Markers for lxr activation

Liver-X-Receptors (LXR) are nuclear hormone receptors that regulate the expression of genes involved in cholesterol and lipid metabolism and bile acid synthesis. LXRs have been implicated in a number of diseases, such as atherosclerosis, dyslipidemia and diabetes. Recent data also suggests a role in inflammation. Several genes have been shown to be regulated by LXR, including ABCA1 (Costet, P., et al., Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem, 2000.275(36): p. 28240-5; Schwartz, K., R. M. Lawn, and D. P. Wade, ABC1 gene expression and ApoA-I-mediated cholesterol efflux are regulated by LXR. Biochem Biophys Res Commun, 2000. 274(3): p. 794-802); WO02/070011 discloses ABCA-1 as a surrogate marker for PPAR activation; ABCG1 (Kennedy, M. A., et al., Characterization of the human ABCG1 Gene; LXR activates an internal promoter that produces a novel transcript encoding an alternative form of the protein. J Biol Chem, 2001. 10: p. 10.); ApoC1 (Stulnig, T. M., et al., Novel roles of liver X receptors exposed by gene expression profiling in liver and adipose tissue. Mol Pharmacol, 2002.62(6): p. 1299-305); ApoE (Laffitte, B. A., et al., LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci USA, 2001. 98(2): p. 507-12); FAS (Joseph, S. B., et al., Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by LXRs. J Biol Chem, 2002. 14: p. 14); LDLR (Stulnig, T. M., et al., Novel roles of liver X receptors exposed by gene expression profiling in liver and adipose tissue. Mol Pharmacol, 2002. 62(6): p. 1299-305); NR1H3; (Laffitte, B. A., et al., Autoregulation of the Human Liver X Receptor alpha Promoter. Mol Cell Biol, 2001. 21(22): p. 7558-68); SREBPF1 (DeBose-Boyd, R. A., et al., Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands. Proc Natl Acad Sci USA, 2001. 98(4):p. 1477-82). Also of interest is US2004/0023276, which discloses LXR-ligand induced genes and proteins.

The present invention relates to surrogate markers for LXR activation, and methods of diagnosing a disease linked to LXR activation, methods of monitoring the treatment of patients suffering from a disease linked to LXR activation, and methods of identifying compounds which modulate LXR activity.

The present invention provides a marker for detecting or monitoring LXR modulation, comprising at least one nucleic acid selected from the group consisting of the nucleic acids listed in tables 2 and/or 3. The term “modulation” as used herein relates to an activation or inhibition of the transcriptional activity of LXR. Thus, said nucleic acids can serve as surrogate markers for modulation of LXR activity.

The term “marker” as used herein refers to a single nucleic acid or polypeptide, or a panel of multiple nucleic acids or polypeptides.

Preferably, said nucleic acids are nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, said nucleic acids are nucleic acids listed in table 2, with the exception of Seq. ID No. 3, 4, 5 and 20. In another more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 6, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises at least one nucleic acid of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises at least one nucleic acid selected from the group consisting of the nucleic acids of Seq ID No. 20, 22 or 26, more preferably at least one nucleic acid selected from the group consisting of the nucleic acids of Seq ID No. 22 or 26 and most preferably, the marker comprises the nucleic acid of Seq ID No. 26.

In another preferred embodiment, the marker comprises at least one nucleic acid listed in table 3. More preferably, said at least one nucleic acid is selected from the group consisting of the nucleic acids of Seq ID No. 16, 17 and 23 to 25. In a most preferred embodiment, said at least one nucleic acid is selected from the group consisting of Seq ID No. 16 or 24.

The present invention also pertains to a marker for diagnosing a disease involving dysregulation of LXR activity, comprising one or more of the nucleic acids selected from the group consisting of the nucleic acids listed in tables 2 and/or 3. Thus, said nucleic acids can serve as surrogate markers for the modulation of LXR activity.

