Abstract
This study aimed to demonstrate the usefulness of human jejunal spheroid-derived differentiated intestinal epithelial cells as a novel in vitro model for clarifying the impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans. Three-dimensional human intestinal spheroids were successfully established from surgical human jejunal specimens and expanded for a long period using L-WRN-conditioned medium, which contains Wnt3a, R-spondin 3, and noggin. The mRNA expression levels of intestinal pharmacokinetics-related genes in the human jejunal spheroid-derived differentiated intestinal epithelial cells were drastically increased over a 5-day period after seeding compared with those in human jejunal spheroids and were approximately the same as those in human jejunal tissue over a culture period of at least 13 days. Activities of typical drug-metabolizing enzymes [cytochrome P450 (CYP) 3A, CYP2C9, uridine 5′-diphospho-glucuronosyltransferase 1A, and carboxylesterase 2] and uptake/efflux transporters [peptide transporter 1/solute carrier 15A1], P-glycoprotein, and breast cancer resistance protein) in the differentiated cells were confirmed. Furthermore, intestinal availability (Fg) values estimated from the apical-to-basolateral permeation clearance across cell monolayer showed a good correlation with the in vivo Fg values in humans for five CYP3A substrate drugs (Fg range, 0.35–0.98). In conclusion, the functions of major intestinal drug-metabolizing enzymes and transporters could be maintained in human jejunal spheroid-derived differentiated intestinal epithelial cells. This model would be useful for the quantitative evaluation of the impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans.
SIGNIFICANCE STATEMENT Limited information is available regarding the quantitative prediction of the impact of drug-metabolizing enzymes and transporters on the human intestinal absorption of substrates using in vitro assays with differentiated cells derived from human intestinal spheroids/organoids. This study confirmed the functions of typical drug-metabolizing enzymes and transporters in human jejunal spheroid-derived differentiated intestinal epithelial cells and demonstrated that intestinal availability (Fg) estimated from apical-to-basolateral permeation clearance across cell monolayers showed a good correlation with in vivo human Fg for CYP3A substrates.
Introduction
Various drug-metabolizing enzymes and drug transporters that play major roles in the oral absorption of drugs are expressed in the small intestine (Xie et al., 2016; Müller et al., 2017). For example, cytochrome P450 (CYP) 3A, which is the most important drug-metabolizing enzyme in the small intestine, and P-glycoprotein (P-gp), which is a multispecific efflux transporter on the apical membrane, are responsible for limiting the intestinal absorption of a wide variety of drugs. They provide the site of saturation, leading to the nonlinear pharmacokinetics of drugs (Takano et al., 2016). Furthermore, inhibition and induction of intestinal CYP3A and P-gp led to significant changes in the bioavailability of their substrate drugs in humans (Holtbecker et al., 1996; Greiner et al., 1999; Kato, 2008; Fenner et al., 2009). Therefore, quantitative evaluation of the impact of drug-metabolizing enzymes and transporters on intestinal drug absorption is important in the process of drug development.
Various in vitro models have been developed to quantitatively assess intestinal drug absorption in humans. Currently, Caco-2 cells, which are an immortalized cell line derived from human colorectal adenocarcinoma, are widely used in drug development as a gold standard for the in vitro models. The apparent permeability coefficient (Papp) of drugs that are absorbed mainly by passive diffusion across Caco-2 cell monolayers were closely correlated with the fraction of orally administered drugs that enter the gut wall (Fa) in humans (Artursson and Karlsson, 1991). However, previous studies showed that expression levels of certain drug-metabolizing enzyme and transporter subtypes, such as CYP3A and peptide transporter (PEPT) 1, in Caco-2 cells are significantly lower than those in the human small intestine (Nakamura et al., 2002; Seithel et al., 2006).
We recently reported freshly isolated human jejunal sections mounted onto the Ussing chamber as an in vitro model to investigate the impact of transporters on the intestinal absorption of substrate drugs in humans (Michiba et al., 2021). The functions of major drug-metabolizing enzymes and uptake/efflux transporters were maintained in this model (Rozehnal et al., 2012; Sjöberg et al., 2013; Michiba et al., 2021). However, some limitations exist with regard to utilizing this experimental system in the process of drug development; for instance, limited and irregular availability of fresh human intestinal tissue, narrow time window to maintain the barrier integrity in epithelial cells and transporter functions (Michiba et al., 2021), and a lack of cryopreservation method of intestinal tissue.
In recent years, rapid progress has been made in research on the generation of human-induced pluripotent stem cell (iPSC)-derived intestinal epithelial cells. The activities of major intestinal drug-metabolizing enzymes and transporters were confirmed in human iPSC-derived intestinal epithelial cells (Kodama et al., 2016a; Yoshida et al., 2021). However, it was suggested that human iPSC-derived intestinal epithelial cells partly showed an immature phenotype compared with adult human small intestinal cells (Finkbeiner et al., 2015). Although some studies reported methods to control the differentiation status (Kodama et al., 2016b; Kabeya et al., 2018), further research is needed to realize the quantitative prediction of intestinal drug absorption in humans, especially for CYP3A substrate drugs (Kabeya et al., 2020).
