Intestinal in vitro transport assay combined with physiologically based kinetic modeling as a tool to predict bile acid levels in vivo

Main Article Content

Véronique M. P. de Bruijn , Willem te Kronnie, Ivonne M. C. M. Rietjens, Hans Bouwmeester
[show affiliations]


Bile acid homeostasis is vital for numerous metabolic and immune functions in humans. The enterohepatic circulation of bile acids is extremely efficient, with ~95% of intestinal bile acids being reabsorbed. Disturbing intestinal bile acid uptake is expected to substantially affect intestinal and systemic bile acid levels. Here, we aimed to predict the effects of apical sodium-dependent bile acid transporter (ASBT)-inhibition on systemic plasma levels. For this, we combined in vitro Caco-2 cell transport assays with physiologically based (PBK) modeling. We used the selective ASBT-inhibitor odevixibat (ODE) as a model compound. Caco-2 cells grown on culture inserts were used to obtain transport kinetic parameters of glycocholic acid (GCA). The apparent Michaelis-Menten constant (Km,app), apparent maximal intestinal transport rate (Vmax,app), and ODE’s inhibitory constant (Ki) were determined for GCA. These kinetic parameters were incorporated into a PBK model and used to predict the ASBT inhibition effects on plasma bile acid levels. GCA is transported over Caco-2 cells in an active and sodium-dependent manner, indicating the presence of functional ASBT. ODE inhibited GCA transport dose-dependently. The PBK model predicted that oral doses of ODE reduced conjugated bile acid levels in plasma. Our simulations match in vivo data and provide a first proof-of-principle for the incorporation of active intestinal bile acid uptake in a bile acid PBK model. This approach could in future be of use to predict the effects of other ASBT-inhibitors on plasma and intestinal bile acid levels.

Plain language summary
Bile acids regulate digestion and immune functions. Too little bile acid reuptake in the gut is related to several diseases, including inflammatory bowel disease. This study investigates how reducing bile acid absorption affects bile acid levels in humans using the drug odevixibat (ODE) as an example. ODE reduces bile acid absorption by blocking the intestinal bile acid transporter protein in gut cells. The transport of a bile acid through a gut cell line commonly used to model the intestinal barrier was measured with and without ODE, and mathematical modeling was used to translate the laboratory results to whole-body effects. This combined approach accurately predicted the known effects of ODE on intestinal and bloodstream bile acid levels in humans. This novel approach could be used to predict the effects of other chemicals on intestinal bile acid absorption and intestinal and bloodstream bile acid levels instead of animal testing.



Article Details

How to Cite
de Bruijn, V. M. P. (2024) “Intestinal in vitro transport assay combined with physiologically based kinetic modeling as a tool to predict bile acid levels in vivo”, ALTEX - Alternatives to animal experimentation, 41(1), pp. 20–36. doi: 10.14573/altex.2302011.

Al-Hilal, T. A., Chung, S. W., Alam, F. et al. (2014). Functional transformations of bile acid transporters induced by high-affinity macromolecules. Sci Rep 4, 4163. doi:10.1038/srep04163

Aldini, R., Roda, A., Montagnani, M. et al. (1996). Relationship between structure and intestinal absorption of bile acids with a steroid or side-chain modification. Steroids 61, 590-597. doi:10.1016/s0039-128x(96)00119-5

Antunes, F., Andrade, F., Araujo, F. et al. (2013). Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs. Eur J Pharm Biopharm 83, 427-435. doi:10.1016/j.ejpb.2012.10.003

Baier, V., Cordes, H., Thiel, C. et al. (2019). A physiology-based model of human bile acid metabolism for predicting bile acid tissue levels after drug administration in healthy subjects and BRIC type 2 patients. Front Physiol 10, 1192. doi:10.3389/fphys.2019.01192

Balakrishnan, A., Wring, S. A. and Polli, J. E. (2006). Interaction of native bile acids with human apical sodium-dependent bile acid transporter (hASBT): Influence of steroidal hydroxylation pattern and c-24 conjugation. Pharm Res 23, 1451-1459. doi:10.1007/s11095-006-0219-4