Preferably, said nucleic acids are nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, said nucleic acids are nucleic acids listed in table 2, with the exception of Seq. ID No. 3, 4, 5 and 20. In another more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 16, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises at least one nucleic acid of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises at least one nucleic 15 acid selected from the group consisting of the nucleic acids of Seq ID No. 20, 22 or 26, more preferably at least one nucleic acid selected from the group consisting of the nucleic acids of Seq ID No. 22 or 26 and most preferably, the marker comprises the nucleic acid of Seq ID No. 26.

In another preferred embodiment, said at least one nucleic acids are selected from the group consisting of the nucleic acids listed in table 3. More preferably, said at least one nucleic acids are selected from the group consisting of the nucleic acids of Seq ID No. 16, 17 and 23 to 25. Most preferably, said at least one nucleic acids are selected from the group consisting of Seq ID No. 16 and/or 24.

The invention also pertains to a marker for diagnosing a disease involving dysregulation of LXR activity, comprising at least one nucleic acid selected from the group consisting of the nucleic acids listed in table 3 and one or more nucleic acids listed selected from the group consisting of the nucleic acids in table 2. Preferably, the marker comprises one nucleic acid listed in table 3 and one nucleic acid listed in table 2.

The polypeptides encoded by the nucleic acids listed in tables 2 and/or 3 can also be used as markers. Thus, the present invention also provides a marker for detecting or monitoring LXR modulation, comprising one or more polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in tables 2 and/or 3.

Preferably, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises at least one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, said polypeptides are the polypeptides encoded by the nucleic acids listed in table 2, with the exception of Seq. ID No. 3, 4, 5 and 20. In another more preferred embodiment, the marker comprises at least one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 1, 6, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises at least one polypeptide encoded by the nucleic acids selected from the group consisting of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises at least one polypeptide encoded by the nucleic acids of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises at least one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 20, 22 or 26, more preferably at least one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 22 or 26 and most preferably, the marker comprises the polypeptides encoded by the nucleic acid of Seq ID No. 26.

In another preferred embodiment, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 3. More preferably, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 16, 17 and 23 to 25. Most preferably, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 16 and/or 24.

In another embodiment, the present invention provides a marker for detecting or monitoring LXR modulation, comprising at least one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acid listed in table 3 and one or more polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. Preferably, the marker comprises one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 3 and one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. More preferably, the marker comprises two polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 3 and two polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. Even more preferably, the marker comprises three polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 3 and three polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. Most preferably, the marker comprises four polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 3 and four polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2.

Further to this, the present invention also provides a marker for diagnosing a disease involving dysregulation of LXR activity, comprising one or more polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in tables 2 and/or 3.

In another preferred embodiment, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 3. More preferably, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 16, 17 and 23 to 25. Most preferably, said said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids of Seq ID No. 16 and/or 24.

The invention also pertains to a marker for diagnosing a disease involving dysregulation of LXR activity, comprising at least one polypeptide selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 3 and one or more polypeptides selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. Preferably, the marker comprises one polypeptide selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 3 and one polypeptide selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 2. More preferably, the marker comprises at least two polypeptides selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 3 and at least two polypeptides selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 2. Even more preferably, the marker comprises at least three polypeptides selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 3 and at least three polypeptides selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 2. Most preferably, the marker comprises at least four polypeptides selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 3 and at least four polypeptides selected from the group consisting of the polypeptides encoded by a nucleic acid listed in table 2.

The nucleic acids and polypeptides which constitute the novel markers hereinbefore described are useful for several processes. A method for screening compounds that modulate LXR activity is provided, comprising the steps of contacting said compounds with a host, and measuring the expression of at least one nucleic acid selected from the group consisting of the nucleic acid listed in tables 2 and/or 3.

Preferably, said nucleic acids are nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, said nucleic acids are nucleic acids listed in table 2, with the exception of Seq. ID No. 3, 4, 5 and 20. In another more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 6, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises at least one nucleic acid selected from the group consisting of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises at least one nucleic acid of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises at least one nucleic acid selected from the group constisting of the nucleic acids of Seq ID No. 20, 22 or 26, more preferably at least one nucleic acid selected from the group constisting of the nucleic acids of Seq ID No. 22 or 26 and most preferably, the marker comprises the nucleic acid of Seq ID No. 26.