Spheroids and organoids, which are three-dimensional (3D) culture systems, have been used in a wide range of research fields as a model that reflects functional properties of organs and maintains cellular characteristics (Corrò et al., 2020). Intestinal spheroid cultures allow the long-term culture of intestinal stem cells with the capability to differentiate into multiple lineages (Sato et al., 2009; VanDussen et al., 2015). Furthermore, two-dimensional (2D) monolayers, which can allow easy evaluation of transcellular drug transport, can be set up from 3D intestinal spheroids (van der Hee et al., 2018). Recently, Yamashita et al. (2021) successfully demonstrated 2D monolayers from biopsy-derived human duodenal organoids showed the functions of CYP3A, carboxylesterase (CES) 2, and P-gp, and its global gene expression profile was closer to that of the human duodenum than of Caco-2 cells. Thus, human intestinal spheroid/organoid-derived intestinal epithelial cells are expected to maintain the function of detoxification occurring in the human intestine. However, limited information is available regarding the overall performance of such an in vitro assay system using differentiated cells derived from human 3D-cultured intestinal stem cells from the viewpoint of quantitative prediction of the impact of drug-metabolizing enzymes and transporters on the human intestinal absorption of substrate drugs.
In the present study, we aimed to demonstrate the usefulness of human jejunal spheroid-derived intestinal epithelial cells for investigating the quantitative impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans.
Materials and Methods
Chemicals
Commercially available compounds, including stable isotope-labeled compounds, were used in all experiments (Supplemental Table 1). All other chemicals and reagents used for analyses were also commercially available and analytical-grade products.
Human Jejunal Tissue
Human intestinal tissues (approximately 5 cm normal jejunal tissue specimens) were obtained from clinical patients with pancreatic head tumors who underwent pancreatoduodenectomy at the University of Tsukuba Hospital, as in our previous study (Michiba et al., 2021). All experimental protocols regarding these jejunal sections were approved by the ethics committees of both the Graduate School of Pharmaceutical Sciences, University of Tokyo and the Faculty of Medicine, University of Tsukuba. Written informed consent prior to surgery was obtained from all patients. The excised jejunum was immediately put in ice-cold Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, Waltham, MA) at the operating theater and transferred to the laboratory at the University of Tsukuba within 30 minutes. For establishing human intestinal spheroid cultures, the excised jejunum was put in an ice-cold washing medium (DMEM/F12; Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 2 mM L-glutamine (Merck), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific), and transferred on ice to the laboratory at the University of Tokyo. Procedures for establishing human intestinal spheroid cultures were immediately performed on the same day. To stabilize mRNA in the freshly isolated human jejunal tissues, a small part of the excised jejunum sample was immediately put into RNAlater solution (Thermo Fisher Scientific). This sample was transferred on dry ice to the laboratory at the University of Tokyo and preserved at −80°C until analyses by reverse transcription–quantitative polymerase chain reaction.
Establishment of Human Jejunal Spheroids
L-WRN cells (CRL-3276), which are L-cells expressing Wnt3a, R-spondin 3, and noggin, were obtained from the American Type Culture Collection (Rockville, VA). We followed protocols for the establishment of human intestinal spheroid cultures and the preparation of L-WRN conditioned medium (L-WRN CM) previously described by Miyoshi and Stappenbeck (2013). Briefly, approximately 1 cm2 of jejunal tissue specimens was minced and incubated with collagenase solution [washing medium supplemented with 2 mg/mL collagenase type I powder (FUJIFILM Wako Pure Chemical, Osaka, Japan) and 50 μg/mL gentamicin (Thermo Fisher Scientific)] at 37°C for 1 hour. Crypts were isolated from tissue fragments by pipetting, filtering through a 100 μm strainer and centrifuging at 200 g for 5 minutes. The pellets were gently resuspended in Matrigel (Corning, Corning, NY), and 60 μL of the cell suspension were placed at the center of each well in a 6-well plate and spread carefully using pipette tips. The plate was incubated at 37°C for 15 minutes to allow Matrigel to be polymerized. A total of 2 mL of 50% L-WRN CM [1:1 dilution with advanced DMEM/F12 (Thermo Fisher Scientific) supplemented with 20% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin] containing 10 μM Y-27632 (a Rho-associated protein kinase inhibitor, FUJIFILM Wako Pure Chemical) and 1 μM SB431542 (a transforming growth factor-β inhibitor, FUJIFILM Wako Pure Chemical) was added to each well. Primary spheroids were generated within 3–5 days. The medium was replaced with fresh 50% L-WRN CM every 2 days.
Passage and Cryopreservation of Human Jejunal Spheroids
Human jejunal spheroids were passaged every 5–7 days at ratios of 1:3–1:5 for maintenance. We followed the protocol for the passage of human intestinal spheroids previously described by Miyoshi and Stappenbeck (2013). Briefly, human jejunal spheroids at 5–7 days after passage were collected and dissociated into small cell clusters by scratching the Matrigel using pipette tips, incubating the Matrigel-cell mixture in TrypLE Express (Thermo Fisher Scientific) at 37°C for 5 minutes and pipetting gently. The small cell clusters were gently resuspended in fresh Matrigel, and 60 μL of the suspension was placed at the center of each well in a 6-well plate and spread carefully using pipette tips. The plate was incubated at 37°C for 15 minutes to allow the Matrigel to be polymerized, and 2 mL of 50% L-WRN CM containing 10 μM Y-27632 and 1 μM SB431542 was added to each well. The medium was replaced with fresh 50% L-WRN CM every 2 days.
