EP1239848A2 - Compositions et methodes permettant de moduler le taux de hdl cholesterol et de triglycerides - Google Patents

Compositions et methodes permettant de moduler le taux de hdl cholesterol et de triglycerides

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Publication number
EP1239848A2
EP1239848A2 EP00974705A EP00974705A EP1239848A2 EP 1239848 A2 EP1239848 A2 EP 1239848A2 EP 00974705 A EP00974705 A EP 00974705A EP 00974705 A EP00974705 A EP 00974705A EP 1239848 A2 EP1239848 A2 EP 1239848A2
Authority
EP
European Patent Office
Prior art keywords
abcl
compound
biological activity
candidate compound
disease
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Withdrawn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
EP00974705A
Other languages
German (de)
English (en)
Inventor
Michael R. Hayden
Angela R. Brooks-Wilson
Simon N. Pimstone
Susanne M. Clee
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.)
University of British Columbia
Xenon Pharmaceuticals Inc
Original Assignee
University of British Columbia
Xenon Genetics Inc
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
Priority claimed from US09/526,193 external-priority patent/US6617122B1/en
Application filed by University of British Columbia, Xenon Genetics Inc filed Critical University of British Columbia
Publication of EP1239848A2 publication Critical patent/EP1239848A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • HDL-C low HDL cholesterol
  • CVD cardiovascular disease
  • CAD coronary artery disease
  • cerebrovascular disease coronary restenosis
  • peripheral vascular disease low HDL cholesterol
  • HDL-C levels are influenced by both environmental and genetic factors.
  • HDL-C concentration is inversely related to the incidence of CAD.
  • HDL-C levels are a strong graded and independent cardiovascular risk factor.
  • HDL-C high-density lipoprotein
  • Coronary disease risk is increased by 2% in men and 3% in women for every 1 mg/dL (0.026 mmol/l) reduction in HDL-C and in the majority of studies this relationship is statistically significant even after adjustment for other lipid and non-lipid risk factors.
  • Decreased HDL-C levels are the most common lipoprotein abnormality seen in patients with premature CAD. Four percent of patients with premature CAD have an isolated form of decreased HDL-C levels with no other lipoprotein abnormalities while 25% have low HDL-C levels with accompanying hypertriglyceridemia.
  • HDL-C levels are important predictors of CAD.
  • those with isolated low HDL-C had a 65% increased death rate compared to diabetics with normal HDL-C levels (>0.9 mmol/l).
  • HDL-C level is an important predictor of CAD.
  • Low HDL-C levels thus constitute a major, independent, risk for CAD.
  • HDL-C values below 0.9 mmol/l confer a significant risk for men and women. As such, nearly half of patients with CAD would have low HDL-C. It is therefore crucial that we obtain a better understanding of factors which contribute to this phenotype. In view of the fact that pharmacological intervention of low HDL- C levels has so far proven unsatisfactory, it is also important to understand the factors that regulate these levels in the circulation as this understanding may reveal new therapeutic targets. Absolute levels of HDL-C may not always predict risk of CAD. In the case of CETP deficiency, individuals display an increased risk of developing CAD, despite increased HDL-C levels.
  • This process has multiple steps which include the binding of HDL to cell surface components, the acquisition of cholesterol by passive abso ⁇ tion, the esterification of this cholesterol by LCAT and the subsequent transfer of esterified cholesterol by CETP, to VLDL and chylomicron remnants for liver uptake. Each of these steps is known to impact the plasma concentration of HDL.
  • TD Tangier disease
  • OMIM 205400 An autosomal recessive trait
  • TD patients have very low HDL-C and ApoAI levels, which have been ascribed to impairment of lipid transport and hypercatabolism of nascent HDL and ApoAI, due to a delayed acquisition of lipid and resulting failure of conversion to mature HDL.
  • TD patients accumulate cholesterol esters in several tissues, resulting in characteristic features, such as enlarged yellow tonsils, corneal opacity, hepatosplenomegaly, peripheral neuropathy, and cholesterol ester deposition in the rectal mucosa.
  • the invention features a method for treating a patient diagnosed as having a lower than normal HDL-cholesterol level or a higher than normal triglyceride level.
  • the method includes administering to the patient a compound that modulates LXR- mediated transcriptional activity.
  • the compound is administered to the patient with a pharmaceutically acceptable carrier.
  • the compound may be selected, for example, from the group consisting of 24-(S),25-epoxycholesterol;
  • the compound is an oxysterol.
