The regulation of genes that are essential to many

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1 GASTROENTEROLOGY 2005;129: NEW CONCEPTS IN GASTROENTEROLOGY Nuclear Receptor Ligands: Rational and Effective Therapy for Chronic Cholestatic Liver Disease? JAMES L. BOYER Liver Center, Department of Medicine, Yale University School of Medicine, New Haven, Connecticut The regulation of genes that are essential to many hepatic metabolic and transport functions are mediated in large part by the action of small molecules that function as nuclear receptor ligands. 1 This process has been labeled chemical genomics. 2 These ligands bind to their specific nuclear receptors, activating the receptor that then binds to specific elements in a gene s promoter resulting in stimulation or inhibition of gene expression. 3 In the liver many drugs, metabolites, and herbal compounds exert their biologic properties as ligands for nuclear receptors. 4,5 Some familiar examples of drugs that activate nuclear receptors include phenobarbital and St John s wort. Both drugs induce hepatic drug metabolizing enzymes by acting as ligands for the pregnane X receptor (PXR), which binds to specific response elements in the promoter of cytochrome P-450 3A (CYP3A), a major hepatic microsomal drug metabolizing enzyme. This induction then indirectly affects the metabolism and systemic effects of a wide variety of other compounds metabolized by CYP3A. An extract of 4 different plants, Yin Zhi Huang, has been used traditionally for treating neonatal jaundice in China and is a ligand for the constitutive androstane receptor (CAR) that up-regulates the expression of several different liver transporters and enzymes involved in the hepatic clearance of serum bilirubin. 5 Indeed, more than 10% of medically useful drugs are known to exert their biologic behavior by binding to ligand-binding domains on specific nuclear receptors. 4 Table 1 shows some of the most important nuclear receptors and their physiologic ligands that determine the hepatic transport and metabolism of a variety of xenobiotics and endogenous substrates. In this issue of GASTROENTEROLOGY, Marschall et al 6 report that rifampicin and ursodeoxycholic acid (UDCA), two well-known drugs used in the treatment of cholestatic liver disease, each stimulate the transcription of a distinct set of genes that together decrease bile acid uptake, enhance bile acid detoxification and excretion, and stimulate the clearance of bilirubin. Rifampicin increased the expression of CYP3A4, uridine 5=-glucuronosyl transferase (UGT1A1), and multidrug-resistance associated protein (MRP2) whereas UDCA stimulated the bile salt export pump (BSEP), and multidrug resistance protein (MDR3), and MRP4. Although these effects were observed in patients undergoing cholecystectomy who otherwise were healthy, these separate but complementary effects on gene and protein expression are predicted to have beneficial effects if they occurred in patients with cholestatic liver disease. These findings thus suggest that there is therapeutic benefit from combining drugs such as rifampicin and UDCA that target nuclear receptors that have coordinated effects on the transcriptional regulation of hepatobiliary excretory mechanisms. 7 How does this occur? The hepatic clearance of bile acids and bilirubin can be divided into 4 phases that include the following: phase 0, hepatic uptake; phase I, metabolism (eg, hydroxylation); phase II, detoxification (eg, conjugation); and phase III, excretion. Nuclear receptors and their ligands that are the major determinants of the functional expression of genes that determine these pathways are shown in Figure 1, together with enzymes that control bile acid synthesis. In the enterohepatic circulation, the hepatic uptake of conjugated bile acids (phase 0) is mediated predominantly by the sodium taurocholate cotransporting polypeptide NTCP (SLC10A1), whereas unconjugated bile acid uptake is facilitated by several organic anion transporting polypeptides (OATPs) on the sinusoidal membrane, but predomi- Abbreviations used in this paper: ABCG5/8, cholesterol export pump; BSEP, bile salt export pump; CAR, constitutive androstane receptor; CYP, cytochrome P-450; FTF, fetal transcription factor; FXR, farnesoid X receptor; GST, glutathione-s-transferase; LXR, liver X receptor; MDR, multidrug resistance protein; MRP, multidrug-resistance associated protein; NTCP, sodium taurocholate co-transporting polypeptide; OATP, organic anion transporting polypeptides; PXR, pregnane X receptor; RAR, retinoic acid receptor; SULT, sulfotransferase; UGT, uridine 5=-glucuronosyl transferase; VDR, vitamin D receptor by the American Gastroenterological Association /05/$30.00 doi: /j.gastro