For cryopreservation, human jejunal spheroids at 5–7 days after passage were collected and dissociated into small cell clusters as described above and centrifuged at 200 g for 5 minutes. The pellets were gently resuspended in BAMBANKER (Lymphotec, Tokyo, Japan), and the suspension was frozen at −80°C. The frozen cell suspension was stored in cryotubes in a liquid nitrogen tank.
Generation of 2D Monolayers of Differentiated Intestinal Cells from Human Jejunal Spheroids
We followed the protocol for the generation of 2D monolayers of differentiated intestinal cells previously described by VanDussen et al. (2015). Human jejunal spheroids at 6 days after passage were collected and dissociated into single cells by scratching the Matrigel using pipette tips, incubating the Matrigel-cell mixture in TrypLE Express at 37°C for 15 minutes, and pipetting vigorously. The single cells were filtered using a 40 μm strainer and centrifuged at 200 g for 5 minutes. The pellets were resuspended in 50% L-WRN CM containing 10 μM Y-27632, and cells were seeded into 24-well plates or 24-well culture inserts (polyester membrane, 0.4 μm pore size; Corning) at a density of 2.5 × 105–7.5 × 105 cells/cm2. The plates or inserts were precoated using 0.5% (vol/vol) Matrigel in advanced DMEM/F12 at 37°C for 1–2 hours. The medium was removed and replaced with 0% L-WRN CM (advanced DMEM/F12 supplemented with 20% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin without Wnt3a, R-spondin 3, and noggin) at 24 hours after seeding. The medium was replaced with fresh 0% L-WRN CM every 2 days.
Transepithelial electrical resistance (TEER) values were measured using Millicell ERS-2 (Merck). Resistance of the monolayer seeded on culture inserts was obtained by subtracting the blank resistance value from the measured resistance value and then multiplying the values by the culture area of a culture insert (0.33 cm2).
Gene Expression Analysis
Total RNA was isolated from the human jejunal tissue sections and human jejunal spheroid-derived intestinal epithelial cells seeded on 24-well plates using ISOGEN II (NIPPON GENE, Tokyo, Japan). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Tokyo, Japan) according to the manufacturer’s instructions. The mRNA expression levels of ATP-binding cassette (ABC) B1 (P-gp), solute carrier (SLC) 15A1 (PEPT1), CYP3A4, and sucrase-isomaltase (SI) were determined by qPCR analyses using THUNDERBIRD SYBR qPCR Master Mix (TOYOBO, Tokyo, Japan) and a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). The mRNA expression levels were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase gene. The sequences of primers for the detection of the above-mentioned mRNAs are presented in Supplemental Table 2.
Measurement of the Bidirectional Transport of Digoxin, Fexofenadine, Sulfasalazine, and Rosuvastatin across Human Jejunal Spheroid-Derived Intestinal Epithelial Cell Monolayer
Digoxin and fexofenadine (5 μM each) were selected as P-gp substrates and sulfasalazine and rosuvastatin (5 μM each) as breast cancer resistance protein (BCRP) substrates to investigate the efflux transport mediated by P-gp and BCRP. PSC833 (5 μM) and Ko143 (5 μM) were selected as specific inhibitors of P-gp and BCRP, respectively. To examine the effects of these inhibitors on passive transcellular transport, antipyrine (1 μM) was selected as a passive transcellular marker. Lucifer yellow (10 μM) was used as a paracellular marker to evaluate the integrity of the cell monolayer.
Prior to the addition of test compounds, the cell monolayers at 7 days after seeding on 24-well culture inserts were preincubated for 30 minutes with transport buffer containing 118 mM NaCl, 1.53 mM CaCl2, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 5.00 mM D-glucose, 23.8 mM NaHCO3, and 12.5 mM MES (MES transport buffer, adjusted to pH 6.5, for apical side) or 12.5 mM HEPES (HEPES transport buffer, adjusted to pH 7.4, for basolateral side) in the presence or absence of P-gp/BCRP inhibitor cocktail (added to both sides). The buffer volumes used for the apical and basolateral compartments were 0.4 mL and 1 mL, respectively. The bidirectional transport assay was initiated by replacing the buffer in the apical and basolateral compartments with fresh MES or HEPES transport buffer with or without test compounds in the presence or absence of P-gp/BCRP inhibitor cocktail (added to both sides). The cell monolayers were incubated at 37°C for 2 hours. At 30, 60, and 120 minutes after the addition of test compounds, aliquots of the buffer in the receiver compartment were collected and an equal volume of fresh MES or HEPES transport buffer was added. The collected samples were preserved at −20°C until liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses.
Measurement of the Apical-to-Basolateral Transport of Cefadroxil across Human Jejunal Spheroid-Derived Intestinal Epithelial Cell Monolayer
Cefadroxil (10 μM) was selected as a PEPT1 substrate to evaluate PEPT1-mediated cellular uptake. An excess concentration of glycylsarcosine (Gly-Sar; 10 mM), which is a typical substrate of PEPT1, was used to saturate PEPT1. As mentioned above, antipyrine (1 μM) and lucifer yellow (10 μM) were added to the buffer in the donor compartment to confirm no effect on passive and paracellular transport.
