Farnesoid X receptor agonist WAY-362450 attenuates liver inflammation and fibrosis in murine model of non-alcoholic steatohepatitis.

BACKGROUND/AIMS: The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, which plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. Here we investigated whether WAY-362450, a synthetic potent FXR agonist, could protect against non-alcoholic steatohepatitis (NASH) in mice fed a methionine and choline-deficient (MCD) diet. METHODS: Male C57BL/6 mice on the MCD diet were treated with or without WAY-362450 (30 mg/kg) for 4 weeks. RESULTS: The elevations of serum ALT and AST activities induced by the MCD diet were decreased with WAY-362450 treatment. In terms of liver histology, while WAY-362450 treatment showed no impact on hepatic triglyceride accumulation, it significantly reduced inflammatory cell infiltration and hepatic fibrosis. The reduction in inflammatory cell infiltration correlated with deceased serum levels of keratinocyte derived chemokine (mKC) and MCP 1 and decreased hepatic gene expression of MCP-1 and VCAM-1. The reduction of hepatic fibrosis by WAY-362450 treatment corresponded to a reduction in hepatic gene expression of fibrosis markers. The positive effects of WAY-362450 were FXR-dependent since no protection was observed in MCD diet-fed FXR deficient mice. CONCLUSIONS: These findings demonstrate that FXR agonists may be useful for the treatment of non-alcoholic steatohepatitis.

Suppression of interleukin-6-induced C-reactive protein expression by FXR agonists.

C-reactive protein (CRP), a human acute-phase protein, is a risk factor for future cardiovascular events and exerts direct pro-inflammatory and pro-atherogenic properties. The farnesoid X receptor (FXR), a member of the nuclear hormone receptor superfamily, plays an essential role in the regulation of enterohepatic circulation and lipid homeostasis. In this study, we report that two synthetic FXR agonists, WAY-362450 and GW4064, suppressed interleukin-6-induced CRP expression in human Hep3B hepatoma cells. Knockdown of FXR by short interfering RNA attenuated the inhibitory effect of the FXR agonists and also increased the ability of interleukin-6 to induce CRP production. Furthermore, treatment of wild type C57BL/6 mice with the FXR agonist, WAY-362450, attenuated lipopolysaccharide-induced serum amyloid P component and serum amyloid A3 mRNA levels in the liver, whereas no effect was observed in FXR knockout mice. These data provide new evidence for direct anti-inflammatory properties of FXR.

Discovery and implementation of transcriptional biomarkers of synthetic LXR agonists in peripheral blood cells.

BACKGROUND: LXRs (Liver X Receptor alpha and beta) are nuclear receptors that act as ligand-activated transcription factors. LXR activation causes upregulation of genes involved in reverse cholesterol transport (RCT), including ABCA1 and ABCG1 transporters, in macrophage and intestine. Anti-atherosclerotic effects of synthetic LXR agonists in murine models suggest clinical utility for such compounds. OBJECTIVE: Blood markers of LXR agonist exposure/activity were sought to support clinical development of novel synthetic LXR modulators. METHODS: Transcript levels of LXR target genes ABCA1 and ABCG1 were measured using quantitative reverse transcriptase/polymerase chain reaction assays (qRT-PCR) in peripheral blood from mice and rats (following a single oral dose) and monkeys (following 7 daily oral doses) of synthetic LXR agonists. LXRalpha, LXRbeta, ABCA1, and ABCG1 mRNA were measured by qRT-PCR in human peripheral blood mononuclear cells (PBMC), monocytes, T- and B-cells treated ex vivo with WAY-252623 (LXR-623), and protein levels in human PBMC were measured by Western blotting. ABCA1/G1 transcript levels in whole-blood RNA were measured using analytically validated assays in human subjects participating in a Phase 1 SAD (Single Ascending Dose) clinical study of LXR-623. RESULTS: A single oral dose of LXR agonists induced ABCA1 and ABCG1 transcription in rodent peripheral blood in a dose- and time-dependent manner. Induction of gene expression in rat peripheral blood correlated with spleen expression, suggesting LXR gene regulation in blood has the potential to function as a marker of tissue gene regulation. Transcriptional response to LXR agonist was confirmed in primates, where peripheral blood ABCA1 and ABCG1 levels increased in a dose-dependent manner following oral treatment with LXR-623. Human PBMC, monocytes, T- and B cells all expressed both LXRalpha and LXRbeta, and all cell types significantly increased ABCA1 and ABCG1 expression upon ex vivo LXR-623 treatment. Peripheral blood from a representative human subject receiving a single oral dose of LXR-623 showed significant time-dependent increases in ABCA1 and ABCG1 transcription. CONCLUSION: Peripheral blood cells express LXRalpha and LXRbeta, and respond to LXR agonist treatment by time- and dose-dependently inducing LXR target genes. Transcript levels of LXR target genes in peripheral blood are relevant and useful biological indicators for clinical development of synthetic LXR modulators.