In one embodiment of the method hereinbefore described, the expression of at least one nucleic acid selected from the group consisting of the nucleic acids herein before described is compared to the expression of said at least one nucleic acid in a control. The control can either be an untreated host, which may be the same host before the treatment or a different host, and/or the same host after an appropriate period of treatment for normalization to pretreatment levels. In a preferred embodiment of the method hereinbefore described, the compound that modulates LXR activity is either an antagonist or an agonist.

Several methods for measuring expression of said nucleic acids can be used. Methods such as Northern Blotting, and quantitation of the bands by densitometry are well known in the art and may be used, although they may not be sufficiently accurate. Other methods include the use of genechips, microarray analysis, dot blotting or different quantitative PCR methodologies. Preferably, Taqman or real time quantitative PCR is used. In a preferred embodiment, expression levels of at least one nucleic acids selected from the group consisting of Seq ID No. 16 to 26 are determined by Taqman quantitative PCR using the forward primers, reverse primers and probes listed in table 1. In a more preferred embodiment, the expression levels of at least one nucleic acid selected from the group consisting of Seq ID No. 20, 21, 22 and 26 are measured by Taqman quantitative PCR. In a most preferred embodiment, the primers and probes used for measuring at least one nucleic acid selected from the group consisting of Seq ID No. 20, 21, 22 and 26 are primers and protes of Seq ID No. 39 to 41 (for measuring expression levels of the nucleic acid of Seq ID No. 20), or Seq ID No. 42 to 44 (for measuring expression levels of the nucleic acid of Seq ID No. 21), or Seq ID No. 45 to 47 (for measuring expression levels of the nucleic acid of Seq ID No. 22), or Seq ID No. 57 to 59 (for measuring expression levels of the nucleic acid of Seq ID No. 26).

Taqman quantitative PCR is performed as follows:

Three oligonucleotides are used: a forward primer, a reverse primer, and a probe. All of them are specific for the target and are able to bind to it. The TaqMan assay uses a probe technology that exploits 5′→3′-nuclease activity of an enzyme, the most commonly used being Taq polymerase. The assay efficiency is largely dependent on this 5′→3′ nuclease activity. In this regard one should be careful in choosing a suitable polymerase. Indeed, some polymerases available on the market appear not to be suitable for real-time RT-PCR, even though the manufacturers claim they possess 5′-exonuclease activity. The probe is an oligonucleotide with a reporter dye at the 5′ end and a quencher dye at the 3′ end. The fluorescent reporter dye is attached covalently to the 5′ end and can be FAM (6-carboxyfluorescein), TET (tetrachloro-6-carboxyfluorescein), JOE (2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein), HEX (hexacholoro-6-carboxyfluorescein), or VIC. The reporter is quenched by TAMRA (6-carboxytetramethylrhodamine), bound to the 3′ end by a linker arm. DABCYL [4-(48-dimethylaminophenylazo)benzoic acid] can also be used as a quencher dye, but its use is much more prevalent in the molecular beacon probes. An advantage of using DABCYL in the TaqMan probes is its reduced autofluorescence compared with TAMRA When the probe is intact the quencher dye absorbs the fluorescence of the reporter dye due to the proximity between both. The proximity between quencher and fluorophore permits FRET, and fluorescence emission does not occur. By the 5′-exonuclease activity of the Taq:polymerase the probe is hydrolyzed and the reporter dye is separated from the quencher, resulting in an increase in fluorescence emission. During PCR amplification, if the target of interest is present, the probe specifically anneals to the target. The Taq polymerase cleaves the probe, allowing an increase in fluorescence emission. This increase in fluorescence is measured cycle by cycle and is a direct consequence of the amplification process (Giulietti, A., et al., An Overview of eal-Time Quantitative PCR: Applications to Quantify Cytokine Gene Expression. Methods 25, 386-401 (2001)).