Prior to the addition of test compounds, the cell monolayers at 7 days after seeding on 24-well culture inserts were preincubated for 30 minutes in MES or HEPES transport buffer in the presence or absence of Gly-Sar (added only to the apical side). Afterward, the transport assay was initiated by replacing the buffer in the apical and basolateral compartments with fresh MES transport buffer with test compounds and HEPES transport buffer without test compounds, respectively, in the presence or absence of Gly-Sar (added only to the apical side). The sampling of the buffer was carried out as described above.
Measurement of the Metabolic Activities of CYP3A, CYP2C9, Uridine 5′-Diphospho-Glucuronosyltransferase 1As, and CES2 in Human Jejunal Spheroid-Derived Intestinal Epithelial Cells
Midazolam (5 μM), diclofenac (10 μM), raloxifene (5 μM), and irinotecan (50 μM) were selected to evaluate the metabolic activities of CYP3A4/5, CYP2C9, uridine 5′-diphospho-glucuronosyltransferase (UGT) 1A, and CES2, respectively. Ketoconazole (10 μM), sulfaphenazole (50 μM), zafirlukast (50 μM), and loperamide (20 μM) were used as specific inhibitors of CYP3A4/5, CYP2C9, UGTs, and CES2, respectively.
Prior to the addition of test compounds, the cells at 5 days after seeding on 24-well plates were preincubated for 30 minutes in HEPES transport buffer in the presence or absence of ketoconazole, sulfaphenazole, zafirlukast, and loperamide. Afterward, metabolism assay was initiated by replacing the buffer with fresh HEPES transport buffer containing the substrates in the presence or absence of the corresponding inhibitor. The cells were incubated at 37°C for 2 hours. After the 2 hour-incubation, the buffer and cells were collected to determine the amount of the major metabolites produced by the corresponding enzyme (1′-hydroxy midazolam, 4′-hydroxy diclofenac, raloxifene-4′-glucuronide, and SN-38). Cellular protein amounts were measured using the bicinchoninic acid method. The metabolic activity was normalized by the cellular protein amount.
Measurement of the Apical-to-Basolateral Transport of Midazolam, Triazolam, Alprazolam, Felodipine, and Nifedipine across Human Jejunal Spheroid-Derived Intestinal Epithelial Cell Monolayer
Midazolam, triazolam, alprazolam, felodipine, and nifedipine (1 μM each) were selected as CYP3A substrates (non-P-gp substrates) to investigate the effects of ketoconazole, which is a potent CYP3A inhibitor, and 1α, 25-dihydroxyvitamin D3, which is a typical CYP3A inducer, on the apical-to-basolateral transport across the cell monolayers. To induce CYP3A expression by 1,25-(OH)2D3, cell monolayers were treated with 10 nM 1,25-(OH)2D3 for 96 hours before starting the transport assay.
Prior to the addition of the substrates, the cell monolayers at 9 days after seeding on 24-well culture inserts were preincubated for 30 minutes in MES or HEPES transport buffer in the presence or absence of ketoconazole (10 μM; added to both sides). The transport assay was initiated by replacing the buffer in the apical and basolateral compartments with fresh MES transport buffer with the substrates and HEPES transport buffer without the substrates in the presence or absence of ketoconazole (added to both sides), respectively. The cell monolayers were incubated at 37°C for 2 hours. At 30, 60, and 120 minutes after the addition of the substrates, aliquots of the buffer in the receiver compartment were collected and an equal volume of fresh HEPES transport buffer was added. After a 2 hour-incubation, the buffer in the donor compartment and cells were collected to determine the amount of the major metabolites formed by CYP3A (1′-hydroxy midazolam, α-hydroxy triazolam, α-hydroxy alprazolam, dehydro-felodipine, and dehydro-nifedipine).
Analytical Methods for Determining the Concentration of Test Compounds
Quantification of all test compounds except lucifer yellow was performed using QTRAP 5500 LC-MS/MS System (AB SCIEX, Foster City, CA) equipped with Nexera ultra-high performance liquid chromatography (Shimadzu, Kyoto, Japan). Samples were mixed with three volumes of acetonitrile containing internal standards. The solution was vortexed and centrifuged at 20,000 g for 5 minutes, and the supernatant was injected into the LC-MS/MS system. An Atlantis T3 C18 column (3 μm, 2.1 mm × 50 mm; Waters Corp., Milford, MA) was used for the chromatographic separation of analytes, and the column temperature was maintained at 40°C. The mobile phase was distilled water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The flow rate was 0.4 mL/min, and the injection volume was 10 μL. The details of the HPLC conditions are summarized in Supplemental Table 3. The mass spectrometer with electrospray ionization was operated in both positive and negative multiple-reaction monitoring modes. The details of the multiple-reaction monitoring conditions are summarized in Supplemental Table 4.
The concentration of lucifer yellow was measured using a fluorescence microplate reader (Infinite 200, excitation wavelength 428 nm, emission wavelength 536 nm; TECAN, Männedorf, Switzerland).
Data Analysis
The apparent permeability coefficient (Papp; cm/s) was calculated according to eq. 1:
where dQ/dt, A, and C0 represent the amount of the test compound transported to the receiver compartment per unit time when the linearity of its time-dependent transport was apparently maintained, the culture area of the culture insert membrane (0.33 cm2), and the initial concentration of test compound in the donor compartment, respectively.
