Taurocholate or Glycocholate – which is the more appropriate Substrate for Assessing Hepatic Salt Transport?

Erica Deibert, Jonathan Jackson, Joy Mitchell, Kirsten Amaral, Rafal Witek, Stephen Ferguson and Jasminder Sahi ADME/Tox Division of Life Technologies, Durham NC USA

ABSTRACT RESULTS Figure 6. Effect of inhibitors on Biliary Clearance in Human Table 1. Relative Concentrations of Sandwich Cultured Hepatocytes Fig 1. Hepatocyte Transporters Purpose: Interactions with the hepatic bile salt transporters NTCP and BSEP can Bile Salts in Healthy Human Rat Bile salt Human 70 Bile salt + Ritonavir 80 contribute to cholestasis and hypercholesterolemia. The initial studies to evaluate Bile salt + Cyclosporin A Subjects 70 60 uptake and biliary excretion via these transporters were developed by Brouwer and Glycocholate Ble salt + Troglitazone 60 50 Glycocholate LeCluyse (1998) in primary sandwich cultured rat hepatocytes, using the bile salt In order of abundance: 50 40 40 30 taurocholate as a substrate. Since then, a majority of in vitro biliary transporter 30 GCDCA 0.696 µM 20 studies have been conducted with taurocholate for preclinical and human GCA 0.232 µM 20 extrapolations. While conjugated bile acids (e.g. taurocholate) are the 10 10 TCDCA 0.228 µM 0 0 BEI % In Vitro Biliary Clearance 90 BEI % In Vitro Biliary Clearance major bile salts in rat, human plasma predominantly contains glycine-conjugated CDCA 0.200 µM (mL/min/kg) (mL/min/kg) bile acids (e.g. glycocholate). To identify the appropriate bile salt for in vitro DCA 0.129 µM 90 80 UDCA 0.118 µM 80 70 transporter evaluations, we compared the substrate and inhibition potential of 70 Taurocholate 60 Taurocholate CA 0.111 µM 60 50 50 taurocholate and glycocholate in human and rat in vitro systems. Methods: GDCA 0.083 µM 40 40 30 Mechanistic studies were conducted using primary rat and human hepatocytes in TCA 0.070 µM 30 TDCA 0.019 µM 20 20 suspension and in primary culture. Hepatocytes were prepared from healthy 10 10 LCA 0.010 µM 0 0 human liver resections and from male Sprague Dawley rats and used freshly -10 BEI % In Vitro Biliary Clearance BEI % In Vitro Biliary Clearance TUDCA 0.008 µM (mL/min/kg) (mL/min/kg) isolated or after cryopreservation. The oil spin method was used to compare GLCA 0.006 µM -20 transporter - mediated uptake of taurocholate and glycocholate in suspension TLCA 0.0005 µM Safer to use appropriate bile salt for each species. hepatocytes. Uptake was also conducted after 24 hours in primary culture on total 1.911 µM collagen coated plates. Additional hepatocytes were overlaid with Geltrex® Matrix Tagliacozzi et al., 2003 (Invitrogen) and used at 5-7 days in culture to assess uptake and biliary efflux of the bile salts. Membrane vesicles over-expressed with the human and rat BSEP Figure 7. Uptake of Bile Salts in Human & Rat BSEP Vesicles transporter (Genomembrane) were also used. Results: Time linearity studies with taurocholate and glycocholate indicate linear uptake at 5 minutes incubation for Fig 2. Uptake in Plated Fig 3. Bile Acid Uptake in Suspension 300 100 Glycocholate Taurocholate the rat and human hepatocytes in culture and a 1 minute incubation time for Human & Rat Hepatocytes Human & Rat Hepatocytes Rat Rat 80 K 18.5µM Km 32.7µM m suspension hepatocytes. In 24-hour plated hepatocytes, the Km for taurocholate 200 60 uptake was similar: 13.3 µM in human and 22.1 µM in rat; while the Km for 8000 15000 Human Rat 2500 6000 glycocholate demonstrated differences between the two species: 23.1 µM (human) 6000 Human Rat 40 10000 2000 100 Human Human pmol/min-mg 4000 pmol/min-mg K 26.5µM Km 11.8µM and 166.8 µM (rat). Using sandwich hepatocytes with intact bile canaliculi, we 4000 1500 m 20 pmol/mg pmol/mg 5000 1000 demonstrated a biliary excretion index (BEI) for taurocholate of 82.5%, in rat and 2000 2000 0 0 K 13.3 M K 22.1 µM 500 m µ m K 311µM 49.6% in human hepatocytes. The BEI for glycocholate was 77.5% for human and m Km 145µM 050100150 0 10 20 30 40 50 Ave.Accum. (pmol/mg) Ave.Accum. 0 0 (pmol/mg) Ave.Accum. 0 0 0 20406080 0 20 40 60 80 0 20 40 60 80 0 20 40 60 80 [GCA] µM [TCA] µM 53.4% for rat hepatocytes. As a whole, in these two culture systems, glycocholate Taurocholate µM Taurocholate µM Taurocholate uM Taurocholate uM 8000 Human is a more efficient substrate in human vs. rat hepatocytes and taurocholate is a 10000 15000 15000 6000 Rat stronger substrate in rat hepatocytes. BSEP vesicles mimicked these trends. 8000 Human Rat