Adipocyte fatty acid-binding protein (aP2), a newly identified LXR target gene, is induced by LXR agonists in human THP-1 cells.

The liver X receptors (LXRalpha and LXRbeta), ligand-activated transcription factors, belong to the superfamily of nuclear hormone receptors and have been shown to play a major role in atherosclerosis by modulating cholesterol and triglyceride metabolism. In this report, we describe a novel LXR target, the adipocyte fatty acid binding protein (aP2), which plays an important role in fatty acid metabolism, adipocyte differentiation and atherosclerosis. While LXR agonists induce aP2 mRNA expression in human monocytes (THP-1 cells) and macrophages in a time- and concentration-dependent manner, they have no effect on aP2 expression in human adipocytes. The increase in aP2 mRNA level was additive when THP-1 cells were treated with LXR and PPARgamma agonists. Also, an RXR agonist induced aP2 expression in these cells. While no additive effect was observed with LXR and RXR agonists, additive effects were observed with RXR and PPARgamma agonists. GW9662, a potent PPARgamma antagonist, inhibited PPARgamma-induced aP2 expression without affecting LXR-mediated aP2 expression indicating the induction is mediated directly through LXR activation. Analysis of human aP2 promoter revealed a potential LXR response element (LXRE). Gel shift data showed that the LXRalpha/RXRalpha heterodimer bound to the LXRE motif in aP2 promoter in vitro in a sequence-specific manner. Deletion and mutation analyses of the proximal aP2 promoter confirm that this is a functional LXRE. These data indicate for the first time that human macrophage aP2 promoter is a direct target for the regulation by LXR/RXR heterodimers.

Further modification on phenyl acetic acid based quinolines as liver X receptor modulators.

A series of phenyl acetic acid based quinolines was prepared as LXR modulators. An SAR study in which the C-3 and C-8 positions of the quinoline core were varied led to the identification of two potent LXR agonists 23 and 27. Both compounds displayed good binding affinity for LXRbeta and LXRalpha, and increased expression of ABCA1 in THP-1 cells. These two compounds also had desirable pharmacokinetic profiles in mice and displayed in vivo efficacy in a 12-week Apo E knockout mouse lesion model.

Discovery of phenyl acetic acid substituted quinolines as novel liver X receptor agonists for the treatment of atherosclerosis.

A structure-based approach was used to optimize our new class of quinoline LXR modulators leading to phenyl acetic acid substituted quinolines 15 and 16. Both compounds displayed good binding affinity for LXRbeta and LXRalpha and were potent activators in LBD transactivation assays. The compounds also increased expression of ABCA1 and stimulated cholesterol efflux in THP-1 cells. Quinoline 16 showed good oral bioavailability and in vivo efficacy in a LDLr knockout mouse model for lesions.

Identification and characterization of triosephosphate isomerase that specifically interacts with the integrin alphaIIb cytoplasmic domain.

Integrin alphaIIb/beta3 (IIb/IIIa), a platelet fibrinogen receptor, has been shown to play a critical role in thrombosis and hemostasis. However, the mechanisms by which ligands interact with the alphaIIb/beta3 receptor is not very clear at this time. The interaction between the ligand, the receptor and the transmission of extracellular signals may involve the cytoplasmic domains of these integrins. The objective of this investigation was to identify novel proteins that interact with the cytoplasmic tail of alphaIIb. Using alphaIIb cytoplasmic tail as the bait and a yeast two-hybrid system, we have identified three separate clones containing inserts that encoded the same protein with different truncated N-terminals. Sequence analysis showed that the inserts of the three clones encoded a previously identified enzyme: triose phosphate isomerase (TPI). In addition, we demonstrated that TPI failed to interact with the integrin alpha2 tail, beta3 tail and lamin, but showed a weak binding to the alphaV tail which shares the highest homology with alphaIIb tail among the integrin alpha family. Site-directed mutagenesis studies around the homology region indicated that the critical peptide sequence necessary for the interaction between TPI and alphaIIb tail is GFFKRNRPPLEE. Using RT-PCR, we have demonstrated the presence of TPI mRNA in platelets. In addition, experiments were also performed to demonstrate specific binding of TPI to alphaIIb using an ELISA and fusion protein. Taken together, these data suggest that TPI specifically interacts with alphaIIb and may play a critical role in alphaIIb/beta3-mediated platelet function.