The efflux ratio (ER) was calculated according to eq. 2:
where Papp,BtoA and Papp,AtoB represent the apparent permeability coefficient in the basolateral-to-apical direction and the apical-to-basolateral direction, respectively.
The intestinal availability (Fg) of CYP3A substrates was calculated using the following equations:
where CLAtoB, PS1, PS2, PS3, and CLmet represent the transcellular transport clearance in the apical-to-basolateral direction, the intrinsic passive permeation clearance in the apical-to-cell direction, the intrinsic passive permeation clearance in the cell-to-apical direction, the intrinsic passive permeation clearance in the cell-to-basolateral direction, and the intrinsic metabolic clearance mediated by CYP3A, respectively. CLAtoB(−inh) and CLAtoB(+inh) represent the apical-to-basolateral transcellular transport clearance in the absence and presence of ketoconazole, respectively. To simplify the mathematical handling, assuming the intrinsic passive permeation clearance in each process was the same (PS1 = PS2 = PS3 = PS) and the metabolic clearance mediated by CYP3A in the presence of ketoconazole was zero (CLmet = 0) in eq. 3 because of its potent inhibitory effect, CLAtoB(−inh), CLAtoB(+inh), and Fg were simply described using eqs. 4–6, respectively.
Statistical Analysis
Results are presented as the mean ±standard deviation. Statistical significance of the differences in the mean values between the data for two groups and among the data for three or more groups was assessed using unpaired Student’s t test and analysis of variance followed by Tukey’s test, respectively. All statistical analyses were performed using GraphPad Prism (version 9.2; GraphPad Software, San Diego, CA). Statistical significance was defined at p < 0.05.
Results
Establishment of Human Intestinal Spheroids and Generation of 2D Cell Monolayers from 3D Spheroids
We successfully established human intestinal spheroids from surgical jejunal specimens and generated 2D cell monolayers from 3D spheroids according to the protocols previously described by Miyoshi and Stappenbeck (2013) and VanDussen et al. (2015), respectively (Fig. 1). Intestinal spheroids were expanded in 50% L-WRN CM containing 10 μM Y-27632 and 1 μM SB431542 and passaged every 5–7 days at ratios of 1:3–1:5. Under our culture condition, intestinal spheroids could be normally cultured over at least 25 passages. To generate 2D monolayer cultures from 3D spheroids, 6-day cultured intestinal spheroids were collected from the Matrigel, enzymatically dissociated into single cells, and plated onto Matrigel-precoated cell culture plates or inserts. To avoid anoikis, the cells were incubated with 10 μM Y-27632 over the first 24 hours after seeding. To differentiate the cells, Wnt3a, R-spondin 3, and noggin were removed at 24 hours after seeding, and the medium was replaced with fresh 0% L-WRN CM every 2 days.
Culture Period-Dependent mRNA Expression Levels of Enzymes and Transporters and TEER Values in Human Jejunal Spheroid-Derived Differentiated Intestinal Epithelial Cells
To investigate the appropriate timing for metabolism/transport assays with human jejunal spheroid-derived differentiated intestinal epithelial cells, mRNA expression levels of ABCB1 (P-gp), which is an efflux transporter on the apical membrane; SLC15A1 (PEPT1), which is an uptake transporter on the apical membrane; CYP3A4, which is the most abundant intestinal CYP enzyme; and SI, which is a typical matured enterocyte marker, were monitored over the culture period until 13 days.
Over a 5-day period after seeding, human jejunal spheroid-derived differentiated intestinal epithelial cells showed a drastic increase in the mRNA expression levels of ABCB1 (3.8-fold), SLC15A1 (21-fold), CYP3A4 (63-fold), and SI (24-fold) compared with those in undifferentiated human jejunal spheroids at 6 days after passage (Fig. 2, A–D). At 5 days after seeding, the ratio of mRNA expression level in differentiated intestinal epithelial cells to that in intact human jejunal tissue was 2.17 for ABCB1, 0.88 for SLC15A1, 1.92 for CYP3A4, and 0.74 for SI. Moreover, their expression levels were maintained at approximately the same levels as 5 days after seeding until 11 days after seeding and started to gradually decrease at 13 days after seeding. These findings suggested that this cell system is expected to maintain the functions of P-gp, PEPT1, and CYP3A4 until 11 days after cell seeding.
In addition, TEER values were measured to determine the integrity of the cell monolayer. For human jejunal spheroid-derived differentiated intestinal epithelial cells, TEER values reached approximately 300 Ω cm2 by 5 days after seeding and remained steady at least until 13 days after seeding (Fig. 2E).
P-gp and BCRP-Mediated Transport in Human Jejunal Spheroid-Derived Differentiated Intestinal Epithelial Cells
To investigate whether the functions of P-gp and BCRP, which are typical intestinal efflux transporters, were maintained in human jejunal spheroid-derived differentiated intestinal epithelial cells, we evaluated the bidirectional transport of digoxin, fexofenadine, sulfasalazine, and rosuvastatin in the presence or absence of a mixture of PSC833 and Ko143. In parallel, the bidirectional transport of antipyrine and lucifer yellow were evaluated to confirm no effect of P-gp/BCRP inhibitor cocktail on the transport of non-P-gp/BCRP substrates across the cell monolayer.
