On the Pharmacology of Farnesoid X Receptor Agonists: Give me an “A”, Like in “Acid”

The Farnesoid X Receptor (FXR) has recently moved into the spotlight through the release of clinical data using Obeticholic Acid, an FXR agonist, that demonstrated effectiveness of this bile acid-like drug in patients with Primary Biliary Cirrhosis and Non-alcoholic Steatohepatitis (NASH). FXR holds the promise to become an attractive drug target for various conditions, from Non-alcoholic Fatty Liver Disease (NAFLD), NASH, liver cirrhosis, portal hypertension and a variety of cholestatic disorders to intestinal diseases including inflammatory bowel disease and bile acid diarrhea. Despite the wide therapeutic potential, surprisingly little is known about the pharmacology, pharmacokinetics and tissue distribution properties of drugs targeting FXR. Are tissue specific FXR agonists preferable for different indications, or might one type of ligand fit all purposes? This review aims to summarize the sparse data which are available on this clinically and pharmacologically relevant topic and provides a mechanistic model for understanding tissue-specific effects in vivo

In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases

AbstractThe results of previous in vitro experiments indicate that a glycosylphosphatidylinositol (GPI)-anchored protein may play an important role in the guidance of temporal retinal axons during the formation of the topographically ordered retinotectal projection. We have purified and cloned a GPI-anchored, 25 kDa glycoprotein that is a good candidate for a molecule involved in this process. During the time of innervation by retinal ganglion cells, this protein is gradedly expressed in the posterior part of the developing tectum. In two different in vitro assay systems, the recombinant protein induces growth cone collapse and repulsion of retinal ganglion cell axons. These phenomena are observed for axons of temporal as well as nasal origin, indicating that an additional activity may be necessary to confer the nasotemporal specificity observed in previous assays. We named the protein RAGS (for repulsive axon guidance signal). The sequence of RAGS shows significant homology to recently identified ligands for receptor tyrosine kinases of the Eph subfamily

FXR Controls the Tumor Suppressor NDRG2 and FXR Agonists Reduce Liver Tumor Growth and Metastasis in an Orthotopic Mouse Xenograft Model

<div><p>The farnesoid X receptor (FXR) is expressed predominantly in tissues exposed to high levels of bile acids and controls bile acid and lipid homeostasis. FXR^−/− mice develop hepatocellular carcinoma (HCC) and show an increased prevalence for intestinal malignancies, suggesting a role of FXR as a tumor suppressor in enterohepatic tissues. The N-myc downstream-regulated gene 2 (NDRG2) has been recognized as a tumor suppressor gene, which is downregulated in human hepatocellular carcinoma, colorectal carcinoma and many other malignancies.</p> <p>We show reduced NDRG2 mRNA in livers of FXR^−/− mice compared to wild type mice and both, FXR and NDRG2 mRNAs, are reduced in human HCC compared to normal liver. Gene reporter assays and Chromatin Immunoprecipitation data support that FXR directly controls NDRG2 transcription via IR1-type element(s) identified in the first introns of the human, mouse and rat NDRG2 genes. NDRG2 mRNA was induced by non-steroidal FXR agonists in livers of mice and the magnitude of induction of NDRG2 mRNA in three different human hepatoma cell lines was increased when ectopically expressing human FXR. Growth and metastasis of SK-Hep-1 cells was strongly reduced by non-steroidal FXR agonists in an orthotopic liver xenograft tumor model. Ectopic expression of FXR in SK-Hep1 cells reduced tumor growth and metastasis potential of corresponding cells and increased the anti-tumor efficacy of FXR agonists, which may be partly mediated via increased NDRG2 expression. FXR agonists may show a potential in the prevention and/or treatment of human hepatocellular carcinoma, a devastating malignancy with increasing prevalence and limited therapeutic options.</p> </div

Activities of the different FXR agonists in different assay systems.

<p>(<b>A</b>) Activities in the biochemical TR-FRET assay using the human FXR-LBD. (<b>B</b>) Activities in Gal4-FXR-LBD fusion cellular reporter assays using mouse (mFXR) and human FXR-LBDs (hFXR). Efficacy is provided as % relative to the maximum effect of GW4064 in the respective assays.</p

<i>In vitro</i> characterization of SK-Hep-1 and SK-GI-18 cells.

