WO2015025180A1 - Modulation du cholestérol - Google Patents

Modulation du cholestérol Download PDF

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Publication number
WO2015025180A1
WO2015025180A1 PCT/GB2014/052585 GB2014052585W WO2015025180A1 WO 2015025180 A1 WO2015025180 A1 WO 2015025180A1 GB 2014052585 W GB2014052585 W GB 2014052585W WO 2015025180 A1 WO2015025180 A1 WO 2015025180A1
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tspo
lxra
ppara
modulators
receptor
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PCT/GB2014/052585
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English (en)
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Annette GRAHAM
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Glasgow Caledonian University
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Priority to EP14761394.7A priority Critical patent/EP3035922A1/fr
Publication of WO2015025180A1 publication Critical patent/WO2015025180A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]

Definitions

  • the present invention provides compounds for use in the modulation of macrophage cholesterol efflux and the treatment of cardiovascular disease.
  • the invention further provides diagnostic tests for cardiovascular disease and/or a susceptibility/predisposition thereto.
  • Mitochondrial dysfunction associated with increased production of reactive oxygen species, accumulation of mitochondrial DNA and progressive loss of respiratory chain function, is found in atherosclerotic lesions in both human studies, and in animal models of atherogenesis 1 .
  • Oxidized low density lipoprotein (OxLDL) ageing, hyperhomocysteinaemia, hyperglycaemia, hypertriglyceridaemia, type 2 diabetes mellitus and cigarette smoking are all associated with mitochondrial damage and increased production of reactive oxygen species 2"4 .
  • Increased arterial oxidative stress modifies LDL to a form recognised by macrophage scavenger receptors, resulting in further mitochondrial damage, and the unregulated accumulation of excess cholesterol and cholesteryl esters within macrophage 'foam cells', are a hallmark of early and developing atheroma 1"5 .
  • Reversal of this process, regressing and stabilising atherosclerotic lesions, can be effected by efficient removal of cholesterol from 'foam cells', to acceptor particles such as apolipoprotein (apo) A-I, apoE or nascent high density lipoproteins (HDL), which then enter the athero-protective reverse cholesterol transport pathway responsible for delivery of excess cholesterol to the liver for excretion in bile and bile acids 6 .
  • apo apolipoprotein
  • HDL high density lipoproteins
  • Sterol 27-hydroxylase generates oxysterol ligands for Liver X Receptors (LXRoc/ ⁇ ), nuclear transcription factors which regulate gene expression of proteins involved in the cholesterol efflux pathway, including ATP binding cassette transporters, ABCA1, ABCG1/ABCG4, which orchestrate transfer of cholesterol and/or phospholipids across the plasma membrane to (apo)lipoproteins, such as apoA-I and apoE 12"14 .
  • Loss of CYP27A1 results in cerebrotendinous xanthomatosis, which can be characterised by increased risk of premature atherosclerosis, despite normal circulating concentrations of plasma LDL 15 . Exploiting the anti- atherogenic functions of StAR may prove problematic, however, due either to induction of lipogenesis 7 or the dearth of small molecules capable of modulating the activity of this protein in vivo.
  • StAR interacts with a protein complex, located at contact sites between outer and inner mitochondrial membranes, which includes 18kDa translocator protein (TSPO/peripheral benzodiazepine receptor/PBR), voltage dependent anion channel (VDAC) and adenine nucleotide transporter (ANT), and associated proteins, including cAMP-dependent protein kinase associated protein (PAP7/ Acyl CoA binding domain-3/ACBD3), PBR-associated protein (PRAXl) and its proposed endogenous ligand, diazepam binding inhibitor (DBI/ACBD1) 16 ' 17 .
  • TSPO translocator protein
  • VDAC voltage dependent anion channel
  • ANT adenine nucleotide transporter
  • associated proteins including cAMP-dependent protein kinase associated protein (PAP7/ Acyl CoA binding domain-3/ACBD3), PBR-associated protein (PRAXl) and its proposed endogenous ligand, diazepam binding inhibitor (DBI/
  • the translocator protein has five transmembrane domains, and a high affinity cholesterol recognition amino acid (CRAC) binding C-terminal domain 16 ' 11 ; the CRAC peptide was recently used as an interchelating agent to reduce cholesterol and aortic lesions in apoE-/- mice 18 .