Another method provided by the present invention is a method for screening compounds that modulate LXR activity, comprising the steps of contacting said compounds with a host, and measuring the expression of at least one polypeptide selected from the group consisting of the polypeptides encoded by the nucleic acids listed in tables 2 and/or 3. Preferably, said polypeptides are selected from the group consisting of the polypeptides encoded by the nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, the marker comprises the nucleic acids, or the polypeptides encoded by the nucleic acids listed in table 2, with the exception of Seq ID No. 3 to 5 and 20. In another more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 6, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 20, 22 or 26, and most preferably, the marker comprises the nucleic acid of, or the polypeptide encoded by Seq ID No. 26.

In another embodiment of the method hereinbefore described, the expression of at least one of the polypeptides is compared to the expression of said at least one polypeptide in a control. The control can be an untreated host or a host treated with a carrier. The carrier may be the solvent in which the compound is dissolved or resuspended. In a preferred embodiment of the method hereinbefore described, the compound that modulates LXR activity is either an antagonist or an agonist.

The present invention also provides a method for monitoring treatment of patients suffering from a disease associated with dysregulation of LXR activity, comprising the steps of purifying mRNA or protein from monocytes/macrophages or from total blood isolated from patients treated with a modulator of LXR activity and measuring the expression of at least one of the nucleic acids, or at least one of the polypeptides encoded by one of the nucleic adds listed in tables 2 and/or 3.

Preferably, said polypeptides are encoded by the nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, the marker comprises the nucleic acids, or the polypeptides encoded by the nucleic acids listed in table 2, with the exception of Seq ID No. 3 to 5 and 20. In another more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 6, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 20, 22 or 26, and most preferably, the marker comprises the nucleic acid of, or the polypeptide encoded by Seq ID No. 26.

The control can either be an untreated host, which may be the same host before the treatment or a different host, and/or the same host after an appropriate period of treatment for normalization to pretreatment levels.

The present invention also provides a method for diagnosing a disease involving dysregulation of LXR activity, comprising the steps of extracting mRNA or protein from total blood or from purified monocytes/macrophages, and measuring the expression of at least one of the nucleic acids, or at least one of the polypeptides encoded by one of the nucleic acids listed in tables 2 or 3.

In another preferred embodiment, said at least one nucleic acids or polypeptides are selected from the group consisting of the nucleic acids, or the polypeptides encoded by the nucleic acids, listed in table 3. More preferably, said at least one nucleic acids or polypeptides are selected from the group consisting of the nucleic acids, or the polypeptides encoded by the nucleic acids of Seq ID No. 16, 17 and 23 to 25. Most preferably, said at least one nucleic acids or polypeptides are selected from the group consisting of the nucleic acids, or polypeptides encoded by the nucleic acids of Seq ID No. 16 and/or 24.

Further to the methods and markers hereinbefore described, the present invention provides a use of one or more nucleic acids, or one or more polypeptides encoded by the nucleic acids listed in tables 2 and/or 3 as a marker for LXR modulation. Preferably, said polypeptides are encoded by the nucleic acids listed in table 2. In a more preferred embodiment, the marker comprises the nucleic, acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 4 to 7, 12, 14, 20 to 22 and 26. In another preferred embodiment, the marker comprises the nucleic acids, or the polypeptides encoded by the nucleic acids listed in table 2, with the exception of Seq ID No. 3 to 5 and 20. In another more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 6, 7, 12, 14, 21, 22 and 26. In an even more preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1, 12 or 21. In a most preferred embodiment, the marker comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 1 or 21. In another embodiment, the marker more preferably comprises the nucleic acids of, or the polypeptides encoded by the nucleic acids of Seq ID No. 20, 22 or 26, and most preferably, the marker comprises the nucleic acid of, or the polypeptide encoded by Seq ID No. 26.

The present invention also pertains to compounds identified by the methods hereinbefore described, and to the use of compounds identified by a method hereinbefore described for the preparation of a medicament for the treatment of a disease involving dysregulation of LXR activity.

EXAMPLES

The invention will now be illustrated by reference to the following examples which should not be construed as a limitation thereto.