2 736 JAMES L. BOYER GASTROENTEROLOGY Vol. 129, No. 2 Table 1. Nuclear Receptors, Important Ligands, and Target Genes Nuclear receptor Ligand(s) Some major target genes RXR (retinoid X receptor) 9-cis-retinoic acid Heterodimeric partner of other class II receptors RXR partners FXR (farnesoid X receptor) Bile acids BSEP, SHP, UGTs, SULTs, MRP2, MDR3 PXR (pregnane X receptor) Xenobiotics, Ursodeoxycholic acid, CYP3A, OATP-C, MRP2, MRP4, GST Rifampacin CAR (constitutive androstane receptor) Xenobiotics, Phenobarbital CYP3A, OATP-C, MRP2, MRP4, UGT, SULTs, GSTs LXR (liver X receptor) Oxysterols (metabolites of cholesterol) CYP7A, CYP8B, ABCG5/8 RAR (retinoic acid receptor) All-trans retinoic acid NTCP, MRP2 Others SHP-1 (short heterodimeric partner) None Inhibits CYP7A, CYP8B, NTCP FTF (fetal transcription factor)? bile acids CYP7A, CYP7A and 8B1, MRP3 VDR (vitamin D receptor) Vitamin D, Lithocholic acid CYP3A, SULTs HNF-1 (hepatocyte nuclear factor 1) None NTCP, CYP7A nantly by OATP1B1 (SLC01B1), formally known as OATP-C. Normally, bile acids then are excreted into bile via BSEP (ABCB11), an adenosine triphosphate binding cassette family member located on the apical canalicular membrane (phase III). Bile acids also are synthesized in the hepatocyte from the conversion of cholesterol via CYP7A1, leading to the synthesis of the 2 primary bile acids: cholic acid and chenodeoxycholic acid. 8 Bilirubin is thought to be taken up by OATP1B1, bind to glutathione-s-transferase, and undergo conjugation by hepatic microsomal UGTs to monoglucuronides and then diglucuronides (phase II). These conjugation reactions increased the hydrophobicity of this hydrophobic end product of heme metabolism, enabling it to be excreted into bile by MRP2 (ABCC2), a member of the multidrug resistance associated protein gene family located at the canalicular membrane of the hepatocyte (phase III). In animal models of cholestasis and in patients with cholestatic liver disease, sodium dependent taurocholate co-transporting polypeptide (NTCP) and most OATPs are down-regulated to minimize bile acid uptake. 9 Bile acid synthesis is diminished by the down-regulation of CYP7A1 and CYP8B, which reduces the conversion of cholesterol to bile acids. The more hydrophobic toxic secondary bile acids, lithocholate and deoxycholate, accumulate in the enterohepatic circulation and undergo phase I 6- hydroxylation reactions in the liver via CYP3A1. Subsequently, phase II detoxifying conjugation reactions form more water-soluble bile acid glucuronides and sulfates, respectively, via the enzymatic activity of UGTs and sulfotransferases (phase II). Under cholestatic conditions, BSEP generally continues to be expressed functionally whereas MRP3 and MRP4, two alternative phase III export pumps for bile acid and bilirubin conjugates, are up-regulated on the basolateral membrane. 10,11 Normally, MRP3 and MRP4 are expressed only weakly but they appear to function during cholestasis to allow these more water-soluble bilirubin and bile acid conjugates to be transported back into the systemic circulation to be excreted by the kidney. Although these adaptive responses in transporter expression are beneficial, they clearly are unable to compensate fully for the primary defects in cholestasis, and progressive liver injury ultimately occurs in many cholestatic diseases. Nevertheless, if these adaptive responses could be enhanced or possibly initiated earlier in the natural history of cholestatic liver disease, they might have additional benefits. This is where nuclear receptor therapy is proposed to play a role, particularly ligands for the farnesoid X receptor (FXR), CAR, and PXR. 12,13 FXR (NR1H4) is activated by bile acids. The hydrophobic bile acid CDCA is its most potent physiologic agonist. FXR is expressed highly in the liver, intestine, adrenal glands, and kidney, and up-regulates many of the steps in the enterohepatic circulation of bile acids including BSEP, the ileal bile acid binding protein (FABP6), the hepatic canalicular membrane phospholipid flippase (MDR3, ABCB4), and MRP2. MDR3 is the export pump for phosphotidylcholine and mutations in MDR3 result in progressive cholestasis. 