Title: FXR activation by obeticholic acid or nonsteroidal agonists induces a human-like lipoprotein cholesterol change in mice with humanized chimeric liver
Abstract: Obeticholic acid (OCA) is a selective farnesoid X receptor (FXR) agonist that regulates bile acid and lipid metabolism. FXR activation induces distinct changes in circulating cholesterol among animal models and humans. The mechanistic basis of these effects has been elusive because of difficulties in studying lipoprotein homeostasis in mice, which predominantly package circulating cholesterol in HDLs. Here, we tested the effects of OCA in chimeric mice whose livers are mostly composed (≥80%) of human hepatocytes. Chimeric mice exhibited a human-like ratio of serum LDL cholesterol (LDL-C) to HDL cholesterol (HDL-C) at baseline. OCA treatment in chimeric mice increased circulating LDL-C and decreased circulating HDL-C levels, demonstrating that these mice closely model the cholesterol effects of FXR activation in humans. Mechanistically, OCA treatment increased hepatic cholesterol in chimeric mice but not in control mice. This increase correlated with decreased SREBP-2 activity and target gene expression, including a significant reduction in LDL receptor protein. Cotreatment with atorvastatin reduced total cholesterol, rescued LDL receptor protein levels, and normalized serum LDL-C. Treatment with two clinically relevant nonsteroidal FXR agonists elicited similar lipoprotein and hepatic changes in chimeric mice, suggesting that the increase in circulating LDL-C is a class effect of FXR activation. Obeticholic acid (OCA) is a selective farnesoid X receptor (FXR) agonist that regulates bile acid and lipid metabolism. FXR activation induces distinct changes in circulating cholesterol among animal models and humans. The mechanistic basis of these effects has been elusive because of difficulties in studying lipoprotein homeostasis in mice, which predominantly package circulating cholesterol in HDLs. Here, we tested the effects of OCA in chimeric mice whose livers are mostly composed (≥80%) of human hepatocytes. Chimeric mice exhibited a human-like ratio of serum LDL cholesterol (LDL-C) to HDL cholesterol (HDL-C) at baseline. OCA treatment in chimeric mice increased circulating LDL-C and decreased circulating HDL-C levels, demonstrating that these mice closely model the cholesterol effects of FXR activation in humans. Mechanistically, OCA treatment increased hepatic cholesterol in chimeric mice but not in control mice. This increase correlated with decreased SREBP-2 activity and target gene expression, including a significant reduction in LDL receptor protein. Cotreatment with atorvastatin reduced total cholesterol, rescued LDL receptor protein levels, and normalized serum LDL-C. Treatment with two clinically relevant nonsteroidal FXR agonists elicited similar lipoprotein and hepatic changes in chimeric mice, suggesting that the increase in circulating LDL-C is a class effect of FXR activation. The farnesoid X receptor (FXR) is a ligand-activated nuclear receptor/transcription factor that regulates bile acid homeostasis, lipid and glucose metabolism, and hepatic inflammation and fibrosis (1.Lefebvre P. Cariou B. Lien F. Kuipers F. Staels B. Role of bile acids and bile acid receptors in metabolic regulation.Physiol. Rev. 2009; 89: 147-191Crossref PubMed Scopus (1167) Google Scholar, 2.Adorini L. Pruzanski M. Shapiro D. Farnesoid X receptor targeting to treat nonalcoholic steatohepatitis.Drug Discov. Today. 2012; 17: 988-997Crossref PubMed Scopus (212) Google Scholar). FXR knockout mice present with elevated cholesterol and triglycerides in serum and liver (3.Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar, 4.Lambert G. Amar M.J. Guo G. Brewer Jr., H.B. Gonzalez F.J. Sinal C.J. The farnesoid X-receptor is an essential regulator of cholesterol homeostasis.J. Biol. Chem. 2003; 278: 2563-2570Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). These mice are prone to an atherogenic phenotype (5.Hanniman E.A. Lambert G. McCarthy T.C. Sinal C.J. Loss of functional farnesoid X receptor increases atherosclerotic lesions in apolipoprotein E-deficient mice.J. Lipid Res. 2005; 46: 2595-2604Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), mediated by the collective contribution of elevated circulating non-HDL cholesterol (non-HDL-C) and triglyceride levels, increased apolipoprotein B-containing lipoprotein synthesis, and increased intestinal cholesterol absorption (5.Hanniman E.A. Lambert G. McCarthy T.C. Sinal C.J. Loss of functional farnesoid X receptor increases atherosclerotic lesions in apolipoprotein E-deficient mice.J. Lipid Res. 2005; 46: 2595-2604Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). By contrast, in mice with intact FXR signaling, FXR ligands target lipid pathways to reduce hepatic and circulating triglycerides by repressing SREBP1c and its lipogenic targets (6.Watanabe M. Houten S.M. Wang L. Moschetta A. Mangelsdorf D.J. Heyman R.A. Moore D.D. Auwerx J. Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP-1c.J. Clin. 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The farnesoid X-receptor is an essential regulator of cholesterol homeostasis.J. Biol. Chem. 2003; 278: 2563-2570Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 9.Li G. Thomas A.M. Williams J.A. Kong B. Liu J. Inaba Y. Xie W. Guo G.L. Farnesoid X receptor induces murine scavenger receptor class B type I via intron binding.PLoS One. 2012; 7: e35895Crossref PubMed Scopus (33) Google Scholar), proteins important in reverse cholesterol transport. Excretion of cholesterol may also occur across the intestine through a pathway dependent on intestinal FXR (10.de Boer J.F. Schonewille M. Boesjes M. Wolters H. Bloks V.W. Bos T. van Dijk T.H. Jurdzinski A. Boverhof R. Wolters J.C. et al.Intestinal farnesoid X receptor controls transintestinal cholesterol excretion in mice.Gastroenterology. 2017; 152: 1126-1138Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Together, these results suggest that activation of FXR exerts significant hypolipidemic effects in both the liver and circulation (3.Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1423) Google Scholar). These hypolipidemic and antiatherogenic effects of FXR activation have been demonstrated across studies in a variety of animal models (2.Adorini L. Pruzanski M. Shapiro D. Farnesoid X receptor targeting to treat nonalcoholic steatohepatitis.Drug Discov. Today. 2012; 17: 988-997Crossref PubMed Scopus (212) Google Scholar, 11.Hambruch E. Miyazaki-Anzai S. Hahn U. Matysik S. Boettcher A. Perovic-Ottstadt S. Schlüter T. Kinzel O. Krol H.D. 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Liver Dis. 2016; 36: 69-86Crossref PubMed Scopus (75) Google Scholar). With the recognition that FXR has pleiotropic actions in regulation of bile acid and lipid metabolism, glucose regulation, and inflammation, FXR agonists are a current focus of therapeutic development in metabolic and inflammatory disease. Obeticholic acid (6-ECDCA; INT-747; OCA; OCALIVA™) is a bile acid analog and selective FXR agonist with limited to no activity on TGR5 (15.Rizzo G. Passeri D. De Franco F. Ciaccioli G. Donadio L. Orlandi S. Sadeghpour B. Wang X.X. Jiang T. Levi M. et al.Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist.Mol. Pharmacol. 2010; 78: 617-630Crossref PubMed Scopus (148) Google Scholar). OCA is indicated for the treatment of primary biliary cholangitis in combination with ursodeoxycholic acid (UDCA) in adults with an inadequate response to UDCA or as monotherapy in adults unable to tolerate UDCA (16.Nevens F. Andreone P. Mazzella G. Strasser S.I. Bowlus C. Invernizzi P. Drenth J.P. Pockros P.J. Regula J. Beuers U. et al.A placebo-controlled trial of obeticholic acid in primary biliary cholangitis.N. Engl. J. Med. 2016; 375: 631-643Crossref PubMed Scopus (661) Google Scholar). OCA is also in late-stage development for nonalcoholic steatohepatitis (NASH) (17.Neuschwander-Tetri B.A. Loomba R. Sanyal A.J. Lavine J.E. Van Natta M.L. Abdelmalek M.F. Chalasani N. Dasarathy S. Diehl A.M. Hameed B. et al.Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): A multicentre, randomised, placebo-controlled trial.The Lancet. 