10000 10000 Inhibition studies were less conclusive and further evaluations are being 4000 6000 pmol/mg

pmol/mg 4000 conducted to understand differences if any, using a wider range of inhibitors in 2000 5000 5000 Figure 8. Inhibition of Bile Salt Uptake in Human & Rat BSEP Vesicles 2000 K 23.1 µM Km 213µM Km 246µM m (pmol/mg) Ave.Accum. both species. Conclusions: In preliminary evaluations, glycocholate is a stronger Km 115.4 µM (pmol/mg) Ave.Accum. 0 0 0 with Cyclosporine A and Troglitazone 0 0 50 100 150 0 50 100 150 0 20406080 0 50 100 150 substrate for human hepatic bile salt uptake and efflux transporters while Glycocholate uM Glycocholate uM Glycocholate µM taurocholate is the appropriate substrate for assessing rat biliary transport. Glycocholate µM Mechanistic studies to further understand differences between these two bile salts hBSEP Vesicles hBSEP Vesicles hBSEP Vesicles in rat and human hepatocytes are currently being conducted. Our data thus far 200 0 uM 50 150 0 uM 0 uM indicates that serious consideration should be given to using glycocholate for in Ki 0.8 µMK0.4 uM i 3.8 µM K 111 µM 150 40 0.4 uM i 0.8 uM 2 uM 2 uM 4 uM vitro studies aimed at studying human bile salt transporters. Fig 4. Inhibition of Bile Salt Uptake in Suspension Hepatocytes by 10 uM 100 30 10 uM 20 uM 100 50 uM 100 uM Cyclosporin A, Ritonovir & Troglitazone 20 pmol/mg-min 50 50 pmol/mg-min INTRODUCTION 10 pmol/mg-min 0 0 10 20 30 40 50 0 0 5 10 15 20 0 Human Human GCA (µM) 0 10 20 30 40 50 Human TCA (uM) Hepatic toxicity is a major factor for discontinuing the development of compounds GCA (µM) Glycocholate IC50 2.7 µM in pharmaceutical preclinical development or Phase I clinical trials. Some Glycocholate IC50 4.4 µM hBSEP Vesicles rBSEP Vesicles rBSEP Vesicles 80 Glycocholate IC50 1.4 µM compounds demonstrate liver toxicity while being tested in experimental animals 200 150 0 uM 0 uM 0 uM 0.8 uM Ki 3.4 µM Ki 79 µM 0.4 uM Ki 3.0 µM 0.4 uM 60 suggesting potential adverse clinical liver effects. In contrast, other compounds 150 4 uM 2 uM 2 uM Taurocholate IC50 3.2 µM 100 20 uM 10 uM 10 uM demonstrate only minor or no signs of hepatotoxicity in the animal species tested, 40 100 uM 100 50 uM Taurocholate IC50 2.3 µM Taurocholate IC 8.0 µM yet cause an increase in serum bile salts and/or liver enzymes in more than 10% of 50 50 pmol/mg-min pmol/mg-min 50 pmol/mg-min 20 humans during early clinical trials. Typically, drugs that result in hepatotoxicity 0 0 0 are preferentially eliminated from the body via the biliary pathway. We and others Rat 0 20 40 60 0 10 20 30 0 5 10 15 20 Rat Rat GCA (µM) TCA (uM) TCA (uM) have hypothesized that hepatotoxicity in humans taking these drugs may be Glycocholate IC 20.7 µM 50 Glycocholate IC 4.0 µM associated with drug-mediated inhibition of active canalicular transport of bile Glycocholate IC50 2.9 µM 50 components including, but not limited to, bile acids (1,2). These drugs are likely substrates for active liver transporter-mediated uptake (NTCP) and efflux (BSEP) CONCLUSIONS into the bile canaliculi. The transporters that participate in biliary drug elimination Taurocholate IC 7 µM Taurocholate IC50 0.1 µM Taurocholate IC50 9.1 µM also transport endogenous bile components. This results in the potential for 50 mutual inhibition of drug and bile acids efflux from the liver, resulting in an • Clinical studies indicate that chenodeoxycholic acid is the major circulating bile salt increase in drug and bile acids retained in the liver over time. Compounds with in human plasma, followed by . greater inhibition of bile acid transport have a higher risk of being hepatotoxic. • Taurocholic acid is a relatively minor bile salt in humans and the major bile acid in rat A majority of studies conducted to evaluate bile salt inhibition mediated hepatotoxicity in vitro, use primary hepatocytes or membrane vesicles over- • In vitro uptake studies in suspension and plated human hepatocytes reveal Fig 5. Biliary Clearance in Human & Rat Sandwich Cultured expressed with the bile salt efflux transporters BSEP. Typically, a competition equivalent uptake for both bile acid. assay between the drug and the bile acid taurocholate is used to test whether Hepatocytes • In vitro uptake studies in suspension and plated rat hepatocytes indicate taurocholic canalicular efflux of taurocholate can be inhibited in a concentration-dependent Human Accumulation (+): 24 pmol/mg 90 acid is a better substrate than glycocholic acid. manner and whether this inhibition would correlate with clinical hepatotoxicity. 80 TCA (-): 4.2 pmol/mg GCA Biliary CL : 15.6 mL/min/Kg However, while taurocholate is the major bile salt in the rat, glycine conjugated 70 60 BEI : 82.5% • B-clear studies demonstrate equivalent biliary excretion in human hepatocytes with bile salts (e.g. glycocholate) and their metabolites (e.g. glycochenodeoxycholic) are 50 40 Accumulation (+): 23 pmol/mg the two bile acids. the predominant bile salts in humans3. In this study, 14 bile acids were 30 (-): 5.1pmol/mg quantitated and glycochenodeoxycholic acid was the most abundant, followed by 20 Biliary CL : 14.1mL/min/Kg • BSEP membrane vesicles studies reveal similar uptake trends. Inhibition needs to be 10 BEI : 77.5% 0 repeated. glycocholic acid. Taurocholic acid was one of the relatively minor bile salts. We BEI % In Vitro Biliary CL (mL/min/kg) hypothesized that glychocholic acid should be the appropriate bile acid for in vitro • Inhibition studies using the B-clear model indicat different profiles using 60 studies that are conducted to predict clinical cholestasis and have conducted Rat TCA Accumulation (+): 38.5 pmol/mg 50 GCA troglitazone as the inhibitor, indicating that to predict human inhibition of bile salts, extensive comparisons of these two bile acids using in vitro models. (-): 16.2 pmol/mg 40 Biliary CL : 15.3 mL/min/Kg the species – appropriate bile salt should be used