14 FXR also activates transcription of the nuclear receptor short heterodimeric protein, which in turn inhibits transcription of CYP7A and CYP8B, thereby providing feedback inhibition of bile acid synthesis (Figure 1). 15 These adaptive changes mediated by FXR protect the liver from the injurious effects of the hepatic accumulation of bile acids during cholestasis. Thus, there has been considerable interest in the development of nontoxic synthetic FXR agonists for the treatment of cholestatic liver disease and several compounds have been shown to be hepatoprotective in rat and mouse models of cholestasis. The administration of a synthetic FXR agonist, GW4064, reduces markers of liver damage, inflammation, and bile duct proliferation induced by -napthylisothiocyanate and

3 August 2005 NUCLEAR RECEPTOR LIGANDS 737 Figure 1. Coordinated ligand-activated regulation of gene expression that determines the hepatic clearance of bile acids, bilirubin, and xenobiotics a rational basis for the therapy of cholestatic liver disease. Some of the major nuclear receptors that regulate the expression of these key genes are shown. Unless otherwise indicated by 2 or symbols, these ligands stimulate gene expression. Normally many of these nuclear receptors form heterodimeric complexes with the retinoid X receptor (RXR). This complex then binds to specific response elements in the gene promoter. Other nuclear receptors such as short heterodimeric protein-1, fetal transcription factor (FTF), and hepatocyte nuclear factor 1 (HNF-1) do not form heterodimers with RXR and do not have specific ligands. bile duct ligation and is associated with predicted changes in FXR mediated expression of bile acid transporters and biosynthetic genes. 16 Both GW4064 and another potent synthetic FXR ligand, 6-ethyl chenodesoxycholic acid, protect rats from ethinyl estradiol induced cholestasis by increasing the expression of short heterodimeric protein, Bsep, and Mrp2, and reducing the synthesis of Cyp7a1 and Cyp8b1 as well as Ntcp. 17 Mice with disruption of FXR are unable to up-regulate Bsep and other FXR-regulated events in response to the feeding of cholic acid, resulting in increased liver toxicity and mortality, 18 further emphasizing that FXR is essential for the adaptive response in transporter function in cholestasis. FXR null mice also develop cholesterol gallstones because of deficiency in the excretion of bile acids and phospholipids. FXR agonists prevent gallstone formation in this model. 19 CAR, the constitutive androstane receptor (NR113), is another important nuclear receptor involved in adaptive responses to cholestasis and hyperbilirubinemia. CAR plays an important role in the detoxification of bile acids by stimulating bile acid sulfation through the activation of steroid sulphotransferases (SULT2A9). CAR also mediates the response of the liver to phenobarbital and other similar compounds and has been shown to be an important determinant of the expression of pathways of bilirubin clearance including hepatic uptake by Oatp2, binding to the intracellular proteins gluthathione-s-transferases (A1 and 2), conjugation with glucuronide by the microsomal bilirubin uridine 5= diphos-

