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EP1137940A1 - Procede d'identification de ligands de recepteurs nucleaires - Google Patents

Procede d'identification de ligands de recepteurs nucleaires

Info

Publication number
EP1137940A1
EP1137940A1 EP99971090A EP99971090A EP1137940A1 EP 1137940 A1 EP1137940 A1 EP 1137940A1 EP 99971090 A EP99971090 A EP 99971090A EP 99971090 A EP99971090 A EP 99971090A EP 1137940 A1 EP1137940 A1 EP 1137940A1
Authority
EP
European Patent Office
Prior art keywords
nuclear receptor
component
marking
binding domain
interaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99971090A
Other languages
German (de)
English (en)
Other versions
EP1137940A4 (fr
Inventor
Steven Gerard Glaxo Wellcome Inc. BLANCHARD
Derek J. Glaxo Wellcome Inc. PARKS
Julie Beth Glaxo Wellcome Inc. STIMMEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Glaxo Group Ltd
Original Assignee
Glaxo Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Glaxo Group Ltd filed Critical Glaxo Group Ltd
Publication of EP1137940A1 publication Critical patent/EP1137940A1/fr
Publication of EP1137940A4 publication Critical patent/EP1137940A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins

Definitions

  • RXR is an orphan nuclear receptor initially identified from a rat liver cDNA library (8) that is most closely related to the insect ecdysone receptor.
  • the ligand binding domain of the receptor was cloned and expressed in support of an effort to develop a robust assay to identify a novel ligand.
  • the availability of ligands for FXR will aid in the elucidation of the physiological role of this receptor.
  • the information gained will further increase understanding of nuclear receptors as a target class.
  • Another aspect of the invention is a new nuclear receptor-peptide assay for identifying ligands.
  • This assay utilizes fluorescence resonance energy transfer (FRET) and was used to test whether putative ligands bound to FXR.
  • FRET fluorescence resonance energy transfer
  • the FRET assay is based upon the principle that ligands induce conformational changes in nuclear receptors that facilitate interactions with coactivator proteins required for transcriptional activation.
  • FRET a fluorescent donor molecule transfers energy via a non-radiative dipole-dipole interaction to an acceptor molecule (which is usually a fluorescent molecule).
  • FRET is a standard spectroscopic technique for measuring distances in the 10-70A range.
  • Fig. 1 As shown in Fig 1 , ligand binding to LXR ⁇ measured by modulation of LXR ⁇ :RXR heterodimer formation.
  • Fig.2. shows ligand binding to FXR measured by modulation of
  • a method for the rapid and simple determination of a ligand for a nuclear receptor which comprises contacting a component to be tested with an isolated nuclear receptor ligand binding domain which may be associated with a marking component, and a dimerization partner for the nuclear receptor ligand binding domain which is also associated with a marker; and measuring the interaction between the marking components to determine whether the component to be tested modifies heterodimerization.
  • markers may be used in the process of the present invention such as radioactive markers.
  • the marker could also be a fluorescent dye. When the marker is radioactive, scintillation proximity may be used to measure the marker. When the marker used is a fluorescent dye, homogenous time-resolved fluorimetry may be used to detect the marker.
  • Other known marking and measuring techniques may be used depending on the marker. However, the markers need to be in close proximity to indicate heterodimerization. That is, to indicate that the component to be tested functions as a ligand for the dimerization pair.
  • This method for the rapid determination of a ligand for a nuclear receptor comprises contacting a component to be tested with an isolated nuclear receptor ligand binding domain which is associated with a first marking component, and a heterodimeric partner for the nuclear receptor ligand binding domain associated with a second marking component, and measuring the interaction between the marking components to determine whether the component to be tested modifies hetero-dimerization.
