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1 Supplemental material to this article can be found at: X/45/7/ $ DRUG METABLISM AND DISPSITIN Drug Metab Dispos 45: , July 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3--Sulfate Conjugates as Potential Novel Biomarkers for rganic Anion-Transporting Polypeptides s Rhishikesh Thakare, ongying Gao, Rachel E. Kosa, Yi-An Bi, Manthena V. S. Varma, Matthew A. Cerny, Raman Sharma, Max Kuhn, Bingshou uang, Yiping Liu, Aijia Yu, Gregory S. Walker, Mark Niosi, Larry Tremaine, Yazen Alnouti, and A. David Rodrigues Pharmacokinetics, Dynamics & Metabolism, Medicine Design (.G., R.E.K., Y.B., M.V.S.V., M.A.C., R.S., G.S.W., M.N., L.T., A.D.R.), and Nonclinical Statistics, Pfizer Inc., Groton, Connecticut (M.K.); Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, maha, Nebraska (R. T., Y. A.); and WuXi AppTec, Waigaoqiao Free Trade Zone, Shanghai, China (B.., Y. L., A.Y.) ABSTRACT In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (ATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free C max values (0.06, 0.66, 2.57, and 7.79 mm, respectively) spanning the reported in vitro IC 50 values for ATP1B1 and ATP1B3 ( 1.7 mm). As expected, the area under the plasma concentration-time curve (AUC) of an ATP probe drug (i.v pitavastatin, 0.2 mg/kg) was increased 1.2-, 2.4-, 3.8-, and 4.5-fold, respectively. Plasma of RIF-dosed cynomolgus monkeys was subjected to a liquid chromatography-tandem mass spectrometry method that supported the analysis of 30 different BAs. Monkey urine was profiled, and we also determined that the impact of RIF on BA renal clearance was minimal. Although sulfated BAs comprised only 1% of Introduction It is now accepted that organic anion-transporting polypeptides (ATPs) mediate the active uptake of numerous drugs into hepatocytes and hence govern their pharmacokinetic profile and liver (free)-toplasma (free) concentration ratio. ATPs can also serve as the loci of important drug-drug interactions (DDIs) leading to changes in systemic and local drug concentrations, possibly resulting in altered efficacy and toxicity profiles (Giacomini et al., 2010; Yoshida et al., 2012). Consequently, tools have been developed to facilitate ATP inhibition s This article has supplemental material available at dmd.aspetjournals.org. Received January 28, 2017; accepted April 5, 2017 the plasma BA pool, a robust RIF dose response (maximal 50-fold increase in plasma AUC) was observed for the sulfates of five BAs [glycodeoxycholate (GDCA-S), glycochenodeoxycholate (GCDCA-S), taurochenodeoxycholate, deoxycholate (DCA-S), and taurodeoxycholate (TDCA-S)].In vitro,rif( 100 mm) did not inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation and cynomolgus monkey hepatocytemediated uptake of representative sulfated BAs (GDCA-S, GCDCA-S, DCA-S, and TDCA-S) was sodium-independent and inhibited ( 70%) by RIF (5 mm); uptake of taurocholic acid was sensitive to sodium removal (74% decrease) and relatively refractory to RIF ( 21% inhibition). We concluded that sulfated BAs may serve as sensitive biomarkers of cynomolgus monkey ATPs and that exploration of their utility as circulating human ATP biomarkers is warranted. screening in vitro, drive model-based DDI in vitro-in vivo extrapolations, and support ATP DDI risk assessment before human dosing (Poirier et al., 2007; Jamei et al., 2014; Vaidyanathan et al., 2016; Yoshikado et al., 2016). The latter is particularly important because ATP activity and expression are also known to be impacted by genotype and liver disease (Gong and Kim, 2013; Clarke et al., 2014). More recently, it has been envisioned that ATP biomarkers will greatly facilitate clinical phenotyping and DDI studies while possibly deferring more formal studies using drug probes (Lai et al., 2016; Yee et al., 2016). For example, Lai et al. (2016) recently evaluated plasma bilirubin, bilirubin glucuronide, and coproporphyrin isomers (I and III) as ATP biomarkers in human subjects after a single dose of rifampicin (RIF). These authors noted a 4.0- and 3.3-fold increase in the Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018 ABBREVIATINS: AUC, area under the plasma concentration-time curve; AUCR plasma, AUC ratio determined by dividing the AUC 0 24,plasma after RIF treatment by the AUC 0 24,plasma after vehicle alone; BA, bile acid; CA, cholic acid; CA-S, cholic acid 3--sulfate; CDCA, chenodeoxycholic acid; CDCA-S, chenodeoxycholic acid 3--sulfate; CL renal ratio, renal clearance; DCA-S, deoxycholic acid 3--sulfate; DDI, drug-drug interaction; DEA, dehydroepiandrosterone; FDR, false discovery rate; GCA, glycocholic acid; GCA-S, glycocholic acid 3--sulfate; GCDCA, glycochenodeoxycholic acid; GCDCA-S, glycochenodeoxycholic acid 3--sulfate; GLCA, glycolithocholic acid; GLCA-S, glycolithocholic acid 3--sulfate; GUDCA, glycoursodeoxycholic acid; GUDCA-S, glycoursodeoxycholic acid 3--sulfate; LCA, lithocholic acid; LCA-S, lithocholic acid 3--sulfate; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NTCP, sodium-taurocholate cotransporting polypeptide; AT3, organic anion transporter 3; ATP, organic anion-transporting polypeptide; PAPS, 39-phosphoadenosine-59-phosphosulfate; QC, quality control; RIF, rifampicin; RIFsv, rifamycin SV; SLC, solute carrier; SULT2A1, sulfotransferase 2A1; TCA, taurocholic acid; TCA-S, taurocholic acid 3-sulfate; TCDCA, taurochenodeoxycholic acid; TCDCA-S, taurochenodeoxycholic acid 3--sulfate; TDCA, taurodeoxycholic acid; TDCA-S, taurodeoxycholic acid 3--sulfate; TLCA, taurolithocholic acid; TLCA-S, taurolithocholic acid 3--sulfate; TUDCA, tauroursodeoxycholic acid; TUDCA-S, tauroursodeoxycholic acid 3--sulfate; UDCA, ursodeoxycholic acid; UDCA-S, ursodeoxycholic acid 3--sulfate. 721

2 722 Thakare et al. coproporphyrin I and III area under the plasma concentration-time curve (AUC), respectively, consistent with in vitro data (Bednarczyk and Boiselle, 2016; Shen et al., 2016). Likewise, Yee et al. (2016) identified the 3--sulfate conjugates of glycochenodeoxycholic acid (GCDCA-S), glycodeoxycholic acid (GDCA-S), and taurolithocholic acid (TLCA-S) as candidate ATP biomarkers after dosing of SLC1B1 genotyped subjects with cyclosporine. Because of the high sequence identity with human ATPs, the cynomolgus monkey has been used increasingly as a model to study ATP inhibition in vivo (Shen et al., 2013; Takahashi et al., 2013; Shen et al., 2015). The utility of the cynomolgus monkey has extended also to the search for ATP biomarkers, which has involved the administration of a single RIF dose and reporting its impact on plasma bilirubin, bilirubin glucuronide, coproporphyrins (I and III), nonsulfated bile acids (BAs), and dehydroepiandrosterone (DEA) 3--sulfate (Chu et al., 2015; Watanabe et al., 2015; Shen et al., 2016). As described, an attempt was made to extend the work of Chu et al. (2015) by profiling 30 different BAs in cynomolgus monkey plasma after single oral doses of RIF (1, 3, 10, and 30 mg/kg). It was possible to prepare synthetic standards of numerous BA 3--sulfates and apply a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method that has been used successfully to profile nonsulfated and sulfated BAs in human serum and urine (Bathena et al., 2013). It should be noted that at the time of the study, BA sulfation in the cynomolgus monkey was not well characterized, although the putative hydroxysteroid sulfotransferase (SULT2A1) that catalyses BA sulfation in humans was known to be expressed in monkey liver (Nishimura et al., 2008; Alnouti, 2009; Nishimura et al., 2009). It was also known that DEA 3--sulfate was detectable in cynomolgus monkeys after DEA administration (Leblanc et al., 2003). In the present study, animals also received a single i.v. dose of pitavastatin to ensure that ATP was inhibited by RIF in a dosedependent manner (Takahashi et al., 2013). In addition, the plasma concentrations of RIF were determined at each of the four dose levels. Sulfated BAs are known to be cleared renally in humans (Alnouti, 2009; Bathena et al., 2013; Tsuruya et al., 2016); therefore, BA profiling was extended to include urine of control and RIF-dosed cynomolgus monkeys. The different BAs were then assessed in terms of their utility as ATP biomarkers: 1) detectability in control animals; 2) magnitude of the RIF dose response; and 3) detectability in human serum. Based on these criteria, seven sulfated BAs [GCDCA-S, GDCA-S, glycolithocholate 3--sulfate (GLCA-S), TLCA-S, taurochenodeoxycholate 3--sulfate (TCDCA-S), taurodeoxycholate 3--sulfate (TDCA-S), and deoxycholate 3--sulfate (DCA-S)] were identified as potential ATP biomarkers. For GCDCA-S, GDCA-S, and TLCA-S, the results are consistent with human plasma metabolomic data from a recent ATP1B1 (SLC1B1) genome-wide association study and earlier reports describing TLCA-S as an ATP substrate in vitro (Meng et al., 2002; Sasaki et al., 2002; Yee et al., 2016). The present study also included an in vitro assessment of BA sulfation by cynomolgus monkey liver cytosol, as well as uptake studies with GDCA-S, GCDCA-S, DCA-S, and TDCA-S (cynomolgus monkey plated primary hepatocytes) to phenotype both in terms of ATP- and sodium-taurocholate co-transporting polypeptide (NTCP)-mediated active uptake. Materials and Methods Chemicals and Reagents 39-Phosphoadenosine-59-phosphosulfate (PAPS), simvastatin, rifamycin SV (RIFsv), and RIF were purchased from Sigma-Aldrich (St. Louis, M). Deuterium-labeled RIF ( 2 8 -RIF) was obtained from ALSACIM (Illkirch, Graffenstaden, France). Pitavastatin was purchased from Sequoia Research Products Ltd. (xford, UK). Deuterium-labeled pitavastatin ( 2 4 -pitavastatin) was purchased from Clearsynth Canada Inc (Mississauga, N, Canada). InVitroGro-T and CP hepatocyte media were purchased from Celsis IVT (Baltmore, MD). Collagen I coated 24-well plates were obtained from BD Biosciences (Franklin Lakes, NJ). Cryopreserved cynomolgus monkey hepatocytes were purchased from In vitro ADMET Laboratories, LLC (Columbia, MD). Bicinchoninic acid protein assay kit was purchased from Pierce (Rockford, IL). Methanol, acetonitrile, water, and ammonium hydroxide were obtained from Fisher Scientific (Fair Lawn, NJ). Cholic acid (CA), glycocholic acid (GCA), taurocholic acid (TCA), deuteriumlabeled taurocholic acid ( 2 4 -TCA), chenodeoxycholic acid (CDCA), deuterium-labeled chenodeoxycholic acid ( 2 4 -CDCA), taurochenodeoxycholic acid (TCDCA), deoxycholic acid (DCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA), lithocholic acid (LCA), glycolithocholic acid (GLCA), taurolithocholic acid (TLCA), ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), glycoursodeoxycholic acid (GUDCA), and deuterium-labeled glycodeoxycholic acid 3--sulfate ( 2 4 -GDCA-S) were purchased from IsoSciences (King of Prussia, PA). Lithocholic acid 3--sulfate (LCA-S), glycolithocholic acid 3--sulfate (GLCA-S), chenodeoxycholic acid 3--sulfate (CDCA-S), glycochenodeoxycholic acid 3--sulfate (GCDCA-S), and deuterium-labeled glycochenodeoxycholic acid 3--sulfate ( 2 5 -GCDCA-S) were purchased from Toronto Research Chemicals (Toronto, N, Canada). Ursodeoxycholic acid 3--sulfate (UDCA-S) was obtained from ALSACIM (Illkirch, Graffenstaden, France). Deuterium labeled glycochenodeoxycholic acid ( 2 4 -GCDCA) was purchased from C/D/N isotopes, Inc. (Pointe-Claire, Quebec, Canada). Taurolithocholic acid 3--sulfate (TLCA-S) was purchased from Sigma- Aldrich (St. Louis, M). Refer to the Supplemental Material for the chemical synthesis of taurochenodeoxycholic acid 3--sulfate (TCDCA-S), deoxycholic acid 3-sulfate (DCA-S), glycodeoxycholic acid 3--sulfate (GDCA-S), taurodeoxycholic acid 3--sulfate (TDCA-S), cholic acid 3--sulfate (CA-S), glycocholic acid 3--sulfate (GCA-S), taurocholic acid 3--sulfate (TCA-S), and biosynthesis of glycoursodeoxycholic acid 3--sulfate (GUDCA-S) and tauroursodeocycholic acid 3--sulfate (TUDCA-S). Biosynthesis of 2 4 -TDCA-S and 2 4 -DCA-S is described also. An alternative method for preparing some of the BA sulfates described herein has been described by Donazzolo et al. (2017). Refer to the Supplemental Material for all BA common names, chemical names, structures, and CAS numbers. Animal andling, Dosing, Plasma Draws, and Urine Collection All experiments involving animals were conducted at the Pfizer Groton (Connecticut) facilities (Association for Assessment & Accreditation of Laboratory Animal Care-Accredited) and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. Male cynomolgus macaque Mauritian monkeys (approximately years of age) were used for these studies. A crossover study design was used, in which the same four animals were dosed over a series of five studies, after a minimum 1-week washout period between each study. ne exception was the 3 mg/kg RIF dose group, in which one of four monkeys was dosed only in that single study. Animals were provided a normal food schedule the day before the study (meals at 8:00 AM and 11:00 AM, with one treat daily) and were allowed free access to water. n the day of the study, monkeys were fed at approximately 1 and 3 hours postdose and allowed water ad libitum. RIF was administered via oral gavage at 0 (blank vehicle), 1, 3, 10, and 30 mg/kg. RIF was given at a dose volume of 2 ml/kg in a 0.5% (w/v) methylcellulose (in water) suspension. Approximately 1 hour and 15 minutes after the oral RIF administration, 2 4 -pitavastatin was administered via an i.v. bolus (cephalic vein) at dose of 0.2 mg/kg in a dosing volume of 0.2 ml/kg, 2% DMS (v/v), and 98% of TRIS-buffered saline (p ;7.7). All i.v. formulations were sterile filtered before administration. Serial blood samples were collected via the femoral vein before collecting in K 2 EDTA tubes and then at 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 5, 6, and 24 hours after i.v. dosing. Blood samples were stored on wet ice before being centrifuged to obtain plasma (3000 RPM, 10 minutes at 4 C; Jouan BR4i refrigerated centrifuge). Urine was also collected (metabolism cages) on wet ice, predose and at intervals of 0 6 hours and 6 24 hours postdose. wing to instability and possible interconversion of lactone to pitavastatin, each plasma and urine sample was equally divided into two Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

3 Bile Acid Sulfates as Potential Novel ATP Biomarkers 723 aliquots before being stored frozen. The first was untreated matrix, and the second was added to an equal volume of 0.1 M sodium acetate buffer (p 4) to stabilize pitavastatin. All urine and plasma samples, treated and untreated, were kept cold during collection, after which they were stored frozen at 220 C. It is known that BAs undergo enterohepatic recirculation, but no attempt was made to collect portal vein blood from the different cynomolgus monkeys. After LC-MS/MS analysis of femoral vein derived plasma, it was apparent that the concentration (total plasma) of the various BA sulfates was low (#30 nm). For some of the BA sulfates (TCDCA-S, GCDCA-S,TDCA-S, and GDCA-S), unbound fraction in cynomolgus monkey plasma was determined (;0.016); maximal free plasma concentration was ;0.5 nm. It is assumed that even if free BA sulfate concentrations were 100-fold higher in the portal vein, such concentrations would still likely be below the apparent K m for cynomolgus monkey ATPs. As described in the Results, RIF dosing brought about robust ($10-fold) dose-dependent increase in the area under the plasma concentration-time curve (AUC) for a number of BA sulfates (LCA-S, GLCA-S, TLCA-S, GCDCA-S, TCDCA-S, DCA-S, GDCA-S, TDCA-S). Such a result is consistent with low substrate concentration-to-atp K m ratios in vivo. LC-MS/MS) Analysis Analysis of BAs and their 3--Sulfate Conjugates. Plasma and urine concentrations of BAs and their 3--sulfate conjugates were measured in untreated matrices by LC-MS/MS, as described previously, with some modifications (Bathena et al., 2013). Briefly, a Waters ACQUITY ultraperformance liquid chromatography (UPLC) system (Waters, Milford, MA) was coupled to an 5500 Q TRAP quadrupole linear ion trap hybrid mass spectrometer (MS) with an electrospray ionization (ESI) source (Applied Biosystems, MDS Sciex, Foster City, CA). Chromatographic separations were performed using an ACQUITY UPLC BE C18 column (1.7 mm, mm) maintained at 25 C and equipped with an in line precolumn filter. The mobile phase consisted of 7.5 mm ammonium bicarbonate, adjusted to p 9.0 using ammonium hydroxide (mobile phase A) and 30% acetonitrile in methanol (mobile phase B), at a total flow rate of 0.2 ml/min. The gradient profile was held at 52.5% mobile phase B for minutes, increased linearly to 68% in 0.25 minute, held at 68% for 8.75 minutes, increased linearly to 90% in 0.25 minute, held at 90% for 1 minute, and finally brought back to 52.5% in 0.25 minute followed by 4.75 minute re-equilibration (total run time of 28 minutes per sample). Ten microliters of sample was injected for analysis. Quantitative data were acquired in multiple reaction monitoring (MRM)-negative ESI mode. MRM transitions and MS parameters for the different BAs and their respective 3--sulfate conjugates are shown in Supplemental Table 1. For preparation of calibration curves, blank plasma and urine were obtained by charcoal stripping as described previously (Bathena et al., 2013). Fourteenpoint calibration curves were prepared in stripped matrices by spiking 10 ml of appropriate standard solution at final concentrations ranging from 0.5 to 2500 ng/ml. For extraction of plasma samples, 1 ml of ice-cold alkaline ACN (5% N 4 ) containing 2 4 -GCDCA and 2 4 -CDCA as internal standards was added to 100 ml of samples. Samples were then vortex-mixed and centrifuged at 16,000g for 10 minutes, and the supernatants were aspirated, evaporated, and reconstituted in 100 ml of 50% Me solution. Urine samples were extracted similarly to plasma samples except Tween 20 was added (final concentration of 0.2% v/v) to reduce nonspecific binding. Analysis of 2 4 -Pitavastatin and RIF. The plasma concentrations of RIF and 2 4 -pitavastatin were measured in plasma samples treated with 0.1 M sodium acetate buffer (p 4) using the LC-MS/MS system listed herein. All standards and quality controls (QCs) were made in blank monkey plasma mixed with an equal volume of 0.1 M sodium acetate buffer (p 4). Standard and QC mixtures of the analytes were made to encompass a range of concentrations ( ng/ml, pitavastatin; ng/ml, RIF). Samples were diluted to be measured in the linear range of the instrument responses, with high specificity of MRM (no interference in the blank matrixes) and a wide dynamic range for each analyte; the dilution integrity was confirmed by independent analysis of the drugs in the samples in separate assays. Aliquots of 50 ml of standards, QCs, and plasma samples were prepared by protein precipitation using 200 ml of acetonitrile containing an internal standard mixture of simvastatin and 2 8 -RIF (100 ng/ml). The plates were vortexed for 2 minutes and centrifuged at 3000 rpm for 10 minutes, and 100-ml supernatants of the mixture were transferred for LC-MS/MS analysis. Chromatographic separation was accomplished on a Waters Acquity UPLC SS T3 C18 column (1.8 mm, mm) maintained at 40 C. The mobile phase consisted of two solvents, solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). The total run time for each injection was 3 minutes. The flow rate was 0.6 ml/min. The gradient was maintained at 5% B for 0.3 minutes, followed by a linear increase to 95% B in 1.8 minutes, and kept at 95% B for 0.3 minute and then a linear decrease to 5% in 0.3 minutes. The column was equilibrated at 5% B for 0.3 minute. A Valco VICI valve (Valco Instruments Co., ouston, TX) was used to divert the first 0.3 minute and the last 0.5 minute of UPLC effluent to waste. The injection volume was 2 ml. The analytes were monitored using MRM with settings listed in Supplemental Table 2. Determination of Uptake Clearance in the Presence of Cynomolgus Monkey Plated Primary epatocytes Thawing and seeding procedure for cynomolgus monkey hepatocytes was the same as that described previously for human hepatocytes (Bi et al., 2006). In brief, cryopreserved cynomolgus monkey primary hepatocytes (male animal; Lot no ; In vitro ADMET Laboratories, LLC. Columbia, MD) were thawed and seeded into 24-well collagen-coated plates using In Vitro-T and In Vitro-CP hepatocyte media at a density of cells/well (0.5 ml/well). After culturing for 6 hours, the uptake study was conducted. To assess the rate of uptake and passive diffusion, the cells were princubated with and without (DMS only) RIFsv (1 mm) or RIF (5 mm) at 37 C for 10 minutes. The uptake was initiated by the addition of 0.5 ml containing 2 4 -TCDCA-S (0.1 mm), 2 4 -TDCA-S (0.1 mm), 2 4 -DCA-S (0.1 mm), 2 5 -GCDCA-S (0.1 and 0.5 mm), 2 4 -GDCA-S (0.1 and 0.5 mm), 2 4 -TCA (0.1 and 0.5 mm), and nonlabeled pitavastatin (0.1 mm). To determine the effect of sodium on substrate uptake, the cells were preincubated in Krebs-enseleit buffer without sodium (NaCl and NaC 3 Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018 TABLE 1 Pharmacokinetic parameters of RIF after oral administration to male cynomolgus monkeys Parameter RIF Dose 1 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg AUC 0 24,plasama (ng.h/ml) a , , , ,900 (1) b (11) (71) (243) Plasma total C max (ng/ml) , ,100 (1) (12) (45) (137) Plasma total C max (mm) Plasma free C max (mm) c T max (h) t 1/2 (h) a Data are presented as mean 6 S.D. (n = 4 animals). b Value in parentheses represents a ratio (vs. RIF at 1 mg/kg). c Plasma total C max corrected for RIF plasma-free fraction (0.265). RIF plasma protein binding was determined using equilibrium dialysis with ascorbic acid as a stabilizer (see Supplemental Material).

4 724 Thakare et al. TABLE 2 Pharmacokinetic parameters of 2 4 -pitavastatin in male cynomolgus monkeys after i.v. administration (0.2 mg/kg) with increasing doses of RIF Parameter ral RIF Dose a Vehicle 1 mg/kg 3 mg/kg 10 mg/kg 30 mg/kg AUC 0 24,plasma (ng.h/ml) * * * b AUCR plasma * 3.8* 4.5* C 0 (ng/ml) t 1/2 (h) * CL (ml/min per kilogram) * * * V dss (liter/kg) * * * % Dose in urine c a Data are presented as mean 6 S.D. (n = 4 animals). b Mean AUCR plasma (RIF versus vehicle control). c % Total dose (as 2 4 -pitavastatin) recovered in urine over 24 hours. *Statistically significant difference versus vehicle control arm; P, 0.05 (one-way ANVA, Dunnett multiple comparison method). replaced with choline chloride and choline bicarbonate, respectively) at 37 C for 10 minutes (o et al., 2004). In all cases, incubations were terminated at 0.5, 1, 2, and 5 minutes by washing the cells three times with ice-cold anks balanced salt solution buffer. The cells were then lysed with 100% methanol containing internal standard (diclofenac), centrifuged, and dried down under nitrogen and reconstituted in 50:50 methanol-to-water ratio. Chromatography was performed on a Waters Acquity UPLC System (Milford, MA). The autosampler and column were kept at 10 C and 50 C, respectively. Separation was achieved with a Waters BE C18 column ( mm, 1.7 mm) and a gradient of 7.5 mm ammonium bicarbonate (mobile phase A) and 70:30 methanol/acetonitrile (mobile phase B) at a flow rate of 0.2 ml/min. An initial mobile-phase composition of 50% B was held for 2 minutes, then ramped to 95% in 1.5 minutes, held at 95% for 1 minute, and returned to initial 50% B for 0.5 minute re-equilibration. The total analysis time for each sample was 5 minutes. Data were collected on an AB Sciex API5500 Fig. 1. Plasma concentration-time profile of (A) GDCA-S, (B) TDCA-S, (C) GCDCA-S, (D) TCDCA-S, (E) GLCA-S, and (F) TLCA-S in male cynomolgus monkeys after oral administration of vehicle alone (open squares) and RIF at 1 mg/kg (open circles), 3 mg/kg (closed triangles), 10 mg/kg (closed circles), and 30 mg/kg (closed squares). Data are presented as mean 6 S.D. (n = 4 animals). Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

5 Bile Acid Sulfates as Potential Novel ATP Biomarkers 725 (QTRAP) mass spectrometer (Foster City, CA) using negative Turbo IonSpray ESI and MRM mode with the according to transitions: 290.6/96.8 ( 2 4 -TDCA-S), 475.2/96.8 ( 2 4 -DCA-S), 533.3/453.4 ( 2 5 -GCDCA-S), 532.3/452.3 ( 2 4 -GDCA-S), and 514.2/79.8 ( 2 4 -TCA). Data acquisition and processing were carried out using Analyst software version (Applied Biosystems/MDS Sciex, Canada). Analysis of pitavastatin was performed as described here. Stability of the various sulfated BA substrates was confirmed after their addition to assay buffer and incubation for 5 minutes at 37 C. BA Sulfation Catalyzed by PAPS-Fortified Cynomolgus Liver Cytosol Refer to the Supplemental Material for details related to incubation conditions and LC-MS/MS analysis. Pharmacokinetics Analysis BAs. For each BA, the plasma AUC from 0 to 24 hours (AUC 0 24,plasma )was derived from the concentration-time profile for each individual animal (trapezoidal rule, Microsoft ffice Excel). Renal clearance (CL renal ) was calculated by dividing the amount excreted in urine from 0 to 24 hours (A e0 24,urine ) by the AUC 0 24,plasma.AUC 0 24,plasma ratios (AUCR plasma ) were determined by dividing the AUC 0 24,plasma after RIF treatment by the vehicle alone AUC 0 24,plasma.The CL renal ratio was determined by dividing the CL renal after RIF treatment by the vehicle alone CL renal. RIF and Pitavastatin. The noncompartmental analyses of 2 4 -pitavastatin and RIF plasma concentration-time data were performed using Watson LIMS version (Thermo Fisher Scientific Inc, Waltham, MA), which supported the generation of the various pharmacokinetic parameters; AUC, t 1/2 (half-life), CL (clearance), V dss (volume of distribution), C max (peak plasma concentration), and t max (time to peak plasma concentration). As done for the different BAs, 2 4 -pitavastatin AUCR plasma values were determined by dividing the AUC 0 24,plasma after RIF treatment by the vehicle alone AUC 0 24,plasma. For RIF, it was possible to calculate RIF plasma-free C max based on a plasma unbound fraction of (refer to Supplemental Material for RIF cynomolgus monkey plasma protein binding). In turn, in vitro IC 50 values for RIF with cynomolgus monkey ATP and NTCP (Shen et al., 2013; Chu et al., 2015) were used to calculate % inhibition; % inhibition = 100 * {[I]/([I] + IC 50 )}. It is assumed that IC 50 ; K i (when substrate concentration, K m ). [I] represents plasma C max of RIF (total or free). Consideration of free and total plasma C max is consistent with Vaidyanathan et al. (2016); in the absence of i.v. RIF pharmacokinetic data, it was not possible to derive an absorption rate constant for RIF and estimate its liver inlet (portal) concentration in the cynomolgus monkey. Statistical Analysis of RIF Dose Response Plasma. Plasma profiles over time for each animal were collapsed into an AUC using the trapezoidal rule and a C max score. The analyses were then conducted using a linear mixed model, such that the within-animal correlations were accounted for in the model. The R computing language was used for these calculations (R Foundation for Statistical Computing, Vienna, Austria. utcomes were analyzed on the log (base 2) scale to make Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018 Fig. 2. AUCR plasma values for (A) unsulfated BAs and (B) BA sulfates after dosing of RIF to cynomolgus monkeys. AUCR plasma was calculated by dividing AUC 0 24, plasma after RIF (1, 3, 10, and 30 mg/kg) by the AUC 0 24, plasma after administration of vehicle control.

6 726 Thakare et al. the residuals more normal. Concentrations were entered into the model on the same scale, and the P value for the linear trend was based on an F-statistic using Satterthwaite s approximation (West et al., 2015). False discovery rate (FDR) estimates were computed using the Benjamini and ochberg procedure (Benjamini and ochberg, 1995). Urine. Renal clearance (CL renal ) values (ml/min/kg) of zero were treated as missing, and analytes with greater than five missing values were excluded. As such, 18 of the 30 BAs were analyzed. As described for plasma, the analysis was conducted using a linear mixed model such that the within-animal correlations were accounted for in the model. The BAs were analyzed on the natural log scale to make the residuals more normal. Similar to plasma, the P value for the linear trend was based on an F-statistic using Satterthwaite s approximation and FDR estimates were computed using the Benjamini and ochberg procedure. Results Pharmacokinetics of RIF in Cynomolgus Monkeys. As expected, a dose-dependent increase in RIF AUC 0 24,plasma and plasma C max was observed over the dose range of 1 30 mg/kg (Table 1; Supplemental Fig. 1); however, at RIF doses of 3, 10, and 30 mg/kg, there was evidence for a greater than proportional increase in AUC 0 24,plasma (11-, 71- and 243-fold versus AUC at 1 mg/kg) and plasma C max (12-, 45-, and 137-fold versus C max at 1 mg/kg). For RIF, plasma unbound fraction was and so the calculated free C max was 0.06, 0.66, 2.57, and 7.79 mmat1,3,10, and 30 mg/kg, respectively. Such concentrations exceed the reported in vitro IC 50 values ( mm) for RIF with cynomolgus monkey ATP1B1 and ATP1B3 (Shen et al., 2013; Chu et al., 2015). Impact of RIF on 2 4 -Pitavastatin Pharmacokinetics. The pharmacokinetic parameters of 2 4 -pitavastatin, an accepted cynomolgus monkey ATP probe drug (Takahashi et al., 2013), were determined after dosing of an i.v. bolus (0.2 mg/kg) to vehicle control and RIF-dosed cynomolgus monkeys (Table 2, Supplemental Fig. 1). Compared with the vehicle control, 2 4 -pitavastatin CL was decreased 21%, 58%, 73%, and 76% with RIF treatment at 1, 3, 10, and 30 mg/kg dose levels, respectively; however, the change was only statistically significant at the three top RIF doses. Furthermore, 2 4 -pitavastatin AUC 0-24,plasma was increased 1.2-, 2.4-, 3.8-, and 4.5-fold at 1, 3, 10 and 30 mg/kg RIF, respectively. Both the V dss and T 1/2 of 2 4 -pitavastatin were decreased (;70%) at the three highest RIF doses. At all the RIF dose levels, recovery of unchanged 2 4 -pitavastatin in urine was less than 5% of the dose. verall, the impact of the lowest RIF dose on 2 4 -pitavastatin pharmacokinetics was not statistically significant. Profiling of Plasma BAs at Different Doses of RIF. As with pitavastatin, the plasma levels of various BAs, as well as their corresponding 3--sulfate conjugates, were measured in cynomolgus monkeys after increasing doses of RIF. In total, 30 different BAs were monitored. Sulfated BAs, in particular GDCA-S, TDCA-S, GCDCA-S, TCDCA-S, GLCA-S, and TLCA-S, presented a marked dose-dependent increase in their plasma concentration-time profile (Fig. 1; Supplemental Table 3). In contrast, the plasma concentration-time profile of nonsulfated BAs, particularly DCA, GDCA, TDCA, GCDCA, TCDCA, UDCA, GUDCA, TUDCA, CA, GCA, and TCA, showed a relatively weak increase at the RIF doses of 10 and 30 mg/kg (Supplemental Fig. 2). Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018 Fig. 3. Plasma C max ratios for (A) unsulfated BAs and (B) BA sulfates after dosing of RIF to cynomolgus monkeys. C max ratio was determined by dividing the C max after each RIF treatment by the C max after administration of vehicle control.