2015; 385: 956-965Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar) and early stage development for other chronic liver diseases. The primary action of OCA in cholestasis is the reduction in bile acid synthesis by repression of the cytochrome P450 gene encoding cholesterol 7α-hydroxylase (CYP7A1) expression and elevated expression of bile acid transporters (15.Rizzo G. Passeri D. De Franco F. Ciaccioli G. Donadio L. Orlandi S. Sadeghpour B. Wang X.X. Jiang T. Levi M. et al.Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist.Mol. Pharmacol. 2010; 78: 617-630Crossref PubMed Scopus (148) Google Scholar) to reduce bile acid toxicity and inflammation (18.Pellicciari R. Fiorucci S. Camaioni E. Clerici C. Costantino G. Maloney P.R. Morelli A. Parks D.J. Willson T.M. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity.J. Med. Chem. 2002; 45: 3569-3572Crossref PubMed Scopus (605) Google Scholar). In rabbit (19.Maneschi E. Vignozzi L. Morelli A. Mello T. Filippi S. Cellai I. Comeglio P. Sarchielli E. Calcagno A. Mazzanti B. et al.FXR activation normalizes insulin sensitivity in visceral preadipocytes of a rabbit model of MetS.J. Endocrinol. 2013; 218: 215-231Crossref PubMed Scopus (53) Google Scholar, 20.Vignozzi L. Filippi S. Comeglio P. Cellai I. Sarchielli E. Morelli A. Morelli A. Morelli A. Morelli A. Morelli A. et al.Nonalcoholic steatohepatitis as a novel player in metabolic syndrome-induced erectile dysfunction: an experimental study in the rabbit.Mol. Cell. Endocrinol. 2014; 384: 143-154Crossref PubMed Scopus (68) Google Scholar) and rodent (21.Dong B. Young M. Liu X. Singh A.B. Liu J. Regulation of lipid metabolism by obeticholic acid in hyperlipidemic hamsters.J. Lipid Res. 2017; 58: 350-363Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 22.Xu Y. Li F. Zalzala M. Xu J. Gonzalez F.J. Adorini L. Lee Y.K. Yin L. Zhang Y. Farnesoid X receptor activation increases reverse cholesterol transport by modulating bile acid composition and cholesterol absorption in mice.Hepatology. 2016; 64: 1072-1085Crossref PubMed Scopus (95) Google Scholar) models of fatty liver disease, OCA reduces circulating cholesterol and is involved in pathways contributing to the resolution of steatosis, inflammation, and fibrosis. In humans, similar hepatic histological improvements are evident; however, the impact on circulating lipoprotein profiles differs from that which is observed in preclinical models (17.Neuschwander-Tetri B.A. Loomba R. Sanyal A.J. Lavine J.E. Van Natta M.L. Abdelmalek M.F. Chalasani N. Dasarathy S. Diehl A.M. Hameed B. et al.Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): A multicentre, randomised, placebo-controlled trial.The Lancet. 2015; 385: 956-965Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar). For example, in healthy volunteers, OCA induced a sustained elevation of serum LDL cholesterol (LDL-C) concentration (+22%) and reduction of HDL-C (−16%), with a small elevation of total cholesterol (23.Pencek R. Marmon T. Roth J.D. Liberman A. Hooshmand-Rad R. Young M.A. Effects of obeticholic acid on lipoprotein metabolism in healthy volunteers.Diabetes Obes. Metab. 2016; 18: 936-940Crossref PubMed Scopus (52) Google Scholar). A similar profile was noted in individuals with type 2 diabetes mellitus (24.Mudaliar S. Henry R.R. Sanyal A.J. Morrow L. Marschall H-U. Kipnes M. Adorini L. Sciacca C.I. Clopton P. Castelloe E. et al.Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease.Gastroenterology. 2013; 145: 574-82.e1Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar) and in patients with biopsy-confirmed NASH (17.Neuschwander-Tetri B.A. Loomba R. Sanyal A.J. Lavine J.E. Van Natta M.L. Abdelmalek M.F. Chalasani N. Dasarathy S. Diehl A.M. Hameed B. et al.Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): A multicentre, randomised, placebo-controlled trial.The Lancet. 