  • Knockdown of TSPO induces arrest of mitochondrial cholesterol transport, which can be rescued by TSPO cDNA, while global deletion of TSPO is embryonic lethal in mice 16 ' 11 .
  • TSPO ligands were recently shown to activate fasting metabolism, reducing lipogenesis and hepatosteatosis in obese mice 19 , suggesting that additional activities associate with this protein.
  • the present invention is based on the finding that through modulation of the function, activity and/or expression of the liver X receptor (LXR) a and/or the peroxisome proliferator-activated receptors (PPAR) a proteins (and/or genes encoding the same), it is possible to modulate cholesterol efflux from macrophages.
  • LXR liver X receptor
  • PPAR peroxisome proliferator-activated receptors
  • PPARa is a nuclear receptor protein which functions as a transcription factor to regulate the expression of genes.
  • the PPARa isoform is encoded by the PPARa gene located at 22ql2-13.1 on the human chromosome.
  • LXR is a member of the nuclear receptor family of transcription factors. The LXRs are important regulators of cholesterol, fatty acid, and glucose homeostasis.
  • the LXRa isoform is encoded by the LXRa gene located at l ip 11.2 on the human chromosome.
  • homologues of both the PPARa and LXRa proteins/genes may exist in other non-human animals and insofar as this invention applies to non-human animals, such homologues should be included within the definition of the terms PPAR/PPARa and LXR LXRa.
  • LXR LXRa
  • PPAR PPARa
  • a first aspect of this invention provides LXRa and/or PPARa modulators, for use in modulating cholesterol efflux from macrophages.
  • the invention provides a method of modulating macrophage cholesterol efflux, said method comprising administering to a subject in need thereof, one or more LXRa and/or PPARa modulators.
  • the one or more LXRa and/or PPARa modulators may be administered in therapeutically effective amounts.
  • TSPO mitochondrial 18kDa translocator protein
  • the present invention finds application in the treatment of cardiovascular disease.
  • the invention may be exploited in the treatment and/or prevention of atherosclerosis.
  • use or administration of one or more modulators of LXRa and/or PPARa gene/protein function, activity and/or expression may provide an effective means of regressing atherosclerotic lesions.
  • modulator may encompass any molecule or compound which when brought into contact with a target molecule, alters some aspect of the expression, function and/or activity of that target molecule. Modulators may increase (enhance) or decrease (inhibit) any aspect of the expression, function and/or activity of a target molecule.
  • the LXRa and/or PPARa modulators may be compounds or molecules which increase, stimulate or enhance some aspect of the expression, function and/or activity of the LXRa and/or PPARa genes/proteins.
  • Modulation of LXRa and/or PPARa expression may manifest as an increase and/or decrease in the level of LXRa and/or PPARa gene expression as determined by an increase in the amount of mRNA/DNA expression or an increase or decrease in LXRa and/or PPARa protein expression.
  • a level of LXRa and/or PPARa function or activity may manifest as an increase and/or decrease in LXRa and/or PPARa function and/or activity.
  • the expression, function and/or activity of the LXRa/PPARa genes and/or proteins may be assessed relative to a system, for example a cell based system, which exhibits a normal or control level of LXRa and/or PPARa expression.
  • Modulators suitable for use in this invention may take the form of small organic or inorganic molecules.
  • Modulators for use in this invention may comprise, for example, nucleic acids (DNA, RNA and/or PNA), proteins, peptides, amino acids, antibodies (or antigen binding fragments thereof), carbohydrates and/or lipids.
  • Suitable modulators may include, for example, fragments and/or portions of the LXRa and/or PPARa genes and/or proteins. Fragments and/or portions for use as modulators may be functional, that is to say they retain the ability to perform one or more of the functions and/or activities of the native (wild type) gene or protein.
  • An LXRa and/or PPARa modulator may comprise a vector which encodes an expressible LXRa and/or PPARa gene or fragment. Again, any fragment encoded by such a vector might be a functional fragment as defined above.
  • PPARa agonists suitable for use in this invention may include, for example, fibrate type drugs which are a class of amphipathic carboxylic acids, including, for example, (clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate).
  • fibrate type drugs which are a class of amphipathic carboxylic acids, including, for example, (clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and fenofibrate).
  • Perfluorooctanoic and perfluorononanoic acid are also known to activate PPARa.