Cell Culture and RNA Preparation

The myelogenous monocytic leukemia cell line (THP1; ATCC TID-202) was cultured in RPMI 1640 medium with Phenol Red containing 2 g/l glucose and 2 mM Glutamine, 10% Fetal Bovine Serum FBS (v/v), (Gibco), 1% Penicillin-Streptomycin P/S (v/v) (Gibco), 50 □M 2-Mercaptoethanol (Bio-Rad) and 1% 100 mM Sodium Pyruvate (v/v) (Gibco). Cells were incubated at 37° C. and 5.2% CO₂. For experimental treatments, cells were seeded at a density of 1×10⁶/ml in 6-well plates in medium containing 100 nM phorbol 12-myristate 13-acetate (PMA) for 72 hours to fully differentiate them into adherent macrophages. The medium was then replaced by culture medium containing PMA with vehicle (DMSO) or test compound for 6 or 24 hours prior to harvesting. Total RNA was isolated using the TriZol reagent (Life Technologies) or the RNeasy mini kit (Qiagen) according to the manufacturer's protocols. To remove contaminating DNA, the samples were treated with RNAse free DNAse (GibCo).

Gene Expression Measurement by DNA Chips

Synthesis of first and second strand cDNA were performed using the SuperScript Choice Gene Chip Kit (Life Technologies) and reagents from Gibco. The double stranded cDNA, containing an incorporated T7 RNA polymerase binding site, was purified by extraction with a mix of phenol:chloroform:isoamylalcohol (Life Technologies). The organic and aqueous phases were separated by Phase Lock Gel (Eppendorf) and double-stranded cDNA was recovered by precipitation according to the manufacturer's protocol and then resuspended in water.

The double-stranded cDNA was converted to biotin-labeled cRNA by in vitro transcription (IVT) using a T7 kit (Ambion) and biotin-containing ribonucleotides (Enzo-LOXO GmbH). The IVT-material was purified from unincorporated ribonucleotides using RNeasy spin columns (Qiagen). Following cleanup, the single-stranded biotin-labeled cRNA were chemically hydrolyzed to smaller fragments in 500 mM calcium acetate, 150 mM magnesium acetate, pH 8.1 for 35 min at 95° C. The reaction was terminated by chilling samples on ice.

Probes were hybridized to the HGU95AV2 A GeneChip Microarray (Affymetrix) which contains features representing ˜12,000 genes. All washing, hybridization, detection, and signal amplification steps were performed using a GeneChip Fluidics Station (Affymetrix). Fluorescence intensity data was collected from the hybridized GeneArrays using a GeneArray scanner (Affymetrix). The raw files containing the fluorescence intensity information were transformed into data files using the Affymetrix Microarray Suite (MAS) software. Differentially expressed genes were identified using the Roche Affymetrix Chip Experiment Analysis (RACE-A) software. Differences between vehicle-treated vs test compound-treated wells (n=4 per group) were evaluated by student t-test and expressed as fold change vs control.

Gene Expression Measurement by Taqman Quantitative PCR

Gene expression levels were measured by Taqman quantitative RT-PCR on an ABI PRISM 7700 sequence detection system (Applied Biosystems). Total RNA was reverse transcribed into cDNA using the Superscript II RT kit (Life Technologies). The primer/probe sequences used are listed in Table I. Primers were designed using PrimerSelect Software (Perkin Elmer). Differences between vehicle-treated vs test compound-treated wells (n=4 per group) were evaluated by student t-test and expressed as fold change vs control. TaqMan quantitative PCR was performed as described (Giulietti, A., et al., An Overview of eal-Time Quantitative PCR: Applications to Quantify Cytokine Gene Expression. Methods 25, 386-401 (2001)).

Example 1

LXRalpha and LXRbeta are nuclear receptors that heterodimerize with RXR and have been shown to regulate expression of several target genes such as ABCA1, ABCG1, SREBF1, FASN, LPL, and CYP7A [1-9]. To identify surrogate genes that could be measured in patients to evaluate efficacy of a treatment regime, THP1 macrophages were treated with a selective LXR ligand. At 200 nM, T0901317 [10] is a selective LXR agonist (EC50=30 nM and 10 nM on human LXRalpha and LXRbeta, respectively). Gene expression changes were evaluated by Gene Chip Microarray analysis. The LXR agonist T0901317 showed strong induction of ABCA1, ABCG1, APOC1, APOE, ASM3A, C3F, CDC42EP4, CXCR4, PASN, LDLR, NR1H3, SREBP1, VLDLR, FADS1, FADS2, CYB561, IL15, PPFIA2, SERPINI1, KIAA0763, PPIC, LASSI, NFATC4, NR4A3, PRODH2, PRKCI (Table 2). Thus, all of these genes are surrogate markers for LXR modulation and their activity can be measured using GeneChip Microarray technology.