7 Bile Acid Sulfates as Potential Novel ATP Biomarkers 727 Chu et al. (2015) have also reported a weak increase in nonsulfated BAs after an oral RIF dose of 18 mg/kg. RIF treatment did not cause significant changes in LCA, GLCA, and TLCA. Unfortunately, GUDCA-S, TUDCA-S, CA-S, TCA-S, and GCA-S plasma levels were low and remained undetectable, even after RIF treatment. verall, the presence of various sulfo-conjugates in plasma was indicative of BA sulfation in the cynomolgus monkey. The pool of cynomolgus monkey BAs in circulation was distinct from that reported for human subjects; however, the percentages of sulfated (1.3% versus 28.4%) and amidated (21.5% versus 75.4%) BAs were low for monkey versus human, respectively (Supplemental Table 4). To complement the in vivo studies, the sulfation of six representative BAs (GCDCA, GUDCA, LCA, GLCA, GCA, and TLCA) was investigated after incubation with cynomolgus monkey liver cytosol (Supplemental Fig. 3); the availability of authentic standards supported identification of the 3--sulfate as the major product of PAPS-fortified monkey liver cytosol. Although a good correlation was obtained between human and cynomolgus monkey (R 2 = 0.905), a low activity ratio (cynomolgus monkey-to-human) was obtained for the formation of LCA-S, GLCA-S, TLCA-S, and GUDCA-S (0.20, 0.25, 0.36, and 0.37, respectively). In comparison, GCDCA-S (activity ratio = 0.89) and GCA-S (activity ratio = 3.0) rendered higher activity ratios comparable to those of DEA 3--sulfate (activity ratio of 1.3). Importantly, RIF (up to 100 mm) was shown not to inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation (Supplemental Fig. 4). Assessment of RIF Dose Response. AUCR plasma values for the various BAs after RIF treatment are shown in Fig. 2. The highest AUCR plasma ($78) was observed for GDCA-S and GCDCA-S, followed by TDCA-S, DCA-S, and TCDCA-S (;50) and GLCA-S and TLCA-S (;30). A relatively modest AUCR plasma (5 10) was observed for CA, GCA, and TCA. The remaining nonsulfated BAs presented low AUCR plasma values (2 5). Fold changes in plasma C max after RIF treatment are shown in Fig. 3. Similar to the AUC changes, the highest (50- to 80-) fold increase was observed for DCA-S, GDCA-S, TCDCA-S, TDCA-S, and GCDCA-S, followed by a 20-fold increase for GLCA-S and TLCA-S at an RIF dose of 30 mg/kg. AUC 0 24, plasma and plasma C max values at each RIF dose and vehicle control are shown in Supplemental Table 3. Statistical analysis for linear trend in AUC 0 24, plasma and C max and FDR for each BA are shown in Table 3. verall, with the exception of CA-S, all BA 3--sulfates were characterized by a significant linear trend (P, 0.01) with a slope of more than and an FDR less than 10% for AUC 0-24, plasma. GDCA-S, GCDCA-S, TDCA-S, TCDCA-S, GLCA-S, and TLCA-S were the most significant sulfate conjugates, with a P value for the linear trend of less than 0.001, a slope of more than , and FDR less than 1% for AUC 0 24, plasma, as well as C max.in contrast, most of the nonsulfated bile acids did not show a statistically significant linear trend, and FDR was more than 10% for AUC 0 24,plasma and C max. nly TCA and TDCA showed a P value less than 0.01 and ;10% FDR. Importantly, the plasma AUCR plasma values showed a good linear correlation (R ) between pitavastatin and GDCA-S, TDCA-S, GCDCA-S, TCDCA-S, GLCA-S, and TLCA-S (Fig. 4). Similarly, a good linear correlation (R ) between RIF plasma-free C max and 2 4 -pitavastatin, GDCA-S, TDCA-S, GCDCA-S, TCDCA-S, GLCA-S, and TLCA-S AUCR plasma was observed (Fig. 5). TABLE 3 P value for the linear trend with RIF dose and FDR estimates for the area under the plasma concentration-time curve (AUC 0 24,plasma ), maximum plasma concentration (C max,plasma ) and renal clearance (CL renal ) for various BAs and their respective 3--sulfate conjugates P value AUC 0 24, plasma C max,plasma CL renal Slope a P Value FDR Slope P Value FDR Slope P Value FDR CA-S NA b NA NA DCA-S c 1.44,0.0001, ,0.0001, TCA-S NA NA NA GDCA-S c 1.13, , , GCA-S NA NA NA TCDCA-S c 1.09,0.0001, ,0.0001, GCDCA-S c 1.07,0.0001, ,0.0001, CDCA-S NA NA NA TDCA-S c 0.91,0.0001, , GLCA-S c 0.81,0.0001, ,0.0001, TLCA-S c 0.78,0.0001, ,0.0001, UDCA-S 0.77,0.0001, , CA LCA-S 0.68,0.0001, NA b NA NA TCA GCA DCA UDCA TDCA GDCA GCDCA NA NA NA TCDCA CDCA TUDCA NA NA NA GUDCA NA NA NA GLCA NA NA NA LCA Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018 NA, not applicable. a Results ranked in terms of slope (AUC 0 24, plasma ). b No baseline (vehicle control) levels; NA: not applicable. c Considered candidate ATP biomarkers (See Fig. 2 and 3).

8 728 Thakare et al. As described previously, an effort was made to administer RIF over a dose range that generated a wide range of plasma total ( mm) and free ( mm) C max values (Table 1). In so doing, it was possible to investigate the dose-dependent inhibitionofatpsandntcp.basedoninvitroic 50 data, dosedependent inhibition of ATP1B1 (16% to $96%) and ATP1B3 (,10% $85%) was expected, with less inhibition of ATP2B1 (,10% 31%) and NTCP (,10% 29%) anticipated (Supplemental Table 5 and 6). Despite the effort to ensure dose-dependent inhibition, however, the AUCR plasma values for the various BA sulfates differed markedly. The highest maximal AUCR plasma values ($78) were obtained with GDCA-S and GCDCA-S, followed by TDCA-S, DCA-S, and TCDCA-S (AUCR plasma ;50), GLCA-S and TLCA-S (AUCR plasma ;30). Impact of RIF on BA Renal Clearance. Because the 3--sulfate conjugates of the various BAs are recovered in human urine (Bathena et al., 2013, 2015; Tsuruya et al., 2016), the present study was extended to include the profiling of urine of control and RIF-dosed cynomolgus monkeys. In this regard, a dose-dependent increase in the amounts of GDCA-S, TDCA-S, GCDCA-S, TCDCA-S, GLCA-S, and TLCA-S excreted in urine was observed (Supplemental Table 3). CL renal ratios Fig. 4. Linear regression of AUCR plasma between 2 4 -pitavastatin and GDCA-S (A), TDCA-S (B), GCDCA-S (C), TCDCA-S (D), GLCA-S (E), and TLCA-S (F) in cynomolgus monkeys. AUCR plasma was determined as described in the legend to Fig. 2. and statistical analysis for each BA are shown in Fig. 6 and Table 3, respectively. A weak dose-dependent increase in CL renal was observed for UDCA, GDCA, and DCA; however, there was no statistically significant effect of RIF treatment on the CL renal of sulfated and nonsulfated bile acids. For the former, this is in marked contrast to the changes in plasma AUC and C max after RIF treatment. Incubation of GCDCA-S, GDCA-S, DCA-S, TDCA-S, TCA, and Pitavastatin with Cynomolgus Monkey Plated epatocytes. Based on the availability of deuterium-labeled material and RIF-dependent AUCR plasma values in vivo, four sulfated BAs (GCDCA-S, GDCA-S, DCA-S, and TDCA-S) were chosen for study as solute carrier (SLC) substrates in vitro after incubation with plated cynomolgus monkey primary hepatocytes (Figs. 7 and 8). TCA and pitavastatin were also incubated as representative cynomolgus monkey NTCP (.ATP) and ATP (.NTCP) substrates, respectively (Chu et al., 2015; Takahashi et al., 2013). To discern the role of NTCP versus ATPs, incubations were performed in the presence and absence of sodium (NTCP is sodium-dependent). In addition, RIF (5 mm) and RIFsv (1 mm) were deployed as cynomolgus monkey ATP-selective (ATP1B1 and ATP1B3 inhibition $75%; NTCP inhibition #13%) and pan-slc (ATP and NTCP $97% inhibition) Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

9 Bile Acid Sulfates as Potential Novel ATP Biomarkers 729 Fig. 5. Linear regression between cynomolgus monkey RIF plasma-free C max (mm) at 1, 3, 10, and 30 mg/kg and AUCR plasma for GDCA-S (A), TDCA-S (B), GCDCA-S (C), TCDCA-S (D), GLCA-S (E), TLCA-S (F), and pitavastatin (G). AUCR plasma was determined as described in the legend to Fig. 2. Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018 inhibitors, respectively (Supplemental Table 5) (Shen et al., 2013; Chu et al., 2015; ong Shen, Bristol-Myers Squibb, personal communication). When incubated with RIFsv, the uptake of TCA, GDCA-S, GCDCA-S, and pitavastatin was markedly inhibited ($92%). For the four substrates, such a result is consistent with relatively high rates of active (versus passive; #8%) uptake (Fig. 7A). As shown in Fig. 7B, the uptake of GCDCA-S and GDCA-S was minimally impacted by the removal of sodium. By contrast, uptake of TCA (74%) and pitavastatin (;44%) was decreased in the presence of sodium-free buffer. As expected, RIF elicited relatively weak inhibition of TCA uptake compared with pitavastatin (14% versus 58%). Uptake of both GCDCA-S (69% inhibition) and GDCA-S (82% inhibition) was sensitive to RIF. Although the exact contribution of ATP1B1 and ATP1B3 was not determined, in the absence of selective inhibitors and established relative activity factors for cynomolgus monkey transporters, it is concluded that uptake of both GCDCA-S and GDCA-S (0.5 mm) in the presence of cynomolgus monkey hepatocytes is dominated by ATPs and that their profile is distinct from that of TCA. Based on the results presented in Fig. 8, the same can be said for two additional BA sulfates (TDCA-S and DCA-S). In this instance, RIF (5 mm) was shown to inhibit the uptake of TCA, pitavastatin, GCDCA-S, GDCA-S, TDCA-S, and DCA-S (0.1 mm) by 21%, 73%, 92%, 83%, 95%, and 80%, respectively.

10 730 Thakare et al. Fig. 6. Cynomolgus monkey CL renal ratios for (A) unsulfated BAs and (B) BA sulfates at increasing doses of RIF. CL renal ratio determined by dividing the CL renal after RIF treatment by the CL renal after vehicle alone. Discussion Metabolism of various BAs is complex and involves oxidation, amidation, glucuronidation, and sulfation. nce conjugated, BAs are also subjected to transporter-mediated uptake (e.g., ATP and NTCP) and efflux (Akita et al., 2001; Sasaki et al., 2002; Zelcer et al., 2003a, 2003b; Tsuruya et al., 2016; Rodrigues et al., 2014). Therefore, the BA pool of most species is complex and subject to enterohepatic recirculation and renal clearance. This means that BA profiling requires robust LC-MS/MS methods with access to a large number of authentic standards (Bathena et al., 2013). To support the present study, therefore, authentic standards of a number of noncommercially available BA 3--sulfates were prepared, and 30 different BAs were profiled in cynomolgus monkey plasma and urine. Although in vivo sulfation of DEA has been reported in cynomolgus monkeys (Leblanc et al., 2003), and SULT2A1 (human sulfotransferase involved in BA 3--sulfation) is known to be expressed in cynomolgus monkey liver (Nishimura et al., 2008; Alnouti, 2009; Nishimura et al., 2009), there have been no reports describing the BA sulfation in the cynomolgus monkey. For the first time, it was possible to report that DEA and various BAs undergo sulfation in vitro (Supplemental Fig. 3). Importantly, the presence of BA sulfates in cynomolgus monkey urine is consistent with human data (Bathena et al., 2013; Tsuruya et al., 2016); however, the fraction of the BA pool in circulation as the sulfated species was low in cynomolgus monkey versus human (28.4% versus 1.3%; Supplemental Table 4) and likely reflects species differences in CL renal and formation clearance. In agreement, the CL renal of GCDCA-S is lower in humans (31 versus 0.05 ml/min per kilogram) (Supplemental Table 3; Tsuruya et al., 2016), and the rate of BA sulfation in vitro was lower in the presence of cynomolgus monkey cytosol (Supplemental Fig. 3). A species difference in ATP-mediated hepatic uptake clearance is also a possibility. GCDCA-S has been shown to undergo active renal secretion and has been proposed as a biomarker for human organic anion transporter 3 (AT3) (Tsuruya et al., 2016). Although cynomolgus monkey AT3 has been expressed and characterized, nothing is known of its ability to transport BA sulfates and its inhibition by RIF (Tahara et al., 2005). Therefore, as part of the present study, it was important to assess the impact of RIF on BA sulfation and renal clearance. This ensured that any observed changes in BA AUC 0-24,plasma were reflective of hepatic transporter inhibition. In vitro data indicated that RIF (up to 100 mm) did not inhibit liver cytosol-catalyzed BA sulfation (Supplemental Fig. 4). Likewise, profiling of cynomolgus monkey urine supported calculation of CL renal for the different BAs and it was determined that the impact of RIF was minimal (Table 3; Supplemental Table 3). Importantly, there is evidence indicating that BA sulfates also serve as substrates of human canalicular multidrug resistance-associated protein (MRP) MRP2 and basolateral MRP3 and MRP4 (Akita et al., 2001; Sasaki et al., 2002; Zelcer et al., 2003a, 2003b). The possibility that single dose RIF can inhibit cynomolgus monkey MRP2 has already been considered by Chu et al. (2015). In this instance, the authors showed that RIF is a relatively Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

11 Bile Acid Sulfates as Potential Novel ATP Biomarkers 731 Fig. 7. Impact of RIF and RIFsv (A) and sodium removal (B) on the uptake of 2 5 -GCDCA-S (0.5 mm), 2 4 -GDCA-S (0.5 mm), 2 4 -TCA (0.5 mm), and non-labeled pitavastatin (0.1 mm) by plated cynomolgus monkey primary hepatocytes. RIF and RIFsv were added at a final concentration of 5 mm and 1 mm, respectively. Data for vdms 2 vi individual duplicates are shown. The mean % decrease is shown also and calculated as *100:v DMS and v i is the mean uptake rate in the presence of DMS alone (plus sodium) and inhibitor (or minus sodium) respectively. weak inhibitor of cynomolgus monkey MRP2 (IC 50 =118mM) and argued for relatively minimal inhibition based on estimated free RIF liver levels (;10 mm). Although no RIF inhibition data are available for monkey MRP3 and MRP4, given the robust AUCR plasma values for the various BA sulfates, it is assumed that neither transporter is inhibited. Alternatively, RIF could induce MRP3 and MRP4 and compromise interpretation of the data (Marschall et al., 2005; Badolo et al., 2015); however, it can be argued that significant and sustained induction of both MRP proteins is unlikely after a single RIF dose. Importantly, BA sulfate plasma concentration-time profiles were consistent with relatively rapid (within 6 hours) dose-dependent inhibition of hepatic uptake. Moreover, plasma concentrations for five of the six BA sulfates were trending toward pre-rif dose levels by 24 hours (Fig. 1). To date, efforts to inhibit cynomolgus monkey ATP in vivo have involved oral administration of a single RIF dose that generates a plasma total C max of ;10 mm (Shen et al., 2013; Takahashi et al., 2013; Chu et al., 2015). As described, it was possible to administer RIF at four dose levels of 1, 3, 10, and 30 mg/kg and obtain mean plasma total C max values of 0.2, 2.5, 9.7, and 29 mm, respectively; corresponding to a plasma-free C max of 0.06, 0.66, 2.57, and 7.79 mm, respectively (Table 1). Evidently, the pharmacokinetic profile of RIF in the cynomolgus monkey was nonlinear, likely reflecting saturation of first pass and consistent with human data (Acocella, 1978). Despite the vdms nonlinearity, there was a good linear correlation (R ) between RIF plasma C max and AUCR plasma for a number of BA sulfates (Fig. 5) and, because expression of hepatic ATP2B1 is relatively low in cynomolgus monkey, such a dose response is more likely reflective of ATP1B1 and ATP1B3 inhibition (Wang et al., 2015). Based on the in vitro IC 50 values reported for both ATPs (#1.7 mm; Supplemental Table 5), and assuming that RIF plasma concentrations support estimates of inhibition (Vaidyanathan et al., 2016), up to.85% (RIF-free C max )and.96% (RIF total C max ) inhibition is anticipated (Supplemental Table 6). In agreement, a clear RIF dose-dependent increase in 2 4 -pitavastatin AUCR plasma (1.2, 2.4, 3.8, and 4.5) was evident (Fig. 5). Because of weaker inhibition by RIF (IC 50 $ 35.1 mm, Supplemental Table 5), less NTCP inhibition (10% 29%) is expected (Supplemental Table 6). This is an important consideration because certain BAs are known to favor NTCP over ATPs (Meier et al., 1997; Maeda et al., 2006; Dong et al., 2015: Suga et al., 2017). For example, the uptake of TCA by primary cynomolgus monkey hepatocytes was shown to be highly sodium dependent and relatively refractory to RIF (Figs. 7 and 8). Although no formal in vitro-in vivo exercise was attempted, it was assumed that the RIF dose-dependent AUCR plasma values for TCA (1.2, 2.0, 1.8, and 5.4) are largely reflective of NTCP inhibition (Supplemental Tables 3 and 6). n the other hand, GCDCA-S, GDCA-S, TDCA-S, and DCA-S behaved as Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