<p><b>A</b>, Expression of FXR in SK-Hep-1 and the FXR overexpressing SK-GI-18 cells. The signals in western blot analysis from aliquots of whole cell extracts representing FXR and GAPDH are indicated. <b>B</b>, Increased expression and inducibility of NDRG2 mRNA by Px20350 in SK-GI-18 cells compared to parental SK-Hep-1 cells. SK-Hep-1 cells and SK-GI-18 cells were grown in 24-well plates (n = 4) in presence of DMSO or 0.5 µM Px20350 for 72 hours. Total RNA was isolated from cells at around 60% confluency. The diagram shows RT-qPCR data for NDRG2 mRNA drawn as relative fold increase over the TBP corrected values for DMSO treated SK-Hep-1 cells using the ΔΔCt method. Statistical significance in a nonpaired Student's t-test: *, ** and *** = p<0.05, <0.01 and <0.001. <b>C</b>, Influence of PX20350 on proliferation of SK-Hep-1 and SK-GI-18 cells <i>in vitro</i>. SK-Hep-1 or SK-GI-18 cells were seeded in a 96 well plate and grown for 4 days in presence of DMSO or increasing concentrations of FXR ligand Px20350. Relative proliferation was measured by flurescence determination of DNA content using the CyQuant Direct Proliferation assay. D. Inhibition of proliferation by PX20606 is blunted in SK-GI-18-sh3-14 cells stably expressing shRNA directed against NDRG2. SK-Hep-1, SK-GI-18 NC-16 expressing a negative control shRNA and SK-GI-18-3-14, expressing an NDRG2 shRNA were seeded at 3000 cells/well in a 96 well plate and grown for 3 days in presence of DMSO or increasing concentrations of PX20606. Relative proliferation was measured by flurescence determination of DNA content using the CyQuant Direct Proliferation assay. <b>E</b>, Migration of SK-GI-18 and SK-Hep-1 cells in presence or absence of Px20350 (1 µM). Cells were seeded at 7000 cells per 96 well (in quadruplicates) of a Oris Fibronectin Coated Plate (Platyplus) and seeding stoppers removed after 14 h and cells grown in medium with DMSO as a vehicle or 1 µM Px20350 in DMSO for another 72 hours before quantification of migrated cells using the Cyquant Direct Cell Proliferation Assay.</p

Time course of tumor growth and metastasis.

<p><b>A and B</b>, Nude mice were inoculated with either 5×10⁶ SK-Hep-1 (A) or SK-GI-18 cells (B), randomized into groups and animals were treated by daily gavage starting on day 4 with vehicle, Px20606 (10 mg/kg/d) or Sorafenib (100 mg/kg/d) as indicated. <i>In vivo</i> imaging pictures for representative animals from each group that received SK-Hep-1 (A) or SK-GI-18 (B) cells at the indicated days are displayed. <b>C</b>, Time course of tumor development in the liver. The <i>in vivo</i> fluorescence intensities at day 7 were set to 100% for each group and data plotted for data gathered at days 7, 14, 21, 28, 35 and 56. The sharp rise of fluorescence in the SK-Hep-1/Px20606 group from day 35 to day 56 is due to sharp increases in tumor sizes in just three individual animals out of a total of nine animals (fluorescence data from these animals on day 56 are circled in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043044#pone-0043044-g006" target="_blank">Fig. 6A</a>).</p

Induction of NDRG2 in human hepatoma cell lines.