  • LXRa agonists suitable for use in this invention may include, for example, synthetic or natural agonists such as, for example oxysterol compounds (oxygenated derivatives of cholesterol, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27- hydroxycholesterol, and cholestenoic acid) and T0901317, GW3965, hypocholamide or N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA)).
  • oxysterol compounds oxygenated derivatives of cholesterol, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27- hydroxycholesterol, and cholestenoic acid
  • T0901317 GW3965
  • hypocholamide or N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA)
  • the PPARa and/or LXRa modulators of this invention may include, for example modulators which indirectly modulate the expression, function and/or activity of PPARa and/or LXRa.
  • suitable modulators may take the form of 18kDa translocator protein (TSPO) modulators. Modulators of this type may modulate any aspect of the expression, function and/or activity of the TSPO gene and/or protein.
  • suitable TSPO modulators may comprise nucleic acid (DNA, RNA and/or PNA), proteins, peptides, amino acids, antibodies (or antigen binding fragments thereof), carbohydrates and/or lipids.
  • TSPO modulators for use in this invention may comprise modulators which increase TSPO expression, function and/or activity - such modulators may be collectively referred to as TSPO agonists.
  • Suitable agonists will be known to those skilled in this field and may include, for example Anthralin (a 16kDa polypeptide that binds to both TSPO receptor and dihydropyridine- sensitive calcium channels with high affinity); Diazepam binding inhibitor (DBI: an l lkDa neuropeptide); DBI 17-50 fragment - the active processing product of DBI.
  • Non peptide TSPO agonists may include, for example, Etifoxine, DAA-1097, DAA-1106, DPA-713, DPA-714, Emapunil, FGIN-127, FGIN-143 and SSR-180,575.
  • TSPO peptide TSPO agonists
  • the inventors have noted that by inducing the overexpression of TSPO, it is possible to affect cholesterol efflux from macrophages. Moreover, it is suggested that the increased expression of TSPO induces activation of LXRoc and/or PPARoc. As such, this invention (and specifically the uses and methods described herein) may exploit, for example TSPO modulators.
  • the invention provides one or more TSPO, PPARoc and/or LXRoc agonists for use in (i) modulating cholesterol efflux from macrophages; (ii) treating or preventing cardiovascular disease and/or (iii) treating or preventing atherosclerosis. Furthermore, the invention provides methods of (i) modulating cholesterol efflux from macrophages; (ii) treating or preventing cardiovascular disease and/or (iii) treating or preventing atherosclerosis, said methods comprising administering one or more TSPO, PPARoc and/or LXRa agonists to a subject in need thereof.
  • a subject in need thereof may be a human or animal subject.
  • the human or animal subject may be suffering from or exhibiting symptoms of a cardiovascular disease for which modulation of macrophage cholesterol efflux might represent a means of treatment. Additionally or alternatively, the subject may be suffering from or exhibiting symptoms of atherosclerosis.
  • the subject may be susceptible or predisposed to developing, a cardiovascular disease and/or atherosclerosis.
  • the TSPO, LXRa and/or PPARa modulators (for example agonists) of this invention may be exploited as a means to reduce macrophage total neutral lipid mass.
  • the invention provides TSPO, LXRa and/or PPARa modulators for use in modulating, for example reducing, macrophage total neutral lipid mass.
  • the invention provides methods for reducing macrophage total neutral lipid mass, said methods comprising administering to a subject in need thereof, a therapeutically effective amount of a TSPO, LXRa and/or PPARa modulator.
  • the uses and methods of this invention may be further applied to the treatment and/or prevention of atherosclerosis.
  • the invention provides TSPO, LXRa and/or PPARa modulators for use in treating or preventing atherosclerosis as well as methods which require the administration of one or more TSPO, LXRa and/or PPARoc modulators to a subject in need thereof, for the purpose of treating or preventing atherosclerosis.
  • the TSPO, LXRa and/or PPARa modulators for use in the instant invention may be used and/or administered alone or in combination with other TSPO, LXRa and/or PPARa modulators or existing treatments for cardiovascular disease.
  • the TSPO, LXRa and/or PPARa modulators for use in this invention may be combined with one or more other compositions, molecules or medicaments including compositions, molecules for the treatment of other (distinct) types of disease, condition or syndrome.
  • the present invention is based on the finding that expression of LXRa and/or PPARa (as well as TSPO expression) is associated with cholesterol efflux from macrophages.