12 732 Thakare et al. Fig. 8. Impact of RIF on the uptake of 2 5 -GCDCA-S (0.1 mm), 2 4 -GDCA-S (0.1 mm), 2 4 -TCA (0.1 mm), 2 4 -TDCA-S (0.1 mm), 2 4 -DCA-S (0.1 mm), and nonlabeled pitavastatin (0.1 mm) by plated cynomolgus monkey primary hepatocytes. RIF was added at a final concentration of 5 mm. Data for individual duplicates are shown. The mean % decrease is shown and was calculated as described in the legend to Fig 7. ATP substrates in the presence of cynomolgus monkey hepatocytes (Figs. 7 and 8), consistent with reports of a fifth sulfated BA (TLCA-S) presenting as an ATP substrate in vitro (Meng et al., 2002; Sasaki et al., 2002). As described, five sulfated BAs in plasma responded robustly to RIF in a dose-dependent manner (maximal AUCR plasma $ 50). Two additional BA sulfates, GLCA-S and TLCA-S, were also found to respond to RIF (maximal AUCR plasma ;30) (Fig. 2). From the standpoint of detectability in control animals, the magnitude of the RIF dose response, and detectability in human plasma (Supplemental Table 4), these seven BA sulfates (GCDCA-S, TCDCA-S, DCA-S, GDCA-S, TDCA-S, GLCA-S and TLCA-S) all present as potential sensitive ATP biomarkers. Importantly, the RIF dose response obtained was far greater than the increases (,10-fold) reported for cynomolgus monkey plasma bilirubin, bilirubin glucuronide, coproporphyrin (I and III), nonsulfated BAs, and DEA sulfate (Chu et al., 2015; Watanabe et al., 2015; Shen et al., 2016). Although the results presented herein showcase various BA sulfates as sensitive cynomolgus monkey ATP biomarkers, it cannot be assumed that the results translate directly to human subjects. For example, the balance of ATP- versus NTCP-mediated liver uptake and the contributions of individual ATPs may not be the same across species. Despite the caveats, the results of the present work are consistent with plasma metabolomic data from an ATP1B1 (SLC1B1) genome-wide association study that identified GCDCA-S, GDCA-S, and TLCA-S as potential ATP1B1 biomarkers (Yee et al., 2016). Such BA sulfates could potentially serve as sensitive human ATP biomarkers and provide the necessary dynamic range to support perpetrator differentiation (weak, moderate, versus potent inhibition), enable the study of ATP genotype-phenotype associations, and facilitate phenotyping of hepatobiliary diseased subjects (Gong and Kim, 2013; Clarke et al., 2014). In this regard, sulfated BAs could be superior human ATP biomarkers compared with plasma coproporphyrin I and III (AUCR plasma ;4.0) (Lai et al., 2016). As discussed previously, because circulating sulfated BAs are substrates of renal transporters also, they may serve as dual liver ATP and renal AT3 biomarkers. In agreement, an increase in urinary sulfated bile acids has been reported in patients with various hepatobiliary diseases, which may in part reflect altered liver ATP function (Kobayashi et al., 2002; Nanashima et al., 2009; Bathena et al., 2015). Consistent with human data, the amount of sulfated BAs in cynomolgus monkey urine increased with RIF dose (Supplemental Table 3). Based on the results of the present study, it is concluded that the cynomolgus monkey can form BA sulfates that comprise only ;1% of the plasma BA pool and present as sensitive hepatic ATP biomarkers. Given the number of similarly sulfated BAs that are detectable in human serum (Bathena et al., 2013), it is envisioned that they will be increasingly studied as human ATP substrates and compared with other biomarkers such as coproporphyrin I and III. Acknowledgments The authors thank Dr. ong Shen (Bristol-Myers Squibb Co., Princeton, NJ) for providing rifamycin SV in vitro inhibition data for cynomolgus monkey ATPs and NTCP expressed in EK293 cells (Supplemental Table 5); Dana Gates and Sweta Modi (Pfizer Inc.) for help preparing the 2 4 -pitavastatin and rifampicin dose formulations; John Deschenes (Pfizer Inc.) for help conducting the cynomolgus monkey pharmacokinetics study; Sangwoo Ryu and Keith Riccardi (Pfizer Inc.) for conducting equilibrium dialysis of RIF with cynomolgus monkey plasma (Supplemental Material); and Brian Rago (Pfizer Inc.) for conducting bioanalysis of the cynomolgus monkey hepatocyte incubates. Authorship Contributions Participated in research design: Rodrigues, Gao, Varma, Tremaine, Thakare, Kosa. Conducted experiments: Thakare, Gao, Kosa, Bi, Cerny, Sharma, Walker, Niosi. Contributed new reagents and analytical procedures: Thakare, Alnouti, uang, Liu., Yu, Walker. Performed data analysis: Thakare, Varma, Bi, Kosa Kuhn, Rodrigues, Niosi. Wrote or contributed to the writing of the manuscript: Thakare, Gao, Kosa, Bi, Cerny, Sharma, Kuhn, Alnuti, Varma, Rodrigues. References Acocella G (1978) Clinical pharmacokinetics of rifampicin. Clin Pharmacokinet 3: Akita, Suzuki, Ito K, Kinoshita S, Sato N, Takikawa, and Sugiyama Y (2001) Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump. Biochim Biophys Acta 1511:7 16. Alnouti Y (2009) Bile acid sulfation: a pathway of bile acid elimination and detoxification. Toxicol Sci 108: Badolo L, Jensen B, Säll C, Norinder U, Kallunki P, and Montanari D (2015) Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, ATP1B1, CT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture. Xenobiotica 45: Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

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Clin Pharmacol Ther 91: Yoshikado T, Yoshida K, Kotani N, Nakada T, Asaumi R, Toshimoto K, Maeda K, Kusuhara, and Sugiyama Y (2016) Quantitative analyses of hepatic ATP-mediated interactions between statins and inhibitors using PBPK modeling with a parameter optimization method. Clin Pharmacol Ther 100: Zelcer N, Reid G, Wielinga P, Kuil A, van der eijden I, Schuetz JD, and Borst P (2003b) Steroid and bile acid conjugates are substrates of human multidrug-resistance protein (MRP) 4 (ATPbinding cassette C4). Biochem J 371: Zelcer N, Saeki T, Bot I, Kuil A, and Borst P (2003a) Transport of bile acids in multidrugresistance-protein 3-overexpressing cells co-transfected with the ileal Na + -dependent bile-acid transporter. Biochem J 369: Address correspondence to: Dr. A. David Rodrigues, Pharmacokinetics, Dynamics, & Metabolism, Medicine Design, Eastern Point Road, Bldg 220/002/2565, Mail Stop , Pfizer Inc., Groton, CT a. david.rodrigues@pfizer.com Downloaded from dmd.aspetjournals.org at ASPET Journals on ctober 17, 2018

14 Supplemental Information Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3--Sulfate Conjugates as Potential Novel Biomarkers for rganic Anion-Transporting Polypeptides Rhishikesh Thakare, ongying Gao, Rachel E. Kosa, Yi-An Bi, Manthena V. S. Varma, Matthew A. Cerny, Raman Sharma, Max Kuhn, Bingshou uang, Yiping Liu, Aijia Yu, Gregory S. Walker, Larry Tremaine, Yazen Alnouti and A. David Rodrigues Inhibition of GDCA and GCDCA Sulfation Catalysed by Cynomolgus Monkey Liver Cytosol. The inhibition of GDCA and GCDCA sulfation by danazol (positive control) and rifampicin was assessed after incubation with cynomolgus monkey cytosol. The incubation contained male monkey liver cytosol (1 mg/ml final concentration), 0.1 M potassium phosphate buffer containing MgCl 2 (3.3 mm), substrate (5 or 50 μm GDCA or GCDCA dissolved in DMS) and inhibitor (0 to 100 μm dissolved in MeCN). Following a 5 min pre-incubation at 37 C, reactions were initiated by addition of PAPS [3'-phosphoadenosine 5'-phosphosulfate] (0.1 mm final concentration) and maintained at 37 C for 30 min. The incubation was quenched with MeCN containing TCA-S as an internal standard. The quenched incubations were centrifuged for 5 min at 3000xg. An aliquot of supernatant (300 μl) was transferred to a microtiter 96-well plate and allowed to evaporate under nitrogen (40-60 psi for 30 minutes at 37ºC), then reconstituted with 50% methanol:plc water (120 μl) and vortexed for 1 minute prior to LC- MS/MS analysis. A high pressure LC/MS/MS system was used for monitoring GDCA, GDCA- S, GCDCA, GCDCA-S, and TCA-S (internal standard). The LC/MS/MS system is comprised of an AB Sciex 6500 triple quadrupole mass spectrometer equipped with an electrospray source AB Sciex (Framingham, MA), Agilent Technologies Infinity 1290 (Santa Clara, CA) LC using 90% acetonitrile / 10% water (containing 0.1% formic acid) as the strong needle wash solvent, 10% acetonitrile / 90% water (containing 0.1% formic acid) as the weak needle wash solvent. A binary gradient was employed with a system flow rate of 0.25 ml/min, using 7.5 mm ammonium bicarbonate in water (p 9.0) as the aqueous mobile phase and 70% methanol:30% acetonitrile 1

15 as the organic phase. The LC gradient profile begins at 52.5% organic which was ramped to 72% organic over 2.80 minutes and held 2.30 minutes then further ramped to 98% organic over 1.00 minute and held for 0.10 minutes and returned to initial conditions (52.5% organic) over 2.00 minutes, for a total run time of 8.00 minutes. The analytical column used was a Waters Acquity UPLC BE C µm, 2.1 x 100 mm (Milford, MA) with an injection volume of 10 µl. The mass spectrometer was run under negative mode condition; source temperature was set to 500ºC, ionization voltage was set to 4.5kV with the following MS/MS transitions: GDCA CE: -65 DP: -130, GDCA-S CE: -44 DP: -120, GCDCA CE: -65 DP: -130, GCDCA-S CE: -44 DP: -120, and TCA-S (m/z -2 ) CE: -35 DP: To determine the product response, peak area ratios of analyte-to-internal standard were used (Analyst software, version 1.6.2); and a signal-to-noise 3 criteria. The formation of the 3-sulfate metabolites of GCDCA and GDCA from incubations with male monkey liver cytosol was confirmed by coincidence of retention time with authentic standards by LC/MS/MS as shown below. 2

16 Measurement of Rifampicin Free Fraction in Cynomolgus Monkey Plasma. Ascorbic acid (Sigma Aldrich, St. Louis, M) was prepared in water at 100mg/ml. Monkey plasma was prepared at a p of 7.4 and pretreated with ascorbic acid (0.1 mg/ml) for 30 minutes before the binding assay (Le Guellec et al., 1997). The dialysis membranes were prepared prior to experimental setup. The cellulose membranes (MWC 12 14K) were immersed in deionized water for 15 minutes, followed by 15 minutes in 30% ethanol/deionized water, then overnight in PBS (Riccardi et al., 2015; Banker and Clark, 2008; Banker et al., 2003). The equilibrium dialysis device was assembled according to manufacturer s instructions ( A dimethylsulfoxide (DMS) stock solution of rifampicin was prepared at 200 μm pretreated with ascorbic acid (0.1 mg/ml) 30 minutes before the binding assay, added to matrices in 1:100 ratio, and mixed thoroughly with an eight-channel pipettor (Eppendorf/VWR, Radnor, PA). The final rifampicin concentration was 2 μm containing 1% DMS. A 150-μl aliquot of plasma spiked with 2 μm rifampicin was added to one side of the membrane (donor) and 150 μl of PBS was added to the other side (receiver). The equilibrium dialysis device was covered with Breathe-Easy gas-permeable membranes (Sigma-Aldrich). Equilibrium dialysis devices were placed on an orbital shaker (VWR) at 200 rpm and incubated for 6 hours in a humidified [75% relative humidity (R)] incubator at 37 C with 5% C 2 /95% 2. At the end of the incubation, 15 μl of matrix samples from the donor wells were added to a 96-well plate containing 45 μl of PBS. Aliquots of 45 μl of dialyzed PBS from the receiver wells were added to 15 μl of blank matrix. Before and after incubation, 15 μl of matrix spiked with 2 μm rifampicin was added to a 96-well plate containing 45 μl of PBS. These samples were used for the recovery calculation and stability evaluation. All the samples were quenched with cold acetonitrile (200 μl) containing internal standard (IS; a cocktail of 0.5 ng/ml tolbutamide and 5 ng/ml terfenadine). The plates were sealed and mixed with a vortex mixer (VWR) for 3 minutes, then centrifuged at 3000 rpm Allegra 6R (Beckman Coulter Life Sciences, Indianapolis, IN) at room temperature for 5 minutes. The supernatant was transferred to a new 96-well plate, dried down, reconstituted, and subsequently analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Sertraline was used as a quality control sample on every plate. 3

17 Le Guellec C, Gaudet, ML, Lamanetre S, and Breteau M (1997) Stability of rifampin in plasma: consequences for therapeutic monitoring and pharmacokinetic studies. Therap Drug Monit 19: Riccardi K, Cawley S, Yates PD, Chang C, Funk C, Niosi M, Lin J, and Di L (2015) Plasma protein binding of challenging compounds. J Pharm Sci 104: Banker MJ and Clark T (2008) Plasma/serum protein binding determinations. Curr Drug Metab 9: Banker MJ, Clark T, and Williams JA (2003) Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding. J Pharm Sci 92: Biosynthesis of GUDCA-S and TUDCA-S. The biosynthesis of the 3--sulfate metabolites of GUDCA and TUDCA was carried out using a published protocol (Walker et al., 2014). The bile acid (25 μm) was incubated with recombinant human sulfotransferase 2A1 (Panvera or Cypex, 0.3 mg/ml), 3.3 mm MgCl 2, and 0.1 mm PAPS in a total volume of 10 ml of 0.1 M potassium phosphate buffer (p 7.4). The incubation was conducted in a 50-ml polypropylene conical tube in a shaking water bath maintained at 37 C for 90 min. At the end of the incubation, 10 ml of acetonitrile was added, and the mixture was transferred to two 10 ml glass test tubes and vigorously mixed on a vortex mixer. The tubes were spun in a Jouan centrifuge at 1700g for 5 minutes, and the supernatant was transferred to a 15 ml conical glass tube. The tubes were subjected to vacuum centrifugation in a Genevac evaporator set at the PLC mixture setting for approximately 1 hour to remove the acetonitrile. To the remaining solution was added acetonitrile (60 μl), formic acid (120 μl), and water bringing the solution to a total volume of ~6 ml. This mixture was subjected to centrifugation in a Beckmann centrifuge at 40,000g for 30 minutes to clarify the supernatant. The supernatant was transferred and directly applied onto an Agilent Polaris C18-A column (4.6 x 250 mm; 5 μm particle size) through a Jasco PLC pump at a flow rate of 0.8 ml/min. The PLC column was then transferred to a Thermo rbitrap Elite PLC-UV-MS system with the mass spectrometer operated in the negative ion mode. The effluent was split between the mass spectrometer and a Gilson fraction collector at a ratio of approximately 1:15. The mobile phase used consisted of 10 mm ammonium acetate p 7.0 (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 1.0 ml/min. The mobile phase composition commenced at 95% A/5% B, was held at that composition for 2 minutes, 4

18 followed by a linear gradient to 40% A/60% B at 25 min, then increased to 5% A/95% B over 1 min and held at this composition for 1 min, then re-equilibrated at initial conditions for 2 min. The mobile phase program and data collection were started by making a dummy injection of water from the autosampler. Fractions were collected every 30 sec into a 96-well block containing 1 ml polypropylene inserts. Fractions collected in the region of interest (i.e., where the mass spectrometer showed the presence of m/z 528 or 578, the deprotonated molecular ion of the sulfate metabolites of GUDCA-S and TUDCA-S, respectively) were injected (5 μl) onto an Acquity UPLC BE C18 column. The mobile phase used consisted of ammonium acetate (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.4 ml/min. The mobile phase composition commenced at 95% A/5% B, was held at that composition for 0.75 min, followed by a linear gradient to 40% A/60% B at 2.75 min, then increase to 5% A/95% B over 0.01 minute and held at this composition for 0.3 minute, then re-equilibrated at initial conditions for 1 min. Those fractions containing the product of interest were combined into a 15 ml conical glass tube, and the solvent was evaporated by vacuum centrifugation in the Genevac evaporator. Upon dryness, samples were prepared for NMR analysis as described below. Synthesis of 2 4 -TDCA-S, and 2 4 -DCA-S. The deuterated bile acid (0.01 mmol), sulfur trioxide-triethylamine (0.03 mmol), and pyridine (1 ml) were combined allowed to stir at room temperature with periodic monitoring by LC/MS. After ~72 hr, solvent was removed under reduced pressure. The resulting residue was dissolved in 2 ml of 10 mm aqueous ammonium acetate and loaded onto a SPE cartridge (Isolute, 500 mg, C18 (EC), 6 ml reservoir volume, part No C, Lot No FA), and the eluant collected by suction taking caution to not let the column run dry. Material was eluted from the cartridge using 0-20% MeCN in 10 mm ammonium acetate in steps of 5% MeCN (5 x 10 ml fractions per step), and fractions were analyzed by LC/MS. Those fractions containing the product of interest were combined into a 15 ml conical glass tube, and the solvent was evaporated by vacuum centrifugation in the Genevac evaporator. Upon dryness, samples were prepared for NMR analysis as described below. NMR Analyses. All samples, including parent compounds, were prepared for NMR analysis as follows. Tubes containing the dried products were transferred to a vacuum chamber which was attached to a Vacuum Atmospheres Nexus II dry box. The samples were left under vacuum (230 5

19 psi) for a minimum of 1 h before entry to the glove box. The glove box was maintained at slightly positive pressure under 100% argon. All samples were dissolved in the highest available purity deuterated DMS (typically 100% grade DMS-d6 which is 99.96% deuterium enriched) using a minimum amount of solvent to achieve proper sample height in the NMR tube. This volume is 40 µl for samples prepared for 1.7 mm tubes and 18 µl for 1.0 mm tubes. After addition of the solvent, samples were vortexed for 15 to 30 seconds, transferred to NMR tubes, capped, and removed from the glove box. All transfer pipettes, pipette tips, and NMR tubes were stored in the glove box to remove adsorbed water from their surfaces to minimize the potential for sample contamination. NMR spectra were recorded on a Bruker Avance 600 Mz instrument controlled by Topspin V3.2 and equipped with a 1.7 mm TCI Cryo probe. The one-dimensional spectra were recorded using an approximate sweep width of 8400 z and a total recycle time of 7 seconds. The resulting time-averaged free induction decays were transformed using an exponential line broadening of 1.0 z to enhance signal to noise. The two-dimensional data were recorded using the standard pulse sequences provided by Bruker. At a minimum a 1K 128 data matrix was acquired using a minimum of 2 scans and 16 dummy scans, with a spectral width of 10,000 z in the f2 dimension. In some cases the data were zero-filled to at least 1K data point. 1 and 13 C spectra were referenced using residual DMS-d6 ( 1 δ = 2.50 relative to tetramethylsilane, δ = 0.00, 13 C δ = relative to tetramethylsilane, δ = 0.00). Quantitative NMR (qnmr) was performed using an external standard of benzoic acid (Walker et al., 2014). In the 1-13 C multiplicity edited SQC of GUDCA the observed δ 1 / 13 C values for the aliphatic methines at positions 3 and 7 were at ( 1 / 13 C) δ3.30/69.34 and δ3.33/69.59, Supplementary Figure NMR 1. In a similarly acquired 1-13 C multiplicity edited SQC spectra for GUDCA-Sulfate the chemical shifts of one of these methines shifted to 1 / 13 C δ3.90/75.11 while the other remained unchanged, Supplementary Figure NMR 1. The predicted 13 C δ value (ACD Labs 2015) for a methine adjacent to a sulfated alcohol on either the 3 or 7 positions is approximately 78 +/- 4 ppm. Therefore the new methine with a 13 C δ75.11 in GUDCA-S was identified as the position adjacent to the sulfated alcohol. Determination of which alcohol was sulfated (either the 3 or 7 position) was made using a combination of the 1-1 CSY and 1-6

20 13 C MBC correlations shown below, Supplementary Figures NMR 2 and 3. Notably correlations in the 1-13 C MBC spectra between positions 4 3, 1 3, 7 8, 6 8 and 6 7 and cross peaks between positions 4 3 in the 1-1 CSY spectra established the 3 alcohol as the site of sulfation. Walker GS, Bauman JN, Ryder TF, Smith EB, Spracklin DK, and bach RS (2014). Biosynthesis of drug metabolites and quantitation using NMR spectroscopy for use in pharmacological and drug metabolism studies. Drug Metab Dispos 42:

21 Supplementary Figure NMR C multiplicity edited SQC of GUDCA and GUDCA-Sulfate 3 and GUDCA GUDCA-Sulfate S 4 C C 27 C N C 3 and 7 C 7 C 3 8

22 Supplementary Figure NMR CSY of GUDCA-Sulfate GUDCA-Sulfate S C C 27 C N

23 Supplementary Figure NMR C MBC of GUDCA-Sulfate GUDCA-Sulfate C8 2 3 S C C 27 C N C8 7 C7 4 C3 1 C3 6 C7 Chemical Synthesis of GCA-S, TCA-S, CA-S, TCDCA-S, GDCA-S, TDCA-S, and DCA-S Follows. 10

24 Synthesis of Glyocholic Acid 3--Sulfate (GCA-S) Scheme Ac2, DMAP pyridine 95% N 2 ATU, DMF 75% N K2C3, Me N PyS3 63.4% pyridine crude S 5 1. General procedure for preparation of compound 2 N aq. K Et 47% N S GCSWID# Glycocholic acid 3--sulfate 20 mg Ac2, DMAP pyridine 1 2 To a suspension of compound 1 (2.0 g; mmol) in acetic anhydride (4 ml), pyridine (6 ml) and DMAP (359 mg; 2.94 mmol) were added. The reaction mixture was stirred for 3.5 h at 20 o C. The solvent was removed under reduced pressure, diluted with MTBE (40 ml) and washed with 1 M Cl (3x30 ml), 5% NaC 3 solution (2x30 ml), and brine (30 ml). The organic layer was dried over Na 2 S 4, filtered, and concentrated to obtain compound 2 (2.5 g; 95.5%) as a white solid. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 4.91 (m, 1, C7-), 4.58 (m, 1, C3-), 2.14 (s, 3, -CC3), 2.09 (s, 3, -CC3), 2.04 (s, 3, -CC3), 0.92 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.73 (s, 3, 18-Me). 2. General procedure for preparation of compound 3 11

25 N 2 ATU, DMF N 2 3 To a solution of compound 2 (300 mg, mmol) in DMF (10 ml), DIPEA (218 mg, 1.68 mmol) and ATU (320 mg, mmol) was added. The resulting mixture was stirred at 20 o C for 30 minutes. Ethyl 2-aminoacetate (117 mg, mmol, Cl salt) was added to the above mixture and stirred at 20 o C for 16 h. TLC (PE/EA=1:1, stained by PMA) showed compound 2 was consumed, and a new main spot was detected. 2 (50 ml) was added and extracted with ethyl acetate (30 ml). The organic layer was washed with brine (30 ml), dried over Na 2 S 4, filtered, concentrated and purified by Biotage (12 g silicon column, eluted with EA/PE from 10-55%) to give compound 3 (0.26 g, 74.8%) as a white solid, which will be used in next step without further purification. NMR (400Mz, CDCl 3 ) δ (ppm): 5.94 (t, 1, N), 5.09 (m, 1, C12-), 4.90 (m, 1, C7- ), 4.58 (m, 1, C3-), 4.22 (q, 2, -C2C3), 4.03 (t, 2, -NC2C), 2.13 (s, 3, - CC3), 2.08 (s, 3, -CC3), 2.04 (s, 3, -CC3), 1.29 (t, 3, -C2C3), 0.92 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.73 (s, 3, 18-Me). 3. General procedure for preparation of compound 4 N K2C3, Me N 3 4 To a solution of compound 3 (0.26 g, mmol) in Me (6 ml), K 2 C 3 (116 mg, mmol) was added and stirred at 20 o C for 2 hours. TLC (pure EA, stained by PMA) showed that compound 3 was consumed, and a new main spot was detected. C 3 C (0.3 ml) was added and after liberation of C 2 resulting solution was evaporated under reduced pressure, then 20 ml 2 was added, extracted with ethyl acetate (3x25 ml), dried over Na 2 S 4, filtered, and concentrated to give crude product 4 (150 mg, 63.4%) as a light yellow solid. NMR showed 12

26 that the acetyl was hydrolyzed, but that the ethyl ester had exchanged to the methyl ester. NMR (400Mz, CDCl 3 ) δ (ppm): 5.92 (m, 1, N), 5.09 (m, 1, C12-), 4.90 (m, 1, C7- ), 4.05 (d, 2, -NC2C), 3.77 (s, 3, CC3), 3.50 (m, 1, C3-), 2.13 (s, 3, - CC3), 2.09 (s, 3, -CC3), 0.91 (s, 3, 19-Me), 0.83 (d, 3, 21-Me), 0.73 (s, 3, 18- Me). 4. General procedure for preparation of compound 5 N N PyS3 pyridine S 5 4 To a solution of compound 4 (150 mg, mmol) in pyridine (4 ml), Py-S 3 complex (127 mg, mmol) was added. The mixture was stirred at 20 o C for 4 h. TLC (DCM/Me=10:1, stained by PMA) showed compound 4 was consumed, and a new main spot was detected. The solvent was evaporated under the reduce pressure to remove most of pyridine. Water (10 ml) was added to dissolve the solid powder, and extracted with ethyl acetate (2x15 ml), dried over Na 2 S 4, filtered, and concentrated to give crude product 5 (150 mg, crude) as a white gum. NMR showed it was a pyridine salt, which was used for the next step without further purification. NMR (400Mz, CDCl 3 ) δ (ppm): 8.70 (m, 8, pyridine), 7.87 (m, 4, pyridine), 7.45 (m, 8, pyridine), 5.96 (m, 1, N), 5.5 (br, 4, S3), 5.08 (m, 1, C12-), 4.89 (m, 1, C7-), 4.32 (m, 1, C3-), 4.04 (d, 2, -NC2C), 3.76 (s, 3, CC3), 2.13 (s, 3, -CC3), 2.08 (s, 3, -CC3), 0.90 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.72 (s, 3, 18-Me). 5. General procedure for preparation of glycocholic acid 3--sulfate S 5 N aq. K Et N S Glycocholic GCSWID# acid 3--sulfate 13

27 To a solution of compound 5 (100 mg, mmol, crude) in Et (2 ml), 0.5 ml 2 and K (87.2 mg, 1.55 mmol) was added. The solution was stirred at 50 o C for 16 h. LCMS showed material 5 was consumed, and desire product was confirmed (Rt=0.926, M-=544). The p of reaction mixture was adjusted with concentrated Cl (35%, 0.1 ml), and then the mixture was purified by preparative PLC (pre-plc). The fraction from the prep-plc was evaporated to dryness by lyophilization and glycocholic acid 3--sulfate (39.8 mg, 47%) was obtained as white solid. Prep-PLC: Column: Agla DuraShell 150mm*25mm*5um; Mobile phase: water (0.05%N4 v/v)/mecn, from 10% to 40%; Flow rate: 25ml/minute; Detection: MS (negative) orient. NMR (400Mz, MeD) δ (ppm): 4.14 (m, 1, C3-), 3.95 (m, 1, C12-), 3.79 (m, 1, C7-), 3.76 (d, 2, -NC2C), 1.04 (d, 3, 21-Me), 0.92 (s, 3, 19-Me), 0.69 (s, 3, 18- Me). LCMS (negative, ELSD): Rt=0.907, M-=544.2, (M/2-). 14

28 Synthesis of Taurocholic Acid 3--Sulfate (TCA-S) Scheme N Ac2, DMAP 2 S 3 pyridine ATU, DMF 95% crude N S 3 K2C3, Me N PyS3 64% S 3 pyridine crude S General procedure for preparation of compound 2 N S 3 aq. K 48% N S S 3 GCSWID# Taurocholic acid 3--sulfate 20 mg Ac2, DMAP pyridine 1 2 To a suspension of compound 1 (2.0 g; mmol) in acetic anhydride (4 ml), pyridine (6 ml) and DMAP (359 mg; 2.94 mmol) was added at 20ºC. The reaction mixture was stirred for 3.5 h at 20 o C. The solvent was removed under reduced pressure, diluted with MTBE (40 ml) and washed with 1 M Cl (3x30 ml), 5% NaC 3 solution (2x30 ml), and brine (30 ml). The organic layer was dried over Na 2 S 4, filtered, concentrated to obtain compound 2 (2.5 g; 95.5%) as a white solid. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 4.91 (m, 1, C7-), 4.58 (m, 1, C3-), 2.14 (s, 3, -CC3), 2.09 (s, 3, -CC3), 2.04 (s, 3, -CC3), 0.92 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.73 (s, 3, 18-Me). 2. General procedure for preparation of compound 3 15

29 N 2 S 3 ATU, DMF N S To a solution of compound 2 (1250 mg, mmol) in DMF (15 ml), DIPEA (906 mg, 7.01 mmol) and then ATU (1330 mg, 3.51 mmol) was added. The mixture was stirred at 20 o C for 30 minutes. Taurine (439 mg, 3.51 mmol) was added and stirred at 20 o C for 16 h. TLC (DCM/Me=4:1, stained by PMA) showed compound 2 was consumed and a new main spot was detected. LCMS showed compound 2 was consumed, and the desire product was confirmed (Rt=1.033, M-=640) as the main peak. DMF was removed under oil pump, water (30 ml) was added, and then the mixture was extracted with ethyl acetate (2x30 ml). The organic layers were dried over Na 2 S 4, concentrated and purified by Biotage (12 g silicon column. eluted with DCM/Me from 0-40%) to give compound 3 (1700 mg, crude) as a yellow solid. The product will be used in the next step without further purification. NMR (400Mz, MeD) δ (ppm): 5.06 (m, 1, C12-), 4.89 (m, 1, C7-), 4.55 (m, 1, C3-), 3.56 (t, 2, -NC2C2S3), 2.93 (t, 2, -NC2C2S3), 2.08 (s, 3, -CC3), 2.06 (s, 3, -CC3), 2.02 (s, 3, -CC3), 0.94 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.75 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.966, M-= General procedure for preparation of compound 4 N S 3 K2C3, Me N S To a solution of compound 3 (850 mg, 1.32 mmol) in Me (10 ml), K 2 C 3 (366 mg, 2.65 mmol) was added and stirred at 20 o C for 16 h. The suspension turned to a clear solution. LCMS showed compound 3 was consumed, and the desire product was confirmed (Rt=0.958, M- 16

30 =598) as the main peak. C 3 C (0.3 ml) was added to the solution to neutralize the K 2 C 3. The solution was concentrated to give a crude product which was purified by Biotage (4 g silica gel, eluted with from DCM to DCM/Me 1:1) to give compound 4 (350 mg, 44.1%) as a white solid, which was used in the next step. NMR (400Mz, MeD) δ (ppm): 5.08 (m, 1, C12-), 4.89 (m, 1, C7-), 3.40 (m, 1, C3-), 3.57 (t, 2, -NC2C2S3), 2.95 (t, 2, -NC2C2S3), 2.09 (s, 3, -CC3), 2.06 (s, 3, -CC3), 0.95 (s, 3, 19-Me), 0.83 (d, 3, 21-Me), 0.78 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.997, M-= General procedure for preparation of compound 5 4 N S 3 PyS3 pyridine S 5 N S 3 In a 50 ml flask, pyridine (5 ml), compound 4 (350 mg, mmol) and Py-S 3 complex (279 mg, 1.75 mmol) was added. The resulting mixture was stirred at 20 o C for 16 h. LCMS showed the desired product was obtained (Rt=0.954, M-=678; M/2-=338.7) along with unreacted compound 4 (Rt=0.954, M-=598). Additional PyS 3 complex (279 mg, 1.75 mmol) was added to the suspension and stirred at 20 o C for additional 16 h. LCMS showed no change in the reaction mixture. TLC (DCM/Me=4:1, stained by PMA) showed a new main spot, and the unreacted compound 4. DCM (15 ml) was added and white solid was precipitated. The suspension was filtered to remove the white solid, and the filtrate was concentrated to give the compound 5 (600 mg, crude) as a colorless oil, which was used in next step without further purification. 5. General procedure for preparation of taurocholic acid 3--sulfate 17

31 N N aq. K S S 3 S S 3 Taurocholic GCSWID# acid 3--sulfate 5 20 mg To a solution of compound 5 (500 mg, 0.44 mmol, crude) in Et (5 ml), 2 (1 ml) and K (248 mg, 4.41 mmol) was added. The resulting solution was stirred at 50 o C for 16 h. LCMS analysis showed desired product along with unreacted compound 5. The reaction mixture was further stirred at 80 o C for 16 h and LCMS analysis showed compound 5 was consumed, and the desire product was confirmed as the main peak (Rt=0.874, M/2-=297). The mixture was concentrated to remove the solvent, and then dissolved in 2 /Me (4/2 ml), purified by prep- PLC. The fraction from the prep-plc was evaporated to dryness by lyophilization. Taurocholic acid 3--sulfate (125 mg, 48%) was obtained as white solid. Prep-PLC: Column: Agla DuraShell 150mm*25mm*5um; Mobile phase: water (0.225%FA v/v)/mecn, from 5% to 95%; Flow rate: 35ml/minute; Detection: MS (negative) orient. NMR (400Mz, MeD) δ (ppm): 4.13 (m, 1, C3-), 3.94 (m, 1, C12-), 3.79 (m, 1, C7-), 3.58 (t, 2, -NC2C2S3), 2.96 (t, 2, -NC2C2S3), 1.02 (d, 3, 21-Me), 0.92 (s, 3, 19-Me), 0.71 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.906, M-=594.2, (M/2-). 18

32 Synthesis of Cholic Acid 3--Sulfate (CA-S) Scheme TMSCN2 toluene/me 100% Ac2, DMAP pyridine 98% K2C3/Me 85% Py-S3 aq. K pyridine Et crude S S 26% GCSWID# mg 1. General procedure for preparation of compound 2 TMSCN2 toluene/me 1 2 To a solution of compound 1 (2.0 g, 4.9 mmol) in PhC 3 /Me (30 ml, V/V=2:1), TMSCN 2 (3.67 ml, 7.34 mmol) was added and stirred at 20 o C for 2 h. TLC (Me/DCM= 1:10) showed compound 1 was consumed and a new spot was detected. The mixture was concentrated to give compound 2 (2.08 g, 100%) as a white foam solid. NMR (400Mz, CDCl 3 ) δ (ppm): 3.98 (m, 1, C12-), 3.85 (m, 1, C7-), 3.45 (m, 1, C3-), 3.66 (s, 3, C3), 0.98 (d, 3, 21-Me), 0.89 (s, 3, 19-Me), 0.69 (s, 3, 18-Me). 2. General procedure for preparation of compound 3 19

33 Ac2, DMAP pyridine 2 3 To a solution of compound 2 (1.1 g, 2.6 mmol) in pyridine (6 ml), acetic anhydride (4 ml) and then DMAP (191 mg, 1.56 mmol) was added. The mixture was stirred at 20 o C for 3 h. TLC (DCM/Me=10:1, PE/EA=1:1, stained by PMA) showed compound 2 was consumed. The mixture was concentrated to give a residue, and then dissolved in 50 ml MTBE, washed with 0.14 M Cl (2x20 ml), 0.5 M NaC 3 (2x20 ml), saturated NaCl (30 ml), dried over Na 2 S 4, filtered, concentrated to give compound 3 (1.4 g, 98%) as a white solid. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 5.07 (m, 1, C7-), 4.58 (m, 1, C3-), 3.65 (s, 3, C3), 2.13 (s, 3, -CC3), 2.08 (s, 3, -CC3), 2.04 (s, 3, - CC3), 0.91 (s, 3, 19-Me), 0.81 (d, 3, 21-Me), 0.72 (s, 3, 18-Me). 3. General procedure for preparation of compound 4 K2C3/Me 3 4 To a solution of compound 3 (1.4 g, 2.6 mmol) in Me (20 ml), K 2 C 3 (705 mg, 5.10 mmol) was added and the mixture was stirred at 20 o C for 4 h. TLC (PE/EA=1:1) showed compound 3 was consumed. C 3 C (0.3 ml) was added to neutralize the K 2 C 3. After C 2 was liberated, the resulting solution was evaporated under reduced pressure. 2 (20 ml) was added, and then extracted with ethyl acetate (3x25 ml). The combined organic layers were dried over Na 2 S 4, filtered, and concentrated to give compound 4 (1.1 g, 85%) as a white solid. NMR (400Mz, CDCl 3 ) δ (ppm): 5.07 (m, 1, C12-), 4.89 (m, 1, C7-), 3.48 (m, 1, 20

34 C3-), 3.65 (s, 3, C3), 2.12 (s, 3, -CC3), 2.08 (s, 3, -CC3), 0.90 (s, 3, 19- Me), 0.80 (d, 3, 21-Me), 0.72 (s, 3, 18-Me). 4. General procedure for preparation of compound 5 PyS3 pyridine S 4 5 To a 50 ml flask, 5 ml pyridine, compound 4 (300 mg, mmol) and Py-S3 complex (283 mg, 1.78 mmol) was added. The mixture was stirred at 20 ºC for 3 h. TLC (DCM/Me=10:1, stained by PMA) showed the compound 4 was consumed, and a new main spot was detected. DCM and water (10 ml/15 ml) was added, and a white solid was formed. The mixture was extracted with DCM (2x15 ml), dried over Na 2 S 4, filtered, and concentrated to give crude compound 5 (400 mg) as a white gum, which was used for the next step without further purification. 5. General procedure for preparation of cholic acid 3--sulfate S aq. Na Et S 5 Cholic acid 3--sulfate GCSWID# To a solution of compound 5 (400 mg, mmol, crude) in Et (5 ml), 2 (2 ml) and K (382 mg, 6.82 mmol) was added. The solution was stirred at 80 o C for 16 h. LCMS showed the compound 5 was consumed, and the desired product was confirmed (Rt=0.918, M-=487) as the 21

35 main peak. About 80% of mixture was purified by prep-plc. The fraction from the prep- PLC was evaporated to dryness via lyophilization. Cholic acid 3--sulfate (69.5 mg, 26%) was obtained as white solid. Prep-PLC: Column: YMC-Actus DS-AQ 150mm*30mm*5um; Mobile phase: water (0.1%TFA)-ACN, from 0% to 40%; Flow rate: 40 ml/minute; Detection: MS (negative) orient. NMR (400Mz, MeD) δ (ppm): 4.16 (m, 1, C3-), 3.95 (m, 1, C12-), 3.79 (m, 1, C7-), 1.00 (d, 3, 21-Me), 0.92 (s, 3, 19-Me), 0.71 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.874, M-=487.1, (M/2-). 22

36 Synthesis of Taurochenodeoxycholic Acid 3--Sulfate (TCDCA-S) Scheme 1 Ac2, DMAP pyridine 87.3% N 2 ATU DMF crude S N S 3 K2C3/Me 85% N PyS3 N aq. K S 3 crude S S 3 48% General procedure for preparation of compound 2 N S S 3 Taurochenodeoxycholic GCSWID# acid 3--sulfate 20 mg mg completed Ac2, DMAP pyridine 1 2 To a cooled suspension of compound 1 (2.0 g; 5.09 mmol) in acetic anhydride (4 ml), pyridine (6 ml) and DMAP (373 mg; 3.06 mmol) were added. The reaction mixture was then stirred for 3.5 h at 25ºC in an oil bath. The solvent was removed under reduced pressure, then diluted with MTBE (40 ml) and washed with 0.15 M Cl (3x30 ml). The organic layer was dried over Na 2 S 4, filtered, and concentrated to obtain compound 2 (2.12 g; 87.3%) as white foam. NMR (400Mz, CDCl 3 ) δ (ppm): 4.88 (m, 1, C7-), 4.61 (m, 1, C3-), 2.05 (s, 3, - CC3), 2.04 (s, 3, -CC3), 0.93 (s, 3, 19-Me), 0.94 (d, 3, 21-Me), 0.65 (s, 3, 18- Me). 2. General procedure for preparation of compound 3 23

37 N 2 ATU DMF S 3 N S To a solution of compound 2 (600 mg, 1.26 mmol), in DMF (15 ml), was added DIPEA (488 mg, 3.78 mmol) and then ATU (718 mg, 1.89 mmol). The mixture was stirred at 20 o C for 60 minutes. Taurine (236 mg, 1.89 mmol) was added and stirred at 20 o C for 16 h. LCMS showed compound 2 was consumed. DMF was removed under oil pump. Water (15 ml) and ethyl acetate (10 ml) were added, the p adjusted to 1 with 1N Cl, and the mixture extracted with ethyl acetate (2x15 ml). The organic layers were dried over Na 2 S 4, filtered, concentrated, and purified by Biotage (12 g silicon column, eluted with DCM/Me from 0-40%) to give compound 3 (800 mg, crude, yield>100%) as a yellow solid. NMR of the product was consistent with its structure, and some ATU was incorporated. LCMS showed the desired MS. The product was used in the next step directly without further purification. NMR (400Mz, MeD) δ (ppm): 4.88 (m, 1, C7-), 4.55 (m, 1, C3-), 3.58 (t, 2, - NC2C2S3), 2.95 (t, 2, -NC2C2S3), 2.05 (s, 3, -CC3), 2.02 (s, 3, - CC3), 0.96 (s, 3, 19-Me), 0.96 (d, 3, 21-Me), 0.70 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=1.038, M-= General procedure for preparation of compound 4 N S 3 K2C3/Me N S To a solution of compound 3 (850 mg, 1.46 mmol) in Me (10 ml), K 2 C 3 (402 mg, 2.91 mmol) was added and stirred 20 o C for 16 h. LCMS showed compound 3 was consumed, and the desired product was confirmed (Rt=0.978, M-=540) as the main peak. C 3 C (1 ml) was 24

38 added to neutralize the K 2 C 3. The solution was concentrated to give a crude product that was purified by Biotage (12 g silica gel, eluted from ethyl acetate to ethyl acetate/me 1:1) to give compound 4 (670 mg, 85%) as a white solid. NMR (400Mz, MeD) δ (ppm): 4.88 (m, 1, C7-), 3.34 (m, 1, C3-), 3.57 (t, 2, - NC2C2S3), 2.95 (t, 2, -NC2C2S3), 2.04 (s, 3, -CC3), 0.95 (s, 3, 19- Me), 0.96 (d, 3, 21-Me), 0.70 (s, 3, 18-Me). 4. General procedure for preparation of compound 5 N S 3 PyS3 S N S To a 50 ml flask, pyridine (8 ml), compound 4 (470 mg, mmol) and Py-S3 complex (690 mg, 4.34 mmol) was added. The mixture was stirred at 20 o C for 16 h. LCMS showed the desire product was formed (Rt=0.877, M-=620; M/2-=310). Some unreacted compound 4 was also detected in the same peak (Rt=0.877, M-=540). The suspension was diluted with DCM (15 ml), and then filtered off the white solid, the filtrate was concentrated to give the crude product 5 (620 mg, crude) as a colorless oil, which was used in the next step. 5. General procedure for preparation of taurochenodeoxycholic acid 3--sulfate S N S 3 aq. K S N S 3 5 Taurochenodeoxycholic acid GCSWID# sulfate 25

39 To a solution of compound 5 (620 mg, 0.80 mmol, crude) in Et (8 ml), 2 (2 ml) and K (448 mg, 7.98 mmol) was added and the solution was stirred at 80 o C for 16 h. LCMS showed compound 5 was consumed, and the desired product was confirmed as the main peak (Rt=0.909, M/2-=289). The mixture was concentrated to remove the solvent, and then dissolved in 2 /Me (4/2 ml) and purified by prep-plc. The fraction from prep-plc was evaporated to dryness by lyophilization. Taurochenodeoxycholic acid 3--sulfate (220.5 mg, 48%) was obtained as white solid. NMR (400Mz, MeD) δ (ppm): 4.13 (m, 1, C3-), 3.79 (m, 1, C7-), 3.58 (t, 2, - NC2C2S3), 2.96 (t, 2, -NC2C2S3), 0.97 (d, 3, 21-Me), 0.94 (s, 3, 19-Me), 0.69 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.917, M-=578.2, (M/2-). 26

40 Synthesis of Glycodeoxycholic Acid 3--Sulfate (GDCA-S) Scheme 2 N Ac2, DMAP pyridine ATU DMF 96.1% 71.3% N K2C3/Me crude N PyS3 N aq. K crude S 43% N S Glycodeoxycholic GCSWID# acid 3--sulfate 20 mg 1. General procedure for preparation of compound 2 Ac2, DMAP pyridine 1 2 To a solution of compound 1 (6.0 g; mmol) in acetic anhydride (12 ml), pyridine (18 ml) and DMAP (1120 mg; 9.17 mmol) were added. The reaction mixture was stirred at 20ºC for 3.5 h. TLC (ethyl acetate, stained by PMA) showed compound 1 was consumed, and new main spot was detected. The solvent was removed under reduced pressure, and then diluted with MTBE (40 ml) and washed with 1 M Cl (3x30 ml), 5% NaC3 solution (2x30 ml), and brine (30 ml). The organic layer was dried over Na 2 S 4, concentrated to obtain compound 2 (7.0 g; 96.1%) as a light yellow solid powder. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 4.70 (m, 1, C3-), 2.10 (s, 3, - CC3), 2.04 (s, 3, -CC3), 0.91 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.73 (s, 3, 18-27

41 Me). 2. General procedure for preparation of compound 3 2 N ATU DMF N 2 3 To a solution of compound 2 (1000 mg, mmol) in DMF (15 ml) was added DIPEA (813 mg, 6.29 mmol) and then ATU (1200 mg, 3.15 mmol). The mixture was stirred at 25 o C for 30 minutes, ethyl 2-aminoacetate (439 mg, 3.15 mmol, Cl salt) was added. The solution was stirred at 25 o C for 3 h. TLC (PE/EA=1:1, stained by PMA) showed the reaction was complete. DMF was removed under oil pump. 2 (30 ml) was added and extracted with ethyl acetate (30 mlx3). The combined organic layers were washed with brine (30 ml), dried over Na 2 S 4, filtered, concentrated and purified by Biotage (12 g silicon column. eluted with EA in PE from 10-55%) to give compound 3 (840 mg, 71.3%) as a white solid. 3. General procedure for preparation of compound 4 N K2C3/Me N 3 4 To a solution of compound 3 (840 mg, 1.50 mmol) in Me (15 ml), K 2 C 3 (413 mg, 2.99 mmol) was added and stirred at 20 o C for 2 hours. TLC (PE/EA=1:1, stained by PMA) showed the reaction was complete. C 3 C (0.5 ml) was added to the solution to neutralize the K 2 C 3. The solution was concentrated, water (15 ml) added, and extracted with ethyl acetate (2x15 ml). The organic layers were dried over Na 2 S 4 and concentrated to give compound 4 28

42 (840 mg, crude, yield >100%) as a white solid. NMR (400Mz, CDCl 3 ) δ (ppm): 6.01 (br, 1, N), 5.08 (m, 1, C12-), 3.62 (m, 1, C3- ), 4.05 (d, 2, -NC2C), 3.77 (s, 3, CC3), 2.09 (s, 3, -CC3), 0.89 (s, 3, 19- Me), 0.81 (d, 3, 21-Me), 0.72 (s, 3, 18-Me). 4. General procedure for preparation of compound 5 N PyS3 S N 4 5 To a 50 ml flask, pyridine (10 ml), compound 4 (500 mg, mmol) and Py-S 3 complex (472 mg, 2.97 mmol) was added. The mixture was stirred at 20 o C for 16 h. LCMS analysis showed compound 4 was consumed, and the desired product was obtained as the main peak (Rt=1.014, M-=584). DCM (20 ml) was added and the white solid was filtered off. The filtrate was concentrated to give compound 5 (780 mg, crude, yield>100%) as an oil, which was used in next step without further purification. 5. General procedure for preparation of glycodeoxycholic acid 3--sulfate S N aq. K S N Glycodeoxycholic acid 3--sulfate 5 GCSWID# To a solution of compound 5 (400 mg, 0.48 mmol, crude) in Et (6 ml), 2 (1 ml) and K (268 mg, 4.78 mmol) was added. The solution was stirred at 80 o C for 16 h. LCMS showed compound 5 was consumed, and the desired product was formed (Rt=0.906, M-=528). The reaction mixture was concentrated to give the crude product, which was dissolved in Me/ 2 (4/2 ml) and purified by prep-plc. The fraction from the prep-plc was evaporated to 29