For all the tested P-gp and BCRP substrate drugs, the basolateral-to-apical transport was significantly higher than that in the opposite direction and was clearly suppressed in the presence of P-gp/BCRP inhibitor cocktail (Fig. 3, A–D and G–J). The ERs of digoxin (26), fexofenadine (5.2), sulfasalazine (22), and rosuvastatin (2.8) decreased to 0.65, 0.97, 1.5, and 0.65 after incubation with P-gp/BCRP inhibitor cocktail, respectively. In contrast, no significant difference in the bidirectional transport of antipyrine and lucifer yellow was observed regardless of the presence of P-gp/BCRP inhibitor cocktail (Fig. 3, E, F, K, and L). These findings suggested that the transport activities of P-gp and BCRP were confirmed in human jejunal spheroid-derived differentiated intestinal epithelial cells.
PEPT1-Mediated Transport in Human Jejunal Spheroid-Derived Differentiated Intestinal Epithelial Cells
To investigate whether the function of PEPT1, which is an intestinal uptake transporter for peptides, was retained in human jejunal spheroid-derived differentiated intestinal epithelial cells, we evaluated the apical-to-basolateral transport of cefadroxil in the presence or absence of excess concentration of Gly-Sar.
The apical-to-basolateral transport of cefadroxil was significantly decreased in the presence of 10 mM of Gly-Sar (Fig. 4, A and D). In contrast, no significant difference was noted in the apical-to-basolateral transport of antipyrine and lucifer yellow regardless of the presence of 10 mM Gly-Sar (Fig. 4, B, C, E, and F). These findings suggested that the transport activity of PEPT1 was retained in human jejunal spheroid-derived differentiated intestinal epithelial cells.
CYP3A4/5, CYP2C9, UGT1As, and CES2-Mediated Metabolic Activities in Human Jejunal Spheroid-Derived Differentiated Intestinal Epithelial Cells
To investigate whether the functions of CYP3A4/5, CYP2C9, UGT1As, and CES2, which are typical intestinal drug-metabolizing enzymes, were maintained in human jejunal spheroid-derived differentiated intestinal epithelial cells, marker reactions for these enzymes (midazolam 1′-hydroxylation for CYP3A4/5, diclofenac 4′-hydroxylation for CYP2C9, raloxifene 4′-glucuronidation for UGT1As, and irinotecan hydrolysis for CES2) were observed in the presence or absence of the corresponding enzyme inhibitors.
The production of metabolites (1′-hydroxy midazolam, 4′-hydroxy diclofenac, raloxifene-4′-glucuronide, and SN-38, respectively) was clearly observed and significantly decreased in the presence of the corresponding enzyme inhibitors (Fig. 5). These findings suggested that the metabolic activities of CYP3A4/5, CYP2C9, UGT1As, and CES2 were confirmed in human jejunal spheroid-derived differentiated intestinal epithelial cells.
Predictability of Human Fg Values of CYP3A Substrate Drugs Estimated Using Human Jejunal Spheroid-Derived Differentiated Intestinal Epithelial Cells
To investigate whether human jejunal spheroid-derived differentiated intestinal epithelial cells would be a useful tool for the quantitative evaluation of Fg values for substrate drugs of CYP3A, which is the most important drug-metabolizing enzyme in terms of intestinal drug metabolism, we evaluated the correlation between the estimated Fg values of CYP3A substrate drugs from in vitro assays using human jejunal spheroid-derived differentiated intestinal epithelial cells and in vivo ones obtained from the literature.
Initially, we investigated the effects of ketoconazole (a potent CYP3A inhibitor) and 1,25-(OH)2D3 (a typical CYP3A inducer) on the apical-to-basolateral transport of five CYP3A substrate (non-P-gp substrate) drugs (midazolam, triazolam, alprazolam, felodipine, and nifedipine). For CYP3A substrate drugs with relatively large first-pass metabolism, such as felodipine and midazolam, the apical-to-basolateral transport was increased in the presence of ketoconazole, whereas it was decreased in the presence of 1,25-(OH)2D3 (Fig. 6, A–J). However, the metabolite production of these CYP3A substrate drugs was significantly decreased in the presence of ketoconazole and significantly increased in the presence of 1,25-(OH)2D3 (Fig. 6, K–O).
Then, Fg values were estimated with eqs. 3–6 using the apical-to-basolateral permeation clearances in the presence or absence of ketoconazole. Table 1 shows a summary of the estimated and in vivo Fg values for the five CYP3A substrate drugs. The data points for these CYP3A substrate drugs mapped close to the identity line. Moreover, the data points were shifted to the left, away from the identity line in the presence of 1,25-(OH)2D3; i.e., the estimated Fg values decreased because of the increase in CYP3A-mediated metabolism (Fig. 7).
Discussion
In this study, we aimed to demonstrate the usefulness of human jejunal spheroid-derived differentiated intestinal epithelial cells for investigating the quantitative impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans.