  • the level of LXRa, PPARa and/or TSPO expression, function or activity may be used as the basis of a diagnostic test for detecting cardiovascular disease, atherosclerosis and/or any other related or associated diseases or conditions.
  • this invention may extend to (in vitro) methods of diagnosing cardiovascular disease (including form example atherosclerosis) and/or or a predisposition or susceptibility thereto, the method comprising the steps of
  • cardiovascular disease may include any disease of the cardiovascular system as well as syndromes and/or conditions related thereto, for example aberrant cholesterol processing and/or levels, presence of foam cells and the like.
  • a "level" of TSPO, LXRa and/or PPARa should be understood as encompassing levels (for example amounts) of the TSPO, LXRa and/or PPARa gene (or transcription products) and/or protein in a sample.
  • levels of TSPO, LXRa and/or PPARa encompasses levels of TSPO, LXRa and/or PPARa expression - as evidenced by an increase and/or decrease TSPO, LXRa and/or PPARa mRNA/DNA expression or TSPO, LXRa and/or PPARa protein as well as increases and/or decreases in levels of TSPO, LXRa and/or PPARa function and/or activity.
  • the term "levels of TSPO, LXRa and/or PPARa” includes increases and/or decreases in TSPO, LXRa and/or PPARa expression, function and/or activity.
  • a sample for use in the method provided by this aspect of the invention may be provided by a subject to be tested for a cardiovascular disease, atherosclerosis and/or a predisposition/susceptibility thereto; subjects of this type may exhibit symptoms characteristic of cardiovascular disease, atheroscleosis and/or associated conditions or syndromes.
  • the sample may be provided by asymptomatic subjects for the purposes of identifying a predisposition/susceptibility thereto.
  • a sample for use in this invention may comprise a quantity of protein and/or nucleic acid.
  • sample should be understood as including samples of bodily fluids such as whole blood, plasma, serum, saliva, sweat and/or semen.
  • a sample may comprise a tissue or gland secretion and washing protocols may be used to obtain samples of fluid secreted into or onto various tissues, including, for example, the skin.
  • samples such as tissue biopsies and/or scrapings may be used.
  • An increased and/or decreased level of TSPO, LXRa and/or PPARa may be identified by comparing levels of TSPO, LXRa and/or PPARa identified in a sample with a reference or control level of TSPO, LXRa and/or PPARa.
  • Reduced levels of TSPO, LXRa and/or PPARa expression, function and/or activity are associated with cardiovascular disease, atherosclerosis and, for example, aberrant cholesterol levels, foam cell production and aberrant macrophage cholesterol efflux.
  • a reduced level of TSPO, LXRa and/or PPARa may be detected and/or identified in a sample by comparing an identified level of TSPO, LXRa and/or PPARa with a control or reference level of TSPO, LXRa and/or PPARa.
  • levels of TSPO, LXRa and/or PPARa genes and/or proteins may be detected and/or identified in samples such as those described herein.
  • molecular or PCR based techniques may be used to detect levels of TSPO, LXRa and/or PPARa gene expression or gene quantity in a sample.
  • Useful techniques may include, for example, polymerase chain reaction (PCR) using genomic DNA as template or reverse transcriptase (RT)-PCR based techniques in combination with real-time PCR (otherwise known as quantitative PCR).
  • real time-PCR may be used to determine a level of TSPO, LXRa and/or PPARa gene expression.
  • RT-PCR may be used to reverse transcribe the relevant mRNA to complementary DNA (cDNA).
  • the reverse transcriptase protocol may use primers designed to specifically amplify an mRNA sequence of interest (in this case TSPO, LXRa and/or PPARa gene derived mRNA). Thereafter, PCR may be used to amplify the cDNA generated by reverse transcription. Typically, the cDNA is amplified using primers designed to specifically hybridise with a certain sequence and the nucleotides used for PCR may be labelled with fluorescent or radiolabelled compounds.
  • mRNA sequence of interest in this case TSPO, LXRa and/or PPARa gene derived mRNA
  • PCR may be used to amplify the cDNA generated by reverse transcription.
  • the cDNA is amplified using primers designed to specifically hybridise with a certain sequence and the nucleotides used for PCR may be labelled with fluorescent or radiolabelled compounds.
  • the amount of labelled amplified nucleic acid may be determined by monitoring the amount of incorporated labelled nucleotide during the cycling of the PCR.
  • Other techniques that may be used to determine the level of TSPO, LXRa and/or PPARa gene expression in a sample include, for example, Northern and/or Southern Blot techniques.
  • a Northern blot may be used to determine the amount of a particular mRNA present in a sample and as such, could be used to determine the amount or level of TSPO, LXRa and/or PPARa gene expression.
  • mRNA may be extracted from, for example, a sample described herein using techniques known to the skilled artisan.
  • the extracted mRNA may then be subjected to electrophoresis and a nucleic acid probe, designed to hybridise (i.e. complementary) to an mRNA sequence of interest - in this case mRNA encoding the TSPO, LXRa and/or PPARa genes, may then be used to detect and/or quantify the amount of a particular mRNA present in a sample.
  • a level of TSPO, LXRa and/or PPARa gene expression may be identified by way of microarray analysis. Such a method would involve the use of a nucleic acid probes/primers derived from the TSPO, LXRa and/or PPARa genes. Microarrays of this type may be used to identify levels of TSPO, LXRa and/or PPARa gene expression, nucleic acid, preferably mRNA, may be extracted from a sample and subjected to an amplification protocol such as, RT- PCR to generate cDNA. Primers specific for sequences encoding the TSPO, LXRa and/or PPARa gene may be used.
  • the amplified (TSPO, LXRa and/or PPARa) cDNA may be subjected to a further amplification step, optionally in the presence of labelled nucleotides (as described above). Thereafter, the optionally labelled amplified cDNA may be contacted with the microarray under conditions which permit binding with the nucleic acid probes of the microarray. In this way, it may be possible to identify a level of TSPO, LXRa and/or PPARa gene expression.
  • immunological detection techniques such as, for example, enzyme-linked immunosorbent assays (ELISAs) and/or immunohistochemical staining may be used to identify levels of TSPO, LXRa and/or PPARa proteins in samples.
  • ELISAs enzyme-linked immunosorbent assays
  • ELISPOT dot blot and/or Western blot techniques may also be used.
  • samples may be probed for levels of one or more TSPO, LXRa and/or PPARa proteins so as to detect aberrant or modulated expression, function and/or activity which may indicate cardiovascular disease, atherosclerosis and/or associated conditions or syndromes such as, for example, aberrant cholesterol levels, foam cell production and aberrant macrophage cholesterol efflux or a susceptibility or predisposition thereto.
  • Antibodies for use in this invention may optionally be conjugated to one or more detectable moieties.
  • an antibody for use in any of the immunological detection techniques described herein may be conjugated to an enzyme capable of being detected via a colourmetric/chemiluminescent reaction.
  • conjugated enzymes may include but are not limited to Horse radish Peroxidase (HRP) and alkaline phosphatise (AlkP).
  • the secondary antibodies may be conjugated to a fluorescent molecule such as, for example, a fluorophore, such as FITC, rhodamine or Texas Red.
  • Other types of detectable moiety include radiolabelled moieties.
  • TSPO TSPO
  • LXRa and/or PPARa proteins/peptides for example TSPO, LXRa and/or PPARa fragments and/or epitopes
  • animal immunisation protocols for the generation of polyclonal antibodies
  • hybridomas for generating monoclonal antibodies
  • Further information on the preparation and use of polyclonal and/or monoclonal antibodies may be obtained from Using Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1999) and Antibodies: A Laboratory Manual by Harlow & Lane (CSHLP: 1988) - both of which are incorporated herein by reference.
  • a further aspect of this invention may provide antibodies (either monoclonal or polyclonal) with an affinity or specificity for the TSPO, LXRa and/or PPARa proteins or fragments or epitopes thereof.
  • Antibodies of this type are of particular use in the diagnostic methods of this invention where the can be used to facilitate the detection of levels of TSPO, LXRa and/or PPARa proteins (of fragments) in a sample.
  • the present invention provides a kit for use in the detection and/or (in vitro) diagnosis of a cardiovascular disease, atherosclerosis and/or associated conditions or syndromes such as, for example, aberrant cholesterol levels, foam cell production and aberrant macrophage cholesterol efflux or a susceptibility or predisposition thereto.
  • the kit may comprise one or more components selected from the group consisting of:
  • a kit of this invention may further comprise reagents and/or receptacles for use in methods of diagnosing a cardiovascular disease, atherosclerosis and/or associated conditions or syndromes such as, for example, aberrant cholesterol levels, foam cell production and aberrant macrophage cholesterol efflux or a susceptibility or predisposition thereto.
  • the reagents of component (c) may comprise buffers and/or or other solutions
  • the antibodies of the kit may be conjugated to one or more detectable moieties.
  • Figure 2 The extent of overexpression (0 ⁇ g pCMV_TSPO) and knockdown ( ⁇ SiRNA) of TSPO protein, relative to GAPDH, in THP-1 macrophages, relative to empty vector (EV) and scrambled SiRNA controls, after 24h, is shown in Figure 2A.
  • Figure 3 The effect of TSPO ligands, PK11195 (30 ⁇ ) and flunitrazepam ( ⁇ ), on the efflux of [ 3 H]cholesterol to apoA-I (2( ⁇ g ml "1 ) and HDL (2( ⁇ g ml "1 ) are shown in Figure 3A and 3B, respectively.
  • Figure 3C shows the effect of treatment with FGIN-1-27 ( ⁇ ) on cholesterol efflux to ApoA-I and HDL at the same concentrations
  • Figure 3D shows the impact of the same treatment in cells transiently transfected with either the empty vector (EV) or TSPO.
  • FIG 4 The impact of TSPO overexpression on nmol incorporation of [ 14 C]acetate ( ⁇ ml "1 ) into phospholipid, triacylglycerol, cholesteryl ester, cholesterol and fatty acid lipid pools, compared with the empty vector (EV) control is shown in Figure 4A.
  • Figure 4B shows the effect of TSPO overexpression on macrophage triacylglycerol and total cholesterol mass.
  • Macrophages were exposed to a cholesterol load (AcLDL, 24h, 50 ⁇ g ml "1 ) before measuring cholesterol efflux to apoA-I (20 ⁇ g ml "1 ; 24h) (4C), and determining total cholesterol mass (dark hatched bars), and the incorporation of [ 3 H]oleate ( ⁇ ml "1 ; 10 ⁇ ; light hatched bars) into the cholesteryl ester pool (4D).
  • Values are the mean+SEM of at least four independent experiments; *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared with the relevant control incubation in the absence of acceptor; other significant differences are indicated on each Figure.
  • FIG. 6 The impact of TSPO overexpression on [ 3 H]cholesterol efflux to apoA- I (20 ⁇ g ml "1 ) is blocked by incubation with PPARoc antagonist GW6471 ( ⁇ ) ( Figure 6A), by treatment with SiRNA ( ⁇ ) directed against PPARoc (Figure 6B) and following inhibition of LXRoc using GGPP (10 ⁇ ) ( Figure 6D).
  • PPARoc and LXRoc antagonism on the effect of TSPO ligand, FGIN-1-27 ( ⁇ ) on [ 3 H]cholesterol efflux to apoA-I (20 ⁇ g ml "1 ) is shown in Figure 6C).
  • Figure 6E shows a schematic model illustrating the putative pathways by which TSPO overexpression may interface with LXRoc and PPARoc pathways to mediate the observed effects on macrophage lipid phenotype.
  • THP-1 Human monocytic cell line
  • murine RAW264.7 macrophages were purchased from the European Cell Culture Collection (Porton Down, UK).
  • Human peripheral blood monocytes were purchased from Clontech, UK and tissue culture reagents were purchased from Lonza (Wokingham, UK); other sources include Amaxa Transfection reagent (Lonza, UK), NuPage gels and buffers (Life Technologies, Paisley, Scotland), antibodies (AbCAM, Cambridge, UK) and primers and probes (Eurogentec, Belgium).
  • Apolipoprotein A-I, high density lipoprotein (HDL) and low density density lipoprotein (LDL) were purchased from Athens Research UK (USA); LDL was acetylated according to Brown et al 20 .
  • Radiochemicals [ 3 H]cholesterol, [ 14 C]acetic acid and [ 3 H]oleic acid) were provided by ICN Biologicals; all other chemicals were provided by Sigma- Aldrich (Poole, Dorset, UK).
  • Mammalian expression vectors pCMV6 encoding TSPO, DBI, VDAC and ANT were supplied by Origene, via Cambridge Biosciences (UK); TriFecta Dicer Substrate RNAi duplexes directed against TSPO and scrambled control sequences were supplied by Integrated DNA Technologies (IDT, Germany). We are greatly indebted to Professor D.J. Mangelsdorf (South Western Medical Center, USA) for his kind gift of LXRE reporter plasmid (pCMX.LXRE).
  • Human THP-1 monocytes were maintained using a split ratio of 1 : 10 in RPMI medium supplemented with foetal bovine serum (FBS, 10%, v/v), L-glutamine (200mM) and penicillin/streptomycin (50 ⁇ g ml "1 ; 50 U ml "1 , respectively).
  • FBS foetal bovine serum
  • 200mM L-glutamine
  • penicillin/streptomycin 50 ⁇ g ml "1 ; 50 U ml "1 , respectively.
  • cells were plated onto 12-well tissue culture dishes at a density of lxlO 6 cells well "1 , in complete RPMI medium (above) supplemented with ⁇ phorbol 12-myristate 13-acetate (PMA) to induce macrophage differentiation.
  • PMA ⁇ phorbol 12-myristate 13-acetate
  • Transient transfections were also performed using the same quantities of vector encoding full length VDAC, ANT and DBI.
  • Macrophages were treated with TSPO ligands for 48h prior to assessment of cholesterol efflux, using DMSO ( ⁇ 0.01%) as vehicle.
  • Transient expression of pCMX.LXRE (0 ⁇ g) was achieved using Fugene6 (6 ⁇ 1: ⁇ g DNA; 24h) and luciferase activity determined (Britelite Plus, Perkin Elmer)
  • Macrophage cell lysates were prepared in RIPA buffer (25mM Tris HC1 pH 7.6, 150mM NaCl, 1% (v/v), NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl suphate) supplemented with CompleteTM protease inhibitor cocktail (Roche), and proteins (20-50 ⁇ g lane "1 ) separated using NuPAGE (10%, w/v) gels.
  • FIG. 1A The levels of mRNA encoding TSPO, ANT and VDAC, relative to GADPH, in human peripheral blood monocytes, human heart aorta, THP-1 macrophages and THP-1 macrophage 'foam cells' are shown in Figure 1A.
  • Exposure of THP-1 macrophages to two doses of 50 ⁇ g ml "1 AcLDL (24h), led to a modest but significant increase (27%; p ⁇ 0.05) in total cholesterol mass (control 58.0+1.7 ⁇ g mg "1 cell protein versus AcLDL 73.7+7.2 ⁇ g mg "1 cell protein; p ⁇ 0.05; n 4), a mild cholesterol loading condition that avoids the overt toxicity associated with exposure to higher concentrations of modified LDL 25 .
  • Analysis (Matlnspector) of the - 3kb upstream promoter region of TSPO revealed a number of putative transcription factor binding sites, including sterol regulatory elements (-2113-2097; -1957-1922; - 1783-1769) and peroxisome proliferator activated receptor (PPAR) responsive elements (-2214-2191; -366-344).
  • Wild type THP-1 macrophages were treated with established TSPO ligands, PK11195, FGIN-1-27 and flunitrazepem, using concentrations commonly utilised to increase mitochondrial cholesterol trafficking and steroidogenesis 16 ' 17 .
  • overexpression of mitochondrial cholesterol trafficking protein, steroidogenic acute regulatory protein (StAR; STARD1) is associated with induction of lipogenesis in macrophages 7 .
  • StAR steroidogenic acute regulatory protein
  • TSPO cholesterol efflux pathway
  • TSPO 18kDa translocator protein
  • PPARa can be regulated by stress, hormones and starvation in rodents, while human PPARa can mediate its own expression, and is induced during macrophage differentiation in response to high glucose levels 33 .
  • the PPARa promoter can be activated by PPAR agonists in an LXRa dependent manner, and LXRa is a PPARa target 35 , so that these transcription factors can work together in a cooperative manner to control ABCA1 expression and cholesterol efflux 32 .
  • TSPO Overexpression of TSPO induces expression of LXRa and PPARa, while use of either SiRNA or an antagonist directed against PPARa (GW6471) (Figure 6) indicates an obligate role for PPARa in mediating TSPO-dependent increases in [ 3 H]cholesterol efflux to apoA-I and possibly other cholesterol acceptors (Figure 2B).
  • Activation of PPARoc is known to induce modest increases in lipogenesis, possibly via participation in the generation of an endogenous LXRoc ligand 35 , and enhances fatty acid oxidation, by inducing the expression of long chain fatty acyl CoA synthetases and carnitine palmitoyl transferase- 1, essential for generating fatty acyl CoA and facilitating the entry of fatty acyl carnitine into mitochondria for ⁇ -oxidation 32 34 .