Example 2

To evaluate an alternative method of measuring surrogate marker genes, THP1 macrophages were treated with T0901317 as described above. Gene expression changes were measured by Taqman quantitative PCR for ABCA1, ABCG1, APOC1, APOE, ASM3A, CDC42EP4, CXCR4, FASN, LDLR, NR1H3, VLDLR. The selective LXR agonist produced a robust and significant increase in expression of each of these marker genes (Table 3). These results, taken together, suggest that measurement of mRNA expression of surrogate marker genes by multiple methods can be effectively used to evaluate therapeutic efficacy of an LXR treatment regimen.

TABLE IList of Taqman primers and probes used in this studySeqSeqGeneIDIDNameForward PrimerNoReverse PrimerNo.A.ABCA1AACCCACCACAGGCATGG27ACACTTAGGGCACAATTCCACA28ABCG1CCCTCCAGTCATGTTCTTCGA30ATGATGGAGCGACCCCCT31APOC1CATGAGGCTCTTCCTGTCGC33TGGGCCTTCCAAGACGATC34APOECGTTGCTGGTCACATTCCTG36GCTCTGTCTCCACCGCTTG37ASM3AGAATCTAAAGGGAGAGTCCATCTGG39TCCGGCTGCAAATCTTCAAT40CDC42EPTCGGGTATGAGCCCCTGAG42GGAGGTGGGTCAGGCTGTT43CXCR4GCAGGACCTGTGGCCAAGT45CGCTCTGGAATGTTCAGTTCC46FASNTTGCATTGCTGGTAGAGACCC48CACACGCTGCCTGAGGAGT49LDLRCCCCAGGGACAAAACACTGT51GCTCCGAAACCAGAAAGGCT52NR1H3TGTAACCGGCGCTCCTTTT54TGGTGCCATGGGCCA55VLDLRCAAATAATACCCCCGTCGGA57CCAGCCGAGAGGAAGAAAAA58SeqGeneIDNameProbeNo.B.ABCA1TCCCAAAGCCCGGCGGTTC29ABCG1CCCTCCAGTCATGTTCTTCGA32APOC1CCCGGTCCTGGTGGTGGTTCTGT35APOECAGGATGCCAGGCCAAGGTGGA38ASM3AAGCTGGAGTATATCCTGACCCAGACCTACGA41CDC42EPTTGACTGCCGGTTATTTTTCTGTCCTGG44CXCR4TTAGTTGCTGTATGTCTCGTGGTAGGACTGTAGAAAA47FASNCAGGCCTGTCCACCCTGCCAA50LDLRCCCCCCAGTGCAGGGAACCG53NR1H3TGACCGGCTTCGAGTCACGCC56VLDLRTGGTAACCGAGCCAGCAGCTGAAGTCT59
All sequences are depicted in the 5′ to 3′ direction. The Taqman probes were labeled with 5′-FAM and 3′-TAMRA and purified by HPLC.

