achieved by the use of calibration curves for the individual metabolites with appropriate concentration ranges.



A total of 10 µL of the incubation sample were diluted with 90 µL of water containing 10–20% ACN or methanol, 0.1% formic acid and the respective internal standard. To analyze the intracellular metabolite concentration, cells on Transwell inserts were washed twice with ice-cold PBS and stored at −80 ◦C for 20 min. Afterwards, 150 µL 50% ACN diluted with transport buffer was added to the cells

and incubated at room temperature for 30 min. Cell lysate was transferred to a fresh 96-well plate and centrifuged at 4 ◦C, 4000 rpm for 10 min. Subsequently, 10 µL of the supernatant were diluted with 90 µl water containing 10–20% ACN or methanol, 0.1% formic acid and the respective internal standard. A total of 2 µL sample was injected into the LC-MS/MS system operated with an electrospray ionization source.

#### *2.5. CES-Mediated Metabolism of Dabigatran Etexilate*

CES-mediated metabolism of dabigatran etexilate was measured in cryopreserved human hepatocytes (BioIVT, West Sussex, UK) and cryopreserved human intestinal mucosa (in vitro ADMET laboratories, Columbia, MD, USA) in suspension and in Caco-2 cells and EpiIntestinal microtissues grown on Transwell inserts. Dabigatran etexilate was diluted in culture media for the respective cells or tissues. The final concentrations of dabigatran etexilate were selected in an earlier experiment to ensure reasonable turnover of the compound within the incubation time: 2 µM for hepatocytes and intestinal mucosa, 10 µM for Caco-2 and EpiIntestinal microtissues. In the experiments with Transwell inserts, dabigatran etexilate was given to the apical compartments, metabolites were measured in the basal (receiver) compartments. Concentrations of the metabolites were determined by HPLC-MS/MS (Section 2.3).

#### *2.6. Metabolite Identification*

Raloxifene or ezetimibe (10 µM) in culture media was added to the apical compartment of EpiIntestinal microtissues or incubated with cryopreserved human intestinal mucosa (HIM). Samples from basal compartment of EpiIntestinal microtissues or lysates of the incubation mixture with mucosa were prepared for metabolite identification as follows: samples were mixed with the same amount of 0.1% formic acid in ACN and subsequently evaporated and resuspended in water containing 25% methanol and 0.1% formic acid. Analysis was performed on a LC-MS system containing a Vanquish UPLC (ThermoFisher Scientific, San Jose, CA, USA) coupled to an Orbitrap FusionTribrid high resolution mass spectrometer (ThermoFisher Scientific). Structure elucidation was based on exact mass measurements in combination with the interpretation of fragment spectra.

#### *2.7. Measurement of Intestinal First-Pass Availability in EpiIntestinal Microtissues and Caco-2*

Compounds were diluted in culture media to a final concentration of 10 µM and added to the apical (donor) compartment (total volume: 100 µL for EpiIntestinal and 200 µL for Caco-2). After the incubation at 37 ◦C for 2, 4, 6, and 24 h, samples (50 µL) were taken from the basal (receiver) compartment (total volume: 5000 µL for EpiIntestinal and 800 µL for Caco-2). After the last timepoint, samples from the donor compartments and the cell lysates were also collected. Compound concentrations in the samples were determined by HPLC-MS/MS (Section 2.3).

GI first-pass availability of the tested compounds was expressed as fraction (%) of the total amount of a compound added to the donor compartment recovered in the receiver compartment.