  • These data provide a plausible explanation for the observed reductions in macrophage triacylglycerol content (Figure 4A) and [ 14 C]fatty acid levels (Fig 4B).
  • Tspo can be regulated by protein kinase Ce, by downstream signalling pathways involving mitogen-activated protein kinase MAPK (Raf-ERKl/2), and by acting on targets such as c-Jun, and the signal transducer and activator of transcription 3 (STAT3) ' .
  • STAT3 gene delivery can reduce atherosclerotic lesions in LDL receptor knockout mice fed a high cholesterol diet 37 , but whether induction of TSPO forms part of that response is not established.
  • TSPO TSPO to moderate gene transcription
  • the bacterial ortholog, TspO outer membrane protein found in Rhodobacter sphaeroides, which is involved in effluxing intermediates of the porphyrin biosynthetic pathway, acts as a co-activator of transcription of a number of genes, encoding enzymes involved in photopigment biosynthesis 39 ' 40 .
  • Arabidopsis TSPO is also linked to porphyrin metabolism, and appears to act as a signal linking water- related stress to transient increases in gene expression of the plant stress hormone, absicisc acid 41 .
  • Papadopoulos V Control of hypercholesterolemia and atherosclerosis using the cholesterol recognition/interaction amino acid sequence of the translocator protein TSPO. Steroids 2012; http://dx.doi.Org/10.1016/i.steroids.2012.10.018.
  • SREBP Sterol responsive element-binding protein

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Abstract

La présente invention repose sur la découverte que, par l'intermédiaire de la modulation de la fonction, de l'activité et/ou de l'expression des protéines de récepteur hépatique X (LXR) α et/ou des récepteurs activés par les proliférateurs de peroxysomes (PPAR) α (et/ou les gènes codant pour celles-ci), il est possible de moduler l'efflux de cholestérol à partir des macrophages. PPARα est une protéine de récepteur nucléaire qui fonctionne comme un facteur de transcription pour réguler l'expression de gènes. L'isoforme de PPARα est codée par le gène PPARα se trouvant à l'emplacement 22q12-13.1 sur le chromosome humain. LXR est un membre de la famille des récepteurs nucléaires de facteurs de transcription. Les LXR sont des régulateurs important de l'homéostasie du cholestérol, des acides gras et du glucose. L'isoforme LXRα est codée par le gène LXRα se trouvant à l'emplacement 11p11.2 sur le chromosome humain.
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CN105232561A (zh) * 2015-10-30 2016-01-13 黄恺 22(r)-羟基胆固醇作为parp1抑制剂的应用
CN105232507A (zh) * 2015-10-30 2016-01-13 黄恺 Gw3965作为parp1抑制剂的应用
CN105250253A (zh) * 2015-10-30 2016-01-20 黄恺 T0901317作为parp1抑制剂的应用
WO2019071169A1 (fr) * 2017-10-05 2019-04-11 National Health Research Institutes Procédé et composition pour traiter un carcinome hépatocellulaire sans infection virale par contrôle de l'homéostasie lipidique
WO2020169472A3 (fr) * 2019-02-18 2020-11-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés d'induction de changements phénotypiques dans des macrophages
CN117007806A (zh) * 2023-09-21 2023-11-07 中国人民解放军军事科学院军事医学研究院 靶向肝巨噬细胞内lxr用于控制慢乙肝进展

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105232561A (zh) * 2015-10-30 2016-01-13 黄恺 22(r)-羟基胆固醇作为parp1抑制剂的应用
CN105232507A (zh) * 2015-10-30 2016-01-13 黄恺 Gw3965作为parp1抑制剂的应用
CN105250253A (zh) * 2015-10-30 2016-01-20 黄恺 T0901317作为parp1抑制剂的应用
WO2019071169A1 (fr) * 2017-10-05 2019-04-11 National Health Research Institutes Procédé et composition pour traiter un carcinome hépatocellulaire sans infection virale par contrôle de l'homéostasie lipidique
WO2020169472A3 (fr) * 2019-02-18 2020-11-05 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés d'induction de changements phénotypiques dans des macrophages
CN117007806A (zh) * 2023-09-21 2023-11-07 中国人民解放军军事科学院军事医学研究院 靶向肝巨噬细胞内lxr用于控制慢乙肝进展

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