We successfully developed human jejunal spheroid-derived differentiated intestinal epithelial cells, which retained the functions of multiple intestinal drug-metabolizing enzymes and transporters, without using any complicated differentiation procedures or specialized devices (Fig. 1). Human jejunal spheroids could be reproducibly established from human jejunal specimens obtained from several independent subjects and efficiently expanded over a long period (>25 passages) in 50% L-WRN CM. In addition, differentiated intestinal epithelial cell monolayers could be prepared just by the withdrawal of Wnt3a, R-spondin 3, and noggin from the medium in a short period (within 1 week) (Fig. 2) compared with the time taken using Caco-2 cells (2–3 weeks) (Markowska et al., 2001) and human iPSC-derived intestinal epithelial cells (3–4 weeks) (Kawai et al., 2019; Kabeya et al., 2020). Most importantly, the functions of a wide variety of drug-metabolizing enzymes, such as CYP enzymes, phase II enzymes, and non-CYP phase I enzymes, as well as efflux/uptake transporters, were clearly confirmed in this system (Figs. 4 and 5). Furthermore, the mRNA expression levels of multiple intestinal drug-metabolizing enzymes and transporters, except for SLC10A2, in this model were within a range of 4.1-fold compared with those in human jejunal tissues (Supplemental Fig. 1). Although primary human enterocytes have also been recently obtained from commercial sources, their utilization as an in vitro model for the characterization of drug intestinal absorption remains impractical because of the limited availability of cells and the difficult preparation of 2D monolayers on culture dishes or inserts. However, our simple protocol enables long-term propagation of intestinal stem cells with differentiation capability and routine use of human differentiated intestinal epithelial cells that are suitable for pharmacokinetic research.
We successfully demonstrated that Fg values estimated in this system were well-correlated with the previously reported values in humans for CYP3A substrate drugs (Fig. 7). Kabeya et al., (2020) reported the correlation between the Papp values of various drugs across human iPSC-derived intestinal epithelial cells and the reported product of fraction absorbed and intestinal availability (FaFg) in humans. However, Papp values of CYP3A substrate drugs markedly deviated from the correlation curve for other drugs, leading to speculation that CYP3A activity in the cells was not high enough to account for the clinically relevant FaFg. Theoretically, when drugs undergo significant intracellular metabolism, luminal flow rate (Q) and absorption clearance (CLab) also need to be considered to associate CLAtoB with FaFg (Supplemental Figs. 2 and 3). For simplicity, we calculated Fg according to eq. 6 under the assumption that passive membrane permeation clearance was identical (PS1 = PS2 = PS3) and metabolic clearance mediated by CYP3A in the presence of ketoconazole was zero (CLmet = 0). Felodipine and midazolam were the only drugs whose apical-to-basal Papp were significantly higher in the presence of ketoconazole under normal conditions (Fig. 6, F and I). Hence, we conducted Fg estimation based on the difference in CLAtoB of all tested drugs in the presence or absence of ketoconazole regardless of its statistical significance (Fig. 6, F–J). Comparison of the total amount of parent drugs transferred to the basolateral compartment and the total amount of metabolites produced during the study is another approach to estimate Fg. Our findings enabled reasonable estimation of Fg values (Supplemental Fig. 4 and Supplemental Table 5). This method assumes that all the major drug metabolites are quantified, and secondary metabolism of metabolites is negligible during the study. However, this approach is sometimes difficult to apply for new drug candidates because of limited information on their whole metabolic pathways.
This system maintained the transport activities of PEPT1, P-gp, and BCRP (Figs. 3 and 4). The ERs of digoxin, fexofenadine, sulfasalazine, and rosuvastatin were 26, 5.2, 22, and 2.8, respectively (Fig. 3). The corresponding values in freshly isolated human jejunum were 4.0, 6.4, 19, and 3.1, respectively (Michiba et al., 2021). Thus, these values, except for that of digoxin, were consistent in both cases. Further accumulation of experimental data is necessary to conclude that this system can be a novel in vivo-relevant cellular model for drug absorption of P-gp and BCRP substrates in humans. P-gp and BCRP substrate drugs showed a drastic decrease in the basolateral-to-apical transport in the presence of the P-gp/BCRP inhibitor cocktail, although almost no changes were observed in the apical-to-basolateral transport of the drugs. Because a previous report suggested that a balance between membrane permeation clearances of drugs across the apical and basolateral membranes can modify the pattern of alteration in the bidirectional transport clearances when the apical efflux transport is inhibited (Mizuno et al., 2003), further attention must be paid to the change in each membrane transport process at both sides of the cells.
Although the present study highlighted the successful application of human intestinal spheroid-derived differentiated intestinal epithelial cells for the characterization of oral absorption of drugs in the process of drug development, this study still has some limitations. First, human intestinal spheroids used in this study were established only from the jejunum. It is well-known that some intestinal drug-metabolizing enzymes and transporters show region-specific gene expression profiles (Drozdzik et al., 2014; Fritz et al., 2019). Several studies suggested that intestinal spheroids/organoids established from different regions of the intestine (i.e., the duodenum, jejunum, and ileum) retained the region-specific gene expression profiles of their origin (Middendorp et al., 2014; Meran et al., 2020). Thus, this system is expected to be a novel cellular model possessing a region-specific gene expression profile. However, more in-depth research regarding this aspect is required. Second, human intestinal spheroids used in this study were established from a limited number of donors. Therefore, further studies are needed to judge the applicability of this system to the evaluation of interindividual variation of intestinal drug absorption in humans by collecting various batches of human intestinal spheroids originating from independent donors. As in the case of human liver microsomes and hepatocytes, this system may serve as an in vitro model for studying the interindividual difference in drug absorption, for instance, the difference due to genetic polymorphisms of BCRP. The area under the plasma concentration versus time curve values after oral administration of BCRP substrate drugs, such as sulfasalazine, rosuvastatin, and topotecan, were approximately 1.3–2.4 times higher in homozygotes of ABCG2 c.421C>A, which is associated with lower protein expression and transport function, than in those without this mutation (Sparreboom et al., 2005; Zhang et al., 2006; Urquhart et al., 2008). When crypts can be collected from the subjects that show interindividual differences in drug absorption, this system can be used as an in vitro model to uncover the underlying mechanisms. However, even with the use of cells from the same origin, interexperimental variation in the CYP3A-mediated metabolic activity was observed (approximately threefold; Supplementary Fig. 5). In future, for precise evaluation of the intrinsic interbatch difference in metabolic/transport activity, the assay protocol needs to be improved to achieve high quantitative reproducibility.