<p><b>A</b>, Ectopic expression of FXR in HepG2-FXR5 compared to HepG2 parental cells. Positions of bands representing FXR and GAPDH proteins in the western blots from aliquots of whole cell extracts are indicated. <b>B</b>, Dose dependent induction of NDRG2 mRNA in HepG2 and HepG2-FXR5 cells by the FXR agonist PX20350. HepG2 or HepG2-FXR5 cells were grown for 18 h in medium containing either DMSO, 0.5 µM of PX20350 or 0.5 µM of PX20606 in 96 well plates as indicated and relative fold induction of NDRG2 mRNA over the DMSO control was determined by RT-qPCR using the ΔΔCt method. <b>C</b>, Induction of NDRG2 protein by the FXR agonists Px20350 and PX20606 in HepG2-FXR5 cells. HepG2-FXR5 cells were grown to a density of around 60% and further cultivated for 18 h in medium supplemented with either DMSO as a vehicle, 0.5 µM PX20350 or 0.5 µM PX20606 as indicated and aliquots of whole cell extracts analysed by western blots. Relative positions of size marker protein bands and the position of NDRG2 and GAPDH proteins are indicated. <b>D</b>, Stable overproduction of FXR in HuH-7-37 compared to HuH-7 cells. Positions of bands representing FXR and GAPDH proteins analysed by western blots from aliquots of whole cell extracts are indicated. <b>E</b>, Dose dependent induction of NDRG2 mRNA in HuH7 and HuH7-37 cells by FXR agonist Px20350. HuH7 or HuH7-37 cells were grown for 18 h in medium containing either DMSO or increasing concentrations of PX20350 in DMSO in 96 well plates and relative fold induction over DMSO control of NDRG2 mRNA was determined by RT- qPCR using the ΔΔCt method. <b>F</b>, NDRG2 is a direct transcriptional target of FXR. HepG2-FXR5 cells, grown in quadruplicates in 12 well plates at a density of around 60%, were further incubated for 4 hours in growth medium supplemented with either DMSO, 0.5 µM PX20350 (PX) or 0.5 µM PX20350 together with 10 µM Cycloheximide (CHX). The relative fold induction of NDRG2- and SHP- mRNAs over DMSO as control were determined by RT- qPCR using the ΔΔCt method and a Student's t-test was performed (*** = p<0.001).</p

Ndrg2 mRNA is induced in wt mice by FXR agonists, reduced in livers of FXR^−/− versus wt mice and both FXR and NDRG2 mRNAs are reduced in human HCC versus normal liver.

<p><b>A</b>, Ndrg2 mRNA induction in livers of C57Bl6 mice by FXR agonists. C57Bl6 mice on normal chow received a bolus of vehicle (n = 9), or 30 mg/kg of Px20606 or 30 mg/kg of GW4064 in vehicle (n = 6 each) by oral gavage. Mice were sacrificed after 4 h and liver RNA isolated for RT-qPCR analysis. Ndrg2 and Shp mRNA expression levels were calculated according to the ΔΔCt method using normalization to Tbp as a house keeping transcript and the mean level of Ndrg2 and Shp expression in vehicle treated mice were set to 1.0. Data are presented as the mean ± standard error of the mean (±SEM). Statistical significance in a nonpaired Student's t-test: *, ** and *** = p<0.05, <0.01 and <0.001. <b>B</b>, Quantitative determination of mRNA's in livers of wild type (wt) and FXK^−/− (KO) mice. Relative mRNA levels of Ndrg2, Shp, Cyp7a1 and Cyclophilin E were determined by RT-qPCR from wt (n = 5) and FXR^−/− (n = 5) mice as indicated. Tbp-normalized relative mRNA levels of wt mice were set to 1.0 respectively. <b>C</b>, Decreased expression of FXR and the FXR target genes NDRG2 and SHP in human HCC. FXR, NDRG2 and SHP mRNA levels were quantified by RT- qPCR in 8 normal, 34 HCC from different stages (7 samples stage I, 8 samples of stages II and IIIA, 3 samples of stage IV) and 12 non-HCC liver disease (LD) derived cDNA's. Relative folds of mRNA expression levels between normal livers and the respective stages of HCC or non-HCC liver disease were calculated according to the ΔΔCt method using normalization to TBP. The mean levels of FXR, NDRG2 and SHP expression in normal livers were set to 1.0 respectively and relative mRNA levels are expressed as mean ± SEM. Statistical significance in a nonpaired Student's t-test: *, P<0.05; **, P<0.01; ***, P<0.001.</p

Number of animals with metastases in lymph nodes (A) and bone (B) and the total numbers of animals in each group surviving till day 56 (in parentheses) are shown.

<p>One animal died in each of the groups receiving Sorafenib, in the course of the experiment.</p