  • the invention features another method for treating a patient diagnosed as having a lower than normal HDL-cholesterol level or a higher than normal triglyceride level. This method includes administering to the patient a compound that modulates RXR-mediated transcriptional activity.
  • RXR-modulating compounds include hetero ethylene derivatives; tricyclic retinoids; trienoic retinoids; benzocycloalkenyl-alka:di- or trienoic acid derivatives; bicyclic-aromatic compounds and their derivatives; bicyclylmethyl-aryl acid derivatives; phenyl-methyl heterocyclic compounds; tetrahydro-napthyl compounds; arylthio-tetrahydro-naphthalene derivatives and heterocyclic analogues; 2,4-pentadienoic acid derivatives; tetralin-based compounds; nonatetraenoic acid derivatives; SRI 1237; dexamethasone; hydroxy, epoxy, and carboxy derivatives of methoprene; bicyclic benzyl, pyridinyl, thiophene, furanyl, and pyrrole derivatives; benzofuran-acrylic acid derivatives; aryl-substituted and aryl and (3-o
  • the invention features a method for determining whether a candidate compound modulates ABCl expression by performing the steps of: (a) providing a nucleic acid molecule that includes an ABCl regulatory region or promoter operably linked to a reporter gene; (b) contacting the nucleic acid molecule with the candidate compound; and (c) measuring expression of the reporter gene, wherein altered reporter gene expression, relative to a control not contacted with the compound, indicates that the candidate compound modulates ABCl expression.
  • the regulatory region includes 50 or more consecutive amino acids selected from nudeotides 5854 to 6694, 7756 to 8318, 10479 to 10825, 15214 to 16068, 21636 to 22111, 27898 to 28721, 32951 to 33743, 36065 to 36847, 39730 to 40577, 4543 to 5287, or 45081 to 55639 of SEQ ID NO: 1.
  • the regulatory region 50 or more consecutive amino acids selected from nudeotides 1 to 28,707 or 29,011 to 53,228 of SEQ ID NO: 1.
  • the regulatory region includes a binding site for a transcription factor selected from a group consisting of LXRs, RXRs, RORs, SREBPs, and PPARs.
  • the invention features a method for determining whether a person has an altered risk for developing cardiovascular disease.
  • This method includes examining the person's ABCl gene for polymo ⁇ hisms or mutations. The presence of a polymo ⁇ hism or mutation associated with cardiovascular disease indicates the person has an altered risk for developing cardiovascular disease.
  • the invention features a method for predicting a person's response to a drug by determining whether the person has a polymo ⁇ hism in an ABCl gene that alters the person's response to the drug.
  • Preferred polymo ⁇ hisms are depicted in Fig. 4.
  • the polymo ⁇ hism is in the 5' regulatory region of ABCl.
  • the invention features a substantially purified LXR response element comprising the nucleotide sequence
  • the LXR response element has the sequence AGATCACTTGAGGTCA (SEQ ID NO: 232). Even more preferably, the LXR response element consists essentially of the nucleotide sequence AGATCANNNNAGGTCA, wherein each N is, independently, C, T, G, or A
  • the invention features a substantially pure nucleic acid molecule that consists essentially of a region that is substantially identical to at least 50, 100, 150, 300, 500, 750, 1000, 2000, 3000, 4000, 5000 or all of the consecutive nudeotides selected from nudeotides 5854 to 6694, 7756 to
  • the invention features a substantially pure nucleic acid molecule that has a region that is substantially identical to nudeotides 5854 to 6694, 7756 to 8318, 10479 to 10825, 15214 to 16068, 21636 to 22111, 27898 to 28721, 32951 to 33743, 36065 to 36847, 39730 to 40577, 45081 to 55639, 4543 to 5287, 59188 to 60306, 60689 to 63548, 63574 to 65110, 65030 to 68312, 68605 to 73375, 73395 to 74692, 75586 to 77103, 74774 to 74920, 77519 to 87679, 87651 to
  • nucleic acid molecules have a region that is substantially identical or identical to nudeotides 1 to 28,707 of SEQ ID NO:
  • the invention features a method of treating a human having a lower than normal HDL-cholesterol level, a higher than normal triglyceride level, or a cardiovascular disease, including administering to the human an ABCl polypeptide, or a cholesterol- or triglyceri de-regulating fragment thereof, or a nucleic acid molecule encoding an ABCl polypeptide, or a cholesterol- or triglyceride-regulating fragment thereof.