We successfully demonstrated that this system maintains the activities of drug transporters such as P-gp and BCRP, and drug metabolizing enzymes such as CYP3A and UGT1As. There are drugs whose FaFg are determined by multiple transporters and metabolic enzymes. For instance, many dual substrates of CYP3A and P-gp have been identified because of their overlapping substrate specificities (Takano et al., 2016), and thus further studies should be needed whether this system would be universally valuable for quantitative prediction of intestinal availability when multiple transporters and/or metabolic enzymes are simultaneously involved in drug absorption. Whether this system can be used for examining clinical drug interactions also remains challenge. For example, the area under the plasma concentration versus time curve values after oral administration of organic anion transporting polypeptide (OATP) 1A2/OATP2B1 substrate drugs, such as fexofenadine and β-blockers (talinolol and nadolol), were reduced by 44%–85% after coadministration of grapefruit juice or green tea (Dresser et al., 2002; Schwarz et al., 2005; Misaka et al., 2014). These drug-food interactions were considered to be attributed to the inhibition of OATP2B1/1A2 by food components (Yu et al., 2017); whereas, some studies suggested that the change of luminal water volume depending on the osmolality by oral administration of fruit juice indirectly affects intestinal drug absorption (Ichijo et al., 2017; Funai et al., 2019). This system may give us a clue of the mechanism of such drug interactions.
Besides the pharmacokinetic analysis, this system has a potential application as in vitro pharmacology model of human intestine. For example, intestinal cholesterol transporter Niemann-Pick C1-like 1 and ileal bile acid transporter are pharmacological targets of ezetimibe (a drug for treatment of hypercholesterolemia) and elobixibat (a drug for treatment of chronic idiopathic constipation), respectively (Yamanashi et al., 2018; Miner, 2018). This system could aid future discovery and optimization of drug candidates with gastrointestinal targets/mechanisms of action.
In conclusion, human intestinal spheroid-derived differentiated intestinal epithelial cells could be useful for investigating the impact of intestinal drug-metabolizing enzymes and transporters on the intestinal absorption of substrate drugs in humans.
Acknowledgments
The authors thank Drs. Hiroyuki Miyoshi and Makoto Mark Taketo, Kyoto University, for their technical assistance with human intestinal spheroid culture. The authors also thank Drs. Hiroyuki Nishiyama and Tomoyo Takeuchi, Tsukuba Human Tissue Biobank Center, and Drs. Satoshi Suzuki and Katashi Fukao, Human and Animal Bridging Research Organization, for their kind assistance with establishing the infrastructure to obtain freshly isolated human intestinal sections.
Authorship Contributions
Participated in research design: Michiba, Maeda, Kusuhara, and Oda.
Conducted experiments: Michiba and Maeda.
Performed data analysis: Michiba, Maeda, and Kusuhara.
Wrote or contributed to the writing of the manuscript: Michiba, Maeda, Kusuhara, and Oda.
Footnotes
- Received November 29, 2021.
- Accepted January 2, 2022.
This study was supported by two Grants-in-Aid for Scientific Research [Grant 20H03402] (to K.Ma. and T.O.) and [Grant 20H03401] (to H.K.) from the Ministry of Education, Culture, Sports, Science, and Technology in Japan.
No author has an actual or perceived conflict of interest with the contents of this article.
↵This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS
- ABC
- ATP-binding cassette
- BCRP
- breast cancer resistance protein
- CES
- carboxylesterase
- CLAtoB
- transcellular transport clearance in the apical-to-basolateral direction
- CYP
- cytochrome P450
- 2D
- two-dimensional
- 3D
- three-dimensional
- DMEM
- Dulbecco’s modified Eagle medium
- ER
- efflux ratio
- Fa
- fraction of orally administrated drugs that enter the gut wall
- Fg
- intestinal availability
- Gly-Sar
- glycylsarcosine
- iPSC
- induced pluripotent stem cell
- LC-MS/MS
- liquid chromatography–tandem mass spectrometry
- OATP
- organic anion transporting polypeptide
- 1,25-(OH)2D3
- 1α, 25-dihydroxyvitamin D3
- Papp
- apparent permeability coefficient
- PEPT1
- peptide transporter 1
- P-gp
- P-glycoprotein
- SI
- sucrase-isomaltase
- SLC
- solute carrier
- TEER
- transepithelial electrical resistance
- UGT
- uridine 5′-diphospho-glucuronosyltransferase
- Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics