CB-839 is a potent, selective, and orally bioavailable glutaminase inhibitor with IC50 of 24 nM for recombinant human GAC. Phase 1.
GKT137831 is a potent, dual NADPH oxidase NOX1/NOX4 inhibitor with Ki of 110 nM and 140 nM, respectivelyl; ~10-fold selectivity towards NOX1, 4 and 5 over NOX2, does not inhibit XO or scavange ROS/RNS.
LB-100 is a water soluble protein phosphatase 2A (PP2A) inhibitor with IC50s of 0.85 μM and 3.87 μM in BxPc-3 and Panc-1 cells.
Turofexorate Isopropyl (XL335)
Turofexorate Isopropyl (XL335) is a potent, selective FXR agonist with EC50 of 4 nM, highly selective versus other nuclear receptors, such as LXR, PPAR, ER and etc. Phase 1.
GW4064 is an agonist of farnesoid X receptor (FXR) with EC50 of 65 nM in CV1 cell line and displays no activity at other nuclear receptors at concentrations up to 1 μM.
Lithocholic acid is a toxic secondary bile acid, causes intrahepatic cholestasis, has tumor-promoting activity, its toxic effect can be protected after it activates the vitamin D receptor, PXR and FXR.
H 89 2HCl
H 89 2HCl is a potent PKA inhibitor with Ki of 48 nM in a cell-free assay, 10-fold selective for PKA than PKG,500-fold greater selectivity than PKC, MLCK, calmodulin kinase II and casein kinase I/II.
Puromycin 2HCl is an aminonucleoside antibiotic, which acts as a protein synthesis inhibitor.
Cyclosporin A is an immunosuppressive agent, binds to the cyclophilin and then inhibits calcineurin with IC50 of 7 nM in a cell-free assay, widely used in organ transplantation to prevent rejection.
Dexamethasone (DHAP) is a potent synthetic member of the glucocorticoid class of steroid drugs, and an interleukin receptor modulator that has anti-inflammatory and immunosuppressant effects.
Bile acids are synthesized from cholesterol metabolism exclusively in the liver (Russell and Setchell 1992). In addition to the conventional roles in digestion and absorption of lipid and lipid soluble nutrients in the small intestine, bile acids are also signaling molecules regulating hepatic lipid, glucose, and energy homeostasis (Watanabe et al. 2006; Thomas et al. 2009; Teodoro et al. 2011; Prawitt et al. 2014). Over a decade of research has proven Farnesoid X receptor (FXR) a key regulator in maintaining bile acid homeostasis (Forman et al. 1995; Parks et al. 1999; Wang et al. 1999). FXR is a ligand-activated nuclear receptor, predominantly expressed in the liver, intestine, kidney, and adrenal gland (Forman et al. 1995; Parks et al. 1999; Wang et al. 1999). Bile acids are endogenous ligands for FXR (Forman et al. 1995; Parks et al. 1999; Wang et al. 1999). Rising bile acid concentrations activate FXR which in turn induces the transcription of small heterodimer partner (SHP/NR0B2) and fibroblast growth factor 19 (FGF-19) which suppress the transcription of the rate-limiting anabolic bile acid enzyme, CYP7A1 (Goodwin et al. 2000; Song et al. 2009; Russell and Setchell 1992). Hepatic clearance of bile acids is controlled by FXR. Bile salt export pump (BSEP/ABCB11), the major bile acid efflux transporter on the canalicular membrane of hepatocytes, is critical for formation of bile acid-dependent bile flow (Strautnieks et al. 1998b). Human BSEP transcription is directly induced by FXR (Ananthanarayanan et al. 2001). Insufficient expression or nonfunctional BSEP causes cholestasis. (Strautnieks et al. 1998a; Jansen et al. 1999; Alissa et al. 2008; Davit-Spraul et al. 2009; Whitington et al. 1994). Transporters, MRP2, BCRP, and P-gp, also efflux bile acids into bile cannaluculi (Dawson et al. 2009). Transport of bile acids from hepatocytes into systemic circulation is mediated by basolateral efflux transporters including MRP3 and MRP4 (Rius et al. 2003; Dawson et al. 2009) and OSTα and OSTβ (Landrier et al. 2006). FXR activation up-regulates transcription of OSTα and OSTβ (Boyer et al. 2006; Frankenberg et al. 2006; Landrier et al. 2006).
Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are chronic, cholestatic, and inflammatory autoimmune liver diseases (Beuers et al. 2015; Lindor et al. 2009; Sarkar and Bowlus 2016). Progressive destruction of bile ducts in PBC and PSC results in bile acid elevation in the liver and the circulation. PBC and PSC patients develop liver cirrhosis and failure eventually requiring liver transplantation; otherwise the diseases are fatal. Studies have demonstrated adaptive and compensatory mechanisms in PBC and PSC patient's livers in response to bile acids overload. Liver transporters including uptake and efflux transporters, and bile acid synthesis enzymes are adaptively changed to reduce accumulation of bile acids in hepatocytes. These compensatory mechanisms are largely regulated by the FXR (Takeyama and Sakisaka 2012).
FXR is a pharmacologically attractive target for the treatment of cholestasis in PBC, PSC, and other cholestatic diseases. Chenodeoxycholic acid (CDCA), is the most potent endogenous FXR activator (Makishima et al. 1999; Parks et al. 1999; Liu et al. 2014). Obeticholic acid (OCA), a semi-synthetic analog of CDCA is approximately 100-fold more potent than CDCA (Pellicciari et al. 2002). OCA was protective in a rat cholestasis model induced by estrogen (Fiorucci et al. 2005). In this model, OCA increased the bile flow and decreased the bile acid synthesis (Fiorucci et al. 2005).
Since OCA, a potent FXR agonist, is approved for the treatment of PBC, understanding its mechanistic action on genes involved in bile acid transport and synthesis is relevant. This was accomplished by utilizing sandwich-cultured human hepatocytes (SCHH). This in vitro technique preserves the in vivo-like bile acid biosynthesis and regulatory pathways (Jackson et al. 2016) including uptake and efflux transporters proper localization (Hoffmaster et al. 2004; Li et al. 2009). SCHH are also capable of determining hepatobiliary distribution of endogenous bile acids (Swift et al. 2010). FXR-regulated gene expression, transporter function, and endogenous bile acid levels were evaluated after OCA treatment in comparison to CDCA.
Materials and Methods
CDCA, tamoxifen, and aflatoxin B were purchased from Sigma Aldrich (St. Louis, MO). OCA and its conjugates (taurine and glycine) were provided by Intercept Pharmaceuticals, Inc. (San Diego, CA). Primary human hepatocyte cultures were seeded and maintained utilizing propriety cell culture media formulations developed at Qualyst Transporter Solutions (Durham, NC). Cell culture base medium and supplements were purchased from Gibco (Carlsbad, CA) and Corning (Tewksbury, MA). CellTiter-Glo® Luminescent Cell Viability Assays were purchased from Promega (Madison, WI). All quantitative real-time polymerase chain reaction (qRT-PCR) reagents were purchased from Life Technologies (Carlsbad, CA). Pierce™ BCA™ Protein Assays were purchased from Thermo Fisher Scientific (Waltham, MA).
Sandwich-cultured human hepatocyte culture and treatment
SCHH were prepared by Qualyst Transporter Solutions using cryopreserved human hepatocytes purchased from Triangle Research Laboratories (RTP, NC) and Xenotech (Lenexa, KS). Hepatocytes were QTS Transporter Certified™. QTS Certification signifies that SCHH reestablishes a functional bile canalicular network capable of supporting hepatic drug uptake and biliary efflux function. Transporter Certified™ cryopreserved hepatocytes in sandwich culture are the optimal system for evaluating hepatic transporter interaction potential of new chemical entities. The preparation and treatment of SCHH are described in the Appendix, Section 1.1.1.
SCHH were treated with CDCA, OCA, glyco-OCA, and tauro-OCA (0.1, 0.316, 1.0, 3.16, 10, 31.6, 100 μmol/L) or cytotoxicity-positive controls (50 μmol/L tamoxifen, 10 μmol/L aflatoxin) for 72 h with daily medium change. Cell morphology was evaluated at 24, 48, and 72 h using phase contrast microscopy for each of the treatment groups on a daily basis. Images were captured using a Zeiss Axiovert 40CFL microscope equipped with phase contrast optics, AxioCam MRc camera, and AxioVision imaging software (V4.6.1).
Morphology of the hepatocyte cultures was compared to controls for any morphological alterations (e.g., changes in cell shape, cytoplasmic alterations, accumulation of vacuoles suggestive of dilated organelles and lipid droplets) indicative of cytotoxicity (Guillouzo et al. 1997; Tyson and Green 1987).
Biochemical evaluation of cell viability
Cell viability of hepatocytes was assessed by determining the amount of ATP present after 72 h of exposure to OCA, CDCA, glyco-OCA tauro-OCA, and the positive controls tamoxifen and aflatoxin B using CellTiter-Glo Luminescent Cell Viability Assays from Promega (Madison, WI) following manufacturer's procedures. Each test condition was performed in triplicate.
Total RNA isolation and qRT-PCR
Following 72 h of treatment with CDCA (0.1, 0.316, 1.0, 3.16, 10, 31.6, 100 μmol/L), or OCA, glyco-OCA, or tauro-OCA (0.00316, 0.01, 0.0316, 0.1, 0.316, 1.0, 3.16 μmol/L), SCHH were washed and lysed for total RNA isolation using Qiagen RNeasy kit following manufacturer's instructions. Details are described in Appendix Section 1.2.1
Bile acid profiling and hepatobiliary disposition assessment
Following 72 h of treatment with CDCA (100 μmol/L) or OCA (1.0 μmol/L), the endogenous bile acid composition and hepatobiliary disposition of d8-TCA in SCHH compartments (cell, bile pocket, and cell culture medium) were determined using B-CLEAR® technology. See Appendix Section 1.3.1 for detailed descriptions of cell culture preparation, experimental procedure, and calculations for biliary accumulation, biliary excretion index (BEI), and intracellular concentration (ICC) of d8-TCA. Protein content was determined using Pierce BCA protein assay kit (Thermo Fisher Scientific) following manufacturer's instructions.
Bioanalytical of bile acid profiling and disposition assessment
Analytes (d8-TCA, CA, tauro-CA, glyco-CA, CDCA, tauro-CDCA, and glyco-CDCA) were extracted from study samples (cell culture medium and hepatocyte lysates). Extraction procedure are detailed in Appendix Section 1.4.1. Prepared samples were filtered and analyzed by LC-MS/MS using a Shimadzu binary HPLC system (Columbia, MD) and tandem mass spectrometry using Thermo Electron TSQ® Quantum Discovery MAX™ (Waltham, MA) with an Ion Max ESI source operating in negative ion electrospray ionization mode using multiple reaction monitoring.
The assays in this study were conducted in SCHH prepared from three individual donors except for the cytotoxicity assay that was conducted in SCHH of one liver donor. Every measurement was performed in triplicate per donor. Data were normalized to the vehicle controls (DMSO) and represent the mean ± SD from three donors. The cytotoxicity data represent means from triplicate wells from one donor. The data were analyzed with GraphPad Prism 6.0 (La Jolla, CA).
No Cytotoxicity Induced by OCA
Compared to the vehicle control treatment, no morphological changes including loss of cuboidal cell shape, loss of cell-to-cell contact, and cell detachment were observed in SCHH after 72 h of exposure to OCA or CDCA (0.1, 0.316, 1.0, 3.16, 10, 31.6, 100 μmol/L) (Appendix Fig. 1.3.1). Consistently, ATP depletion studies demonstrated no meaningful reduction in ATP cellular content in hepatocytes exposed to 0.1-100 μmol/L OCA or CDCA for 72 h (Fig. 1). These data suggested that OCA or CDCA were not cytotoxic up to 100 μmol/L.
No marked morphological changes were observed after 72 h of treatment with 0.1-31.6 μmol/L glyco-OCA and tauro-OCA (Appendix Fig. 1.3.1.) indicating that glyco-OCA or tauro-OCA at concentrations from 0.1 to 31.6 μmol/L were not cytotoxic to hepatocytes. Marked morphological changes were observed in SCHH exposed to 100 μmol/L glyco-OCA or 100 μmol/L tauro-OCA for 72 h (Appendix Fig. 1.3.1.). Cell morphology data were supported by ATP depletion studies that demonstrated ATP content in SCHH was reduced to 66.1% and 69.3% relative to control following exposure to 100 μmol/L glyco-OCA and 100 μmol/L tauro-OCA, respectively (Fig. 1). These changes indicated that glyco-OCA and tauro-OCA at 100 μmol/L were toxic to hepatocytes.
The positive control toxicants, tamoxifen (Appendix Fig. 1.3.1) and aflatoxin B (data not shown) caused marked changes in cell morphology (e.g., loss of cuboidal cell shape and loss of cell-to-cell contact). The severity of morphological changes increased over time after 24, 48, and 72 h of exposure. ATP cellular content was reduced to 1.3% and 2.1% relative to control after 72 h exposure to tamoxifen and aflatoxin, respectively (Fig. 1).
OCA activates FXR-mediated bile acid homeostasis feedback mechanism
Using B-CLEAR® technology, the effect of 1 μmol/L OCA exposure for 72 h was evaluated on total endogenous bile acid content, disposition, and bile acid synthesis in the hepatocyte, bile, and cell culture medium (CCM) from three donors in SCHH.
Total endogenous bile acid content comprised the sum of CA, glyco-CA, tauro-CA, CDCA, glyco-CDCA, and tauro-CDCA. OCA decreased the total bile acid content to 42.7 ± 20.5%, relative to control (Fig. 2). OCA correspondingly decreased total endogenous bile acid content in cell, bile, and CCM to 16.6 ± 7.2%, 5.4 ± 1.7%, and 54.6 ± 26.3%, respectively, relative to the control.
The expression of genes involved in bile acid synthesis including SHP, FGF-19, CYP7A1, Cytochrome P450 Family 7 Subfamily B Member 1 (CYP7B1), Cytochrome P450 Family 8 Subfamily B Member 1 (CYP8B1), Bile Acid-CoA:Amino Acid N-Acyltransferase (BAAT), and bile acid acyl-CoA synthetase (BACS), was evaluated in SCHH from three donors following 72 h exposure to increasing concentrations of OCA (0.00316–3.16 μmol/L) or CDCA (0.1-100 μmol/L).
Figure 3 illustrates the effect of OCA and CDCA on the mRNA levels of SHP, FGF-19, and CYP7A1. SHP and FGF-19 are modulators of CYP7A1 activity. CYP7A1 is the rate-limiting enzyme of bile acid synthesis. As OCA and CDCA cell culture concentration were increased, the genes encoding SHP and FGF-19 mRNA levels increased; as postulated, CYP7A1 mRNA deceased. Specifically, OCA at 1 μmol/L increased SHP mRNA to 3.7 ± 0.2-fold and FGF-19 mRNA to 735 ± 63-fold above vehicle control. Similarly, CDCA at 100 μmol/L increased SHP and FGF-19 mRNA levels to 4.5 ± 0.9-fold and 1430 ± 712-fold, respectively, above control. Correspondingly, increased concentration of OCA and CDCA reduced the expression of CYP7A1 by 99%.
Dose proportionality determinations corroborate the effect of OCA and CDCA on SHP, FGF-19, and CYP7A1 (Appendix Fig. 1.3.2; Appendix Table 1.2.1). Slopes were considered dose linear if the 0.95 CI did not cross through zero and contained the slope value. Meeting these requirements, a slope of 1 is dose proportional; a slope less than 1 is linear but less than dose proportional; and a slope greater than 1 is linear but more than dose proportional. With increasing doses of OCA, significant positive slopes (0.95 CI) of 0.2641 (0.2235–0.2993) and 1.333 (1.191–1.474), were determined for SHP and FGF-19, respectively. Similar trends were observed for CDCA in which SHP and FGF-19 slopes were 0.4003 (0.3295–0.4711) and 2.039 (1.683–2.395), respectively. In contrast, increased doses of OCA and CDCA decreased the production of CYP7A1 mRNA in a dose proportional manner; −1.129 (−1.348 to −0.9095) and −2.48 (−3.499 to −1.462), respectively. In addition, correlation plots were constructed between SHP and CYP7A1 mRNA levels as OCA or CDCA dose increased. It is understood that as SHP expression rises, there is a concomitant suppression of CYP7A1. Correlation statistics confirmed this assumption; significant correlations (R2) between SHP and CYP7A1 were 0.8491 and 0.7708 for OCA and CDCA treatment, respectively (Appendix Fig. 1.3.3; Appendix Table 1.2.2.).
No marked changes were observed in CYP7B1, CYP8B1, BAAT, and BACS mRNA (Appendix Fig. 1.3.4.).
Hepatobiliary disposition of d8-TCA
Using B-CLEAR® technology, the hepatobiliary disposition of d8-TCA, a model bile acid, was determined in the hepatocyte and bile following 72 h exposure to 1 μmol/L OCA or 100 μmol/L CDCA. Compared with control, the BEI of d8-TCA in SCHH was unchanged after 72 hour exposure to OCA or CDCA (109.8 ± 18.0% and 96.6 ± 4.1% relative to control, respectively, Fig. 4A). However, exposure to OCA or CDCA decreased d8-TCA hepatic intracellular concentrations ICC to 39.6 ± 8.9% and 26.4 ± 7.5%, respectively, relative to control (Fig. 4B; Appendix Section 1.3.1. for calculations). Total disposition of d8-TCA was reduced to 43.8 ± 2.8% and 24.7 ± 5.7%, relative to control following OCA or CDCA exposure, respectively (Fig. 4C).
OCA increases bile acid efflux transporter gene expression
The gene expression of uptake transporters on the hepatocyte basolateral membrane (sodium-taurocholate cotransporting polypeptide [NTCP]; organic anion transporting polypeptides 1B1, 1B3, 2B1 [OATP1B1, OATP1B3, and OATP2B1]); hepatocyte basolateral efflux transporters (multidrug resistance-associated protein 3 and 4 [MRP3 and MRP4], OSTα, OSTβ; canalicular efflux transporters (multidrug resistance-associated protein 2 [MRP2], breast cancer resistance protein [BCRP], P-glycoprotein [P-gp], and BSEP), were determined following 72 h of exposure to increasing concentrations of OCA or CDCA).
Exposure to OCA at 1 μmol/L increased expression of basolateral efflux heterodimers OSTα mRNA and OSTβ mRNA by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively, relative to control (Fig. 5A and C). Similarly, increases in OSTα and OSTβ expression were observed following CDCA exposure at the highest dose (100 μmol/L) [9.1 ± 1.3-fold and 93.6 ± 23.8-fold relative to control, respectively (Fig. 5B and D)]. Expression of the canalicular efflux transporter, BSEP, was 6.4 ± 0.8-fold greater than the vehicle control following exposure to 1 μmol/L OCA (Fig. 5E). Likewise, exposure to 100 μmol/L CDCA increased the expression of BSEP mRNA to 8.9 ± 0.6-fold above control (Fig. 5F). These data were corroborated using slope determinations for OCA and CDCA with respect to OSTα, OSTβ, and BSEP mRNA expression (Appendix Fig. 1.3.5 and Appendix Table 1.2.3.). In each treatment, as analyte concentration increased, there was a corresponding incremental increase in mRNA levels. Dose–response slopes were linear but less than dose proportional with the exception of the dose proportional slope for CDCA-OSTβ curve.
No marked changes were observed in the expression of basolateral bile acid uptake transporters NTCP, OATP1B1, OATP1B3, and OATP2B1 in SCHH following 72 h exposure to OCA or CDCA. These results suggest that uptake may not contribute to the decrease observed in total bile acid accumulation or bile acid ICC (e.g. d8-TCA). Similarly, the expression of other efflux transporters, P-gp, MRP2, MRP3, MRP4, and BCRP, were unchanged in SCHH following treatment with either OCA or CDCA (Appendix Fig. 1.3.6.).
Using binding assays, glyco-OCA and tauro-OCA have been shown to be equipotent agonist compared to parent at FXR. (data not shown). Confirmation of similar agonist effect of glyco-OCA and tauro-OCA at FXR was demonstrated using a pharmacological platform measuring mRNA levels for CYP7A1, SHP, FGF-19, BSEP, OSTα, OSTβ, other CYP enzymes, and transporters (Appendix Fig. 1.3.7.). For each parameter examined, the conjugated metabolites produced the same level of mRNA expression as OCA.