30 BEI : 52.7% MATERIALS AND METHODS 20 Accumulation (+): 185 pmol/mg REFERENCES 10 (-): 72.3pmol/mg Biliary CL : 16.6mL/min/Kg (1) Kostrubsky, V. E.; Strom, S. C.; Hanson, J.; Urda, E.; Rose, K.; Burliegh, J.; Zocharski, P.; Cai, H.; Sinclair, J. F.; 0 BEI % In Vitro Biliary Clearance BEI : 54.6% Sahi, J. Toxicol Sci 2003, 76, 220-8. Transporter-Mediated Uptake: Uptake and inhibition of uptake was evaluated in (mL/min/kg) (2) Sahi, J.; Sinz, M. W.; Campbell, S.; Mireles, R.; Zheng, X.; Rose, K. A.; Raeissi, S.; Hashim, M. F.; Ye, Y.; de suspension and primary cultured hepatocytes by standard methodologies4. Morais, S. M.; Black, C.; Tugnait, M.; Keller, L. H. Chem Biol Interact 2006, 159, 156-68. Biliary Excretion” This was evaluated in hepatocytes after 5-7 days in culture (3) Tagliacozzi, D.; Mozzi, A. F.; Casetta, B.; Bertucci, P.; Bernardini, S.; Di Ilio, C.; Urbani, A.; Federici, G. Clin Chem Lab Med 2003, 41, 1633-41. using standard methodologies1. (4) Shitara, Y.; Li, A. P.; Kato, Y.; Lu, C.; Ito, K.; Itoh, T.; Sugiyama, Y. Drug Metab Pharmacokinet 2003, 18, 33- BSEP Vesicles: Mechanistic studies to evaluate the efflux and inhibition potential 41. of inhibitors were evaluated using Genomembrane membrane vesicles as (5) Yabuuchi, H.; Tanaka, K.; Maeda, M.; Takemura, M.; Oka, M.; Ohashi, R.; Tamai, I. Biopharm Drug Dispos described before (5). 2008, 29, 441-8.

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