4 738 JAMES L. BOYER GASTROENTEROLOGY Vol. 129, No. 2 phate-glucuronosyltransferase (UGT1a1), and excretion of bilirubin diglucuronide into bile by Mrp2. Activation of Car is necessary and sufficient to mediate resistance to the hepatotoxicity of lithocholic acid. 20 All of these effects are abrogated in the Car knock-out mice. Car also coordinately up-regulates both Sult2a1, which sulfates hydroxy-bile acids, and Mrp4, which functions to extrude bile acid sulfates. 21 In humans, CAR agonists including the traditional herbal medicine Yin Shi Huang and phenobarbital reduce jaundice in neonates and both serum bilirubin and bile acid levels in patients with primary biliary cirrhosis, respectively, 22,23 presumably by stimulating the expression of these beneficial detoxification pathways. PXR (NR112) is related closely to CAR and both nuclear receptors coordinately regulate a similar group of genes that determine hepatic oxidative metabolism, conjugation reactions, and transmembrane transport. 7,13,24 Examples of these genes include many of the cytochrome P450s (CYPs), the glutathione-s-transferases, the uridine 5= diphosphate-glucuronosyltransferases (UGTs), the sulfotransferases (SULTs), the MRPs, and the OATPs, all of which are important in the elimination of xenobiotics and the hepatic clearance of endogenous compounds such as bile acids and bilirubin. Rifampicin is a typical ligand for PXR and therefore it is not surprising that treatment with this drug resulted in up-regulation of CYP3A4, UGT1A1, and MRP2 and 3 in the study by Marschall et al. Rifampicin also has been shown to induce CYP3A metabolism in patients with primary biliary cirrhosis. 25 Rifampicin is an effective treatment for severe pruritus in cholestatic patients 26,27 and has been associated with increased urinary excretion of bile acid glucuronides. 28 Long-term treatment of patients with primary biliary cirrhosis with rifampicin tends to decrease serum bilirubin levels and induces remission in some patients with benign recurrent intrahepatic cholestasis. 29 The beneficial effects on pruritus have been postulated to be mediated by phase I hydroxylation reactions of the hydrophobic bile acids lithocholic and deoxycholic acid via CYP3A4 followed by glucuronidation (UGTs) 28 or sulfation (SULTs). Based on animal models and in vitro transport assays, export of these bile-acid conjugates into the blood then would be expected to occur via MRP3 or 4 at the sinusoidal membrane. Both MRP3 and MRP4 have been reported to be up-regulated in patients with primary biliary cirrhosis 10 and cholestatic infants with genetic defects in BSEP and MDR3. 11 Whether these bile-salt conjugates then enter the urine solely by glomerular filtration or also undergo excretion from the renal tubular epithelium is not clear although MRP2 and MRP4 both are located on the luminal membranes of the renal proximal tubular epithelium and both are upregulated in mice kidneys after treatment with UDCA. 30 UDCA is a weak FXR agonist but a strong ligand for PXR. 31 However, in the study by Marschall et al, UDCA was only a weak inducer of CYP34A activity but a significant enhancer of the expression of BSEP and MDR3 on the canalicular membrane and MRP4 on the sinusoidal membrane. Other studies have found no effects of UDCA on CYP3A metabolic activity in patients with primary biliary cirrhosis. 25 UDCA may have other beneficial effects. Animal studies previously have shown that UDCA enhances the expression of Bsep and Mrp2 protein and stimulates the insertion of Bsep and Mrp2 into the canalicular membrane. 32 UDCA feeding in mice also induces hepatic Mrp3 and Mrp4 expression. 10 Differences in nuclear-receptor specificity in different species and other regulatory posttranscriptional mechanisms may account for some of these reported discrepancies between animal and human studies. Ursodeoxycholic acid therapy also increases the hydrophobicity of the bile acid pool and has anti-apoptotic and anti-inflammatory effects that could be affecting the clinical outcome. Thus, it is not clear to what extent UDCA s beneficial effects in cholestasis relate to its properties as a nuclear-receptor ligand. In addition to FXR, CAR, and PXR, which have coordinated protective effects in cholestasis, 12 other receptors such as the retinoic acid receptor, the vitamin D receptor, and the glucocorticoid receptor, among others, also may be involved in regulating some of these pathways, the latter possibly accounting in some instances for beneficial effects of corticosteroids in cholestasis. 