FXR acts as a master regulator of bile acid homeostasis (Makishima et al. 1999; Chiang 2009) as illustrated in Figure 6. Identification of FXR as a therapeutic target for the treatment of chronic liver diseases (e.g., PBC) has led to the development of more potent FXR agonists including OCA (Ali et al. 2015). OCA has higher selectivity and is approximately 100-fold more potent on FXR than CDCA, the endogenous ligand. Similar to endogenous bile acids, OCA is metabolized to glycine and taurine conjugates, glyco-OCA and tauro-OCA, respectively. It has also been determined that the OCA conjugates have nearly identical activity on FXR as does OCA. To better understand the mechanism-of-action of OCA in humans, a validated in vitro human hepatocyte sandwich-cultured (SCHH) model was employed to investigate gene regulation of bile acid synthesis and bile acid elimination. In addition, the OCA hepatic effects were compared to the natural occurring bile salt ligand CDCA.
Foremost, OCA and CDCA bile acids at concentrations up to 100 μmol/L for 72 h did not damage sandwich-cultured hepatocytes as evidenced by lack of cellular morphological alterations or cellular reduction in ATP (Appendix Figure 1.3.1.). Compared to conventional cultured primary hepatocytes, where OCA concentrations <3 μmol/L produced cytotoxicity (data not shown). The OCA conjugates were not cytotoxic up to 31.6 μmol/L; they did, however, show clear morphological changes and ATP cellular loss at 100 μmol/L. Positive control toxicants damaged hepatocytes in a time-dependent manner adding to the confidence that OCA and metabolites were not toxic at expected therapeutic concentrations. The absence of OCA cellular toxicity may likely be due to polarization of SCH hepatocytes resulting in the appropriate localization and function of bile acid transporters (Li et al. 2009; Swift et al. 2010), thus allowing natural bile acids and bile acid analogues to efflux into bile pockets and reduce intracellular accumulation and cytotoxicities (Jackson et al. 2016).
OCA at 1 μmol/L for 72 h decreased total endogenous bile acid content (CA, glyco-CA, tauro-CA, CDCA, glyco-CDCA, and tauro-CDCA) by approximately 57%. Sampling separate compartments of the model, reductions in total bile acid were observed in hepatocytes, bile, and CCM (Fig. 2). In a separate experiment, total disposition of d8-TCA (a prototypical bile acid) was reduced to 43.8 ± 2.8% and 24.7 ± 5.7%, relative to control following OCA or CDCA exposure, respectively (Fig. 4C). These data support the hypothesis that OCA and CDCA downregulate bile acid production in human hepatocytes. As discussed below, further work using gene biomarkers confirmed this theory.
There are numerous, complex biological cascades triggered by OCA and CDCA that regulate bile acid homeostasis. These include but are not limited to bile acid synthesis and bile acid uptake and efflux transporters. Expression of relevant genes involved in bile acid synthesis stimulated by OCA and CDCA include SHP, FGF-19, and CYP7A1. OCA and CDCA function as FXR agonists resulting in the inhibition of bile acid synthesis. Stimulation of FXR leads to increased levels of SHP and FGF-19. These in turn suppress the production of CYP7A1, the rate-limiting enzyme of bile acid synthesis thereby reducing bile acid levels. Addition of either agonist to the SCHH plates increased SHP and FGF-19 mRNA. OCA at 1 μmol/L, increased mRNA levels approximately 4- and 735-fold, SHP and FGF-19, respectively, above vehicle control (Fig. 3). CDCA concentrations of 100 μmol/L achieved similar effects as OCA. Concentration of OCA and CDCA at 1 and 100 μmol/L, respectively, reduced expression of CYP7A1 by 99%. Dose–response analysis determined that SHP, FGF-19, and CYP7A1 mRNA levels increased in a linear fashion with changing dose (Appendix Fig. 1.3.2). In addition, correlation plots of SHP versus CYP7A1 mRNA (Appendix Fig. 1.3.3) as OCA or CDCA dose increased, showed good correlations (R2) in which an increase in SHP led to a decrease in CYP7A1 mRNA levels after exposure to OCA (0.849) or CDCA (0.771). These data confirm that FXR activation by OCA or CDCA results in predictable pharmacological regulation of bile acid homeostasis.