Balakrishnan, A., Hussainzada, N., Gonzalez, P. et al. (2007). Bias in estimation of transporter kinetic parameters from overexpression systems: Interplay of transporter expression level and substrate affinity. J Pharmacol Exp Ther 320, 133-144. doi:10.1124/jpet.106.107433

Barter, Z. E., Bayliss, M. K., Beaune, P. H. et al. (2007). Scaling factors for the extrapolation of in vivo metabolic drug clearance from in vitro data: Reaching a consensus on values of human micro-somal protein and hepatocellularity per gram of liver. Curr Drug Metab 8, 33-45. doi:10.2174/138920007779315053

Bathena, S. P. R., Mukherjee, S., Olivera, M. et al. (2013). The profile of bile acids and their sulfate metabolites in human urine and serum. J Chromatogr B Analyt Technol Biomed Life Sci 942, 53-62. doi:10.1016/j.jchromb.2013.10.019

Begley, M., Gahan, C. G. M. and Hill, C. (2005). The interaction between bacteria and bile. Fems Microbiol Rev 29, 625-651. doi:10.1016/j.femsre.2004.09.003

Bruck, S., Strohmeier, J., Busch, D. et al. (2017). Caco-2 cells – Expression, regulation and function of drug transporters compared with human jejunal tissue. Biopharm Drug Dispos 38, 115-126. doi:10.1002/bdd.2025

Chiang, J. Y. and Ferrell, J. M. (2022). Discovery of farnesoid x receptor and its role in bile acid metabolism. Mol Cell Endocrinol 548, 111618. doi:10.1016/j.mce.2022.111618

Dawson, P. A., Lan, T. and Rao, A. (2009). Bile acid transporters. J Lipid Res 50, 2340-2357. doi:10.1194/jlr.R900012-JLR200

Dawson, P. A. (2011). Role of the intestinal bile acid transporters in bile acid and drug disposition. Handb Exp Pharmacol, 169-203. doi:10.1007/978-3-642-14541-4_4

de Bruijn, V. M. P., Rietjens, I. and Bouwmeester, H. (2022a). Population pharmacokinetic model to generate mechanistic insights in bile acid homeostasis and drug-induced cholestasis. Arch Toxicol 96, 2717-2730. doi:10.1007/s00204-022-03345-8

de Bruijn, V. M. P., Wang, Z., Bakker, W. et al. (2022b). Hepatic bile acid synthesis and secretion: Comparison of in vitro methods. Toxicol Lett 365, 46-60. doi:10.1016/j.toxlet.2022.06.004

Deeks, E. D. (2021). Odevixibat: First approval. Drugs 81, 1781-1786. doi:10.1007/s40265-021-01594-y

Duan, S., Li, X., Fan, G. et al. (2022). Targeting bile acid signaling for the treatment of liver diseases: From bench to bed. Biomed Pharmacother 152, 113154. doi:10.1016/j.biopha.2022.113154

EMA – European Medicines Agency (2021). Assessment report: Bylvay. Committee for Medicinal Products for Human Use (CHMP). EMA/319560/2021.

Evans, M. V. and Andersen, M. E. (2000). Sensitivity analysis of a physiological model for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): Assessing the impact of specific model parameters on sequestration in liver and fat in the rat. Toxicol Sci 54, 71-80. doi:10.1093/toxsci/54.1.71

Falany, C. N., Johnson, M. R., Barnes, S. et al. (1994). Glycine and taurine conjugation of bile acids by a single enzyme. Molecular cloning and expression of human liver bile acid CoA:Amino acid N-acyltransferase. J Biol Chem 269, 19375-19379.

Fasano, A., Budillon, G., Guandalini, S. et al. (1990). Bile acids reversible effects on small intestinal permeability. An in vitro study in the rabbit. Dig Dis Sci 35, 801-808. doi:10.1007/BF01536791

Fiamoncini, J., Curi, R. and Daniel, H. (2016). Metabolism of bile acids in the post-prandial state. Essays Biochem 60, 409-418. doi:10.1042/EBC20160052

Fitzpatrick, L. R. and Jenabzadeh, P. (2020). IBD and bile acid absorption: Focus on pre-clinical and clinical observations. Front Physiol 11, 564. doi:10.3389/fphys.2020.00564

Fuchs, C. D. and Trauner, M. (2022). Role of bile acids and their receptors in gastrointestinal and hepatic pathophysiology. Nat Rev Gastroenterol Hepatol 19, 432-450. doi:10.1038/s41575-021-00566-7

Garcia-Canaveras, J. C., Donato, M. T., Castell, J. V. et al. (2012). Targeted profiling of circulating and hepatic bile acids in human, mouse, and rat using a UPLC-MRM-MS-validated method. J Lipid Res 53, 2231-2241. doi:10.1194/jlr.D028803

Gijbels, E. and Vinken, M. (2019). Mechanisms of drug-induced cholestasis. Methods Mol Biol 1981, 1-14. doi:10.1007/978-1-4939-9420-5_1

Gilbert-Sandoval, I., Wesseling, S. and Rietjens, I. (2020). Predicting the acute liver toxicity of aflatoxin B1 in rats and humans by an in vitro-in silico testing strategy. Mol Nutr Food Res 64, e2000063. doi:10.1002/mnfr.202000063

Graffner, H., Gillberg, P. G., Rikner, L. et al. (2016). The ileal bile acid transporter inhibitor A4250 decreases serum bile acids by interrupting the enterohepatic circulation. Aliment Pharmacol Ther 43, 303-310. doi:10.1111/apt.13457

Hendriksen, B. A., Felix, M. V. and Bolger, M. B. (2003). The composite solubility versus pH profile and its role in intestinal absorption prediction. AAPS PharmSci 5, E4. doi:10.1208/050104

Hepner, G. W. and Demers, L. M. (1977). Dynamics of the enterohepatic circulation of the glycine conjugates of cholic, chenodeoxycholic, deoxycholic, and sulfolithocholic acid in man. Gastroenterology 72, 499-501.

Hofmann, A. F., Molino, G., Milanese, M. et al. (1983). Description and simulation of a physiological pharmacokinetic model for the metabolism and enterohepatic circulation of bile acids in man. Cholic acid in healthy man. J Clin Invest 71, 1003-1022. doi:10.1172/jci110828

Jamei, M., Bajot, F., Neuhoff, S. et al. (2014). A mechanistic framework for in vitro-in vivo extrapolation of liver membrane transporters: Prediction of drug-drug interaction between rosuvastatin and cyclosporine. Clin Pharmacokinet 53, 73-87. doi:10.1007/s40262-013-0097-y

Jani, M., Beéry, E., Heslop, T. et al. (2018). Kinetic characterization of bile salt transport by human NTCP (SLC10A1). Toxicol In Vitro 46, 189-193. doi:10.1016/j.tiv.2017.10.012

Jazrawi, R. P., Pazzi, P., Petroni, M. L. et al. (1995). Postprandial gallbladder motor function: Refilling and turnover of bile in health and in cholelithiasis. Gastroenterology 109, 582-591. doi:10.1016/0016-5085(95)90348-8

Jia, W., Xie, G. and Jia, W. (2018). Bile acid-microbiota crosstalk in gastrointestinal inflammation and carcinogenesis. Nat Rev Gastroenterol Hepatol 15, 111-128. doi:10.1038/nrgastro.2017.119

Kararli, T. T. (1995). Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm Drug Dispos 16, 351-380. doi:10.1002/bdd.2510160502

Kato, T., Mikami, Y., Sun, L. et al. (2021). Reuse of cell culture inserts for in vitro human primary airway epithelial cell studies. Am J Respir Cell Mol Biol 64, 760-764. doi:10.1165/rcmb.2021-0033LE

Kimura, T. and Higaki, K. (2002). Gastrointestinal transit and drug absorption. Biol Pharm Bull 25, 149-164. doi:10.1248/bpb.25.149

Kis, E., Ioja, E., Nagy, T. et al. (2009). Effect of membrane cholesterol on BSEP/Bsep activity: Species specificity studies for substrates and inhibitors. Drug Metab Dispos 37, 1878-1886. doi:10.1124/dmd.108.024778

Korjamo, T., Heikkinen, A. T. and Monkkonen, J. (2009). Analysis of unstirred water layer in in vitro permeability experiments. J Pharm Sci 98, 4469-4479. doi:10.1002/jps.21762

Kouzuki, H., Suzuki, H., Ito, K. et al. (1998). Contribution of sodium taurocholate co-transporting polypeptide to the uptake of its possible substrates into rat hepatocytes. J Pharmacol Exp Ther 286, 1043-1050.

Krag, E. and Phillips, S. F. (1974). Active and passive bile acid absorption in man. Perfusion studies of the ileum and jejunum. J Clin Invest 53, 1686-1694. doi:10.1172/JCI107720

Kullak-Ublick, G. A., Stieger, B. and Meier, P. J. (2004). Enterohepatic bile salt transporters in normal physiology and liver disease. Gastroenterology 126, 322-342. doi:10.1053/j.gastro.2003.06.005

Kumar, A. R., Prasad, B., Bhatt, D. K. et al. (2021). In vitro-to-in vivo extrapolation of transporter-mediated renal clearance: Relative expression factor versus relative activity factor approach. Drug Metab Dispos 49, 470-478. doi:10.1124/dmd.121.000367

Lamaziere, A., Rainteau, D., Kc, P. et al. (2020). Distinct postprandial bile acids responses to a high-calorie diet in men volunteers underscore metabolically healthy and unhealthy phenotypes. Nutrients 12, 3545. doi:10.3390/nu12113545

Law, V., Knox, C., Djoumbou, Y. et al. (2014). Drugbank 4.0: Shedding new light on drug metabolism. Nucleic Acids Res 42, D1091-1097. doi:10.1093/nar/gkt1068

Leschelle, X., Robert, V., Delpal, S. et al. (2002). Isolation of pig colonic crypts for cytotoxic assay of luminal compounds: Effects of hydrogen sulfide, ammonia, and deoxycholic acid. Cell Biol Toxicol 18, 193-203. doi:10.1023/a:1015515821390

Li, M., Vokral, I., Evers, B. et al. (2018). Human and rat precision-cut intestinal slices as ex vivo models to study bile acid uptake by the apical sodium-dependent bile acid transporter. Eur J Pharm Sci 121, 65-73. doi:10.1016/j.ejps.2018.05.005

Li, S., Qu, X., Zhang, L. et al. (2022). Serum total bile acids in relation to gastrointestinal cancer risk: A retrospective study. Front Oncol 12, 859716. doi:10.3389/fonc.2022.859716

Lin, S., Wang, S., Wang, P. et al. (2023). Bile acids and their receptors in regulation of gut health and diseases. Prog Lipid Res 89, 101210. doi:10.1016/j.plipres.2022.101210

Lineweaver, H. and Burk, D. (1934). The determination of enzyme dissociation constants. J Am Chem Soc 56, 658-666.

Lobell, M. and Sivarajah, V. (2003). In silico prediction of aqueous solubility, human plasma protein binding and volume of distribution of compounds from calculated pKa and AlogP98 values. Mol Div 7, 69-87. doi:10.1023/b:modi.0000006562.93049.36

Lock, J. Y., Carlson, T. L. and Carrier, R. L. (2018). Mucus models to evaluate the diffusion of drugs and particles. Adv Drug Deliv Rev 124, 34-49. doi:10.1016/j.addr.2017.11.001

Lu, Z. N., He, H. W. and Zhang, N. (2022). Advances in understanding the regulatory mechanism of organic solute transporter alpha-beta. Life Sci 310, 121109. doi:10.1016/j.lfs.2022.121109

Marasco, G., Cremon, C., Barbaro, M. R. et al. (2022). Pathophysiology and clinical management of bile acid diarrhea. J Clin Med 11, 3102. doi:10.3390/jcm11113102

Martinez-Augustin, O. and Sanchez de Medina, F. (2008). Intestinal bile acid physiology and pathophysiology. World J Gastroenterol 14, 5630-5640. doi:10.3748/wjg.14.5630