  • the first marking component may be a radioactive marker and the second marking component (or second marker) may be a SPA bead.
  • the interaction of the markers in this case is determined by scintillation proximity.
  • the first marking component may be a first fluorescent dye emitting at an emitting wavelength which excites the second marking component which may be a second fluorescent dye.
  • the interaction of the markers in this case is determined by homogenous time-resolved fluorimetry.
  • the interaction of the marking components in either case is measured by comparing a signal produced by a combination of the heterodimeric partner, the isolated nuclear receptor binding domain and the component to be tested with a signal produced by a combination of the heterodimeric partner and the isolated nuclear receptor ligand binding domain in the absence of the component to be tested.
  • Liver X receptor alpha (LXR ⁇ ) is an orphan nuclear receptor initially identified from a rat liver cDNA library (1 ). Human LXR ⁇ (2) and LXR ⁇ (3) have also been identified. The ligand binding domains of these receptors were cloned and expressed in support of an effort to develop a robust assay to identify a novel ligand. Oxysterols, including 24(S),25- epoxycholesterol have been identified as weak activators for these receptors (4,5). The availability of more potent and selective ligands for the LXRs may aid in the elucidation of the physiological role(s) of these receptors. In addition, the information gained will further increase understanding of nuclear receptors as a target class.
  • LXR ⁇ nuclear receptor Liver X Receptor beta
  • the method measures the ability of putative ligands to mediate the heterodimerization between the purified bacterial expressed LXR ⁇ , and RXR ⁇ , ligand binding domains (LBD). Detection of the associated LBD's are measured by time resolved fluorimetry (TRF).
  • TRF time resolved fluorimetry
  • the purified LBD of LXR ⁇ is labeled with biotin then mixed with stoichiometric amounts of europium labeled streptavidin (Wallac Inc).
  • the purified LBD of RXR ⁇ is labeled with CY5 Tm .
  • Equimolar amounts of each modified LBD are mixed together and allowed to equilibrate for at least one hour prior to the addition to either variable or constant concentrations of the sample for which the affinity is to be determined.
  • the time-resolved fluorescent signal is quantitated using a fluorescent plate reader.
  • the affinity of the test compound is estimated from a plot of fluorescence versus concentration of test compound added. A basal level of LXR ⁇ :RXR ⁇ heterodimer formation is observed in the absence of added ligand. Ligands that promote heterodimer formation induce a concentration-dependent increase in time-resolved fluorescent signal.
  • LXR ⁇ LBD Human LXR ⁇ Ligand Binding Domain
  • Genbank accession number U 07132, amino acids 185-461 was expressed in E.coli strain BL21 (DE3) as an amino-terminal polyhistidine tagged fusion protein. Expression was under the control of an IPTG inducible T7 promoter. DNA encoding this recombinant protein and a modified polyhistidine tag was subcloned into the expression vector pRSETa (Invitrogen).
  • This lysate was loaded onto a column (6 x 8 cm) packed with Sepharose (Ni ++ charged) Chelation resin (Pharmacia) and pre-equilibrated with TBS pH 8.5/ 50mM imidazole. After washing to baseline absorbance with equilibration buffer, the column was developed with a linear gradient of 50 to 275 mM imidazole in TBS, pH 8.5. Column fractions were pooled and dialyzed against TBS, pH 8.5, containing 5% 1 ,2-propanediol, 5mM DTT and 0.5mM EDTA.
  • the protein sample was concentrated using Centri-prep 10K (Amicon) and subjected to size exclusion, using a column (3 x 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS, pH 8.5, containing 5% 1 ,2-propanediol, 5mM DTT and 0.5mM EDTA.
  • Biotinylation of LXR ⁇ was concentrated using Centri-prep 10K (Amicon) and subjected to size exclusion, using a column (3 x 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS, pH 8.5, containing 5% 1 ,2-propanediol, 5mM DTT and 0.5mM EDTA.
  • LXR ⁇ LBD was desalted/buffer exchanged using PD-10 gel filtration columns into PBS [100mM Na Phosphate, pH 7.2, 150 mM NaCI].