2015; 385: 956-965Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar). In the context of primary biliary cholangitis, the rise in LDL-C was smaller and transient, but was still accompanied by a decrease in HDL-C (16.Nevens F. Andreone P. Mazzella G. Strasser S.I. Bowlus C. Invernizzi P. Drenth J.P. Pockros P.J. Regula J. Beuers U. et al.A placebo-controlled trial of obeticholic acid in primary biliary cholangitis.N. Engl. J. Med. 2016; 375: 631-643Crossref PubMed Scopus (661) Google Scholar). Human studies using the endogenous bile acid FXR agonist chenodeoxycholic acid (CDCA) have reported elevated LDL-C (25.Schoenfield L.J. Lachin J.M. Chenodiol (chenodeoxycholic acid) for dissolution of gallstones: the National Cooperative Gallstone Study. A controlled trial of efficacy and safety.Ann. Intern. Med. 1981; 95: 257-282Crossref PubMed Scopus (386) Google Scholar, 26.Nilsson L.M. Abrahamsson A. Sahlin S. Gustafsson U. Angelin B. Parini P. Einarsson C. Bile acids and lipoprotein metabolism: effects of cholestyramine and chenodeoxycholic acid on human hepatic mRNA expression.Biochem. Biophys. Res. Commun. 2007; 357: 707-711Crossref PubMed Scopus (53) Google Scholar, 27.Laskar M.G. Eriksson M. Rudling M. Angelin B. Treatment with the natural FXR agonist chenodeoxycholic acid reduces clearance of plasma LDL whilst decreasing circulating PCSK9, lipoprotein(a) and apolipoprotein C–III.J. Intern. Med. 2017; 281: 575-585Crossref PubMed Scopus (35) Google Scholar) similar to that reported with OCA, with no observed changes in serum HDL-C. To date, animal studies demonstrating FXR-mediated reductions across lipoprotein cholesterol fractions (1.Lefebvre P. Cariou B. Lien F. Kuipers F. Staels B. Role of bile acids and bile acid receptors in metabolic regulation.Physiol. Rev. 2009; 89: 147-191Crossref PubMed Scopus (1167) Google Scholar, 11.Hambruch E. Miyazaki-Anzai S. Hahn U. Matysik S. Boettcher A. Perovic-Ottstadt S. Schlüter T. Kinzel O. Krol H.D. Deuschle U. et al.Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (−/−) mice.J. Pharmacol. Exp. Ther. 2012; 343: 556-567Crossref PubMed Scopus (87) Google Scholar, 13.Miyazaki-Anzai S. Levi M. Kratzer A. Ting T.C. Lewis L.B. Miyazaki M. Farnesoid X receptor activation prevents the development of vascular calcification in ApoE−/− mice with chronic kidney disease.Circ. Res. 2010; 106: 1807-1817Crossref PubMed Scopus (69) Google Scholar, 21.Dong B. Young M. Liu X. Singh A.B. Liu J. Regulation of lipid metabolism by obeticholic acid in hyperlipidemic hamsters.J. Lipid Res. 2017; 58: 350-363Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 28.Mencarelli A. Renga B. Distrutti E. Fiorucci S. Antiatherosclerotic effect of farnesoid X receptor.Am. J. Physiol. Heart Circ. Physiol. 2009; 296: H272-H281Crossref PubMed Scopus (154) Google Scholar, 29.Gardés C. Chaput E. Staempfli A. Blum D. Richter H. Benson G.M. Differential regulation of bile acid and cholesterol metabolism by the farnesoid X receptor in Ldlr−/− mice versus hamsters.J. Lipid Res. 2013; 54: 1283-1299Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 30.Hartman H.B. Gardell S.J. Petucci C.J. Wang S. Krueger J.A. Evans M.J. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice.J. 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The similarity in HDL-C reduction observed in humans and animals suggests underlying mechanisms common to animals and humans, postulated to result in part from induction of SR-B1 (4.Lambert G. Amar M.J. Guo G. Brewer Jr., H.B. Gonzalez F.J. Sinal C.J. The farnesoid X-receptor is an essential regulator of cholesterol homeostasis.J. Biol. Chem. 2003; 278: 2563-2570Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 9.Li G. Thomas A.M. Williams J.A. Kong B. Liu J. Inaba Y. Xie W. Guo G.L. Farnesoid X receptor induces murine scavenger receptor class B type I via intron binding.PLoS One. 2012; 7: e35895Crossref PubMed Scopus (33) Google Scholar). However, the effects of FXR activation on LDL-C vary in different assay systems. FXR activation by CDCA or the nonsteroidal agonist GW-4064 increased LDL receptor (LDLR) mRNA in human hepatocyte cell culture (33.Langhi C. Le May C. Kourimate S. Caron S. Staels B. Krempf M. Costet P. Cariou B. 