43 dryness via lyophilization to give glycodeoxycholic acid 3--sulfate (110 mg, 43%) as a white solid. Prep-PLC: Column: CC YMC-Actus Pro C18 150mm*30mm*5um; Mobile phase: water (0.05%N4 v/v)/mecn, from 5% to 95%; Flow rate: 35ml/minute; Detection: MS (negative) orient. NMR (400Mz, MeD) δ (ppm): 4.28 (m, 1, C3-), 3.95 (m, 1, C12-), 3.76 (s, 2, - NC2C), 1.02 (d, 3, 21-Me), 0.94 (s, 3, 19-Me), 0.71 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.900, M-=528.2, (M/2-1) 30

44 Synthesis of Taurodeoxycholic Acid 3--Sulfate (TDCA-S) Scheme Ac2, DMAP pyridine 96.1% N 2 S 3 ATU, DMF 76.2% N S K2C3, Me crude N S 3 PyS3 pyridine crude S 5 N S 3 aq. K 34% N S S 3 GCSWID# Taurodeoxycholic acid 3--sulfate 20 mg 1. General procedure for preparation of compound mg completed Ac2, DMAP pyridine 1 2 To a solution of compound 1 (6.0 g; mmol) in acetic anhydride (12 ml), pyridine (18 ml) and DMAP (1120 mg; 9.17 mmol) were added. The reaction mixture was stirred at 20 o C for 3.5 h. TLC (ethyl acetate, stained by PMA) showed compound 1 was consumed and a new main spot was obtained. The solvent was removed under reduced pressure, and then diluted with MTBE (40 ml) and wash with 1 M Cl (3x30 ml), 5% NaC3 solution (2x30 ml), and brine (30 ml). The organic layer was dried over Na 2 S 4, filtered, and concentrated to obtain compound 2 (7.0 g; 96.1%) as a light yellow solid. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 4.70 (m, 1, C3-), 2.10 (s, 3, - CC3), 2.04 (s, 3, -CC3), 0.91 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.73 (s, 3, 18- Me). 31

45 2. General procedure for preparation of compound 3 N 2 S 3 ATU, DMF N S To a solution of compound 2 (1500 mg, mmol), in DMF (20 ml), was added DIPEA (1220 mg, 9.44 mmol) and then ATU (1790 mg, 4.72 mmol). The mixture was stirred at 20ºC for 30 minutes, and then taurine (591 mg, 4.72 mmol) was added. The solution was stirred at 20ºC for 16 h. LCMS analysis showed compound 2 was consumed, and the desire product was confirmed as the main peak (Rt=1.038, M-=582.2). DMF was removed under oil pump. Water (15 ml) was added, the p of the solution adjusted to 1 with 1 N Cl, followed by extraction with ethyl acetate (3x30 ml). The organic layers were dried over Na 2 S 4, filtered, concentrated and purified by Biotage (12 g silica column, eluted from ethyl acetate to EA/Me 1:1) to give compound 3 (1.4 g, 76.2%) as a light-yellow solid. Compound 3 was used to the next step without further purification. NMR (400Mz, MeD) δ (ppm): 5.07 (m, 1, C12-), 4.67 (m, 1, C3-), 3.58 (t, 2, - NC2C2S3), 2.95 (t, 2, -NC2C2S3), 2.09 (s, 3, -CC3), 2.01 (s, 3, - CC3), 0.95 (s, 3, 19-Me), 0.84 (d, 3, 21-Me), 0.78 (s, 3, 18-Me). 3. General procedure for preparation of compound 4 N S 3 K2C3, Me N S To a solution of compound 3 (1400 mg, mmol) in Me (15 ml), K 2 C 3 (663 mg, 4.80 mmol) was added and stirred 20ºC for 16 h. The reaction was heated to 60ºC and stirred for 16 h. LCMS analysis showed compound 3 was consumed, and the desire product was confirmed 32

46 (Rt=1.002, M-=540) as the main peak. C 3 C (1 ml) was added to the solution to neutralize the K 2 C 3. The solution was concentrated to give a residue. DCM (30 ml) was added to dissolve the residue, and the undissolved mass was filtered off. The filtrate was concentrated to give the crude product (3.5 g), which was purified by Biotage (12 g silica gel, eluted from DCM to DCM/ME 1:1) to give compound 4 (1500 mg, crude) as a light yellow gum. NMR (400Mz, MeD) δ (ppm): 5.05 (m, 1, C12-), 3.70 (m, 1, C3-), 3.55 (t, 2, - NC2C2S3), 2.93 (t, 2, -NC2C2S3), 2.06 (s, 3, -CC3), 0.91 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.75 (s, 3, 18-Me). 4. General procedure for preparation of compound 5 N S 3 PyS3 pyridine S N S 3 4 To a 50 ml flask, pyridine (10 ml), compound 4 (600 mg, 1.11 mmol) and Py-S3 complex (1060 mg, 6.65 mmol) was added. The mixture was stirred at 20ºC for 16 h. LCMS analysis showed compound 4 was consumed and the desired product was confirmed as the main peak (Rt=0.800, M-=620). DCM (20 ml) was added, and the white solid was filtered off. The filtrate was concentrated to give compound 5 (720 mg, crude, yield>100%) as an oil, which was used in next step without further purification. 5. General procedure for preparation of taurodeoxycholic acid 3--sulfate S 5 N S 3 aq. K S Taurodeoxycholic GCSWID# acid 3--sulfate N S 3 To a solution of compound 5 (720 mg, 1.16 mmol, crude) in Et (10 ml), K (650 mg,

47 mmol) in 2 ( 2 ml) was added. The solution was stirred at 80ºC for 16 h. LCMS showed compound 5 was consumed, and the desired product was formed (Rt=0.896, M-=578). The reaction mixture was concentrated to give the crude product which was dissolved in Me/2 (4/2 ml) and purified by prep-plc. The fraction from the prep-plc was evaporated to dryness via lyophilization to give taurodeoxycholic acid 3--sulfate (210.5 mg, 34%) as a white solid. Prep-PLC: Column: CC YMC-Actus Pro C18 150mm*30mm*5um; Mobile phase: water (0.05%N4 v/v)/mecn, from 5% to 95%; Flow rate: 35ml/minute; Detection: MS (negative) orient. NMR (400Mz, MeD) δ (ppm): 4.28 (m, 1, C3-), 3.95 (m, 1, C12-), 3.57 (t, 2, - NC2C2S3), 2.95 (t, 2, -NC2C2S3), 1.01 (d, 3, 21-Me), 0.94 (s, 3, 19-Me), 0.71 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.880, M-=578.1, (M/2-1). 34

48 Synthesis of Deoxycholic Acid 3--Sulfate (DCA-S) Scheme Ac2, DMAP TMSCN2 pyridine toluene/me 96.1% 71.2% K2C3/Me crude PyS3 aq. K Py Et S crude S 11.4% GCSWID# Deoxycholic acid 3--sulfate mg 63.0 mg completed 1. General procedure for preparation of compound 2 Ac2, DMAP pyridine 1 2 To a solution of compound 1 (6.0 g; mmol) in acetic anhydride (12 ml), pyridine (18 ml) and DMAP (1120 mg; 9.17 mmol) were added. The reaction mixture was stirred at 20 o C for 3.5 h. TLC (ethyl acetate, stained by PMA) showed compound 1 was consumed, and a new main spot was obtained. The solvent was removed under reduced pressure, and then diluted with MTBE (40 ml) and wash with 1 M Cl (3x30 ml), 5% NaC 3 solution (2x30 ml), and brine (30 ml). The organic layer was dried over Na 2 S 4, filtered, and concentrated to obtain compound 2 (7.0 g; 96.1%) as a light yellow solid. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 4.70 (m, 1, C3-), 2.10 (s, 3, - CC3), 2.04 (s, 3, -CC3), 0.91 (s, 3, 19-Me), 0.82 (d, 3, 21-Me), 0.73 (s, 3, 18- Me). 35

49 2. General procedure for preparation of compound 3 TMSCN2 toluene/me 2 3 To a solution of compound 2 (1500 mg, mmol) in PhC 3 /Me (12 ml/5 ml), TMSCN 2 (6.3 ml, 12.6 mmol) was added and then stirred at 20 o C for 16 h. TLC (PE/EA=3:1, stained by PMA) showed unreacted compound 2 and a new main spot. The mixture was concentrated to give the crude product (2.5 g), which was purified by a flash column (12 g silica gel, eluted with PE/EA from 20:1 to 5:1) to give the compound 3 (1100 mg, 71.2%) as an oil. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 4.70 (m, 1, C7-), 3.66 (s, 3, C3), 2.10 (s, 3, -CC3), 2.04 (s, 3, -CC3), 0.91 (s, 3, 19-Me), 0.80 (d, 3, 21- Me), 0.72 (s, 3, 18-Me). 3. General procedure for preparation of compound 4 K2C3/Me 4 3 To a solution of compound 3 (640 mg, 1.30 mmol) in Me (15 ml), K 2 C 3 (270 mg, 1.96 mmol) was added and then stirred 20 o C for 2 h. TLC (PE/EA=3:1, stained by PMA) showed compound 3 was consumed and a new main spot was obtained. C 3 C (0.5 ml) was added to the solution to neutralize the K 2 C 3. The solution was concentrated to give a residue, and water (15 ml) was added, and extracted with ethyl acetate (2x15 ml). The organic layers were dried over Na 2 S 4, filtered, and concentrated to give compound 4 (630 mg, crude, yield >100%) as an oil. NMR (400Mz, CDCl 3 ) δ (ppm): 5.08 (m, 1, C12-), 3.60 (m, 1, C3-), 3.66 (s, 3, 36

50 C3), 2.08 (s, 3, -CC3), 0.89 (s, 3, 19-Me), 0.80 (d, 3, 21-Me), 0.72 (s, 3, 18-Me). 4. General procedure for preparation of compound 5 PyS3 S 4 5 To a 50 ml flask, pyridine (15 ml), compound 4 (630 mg, 1.40 mmol) and Py-S 3 complex (894 mg, 5.62 mmol) was added. The mixture was stirred at 20 o C for 16 h. LCMS showed compound 4 was consumed, and the desire product was confirmed as the main peak (Rt=0.993, M-=528). DCM (20 ml) was added and the white solid was filtered off. The filtrate was concentrated to give crude compound 5 (700 mg, crude, yield>100%) as an oil, which was used in next step without further purification. 5. General procedure for preparation of deoxycholic acid 3--sulfate S aq. Na Et S Deoxycholic acid 3--sulfate GCSWID# To a solution of compound 5 (700 mg, 1.32 mmol, crude) in Et (10 ml), 2 (3 ml) and K (743 mg, 13.2 mmol) was added. The solution was stirred at 80 o C for 16 h. LCMS analysis showed compound 5 was consumed and the desire product was confirmed (Rt=0.900, M- =471). The reaction mixture was concentrated to give the crude product, which was dissolved in Me/2 (4/2 ml) and purified by prep-plc. The fraction from the prep-plc was evaporated to dryness via lyophilization to give deoxycholic acid 3--sulfate (71.5 mg, 11.4%) 37

51 as a white solid. Prep-PLC: Column: Agla DuraShell 150mm*25mm*5um; Mobile phase: water (0.225%FA v/v)/mecn, from 5% to 95%; Flow rate: 35ml/minute; Detection: MS (negative) orient. NMR (400Mz, MeD) δ (ppm): 4.28 (m, 1, C3-), 3.95 (m, 1, C12-), 1.01 (d, 3, 21- Me), 0.93 (s, 3, 19-Me), 0.71 (s, 3, 18-Me). LCMS (negative, ELSD): Rt=0.945, M-=471.1, (M/2-1) 38

52 Structural Information for Non-Sulfated Bile Acids Abbreviation Common Name Chemical Name Structure CAS# LCA Lithocholic Acid 3-hydroxy-(3α,5β)-cholan-24-oic acid GLCA Glycolithocholic Acid N-[(3α,5β)-3-hydroxy-24-oxocholan-24-yl]- glycine TLCA Taurolithocholic Acid 2-[[(3α,5β)-3-hydroxy-24-oxocholan-24-yl] amino]-ethanesulfonic acid UDCA Ursodeoxycholic Acid 3,7-dihydroxy-(3α,5β,7β)-cholan-24-oic acid GUDCA Glycoursodeoxycholic Acid N-[(3α,5β,7β)-3,7-dihydroxy-24-oxocholan-24- yl]-glycine TUDCA Tauroursodeoxycholic Acid 2-[[(3α,5β,7β)-3,7-dihydroxy-24-oxocholan-24- yl]amino]-ethanesulfonic acid CDCA Chenodeoxycholic Acid 3,7-dihydroxy-(3α,5β,7α)- cholan-24-oic acid GCDCA Glycochenodeoxycholic Acid N-[(3α,5β,7α)-3,7-dihydroxy-24-oxocholan-24- yl]-glycine TCDCA Taurochenodeoxycholic Acid 2-[[(3α,5β,7α)-3,7-dihydroxy-24-oxocholan-24- yl]amino]-ethanesulfonic acid DCA Deoxycholic Acid 3,12-dihydroxy-(3α,5β,12α)-cholan-24-oic acid

53 GDCA Glycodeoxycholic Acid N-[(3α,5β,12α)-3,12-dihydroxy-24-oxocholan- 24-yl]-glycine TDCA Taurodeoxycholic Acid 2-[[(3α,5β,12α)-3,12-dihydroxy-24-oxocholan- 24-yl]amino]-ethanesulfonic acid CA Cholic Acid 3,7,12-trihydroxy-(3α,5β,7α,12α)-cholan-24-oic acid GCA Glycocholic Acid N-[(3α,5β,7α,12α)-3,7,12-trihydroxy-24- oxocholan-24-yl]-glycine TCA Taurocholic Acid 2-[[(3α,5β,7α,12α)-3,7,12-trihydroxy-24- oxocholan-24-yl]amino]-ethanesulfonic acid

54 Structural Information for Sulfated Bile Acids Abbreviation Common Name Chemical Name Structure CAS# LCA-S Lithocholic Acid 3--Sulfate 3-(sulfooxy)-(3α,5β)-cholan-24-oic acid GLCA-S Glycolithocholic Acid 3--Sulfate N-[(3α,5β)-24-oxo-3-(sulfooxy)cholan- 24-yl]-glycine TLCA-S Taurolithocholic Acid 3--Sulfate 2-[[(3α,5β)-24-oxo-3-(sulfooxy)cholan- 24-yl]amino]-ethanesulfonic acid UDCA-S Ursodeoxycholic Acid 3--Sulfate 7-hydroxy-3-(sulfooxy)-(3α,5β,7β)- cholan-24-oic acid GUDCA-S Glycoursodeoxycholic Acid 3-- Sulfate N-[(3α,5β,7β)-7-hydroxy-24-oxo-3- (sulfooxy)cholan-24-yl]-glycine TUDCA-S Tauroursodeoxycholic Acid 3--Sulfate 2-[[(3α,5β,7β)-7-hydroxy-24-oxo-3- (sulfooxy)cholan-24-yl]amino]- ethanesulfonic acid CDCA-S Chenodeoxycholic Acid 3--Sulfate 7-ydroxy-3-(sulfooxy)-(3α,5β,7α)- cholan-24-oic acid GCDCA-S TCDCA-S Glycochenodeoxycholic Acid 3-- Sulfate Taurochenodeoxycholic Acid 3-- Sulfate N-[(3α,5β,7α)-7-hydroxy-24-oxo-3- (sulfooxy)cholan-24-yl]-glycine [[(3α,5β,7α)-7-hydroxy-24-oxo-3- (sulfooxy)cholan-24-yl]amino]- ethanesulfonic acid

55 DCA-S Deoxycholic Acid 3--Sulfate 12-hydroxy-3-(sulfooxy)-(3α,5β,12α)- cholan-24-oic acid GDCA-S Glycodeoxycholic Acid 3--Sulfate N-[(3α,5β,12α)-12-hydroxy-24-oxo-3- (sulfooxy)cholan-24-yl]-glycine TDCA-S Taurodeoxycholic Acid 3--Sulfate 2-[[(3α,5β,12α)-12-hydroxy-24-oxo-3- (sulfooxy)cholan-24-yl]amino]- ethanesulfonic acid CA-S Cholic Acid 3--Sulfate 7,12-dihydroxy-3-(sulfooxy)-, (3α,5β,7α, 12α)-cholan-24-oic acid GCA-S Glycocholic Acid 3--Sulfate N-[(3α,5β,7α,12α)-7,12-dihydroxy-24- oxo-3-(sulfooxy)cholan-24-yl]-glycine TCA-S Taurocholic Acid 3--Sulfate 2-[[(3α,5β,7α,12α)-7,12-dihydroxy-24- oxo-3-(sulfooxy)cholan-24-yl]amino]- ethanesulfonic acid