  • LXR ⁇ LBD was diluted to approximately 10 ⁇ M in PBS and five-fold molar excess of NHS-LC-Biotin (Pierce) was added in a minimal volume of PBS. This solution was incubated with gentle mixing for 30 minutes at room temperature. The biotinylation modification reaction was stopped by the addition of 2000x molar excess of Tris-HCI, pH 8.
  • the modified LXR ⁇ LBD was dialyzed against 4 buffer changes, each of at least 50 volumes, PBS containing 5mM DTT 2mM EDTA and 2% sucrose.
  • RXR ⁇ LBD Human Retinoid X Receptor alpha Ligand Binding Domains RXR-alpha LBD (amino acids 225-462) was expressed in E. coli strain BL21 (DE3) as an amino-terminal polyHistidine tagged fusion protein. Expression was under the control of an IPTG inducible T7 promoter. DNA encoding this recombinant protein and a modified histidine tag was subcloned into the expression vector pRSETa (Invitrogen). The sequence used in the construction of RXR-alpha LBD was derived from Genbank accession number X52773.
  • RXR ⁇ LBD Purified RXR ⁇ LBD was diluted to approximately 10 ⁇ M in PBS and approximately five-fold molar excess of Cy5TM monofunctional reactive dye [NHS ester] (Amersham Life Sciences) was added in a minimal volume of PBS. This solution was incubated in the dark with mixing for 30 minutes at ambient room temperature (approximately 23°C). The modification reaction was stopped by the addition of an excess of Tris-HCI, pH 8. Fluorescent dye modified RXR ⁇ LBD was dialyzed at 4°C, with minimal exposure to light, against 4 buffer changes, each of at least 50 volumes, PBS containing 5mM DTT, 2mM EDTA, and 2% (w/v) sucrose. Aliquots were frozen on dry ice and stored at -80°C.
  • Assay Buffer 50 mM KCI, 0.1 mg/mL BSA, 10 mM DTT and 50 mM Tris (pH 8)
  • the stock buffer is made by dissolving 2.853g Tris base, 4.167 g Tris hydrochloride, 3.73 g KCI, and 0.1 g fatty acid free bovine serum albumin, in 1 L of deionized water. The pH is checked and adjusted to 8.0, if necessary, before adjusting to final volume. 0.154 g of solid DTT is added per 100 mL of buffer just before the start of an experiment.
  • F sample is the signal observed in a particular sample well
  • F tota is the signal observed in the presence of control inhibitor
  • is the count rate observed in the presence of no ligand.
  • were averages of the corresponding control wells included on every plate.
  • the data were first normalized to % of control using eq. (1 ).
  • a plot of C L the % of control observed at ligand concentration L, versus ligand concentration, L was constructed.
  • the data were fit to equation (2) to obtain best-fit parameters for the EC 50 , F max and ' basal-
  • F max the maximal amplitude observed at saturating ligand concentrations, can be either a positive or negative value.
  • the sign of this parameter indicates whether a particular test compound favors binding to the LXR:RXR complex (positive F max ) or to either of the component receptors in a non-heterodimeric state (negative F max ).
  • both F max and F basa) are expressed in units of % of a standard compound.
  • FXR ⁇ LBD Human Famasoid X Receptor alpha Ligand Binding Domain Human FXR ⁇ Ligand Binding Domain
  • cell paste (equivalent to 2-3 liters of the fermentation batch) was resuspended in 200-250 mL TBS, pH 7.2 (25mM Tris, 150 mM NaCI). Cells were lysed by passing 3 times through a French Press and cell debris was removed by centrifugation (30 minutes, 20,000g, 4°C). The cleared supernatant was filtered through course pre-filters, and TBS, pH 7.2, containing 500 mM imidazole was added to obtain a final imidazole concentration of 50mM.
  • This lysate was loaded onto a column (6 x 8 cm) packed with Sepharose [Ni ++ charged] Chelation resin (Pharmacia) and pre- equilibrated with TBS pH 7.2/ 50mM imidazole. After washing to baseline absorbance with equilibration buffer, the column was washed with one column volume of TBS pH 7.2 containing 90mM imidazole. FXR ⁇ LBD was eluted directly with 365 mM imidazole. Column fractions were pooled and dialyzed against TBS, pH 7.2, containing 0.5mM EDTA and 5mM DTT.
  • the dialyzed protein sample was concentrated using Centri-prep 10 K (Amicon) and subjected to size exclusion, using a column (3 x 90 cm) packed with Sepharose S-75 resin (Pharmacia) pre-equilibrated with TBS, pH 7.2, containing 0.5mM EDTA and 5mM DTT.