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These actions of FXR potentiated the effect of pravastatin by counteracting the statin-induced increase in proprotein convertase subtilisin/kexin type 9 (PCSK9) expression, resulting in increased LDLR activity (33.Langhi C. Le May C. Kourimate S. Caron S. Staels B. Krempf M. Costet P. Cariou B. Activation of the farnesoid X receptor represses PCSK9 expression in human hepatocytes.FEBS Lett. 2008; 582: 949-955Crossref PubMed Scopus (83) Google Scholar). These in vitro responses are consistent with an LDL-lowering effect of FXR. However, they contrast with clinical observations where CDCA administration for 3 weeks to healthy volunteers and surgical patients reduced mRNA expression of LDLR in liver biopsy (26.Nilsson L.M. Abrahamsson A. Sahlin S. Gustafsson U. Angelin B. Parini P. Einarsson C. Bile acids and lipoprotein metabolism: effects of cholestyramine and chenodeoxycholic acid on human hepatic mRNA expression.Biochem. Biophys. Res. 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Hino H. et al.Near completely humanized liver in mice shows human-type metabolic responses to drugs.Am. J. Pathol. 2004; 165: 901-912Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 46.Tateno C. Kawase Y. Tobita Y. Hamamura S. Ohshita H. Yokomichi H. Sanada H. Kakuni M. Shiota A. Kojima Y. et al.Generation of novel chimeric mice with humanized livers by using hemizygous cDNA-uPA/SCID mice.PLoS One. 2015; 10: e0142145Crossref PubMed Scopus (89) Google Scholar). PhoenixBio Japan provided and performed studies on PXB mice and SCID mice (nonchimeric; same SCID background as PXB mice). The replacement index of human hepatocytes as determined by human albumin levels in serum from PXB mice was ≥80% in mice used throughout the study. All mice were male and 12–18 weeks old on day 1. Mice were fed a chow diet [CRF-1 (supplemented with 300 mg of vitamin C per 100 g of chow); Oriental Yeast Co. Ltd., Japan] and were fasted for 4 h prior to euthanizing. All procedures involving animals were performed humanely. All animal experiments have been approved by the Animal Ethics Committee of PhoenixBio. The tool compounds studied include 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid (OCA; INT-747), 2-[3-({5-cyclopropyl-3-[2-(trifluoromethoxy)phenyl]1,2-oxazol-4-yl}methoxy)-8-azabicyclo[3 .2. l] octan-8-yl]4-fluoro-l,3-benzothiazole-6-carboxylic acid (1-lB; INT-2228), and 2-(3-(2-chloro-4-((5-cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl)methoxy)phenyl)-3-hydroxyazetidin-1-yl)isonicotinic acid (13/9; INT-2231) (see Fig. 5A). The synthesis of OCA can be performed following previously reported procedures by Pellicciari et al. (18.Pellicciari R. Fiorucci S. Camaioni E. Clerici C. Costantino G. Maloney P.R. Morelli A. Parks D.J. Willson T.M. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity.J. Med. Chem. 2002; 45: 3569-3572Crossref PubMed Scopus (605) Google Scholar). Nonsteroidal tool compounds INT-2228 and INT-2231were prepared following detailed published procedures by Tully et al. (43.Tully D.C. Rucker P.V. Alper P.B. Mutnick D. Chianelli D. Inventors; Novartis AG, assignee. Compositions and methods for modulating FXR.Patent no. US009150568B2. 2015; Google Scholar, 44.Tully D.C. Rucker P.V. Chianelli D. Williams J. Vidal A. Alper P.B. Mutnick D. Bursulaya B. Schmeits J. Wu X. et al.Discovery of tropifexor (LJN452).A highly potent non-bile acid FXR agonist for the treatment of cholestatic liver diseases and nonalcoholic steatohepatitis (NASH). J. Med. Chem. 2017; 60: 9960-9973Google Scholar) and Kinzel et al. (42.Kinzel O. Steeneck C. Kremoser C. inventors; Gilead Sciences, Inc., assignee. Novel fxr (nr1h4) binding and activity modulating compounds..US Patent no. US20140221659A1. 2014; Google Scholar). Analytical data for the synthesized tool compounds conformed with previously reported analytical information. Mice were dosed orally (p.o.) once per day (QD) for 14 days with vehicle (0.5% carboxymethyl cellulose) or 10 mg/kg/day OCA, 10 mg/kg/day atorvastatin, or both in combination. In the follow-up study with nonsteroidal agonists, PXB mice were dosed p.o. QD for 14 days wit