56 Supplementary Table S1, S2, S3, S4, S5 and S6 Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3--Sulfate Conjugates as Potential Novel Biomarkers for rganic Anion-Transporting Polypeptides Rhishikesh Thakare, ongying Gao, Rachel E. Kosa, Yi-An Bi, Manthena V. S. Varma, Matthew A. Cerny, Raman Sharma, Max Kuhn, Bingshou uang, Yiping Liu, Aijia Yu, Gregory S. Walker, Larry Tremaine, Yazen Alnouti, and A. David Rodrigues 43

57 Table S1 MRM transitions and MS parameters for bile acids and their 3--sulfate conjugates Analyte MRM DP (V) CE (ev) CXP (V) transition 2 4 -GCDCA 452.2/ CDCA 395.2/ LCA 375.2/ GLCA 432.2/ TLCA 482.2/ LCA-S 455.2/ GLCA-S 512.2/ TLCA-S 280.6/ UDCA 391.2/ CDCA 391.2/ DCA 391.2/ GUDCA 448.2/ GCDCA 448.2/ GDCA 448.2/ TUDCA 498.2/ TCDCA 498.2/ TDCA 498.2/ UDCA-S 471.2/ CDCA-S 471.2/ DCA-S 471.2/ GUDCA-S 528.2/ GCDCA-S 528.2/ GDCA-S 528.2/ TUDCA-S 288.6/ TCDCA-S 288.6/ TDCA-S 288.6/ CA 407.2/ GCA 464.2/ TCA 514.2/ CA-S 487.2/ GCA-S 544.2/ TCA-S 296.6/

58 Table S2 MRM transitions and MS parameters for 2 4 -pitavastatin, rifampicin, and internal standards Analyte MRM DP (V) CE (ev) CXP (V) transition 2 4 -Pitavastatin 426.2/ Rifampicin 823.4/ Rifampicin 831.4/ Simvastatin 419.2/

59 Table S3 Plasma AUC (AUC 0-24,plasma ), maximum concentration in plasma (C max,plasma ), amount recovered in urine, and renal clearance (CL renal ) of bile acids in male cynomolgus monkeys (mean ± SD, N Bile acid LCA GLCA TLCA UDCA GUDCA TUDCA CDCA GCDCA = 4 animals) following the oral administration of dose vehicle and rifampicin (1, 3, 10, and 30 mg/kg) RIF dose (mg/kg) AUC 0-24,plasma (ng.h/ml) C max,plasma (ng/ml) Amount recovered in urine (ng) CL renal (ml/min/kg) Vehicle 3211 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.16 Vehicle 434 ± ± 17 - b ± ± ± ± ± ± ± ± Vehicle 1473 ± ± ± ± ± ± ± ± ± ± Vehicle 123 ± ± ± ± ± ± ± ± ± 11 6 ± ± ± ± ± ± ± ± ± ± ± 1.24 Vehicle 37 ± ± ± 66 7 ± ± 66 6 ± ± ± ± ± Vehicle 57 ± ± ± ± ± ± ± ± ± ± a 0.1 Vehicle 4514 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.13 Vehicle 1734 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

60 TCDCA DCA GDCA TDCA CA GCA TCA LCA-S GLCA-S TLCA-S Vehicle 5669 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.01 Vehicle ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.85 Vehicle 4847 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.24 Vehicle ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 Vehicle 8917 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.96 Vehicle 2769 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.55 Vehicle 6208 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.07 Vehicle 29 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.41 Vehicle 241 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.65 Vehicle 588 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

61 UDCA-S GUDCA-S TUDCA-S CDCA-S GCDCA-S TCDCA-S DCA-S GDCA-S TDCA-S CA-S Vehicle 59 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.00 Vehicle ± ± ± ± ± ± 0.37 Vehicle ± ± ± ± ± ± ± ± ± ± 2.11 Vehicle ± ± ± ± ± ± ± ± 2.69 Vehicle 13 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.48 Vehicle 32 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.51 Vehicle 6.5 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 22 Vehicle 93 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.93 Vehicle 253 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 4.86 Vehicle ± ± ± ± ±

62 Vehicle GCA-S 3 23 ± ± ± ± ± ± ± ± ± ± ± ± 4.60 Vehicle TCA-S 3 31 ± ± ± ± ± ± ± ± ± ± ± ± 3.60 a Data from one animal only. b Not detectable. 49

63 Table S4 Concentration (nm) of circulating bile acids in human and cynomolgus monkey (mean ± SEM) Bile acid uman Serum a Monkey Plasma b LCA 7.50 ± 0.38 (0.21%) c ± 26.7 (7.62%) GLCA 10.7 ± 0.8 (0.29%) 12.5 ± 4.4 (0.27%) TLCA 1.64 ± 0.15 (0.05%) 43.4 ± 6.7 (0.93%) DCA ± 19.9 (10.9%) 1627 ± 46.6 (35%) GDCA ± 22.1 (7.78%) 71.4 ± 7.3 (1.54%) TDCA 79.3±16.9 (2.18%) ± 18 (8.89%) CDCA ± 21.0 (5.28%) ± 26.8 (13.1%) GCDCA ± 45.2 (16.0%) 41.3 ± 5.5 (0.89%) TCDCA ± 22.3 (6.15%) ± 10.7 (2.62%) UDCA 77.3 ± 7.7 (2.13%) 12.5 ± 3.7 (0.27%) GUDCA ± 10.4 (2.92%) 0.9 ± 0.7 (0.02%) TUDCA 6.18 ± 0.90 (0.17%) 1.7 ± 0.90 (0.04%) CA ± 26.6 (5.33%) ± 26.8 (22.4%) GCA ± 50.3 (8.87%) 80 ± 7.8 (1.72%) TCA ± 35.1 (3.49%) ± 10 (3.43%) LCA-S 5.36 ± 0.54 (0.15%) 3.5 ± 2.1 (0.08%) GLCA-S ± 15.0 (7.57%) 13.6 ± 4.5 (0.29%) TLCA-S 83.0 ± 5.1 (2.28%) 29.3 ± 9.0 (0.63%) DCA-S 0.82 ± 0.08 (0.02%) 0.7 ± 0.6 (0.02%) GDCA-S ± 10.4 (5.58%) 3 ± 1.8 (0.06%) TDCA-S 29.3 ± 1.9 (0.81%) 6 ± 3.6 (0.13%) CDCA-S 5.10 ± 0.42 (0.14%) 0.3 ± 0.3 (0.01%) GCDCA-S ± 12.9 (6.81%) 0.2 ± 0.3 (0.01%) TCDCA-S 13.9 ± 0.9 (0.38%) 0.5 ± 0.6 (0.01%) 50

64 UDCA-S 18.2 ± 1.0 (0.50%) 3.9 ± 2.8 (0.08%) GUDCA-S ± 7.3 (3.71%) - c TUDCA-S 3.15 ± 0.18 (0.09%) - CA-S 0.23 ± 0.03 (0.01%) - GCA-S 1.35 ± 0.15 (0.04%) - TCA-S 10.1 ± 0.7 (0.28%) - % Sulfated 28.4% d 1.3% % Non-sulfated 71.6% 98.7% % Amidated 75.4% 21.5% % Non-amidated 24.6% 78.5% % Amidated + sulfated 27.2% 1.1% % Non-amidated + sulfated 0.8% 0.2% % Amidated + non-sulfated 47.8% 20.3% % Non-amidated + non-sulfated 23.8% 78.3% % Glycine amidated 59.5% 4.8% % Taurine amidated 15.9% 16.7% % Glycine amidated + sulfated 23.7% 0.4% % Glycine amidated + non-sulfated 35.8% 4.4% % Taurine amidated + sulfated 3.8% 0.8% % Taurine amidated + non-sulfated 12.0% 15.9% a Bathena SP, Mukherjee S, livera M, and Alnouti Y (2013) The profile of bile acids and their sulfate metabolites in human urine and serum. J Chromatogr B Analyt Technol Biomed Life Sci : b Present study. c Not detectable d Percent of circulating bile acids analysed.. 51

65 Inhibitor Substrate (µm) Table S5 RIF and RIFsv as inhibitors of cynomolgus monkey ATPs and NTCP expressed in EK293 cells Reported IC 50 (μm) a ATP1B1 ATP1B3 ATP2B1 NTCP Reference RIFsv RSV (0.1) 0.05 ± 0.02 (100%) 0.45 ± 0.03 (100%) - b -. Shen (personal communication) TCA (0.1) ± 0.9 (97%) RIF RSV (0.1) 0.42 ± ± ± (Shen et al., 2013) (92%) (75%) (7%) RSV (0.1) 0.25 ± ± ± ± 7.6 (Chu et al., 2015) (95%) (79%) (7%) (6%) ATV (0.01) 0.14 ± 0.01 (97%) 0.75 ± 0.08 (87%) 62.7 ± 10.7 (7%) 93.4 ± 10.4 (5%) E17G (1.0) 0.38 ± ± (93%) (76%) TCA (1.0) ± 6.3 (13%) Mean (all substrates) RSV, rosuvastatin; RIFsv, rifamycin SV; RIF, rifampicin; TCA, taurocholic acid; E17G, estradiol 17β-Dglucuronide; ATV, atorvastatin. a Mean ± SD. Data in parentheses represent % inhibition based on the reported IC 50 and a final RIF and RIFsv inhibitor concentration [I] of 5 μm and 1 mm, respectively. % inhibition = 100 * {[I]/([I] + IC 50 )}, assuming IC 50 ~ K i (where substrate concentration < K m ). b Not reported. Chu X, Shih SJ, Shaw R, entze, Chan G, wens K, Wang S, Cai X, Newton D, Castro-Perez J, Salituro G, Palamanda J, Fernandis A, Ng CK, Liaw A, Savage MJ, and Evers R (2015) Evaluation of cynomolgus monkeys for the identification of endogenous biomarkers for hepatic transporter inhibition and as a translatable model to predict pharmacokinetic interactions with statins in humans. Drug Metab Dispos 43: Shen, Yang Z, Mintier G, an Y, Chen C, Balimane P, Jemal M, Zhao W, Zhang R, Kallipatti S, Selvam S, Sukrutharaj S, Krishnamurthy P, Marathe P, and Rodrigues AD (2013) Cynomolgus monkey as a potential model to assess drug interactions involving hepatic organic anion transporting polypeptides: in vitro, in vivo, and in vitro-to-in vivo extrapolation. J Pharmacol Exp Ther 344:

66 RIF Dose (mg/kg) Table S6 Estimation of cynomolgus monkey ATP and NTCP inhibition at increasing RIF Free C max (µm) doses of rifampicin (RIF) Predicted % Inhibition a ATP1B1 ATP1B3 ATP2B1 NTCP RIF Dose (mg/kg) RIF Total C max (µm) Predicted % Inhibition a ATP1B1 ATP1B3 ATP2B1 NTCP a Based on the mean IC 50 (across substrates) determined in vitro (Supplemental Table 5); % inhibition = 100 * {[I]/([I] + IC 50 )}, assuming IC 50 ~ K i (where substrate concentration < K m ). [I] represents plasma C max of RIF (total or free). Consideration of free and total plasma C max is consistent with Vaidyanathan et al (2016). In the absence of i.v. RIF pharmacokinetic data, it was not possible to derive an absorption rate constant for RIF and estimate its liver inlet (portal) concentration. Vaidyanathan J, Yoshida K, Arya V, and Zhang L (2016) Comparing various in vitro prediction criteria to assess the potential of a new molecular entity to inhibit organic anion transporting polypeptide 1B1. J Clin Pharmacol 56 Suppl 7:S59-S72. 53

67 Supplementary Figure S1, S2, S3, and S4 Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3--Sulfate Conjugates as Potential Novel Biomarkers for rganic Anion-Transporting Polypeptides Rhishikesh Thakare, ongying Gao, Rachel E. Kosa, Yi-An Bi, Manthena V. S. Varma, Matthew A. Cerny, Raman Sharma, Max Kuhn, Bingshou uang, Yiping Liu, Aijia Yu, Gregory S. Walker, Larry Tremaine, Yazen Alnouti, and A. David Rodrigues 54

68 Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) Concentration (ng/ml) (A) Pitavastatin (A) Pitavastatin RIF 0 mg/kg RIF 1 mg/kg RIF 3 mg/kg RIF 10 mg/kg RIF 30 mg/kg Figure S Time (hr) Time (hr) Plasma concentration-time profile of (A) 2 4 -pitavastatin administered, intravenously (0.2 mg/kg), approximately one hour and 15 minutes after oral administration of vehicle (open circles), and rifampicin at 1 mg/kg (closed triangles), 3 mg/kg (open triangles), 10 mg/kg (open square) and 30 mg/kg (closed circles) and (B) rifampicin after oral administration at a dose of 1 mg/kg (open circles), 3 mg/kg (closed triangles), 10 mg/kg (open triangles) and 30 mg/kg (closed circles). Data are presented as the mean ± SD (n = 4 different cynomolgus monkeys) (B) Rifampicin RIF 1 mg/kg RIF 3 mg/kg RIF 10 mg/kg RIF 30 mg/kg Time (hr) Time (hr) 55

69 n g /m l n g /m l n g /m l n g /m l Concentrations (ng/ml) n g /m l n g /m l n g /m l n g /m l n g /m l n g /m l n g /m l Figure S L C A R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g G -L C A (A) LCA (B) GLCA (C) TLCA (D) LCA-S R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T -L C A R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g L C A -S R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T U im D e C (h A r ) T G im -U e D (h C A r ) Tim -Ue D(h C Ar ) (E) UDCA (F) GUDCA (G) TUDCA () UDCA-S R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g UT im D Ce A(h -S r) GT im -U De C(h Ar )-S T -U imde C(h Ar -S ) T im C De C(h Ar ) (I) GUDCA-S (J) TUDCA-S (K) CDCA (L) GCDCA R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T G im -C e D (h C r) A T im e (h r ) T im e (h r ) Time (hr) T im e (h r ) T im e (h r )

70 n g /m l n g /m l n g /m l Concentrations n g /m l (ng/ml) n g /m l n g /m l n g /m l n g /m l n g /m l R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T -C D C A R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g C D C A -S (M) TCDCA (N) CDCA-S () DCA (P) GDCA D C A R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g G -D C A T im e (h r ) T -D C A T im e (h r ) T -C A R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T im e (h r ) D C A -S T im C A e -S (h r ) R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T im e (h r ) C A (Q) TDCA (R) DCA-S (S) CA (T) GCA TG im-c e A(h -Sr ) (U) TCA (V) CA-S (W) GCA-S (X) TCA-S R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T im e (h r ) G -C A R IF 0 m g /k g R IF 1 m g /k g R IF 3 m g /k g R IF 1 0 m g /k g R IF 3 0 m g /k g T im e (h r ) T -C A -S T im e (h r ) T im e (h r ) Plasma concentration-time profile of (A) LCA, (B) GLCA, (C) TLCA, (D) LCA-S, (E) UDCA, (F) GUDCA, (G) TUDCA, () UDCA-S, (I) GUDCA-S, (J) TUDCA-S, (K) CDCA, (L) GCDCA, (M) TCDCA, (N) CDCA-S, () DCA, (P) GDCA, (Q) TDCA, (R) DCA-S, (S) CA, (T) GCA, (U) TCA, (V) CA-S, (W) GCA-S, and (X) TCA-S in cynomolgus monkeys (mean ± SD, n = 4 animals) after oral administration of dose vehicle (open squares), and RIF at 1 mg/kg (open circles), 3 mg/kg (closed triangles), 10 mg/kg (closed circles), and 30 mg/kg (closed squares) Time (hr) T im e (h r ) T im e (h r )

71 Figure S3 Correlation of sulfation rate (pmol of bile acid sulfate formed per pmol of parent bile acid) between PAPS-fortified human liver and cynomolgus monkey liver cytosol (six different bile acids are considered). Dotted line represents the line of identity. Under the same incubation conditions, the rate of DEA sulfation in the presence of cynomolgus liver cytosol (0.35 pmol sulfate per pmol DEA) was similar to that of PAPS-fortified human liver cytosol (0.27 pmol sulfate per pmol DEA). The ratio of sulfation (cynomolgus monkey-to-human) is 0.20, 0.25, 0.36, 0.37, 0.89 and 3.0 for LCA-S, GLCA-S, TLCA-S, GUDCA-S, GCDCA-S, and GCA-S, respectively. 58