  • Biotinylation of FXR Purified FXR ⁇ LBD was desalted/buffer exchanged using PD-10 gel filtration columns into PBS [100mM NaPhosphate, pH 7.2, 150mM NaCI].
  • FXR ⁇ LBD was diluted to approximately 10 ⁇ M in PBS and five-fold molar excess of NHS-LC-Biotin (Pierce) was added in a minimal volume of PBS. This solution was incubated with gentle mixing for 30 minutes at room temperature. The biotinylation modification reaction was stopped by the addition of 2000x molar excess of Tris-HCI, pH 8. The modified FXR ⁇ LBD was dialyzed against 4 buffer changes, each of at least 50 volumes, PBS containing 5mM DTT, 2mM EDTA and 2% sucrose. The biotinylated FXR ⁇ LBD was subjected to mass spectrometric analysis to reveal the extent of modification by the biotinylation reagent. In general, approximately 95% of the protein had at least a single site of biotinylation; and the overall extent of biotinylation followed a normal distribution of multiple sites, ranging from one to nine.
  • RXR ⁇ LBD was prepared and labeled with CY5 Tm in accordance with the procedures set forth in example 1.
  • Assay Buffer 50 mM KCI, 0. 1 mg/mL BSA, 10 mM DTT, and 50 mM Tris
  • the stock buffer is made by dissolving 2.853g Tris base, 4.167 g Tris hydrochloride, 3.73 g KCI, and 0. 1 g fatty acid free bovine serum albumin, in 1 L of deionized water. The pH is checked and adjusted to 8.0, if necessary, before adjusting to final volume. 0.154 g of solid DTT is added per 100 mL of buffer just before the start of an experiment.
  • 96 well plates polypropylene for intermediate dilutions (Costar #3794) and either a clear-bottomed white SPA plates (Costar #3632) or a black Polyfiltronics plate (UP350 PSB) for assays.
  • FRET fluorescence resonance energy transfer
  • the ability of ligand to induce changes in the degree of this complex was then used as a basis for an inventive assay for the discovery of nuclear receptor ligands. Certain sequences of the cofactor may only be required to interact with the nuclear receptor.
  • SRC-1 and CBP were synthesized and tested in HTRF and Biacore to determine the best sequences to use.
  • the peptide, CPSSHSSLTERHKILHRLLQEGSPS-CONH 2 (SEQ ID NO.:1 ), i.e., SRC-1 (LCD2,676-700) was used in screening efforts with FXR and this forms a further aspect of this invention.
  • Coactivator proteins interact with nuclear receptors in a ligand-dependent manner and augment transcription (9).
  • a short amphipathic ⁇ - helical domain that includes the amino acid motif LXXLL (L is Leu and X is any other amino acid) serves as the interaction interface between these coactivator molecules and the ligand-dependent activation function (AF-2) located in the COOH-terminus of the nuclear receptor LBD (10).
  • FRET fluorescence resonance energy transfer
  • Human FXR LBD was prepared and fluorescently labeled as described in Example 2.
  • the LBD of human FXR was labeled with the fluorophore allophycocyanin and incubated with a peptide derived from the second LXXLL (SEQ ID NO.:1 ) motif of SRC1 (amino acids 676 to 700) that was labeled with europium chelate.
  • the FRET ligand-sensing assay was performed by incubating 10 nM of the biotinylated FXR LBD that was labeled with streptavidin-conjugated allophycocyanin (Molecular Probes) and 10 nM of the SRC1 peptide [amino acids 676 to 700, 5'-biotin-
  • Biotinylated SRC-1 (LDC2,676-700):Biotin-CPSSHSSLTERHKILHRLL- QEGSPS-CONH 2 (SynPEP) Assay Buffer: 50 mM KCI, 2mM EDTA, 0.1 mg/mL BSA, 10 mM DTT, and 50 mM Tris (pH 8).
  • the stock buffer is made by dissolving 2.853g Tris base, 4.167 g Tris hydrochloride, 3.73 g KCI, 0.74 g EDTA (disodium salt, dihydrate) and 0.1 g fatty acid free bovine serum albumin, in 1 L of deionized water. The pH is checked and adjusted to 8.0, if necessary, before adjusting to final volume. 0.154 g of solid DTT is added per 100 mL of buffer just before the start of an experiment. BSA, fatty acid free DTT
  • 96 well plates polypropylene for intermediate dilutions (Costar #3794) and either a clear-bottomed white SPA plates (Costar #3632) or a black Polyfiltronics plate (UP350 PSB) for assays.
  • Ligands increased the interaction between FXR and the SRC1 peptide as determined with time-resolved FRET. Dose response analysis showed that the ligands increased the amount of SRC1 peptide bound to the FXR

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Abstract

L'invention concerne de nouveaux procédés d'identification de peptides de récepteurs-coactivateurs nucléaires et d'hétérodimères de récepteurs nucléaires, ce qui permet d'identifier des ligands de récepteurs nucléaires au moyen de techniques de proximité de scintillation et de transfert d'énergie de résonance de fluorescence (FRET).
EP99971090A 1998-10-23 1999-10-22 Procede d'identification de ligands de recepteurs nucleaires Withdrawn EP1137940A4 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US10539098P 1998-10-23 1998-10-23
US105390P 1998-10-23
US13509798P 1998-12-23 1998-12-23
US135097P 1998-12-23
US13483699P 1999-05-19 1999-05-19
US134836P 1999-05-19
PCT/US1999/024956 WO2000025134A1 (fr) 1998-10-23 1999-10-22 Procede d'identification de ligands de recepteurs nucleaires

Publications (2)

Publication Number Publication Date
EP1137940A1 true EP1137940A1 (fr) 2001-10-04
EP1137940A4 EP1137940A4 (fr) 2004-06-02

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EP99971090A Withdrawn EP1137940A4 (fr) 1998-10-23 1999-10-22 Procede d'identification de ligands de recepteurs nucleaires

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EP (1) EP1137940A4 (fr)
JP (1) JP2002528721A (fr)
AU (1) AU1229000A (fr)
WO (1) WO2000025134A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7786102B2 (en) 2001-03-12 2010-08-31 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US7994352B2 (en) 2005-05-19 2011-08-09 Intercept Pharmaceuticals, Inc. Process for preparing 3a(β)-7a(β)-dihydroxy-6a(β)-alkyl-5β-cholanic acid
US8796249B2 (en) 2008-07-30 2014-08-05 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US9238673B2 (en) 2012-06-19 2016-01-19 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US9982008B2 (en) 2012-06-19 2018-05-29 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid

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EP1140079B1 (fr) 1998-12-23 2009-06-03 Glaxo Group Limited Methodes de titrage de ligands de recepteurs nucleaires
WO2002077229A2 (fr) * 2001-03-01 2002-10-03 Lion Bioscience Ag Nouveaux cofacteurs du recepteur alpha x du foie et techniques d'utilisation
EP1465882B1 (fr) 2001-12-21 2011-08-24 X-Ceptor Therapeutics, Inc. Modulateurs heterocycliques de recepteurs nucleaires
EP1465869B1 (fr) 2001-12-21 2013-05-15 Exelixis Patent Company LLC Modulateurs de lxr
US7482366B2 (en) 2001-12-21 2009-01-27 X-Ceptor Therapeutics, Inc. Modulators of LXR
US6987121B2 (en) 2002-04-25 2006-01-17 Smithkline Beecham Corporation Compositions and methods for hepatoprotection and treatment of cholestasis
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US10987362B2 (en) 2004-03-12 2021-04-27 Intercept Pharmaceuticals, Inc. Treatment of fibrosis using FXR ligands
PT1734970E (pt) 2004-03-12 2015-03-11 Intercept Pharmaceuticals Inc Tratamento de fibrose utilizando ligandos de rfx
CA2669088C (fr) * 2006-11-10 2016-04-05 Dimerix Bioscience Pty Ltd Systeme de detection et ses utilisations
CA2928178C (fr) 2007-01-19 2019-09-10 Intercept Pharmaceuticals, Inc. Modulateurs de tgr5 et leurs procedes d'utilisation
DE09780481T1 (de) * 2008-07-11 2012-01-26 Medizinische Universität Innsbruck Agonisten von nr2f6 zur immunsuppression
AU2009316566B9 (en) 2008-11-19 2014-05-15 Intercept Pharmaceuticals, Inc. TGR5 modulators and method of use thereof
CN107899012A (zh) 2011-01-11 2018-04-13 戴麦里克斯生物科学有限公司 联合疗法
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* Cited by examiner, † Cited by third party
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US10421772B2 (en) 2001-03-12 2019-09-24 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US8058267B2 (en) 2001-03-12 2011-11-15 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US8377916B2 (en) 2001-03-12 2013-02-19 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US8969330B2 (en) 2001-03-12 2015-03-03 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
USRE48286E1 (en) 2001-03-12 2020-10-27 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US7786102B2 (en) 2001-03-12 2010-08-31 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US9732117B2 (en) 2001-03-12 2017-08-15 Intercept Pharmaceuticals, Inc. Steroids as agonists for FXR
US7994352B2 (en) 2005-05-19 2011-08-09 Intercept Pharmaceuticals, Inc. Process for preparing 3a(β)-7a(β)-dihydroxy-6a(β)-alkyl-5β-cholanic acid
US8796249B2 (en) 2008-07-30 2014-08-05 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US9540414B2 (en) 2008-07-30 2017-01-10 Intercept Pharmaceuticals, Inc. TGR5 modulators and methods of use thereof
US9732116B2 (en) 2012-06-19 2017-08-15 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US10047117B2 (en) 2012-06-19 2018-08-14 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US10155787B2 (en) 2012-06-19 2018-12-18 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US10174073B2 (en) 2012-06-19 2019-01-08 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US9982008B2 (en) 2012-06-19 2018-05-29 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid
US9238673B2 (en) 2012-06-19 2016-01-19 Intercept Pharmaceuticals, Inc. Preparation and uses of obeticholic acid

Also Published As

Publication number Publication date
AU1229000A (en) 2000-05-15
EP1137940A4 (fr) 2004-06-02
WO2000025134A1 (fr) 2000-05-04
JP2002528721A (ja) 2002-09-03

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