TABLE II

Identification of surrogate marker genes of LXR activation by Gene

Chip Microarrays

Treatment
Seq ID

Gene Name
Unigene ID
Accession
Fold-change
p-Value
Time (hrs)
No

ASM3A
Hs.42945
NM 006714.1
+2.84
<0.001**
6
20

C3F
Hs.300423
NM 005768.3
+9.5
0.038*
6
1

CDC42EP4
Hs.3903
AF099664.1
+39.62
<0.001**
24
21

CXCR4
Hs.89414
NM 003467.1
+3.05
<0.001**
6
22

VLDLR
Hs.73729
D16493.1
+3.88
0.001**
24
26

FADS1
Hs.132898
NM 013402.3
+2.11
<0.001**
24
3

FADS2
Hs.184641
NM 004265.2
+3.24
0.012*
24
4

CYB561
Hs.355264
BC002976.1
+3.16
0.002**
24
5

IL15
Hs.168132
NM 000585.2
+3.44
0.03*
24
6

PPFIA2
Hs.30881
AF034799.1
+3.1
0.016*
24
7

SERPINI1
Hs.78589
NM 005025.1
+2
<0.001**
24
8

KIAA0763
Hs.4764
AB018306.1
+2.38
<0.001**
24
9

PPIC
Hs.110364
NM 000943.2
+2.99
0.001**
24
10

LASS1
Hs.348258
NM 021267.1
+2.26
<0.001**
24
11

NFATC4
Hs.77810
NM 004554.1
+5.62
0.01*
24
12

NR4A3
Hs.80561
X89894.1
+1.99
<0.001**
24
13

PRODH2
Hs.128834
NM 021232.1
+3.87
0.001**
24
14

PRKCI
Hs.1904
NM002740.1
+2.28
<0.001**
24
15

Fold change in mRNA levels in THP1 macrophages treated with LXR agonist T0901317 as measured by Gene Chip Microarray Analysis.

All values are fold-change in mRNA and represent the difference between the means of 4 treated vs 4 controls.

Time of treatment is 6 or 24 hours.

*significant p < 0.05;

**highly significant, p < 0.01.

TABLE III

Identification of surrogate marker genes of LXR activation by Gene

Chip Microarrays

Treatment
Seq ID

Gene Name
Unigene ID
Accession
Fold-change
p-Value
Time (hrs)
No

ABCA1
Hs.211562
AJ012376.1
+3.95
<0.001**
6
16

ABCG1
Hs.10237
NM 004915.2
+12.85
<0.001**
6
17

APOC1
Hs.268571
NM 001645.2
+2.77
0.003**
24
18

APOE
Hs.169401
NM 000041.1
+1.83
0.04*
24
19

FASN
Hs.83190
NM 004104.3
+2.09
0.009**
24
23

LDLR
Hs.213289
NM 000527.2
+1.78
0.001**
24
24

NR1H3
Hs.347353
NM 005693.1
+4.1
0.001**
6
25

SREBF1
Hs.166
NM 004176.2
+1.92
0.001**
24
2

Fold change in mRNA levels in THP1 macrophages treated with LXR agonist T0901317 as measured by Gene Chip Microarray Analysis.

All values are fold-change in mRNA and represent the difference between the means of 4 treated vs 4 controls.

Time of treatment is 6 or 24 hours.

*significant p < 0.05;

**highly significant, p < 0.01.

TABLE IV

Evaluation of surrogate marker genes of LXR activation by Taqman

quantitative RT-PCR

Fold-

Treatment
Seq ID

Gene Name
Unigene ID
change
p-Value
Time (hrs)
No

ABCA1
Hs.211562
+9.52
<0.001**
6
16

ABCG1
Hs.10237
+110.02
<0.001**
6
17

APOC1
Hs.268571
+4.31
<0.001**
24
18

APOE
Hs.169401
+2.2
<0.001**
24
19

ASM3A
Hs.42945
+6.9
0.01*
6
20

CDC42EP4
Hs.3903
+1.86
<0.001**
24
21

CXCR4
Hs.89414
+3.29
<0.001**
6
22

FASN
Hs.83190
+1.42
0.02*
24
23

LDLR
Hs.213289
+2.02
0.01*
24
24

NR1H3
Hs.347353
+5.02
0.001**
6
25

VLDLR
Hs.73729
+6.63
0.001**
24
26

Fold change in mRNA levels in THP1 macrophages treated with LXR agonist T0901317 as measured by Taqman Quantitative RT-PCR.

All values are fold-change in mRNA and represent the difference between the means of 4 treated vs 4 controls.

Time of treatment is 6 or 24 hours.

*significant p < 0.05;

**highly significant, p < 0.01.

Markers for lxr activation

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