McCright, J., Sinha, A. and Maisel, K. (2022). Generating an in vitro gut model with physiologically relevant biophysical mucus properties. Cell Mol Bioeng 15, 479-491. doi:10.1007/s12195-022-00740-0

Miele, L., Valenza, V., La Torre, G. et al. (2009). Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology 49, 1877-1887. doi:10.1002/hep.22848

Molino, G., Hofmann, A. F., Cravetto, C. et al. (1986). Simulation of the metabolism and enterohepatic circulation of endogenous chenodeoxycholic acid in man using a physiological pharmacokinetic model. Eur J Clin Invest 16, 397-414. doi:10.1111/j.1365-2362.1986.tb01015.x

Mouzaki, M., Wang, A. Y., Bandsma, R. et al. (2016). Bile acids and dysbiosis in non-alcoholic fatty liver disease. PLoS One 11, e0151829. doi:10.1371/journal.pone.0151829

Notenboom, S., Weigand, K. M., Proost, J. H. et al. (2018). Development of a mechanistic biokinetic model for hepatic bile acid handling to predict possible cholestatic effects of drugs. Eur J Pharm Sci 115, 175-184. doi:10.1016/j.ejps.2018.01.007

Olander, M., Wisniewski, J. R., Matsson, P. et al. (2016). The proteome of filter-grown caco-2 cells with a focus on proteins involved in drug disposition. J Pharm Sci 105, 817-827. doi:10.1016/j.xphs.2015.10.030

Peyret, T., Poulin, P. and Krishnan, K. (2010). A unified algorithm for predicting partition coefficients for pbpk modeling of drugs and environmental chemicals. Toxicol Appl Pharmacol 249, 197-207. doi:10.1016/j.taap.2010.09.010

Pontier, C., Pachot, J., Botham, R. et al. (2001). HT29-MTX and Caco-2/TC7 monolayers as predictive models for human intestinal absorption: Role of the mucus layer. J Pharm Sci 90, 1608-1619. doi:10.1002/jps.1111

Punt, A., Pinckaers, N., Peijnenburg, A. et al. (2020). Development of a web-based toolbox to support quantitative in-vitro-to-in-vivo extrapolations (QIVIVE) within nonanimal testing strategies. Chem Res Toxicol 34, 460-472. doi:10.1021/acs.chemrestox.0c00307

Punt, A., Louisse, J., Beekmann, K. et al. (2022). Predictive performance of next generation human physiologically based kinetic (PBK) models based on in vitro and in silico input data. ALTEX 39, 221-234. doi:10.14573/altex.2108301

Rietjens, I. M., Louisse, J. and Punt, A. (2011). Tutorial on physiologically based kinetic modeling in molecular nutrition and food research. Mol Nutr Food Res 55, 941-956. doi:10.1002/mnfr.201000655

Ritz, C., Baty, F., Streibig, J. C. et al. (2015). Dose-response analysis using R. PLoS One 10, e0146021. doi:10.1371/journal.pone.0146021

Roda, A., Minutello, A., Angellotti, M. A. et al. (1990). Bile acid structure-activity relationship: Evaluation of bile acid lipophilicity using 1-octanol/water partition coefficient and reverse phase HPLC. J Lipid Res 31, 1433-1443.

Rodgers, T. and Rowland, M. (2006). Physiologically based pharmacokinetic modelling 2: Predicting the tissue distribution of acids, very weak bases, neutrals and zwitterions. J Pharm Sci 95, 1238-1257. doi:10.1002/jps.20502

Sabino, J., Vieira-Silva, S., Machiels, K. et al. (2016). Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD. Gut 65, 1681-1689. doi:10.1136/gutjnl-2015-311004

Sambuy, Y., Angelis, I., Ranaldi, G. et al. (2005). The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21, 1-26. doi:10.1007/s10565-005-0085-6

Schwarz, M. A., Neubert, R. H. and Rüttinger, H. H. (1996). Application of capillary electrophoresis for characterizing interactions between drugs and bile salts. Part I. J Chromatogr A 745, 135-143. doi:10.1016/0021-9673(96)00396-2

Shrivastava, A. and Gupta, V. B. (2011). Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chron Young Sci 2, 21-25.