9 Gene transcriptional regulation is enormously complex and multiple and redundant nuclear factors almost always are involved. This suggests that combinations of specific nuclear receptor ligands may be useful to maximize induction of gene expression. In addition, other cofactors, activators, repressors, histone acetylators, and methylation reactions also influence gene transcription and may become appropriate targets in the future for therapeutic intervention with small molecules as their specific actions are defined. The findings of Marschall et al that rifampicin and UDCA have multiple but separate beneficial effects on gene expression and that nuclear-receptor activators such as phenobarbital and Yin Zhi Huang have had positive results in patients with jaundice suggest that clinical trials of combinations of small molecules that could target multiple but complementary receptors such as FXR, CAR, and PXR should be considered in patients with chronic cholestasis. What are the concerns? Chole-

5 August 2005 NUCLEAR RECEPTOR LIGANDS 739 static liver injury already results in spontaneous major but incompletely protective adaptations in the expression of key transporters and enzymes that regulate the hepatic excretion of endogenous toxins such as bile acids and bilirubin Thus, it remains to be determined whether further significant beneficial adaptive responses can be obtained by targeted nuclear-receptor therapy. Furthermore, there always is concern that nonspecific stimulation of receptors that also regulate other metabolic processes, not only in the liver but also in other tissues including the intestine and kidney, could have unanticipated deleterious effects. Nevertheless, it seems likely that further studies based on mechanistic explanations at the molecular level will need to be performed, particularly in early stages of cholestatic liver disease in which these endogenous adaptive responses may not yet be maximal. Suitable candidates might include patients with primary biliary cirrhosis and other chronic cholestatic disorders such as sclerosing cholangitis, cystic fibrosis, parenteral alimentation induced cholestasis, and patients at risk for formation of cholesterol gallstones. The genomic revolution has provided molecular explanations for long-standing, even ancient, empiric medical practices that we now recognize as nuclear-receptor therapy. Novel strategies for the treatment of cholestatic liver disease should emerge from this new understanding. References 1. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science 2001;294: Willson TM, Jones SA, Moore JT, Kliewer SA. Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism. Med Res Rev 2001;21: Karpen SJ. Nuclear receptor regulation of hepatic function. J Hepatol 2002;36: Goodwin B, Moore JT. CAR: detailing new models. 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6 740 JAMES L. BOYER GASTROENTEROLOGY Vol. 129, No Cancado EL, Leitao RM, Carrilho FJ, Laudanna AA. Unexpected clinical remission of cholestasis after rifampicin therapy in patients with normal or slightly increased levels of gamma-glutamyl transpeptidase. Am J Gastroenterol 1998;93: Zollner G, Fickert P, Fuchsbichler A, Silbert D, Wagner M, Arbeiter S, Gonzalez FJ, Marschall H-U, Zatloukal K, Denk H, Trauner M. Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine. J Hepatol 2003;39: Schuetz EG, Strom S, Yasuda K, Lecureur V, Assem M, Brimer C, Lamba J, Kim RB, Ramachandran V, Komoroski BJ, Venkataramanan R, Cai H, Sinal CJ, Gonzalez FJ, Schuetz JD. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450. J Biol Chem 2001;276: Paumgarnter GABU. Mechanisms of action and therapeutic efficacy of ursodeoxycholic acid in cholestatic liver disease. Clin Liver Dis 204;8: Lee J, Boyer JL. Molecular alterations in hepatocyte transport mechanisms in acquired cholestatic liver disorders. Semin Liver Dis 2000;20: Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function and regulation. Physiol Rev 2003;83: Address requests for reprints to: James L. Boyer, M.D., Department of Medicine, Yale University School of Medicine, PO Box , 33 Cedar Street, 1080 LMP, New Haven, Connecticut e- mail: james.boyer@yale.edu; fax: (203) Dr. Boyer is a consultant for Intercept Pharmaceuticals, a nuclear receptor company.