Sips, F. L. P., Eggink, H. M., Hilbers, P. A. J. et al. (2018). In silico analysis identifies intestinal transit as a key determinant of systemic bile acid metabolism. Front Physiol 9, 631. doi:10.3389/fphys.2018.00631

Smirnova, E., Muthiah, M. D., Narayan, N. et al. (2022). Metabolic reprogramming of the intestinal microbiome with functional bile acid changes underlie the development of NAFLD. Hepatology 76, 1811-1824. doi:10.1002/hep.32568

Soars, M., Burchell, B. and Riley, R. (2002). In vitro analysis of human drug glucuronidation and prediction of in vivo metabolic clearance. J Pharmacol Exp Ther 301, 382-390. doi:10.1124/jpet.301.1.382

Soetaert, K. and Petzoldt, T. (2010). Inverse modelling, sensitivity and monte carlo analysis in R using package FME. J Stat Softw 33, 1-28. doi:10.18637/jss.v033.i03

Steiner, C., Othman, A., Saely, C. H. et al. (2011). Bile acid metabolites in serum: Intraindividual variation and associations with coronary heart disease, metabolic syndrome and diabetes mellitus. PLoS One 6, e25006. doi:10.1371/journal.pone.0025006

Toke, O. (2022). Structural and dynamic determinants of molecular recognition in bile acid-binding proteins. Int J Mol Sci 23, 505. doi:10.3390/ijms23010505

van der Mark, V. A., de Waart, D. R., Ho-Mok, K. S. et al. (2014). The lipid flippase heterodimer ATP8B1-CDC50A is essential for surface expression of the apical sodium-dependent bile acid transporter (SLC10A2/ASBT) in intestinal Caco-2 cells. Biochim Biophys Acta 1842, 2378-2386. doi:10.1016/j.bbadis.2014.09.003

van der Valk, J., Bieback, K., Buta, C. et al. (2018). Fetal bovine serum (FBS): Past – Present – Future. ALTEX 35, 99-118. doi:10.14573/altex.1705101

Vinken, M., Landesmann, B., Goumenou, M. et al. (2013). Development of an adverse outcome pathway from drug-mediated bile salt export pump inhibition to cholestatic liver injury. Toxicol Sci 136, 97-106. doi:10.1093/toxsci/kft177

Voronova, V., Sokolov, V., Al-Khaifi, A. et al. (2020). A physiology-based model of bile acid distribution and metabolism under healthy and pathologic conditions in human beings. Cell Mol Gastroenterol Hepatol 10, 149-170. doi:10.1016/j.jcmgh.2020.02.005

Wanes, D., Naim, H. Y. and Dengler, F. (2021). Proliferation and differentiation of intestinal Caco-2 cells are maintained in culture with human platelet lysate instead of fetal calf serum. Cells 10, 3038. doi:10.3390/cells10113038

Wang, J., Bakker, W., Zheng, W. et al. (2022). Exposure to the mycotoxin deoxynivalenol reduces the transport of conjugated bile acids by intestinal Caco-2 cells. Arch Toxicol 96, 1473-1482. doi:10.1007/s00204-022-03256-8

Wang, Q., Strab, R., Kardos, P. et al. (2008). Application and limitation of inhibitors in drug-transporter interactions studies. Int J Pharm 356, 12-18. doi:10.1016/j.ijpharm.2007.12.024

Yang, N., Dong, Y. Q., Jia, G. X. et al. (2020). ASBT(SLC10A2): A promising target for treatment of diseases and drug discovery. Biomed Pharmacother 132, 110835. doi:10.1016/j.biopha.2020.110835

Zhang, N., Wang, J., Bakker, W. et al. (2022). In vitro models to detect in vivo bile acid changes induced by antibiotics. Arch Toxicol 96, 3291-3303. doi:10.1007/s00204-022-03373-4

Zhu, Q., Komori, H., Imamura, R. et al. (2021). A novel fluorescence-based method to evaluate ileal apical sodium-dependent bile acid transporter ASBT. J Pharm Sci 110, 1392-1400. doi:10.1016/j.xphs.2020.11.030

Most read articles by the same author(s)