  • the human has a low cholesterol or high triglyceride level relative to normal.
  • the ABCl polypeptide is wild-type ABCl, or has a mutation that increases its stability or its biological activity.
  • the nucleic acid molecule is operably linked to a promoter and contained in an expression vector.
  • a preferred biological activity is improved regulation of cholesterol transport.
  • the invention features a method of treating or preventing a lower than normal HDL-cholesterol level, a higher than normal triglyceride level, or a cardiovascular disease, including administering to an animal (e.g., a human) a compound that mimes the activity of wild-type ABCl, R219K ABCl, or V399A ABCl or modulates the biological activity of ABCl.
  • cardiovascular disease One preferred cardiovascular disease that can be treated using the methods of the invention is coronary artery disease. Others include cerebrovascular disease and peripheral vascular disease.
  • the ABCl gene and protein are involved in cholesterol transport that affects serum HDL levels allows the ABCl protein and gene to be used in a variety of diagnostic tests and assays for identification of HDL-increasing, triglyceride-lowering, or CVD-inhibiting drugs.
  • diagnostic tests and assays for identification of HDL-increasing, triglyceride-lowering, or CVD-inhibiting drugs.
  • the ability of domains of the ABCl protein to bind ATP is utilized; compounds that enhance this binding are potential HDL-increasing or triglyceride-lowering drugs.
  • the anion transport capabilities and membrane pore-forming functions in cell membranes can be used for drug screening.
  • ABCl expression can also serve as a diagnostic tool for a lower than normal HDL-cholesterol level, a higher than normal triglyceride level, or CVD; determination of the genetic subtyping of the ABCl gene sequence can be used to subtype individuals or families with lower than normal HDL levels or higher than normal triglyceride levels to determine whether the lower than normal HDL or higher than normal triglyceride phenotype is related to ABCl function.
  • This diagnostic process can lead to the tailoring of drug treatments according to patient genotype (referred to as pharmacogenomics), including prediction of the patient's response (e.g., increased or decreased efficacy or undesired side effects upon administration of a compound or drug).
  • Antibodies to an ABCl polypeptide can be used both as therapeutics and diagnostics. Antibodies are produced by immunologically challenging a B -cell-containing biological system, e.g., an animal such as a mouse, with an ABCl polypeptide to stimulate production of anti- ABCl protein by the B- cells, followed by isolation of the antibody from the biological system. Such antibodies can be used to measure ABCl polypeptide in a biological sample such as serum, by contacting the sample with the antibody and then measuring immune complexes as a measure of the ABCl polypeptide in the sample. Antibodies to ABCl can also be used as therapeutics for the modulation of ABCl biological activity.
  • a biological sample such as serum
  • the invention features a purified antibody that specifically binds to ABCl.
  • the antibody modulates cholesterol or triglyceride levels when administered to a mammal.
  • the invention features a method for determining whether candidate compound is useful for modulating cholesterol or triglyceride levels, the method including the steps of: (a) providing an ABCl polypeptide; (b) contacting the polypeptide with the candidate compound; and (c) measuring binding of the ABCl polypeptide, wherein binding of the ABCl polypeptide indicates that the candidate compound is useful for modulating cholesterol or triglyceride levels.
  • the invention features a method for determining whether a candidate compound is useful for the treatment of a lower than normal HDL-cholesterol level, a higher than normal triglyceride level, or a cardiovascular disease.
  • the method includes (a) providing an ABC transporter (e.g., ABCl); (b) contacting the transporter with the candidate compound; and
  • the ABC transporter is in a cell or a cell free assay system.
  • the invention features a method for determining whether candidate compound is useful for modulating cholesterol or triglyceride levels.
  • the method includes (a) providing a nucleic acid molecule comprising an ABC transporter promoter operably linked to a reporter gene; (b) contacting the nucleic acid molecule with the candidate compound; and (c) measuring expression of the reporter gene, wherein increased expression of the reporter gene, relative to a nucleic acid molecule not contacted with the compound, indicates that the candidate compound is useful for modulating cholesterol or triglyceride levels.
  • the invention features a non-human mammal having a transgene comprising a nucleic acid molecule encoding a mutated ABCl polypeptide.
  • the mutation is a dominant-negative mutation, such as the M >T mutation at position 1091 of ABCl.
  • the invention features an expression vector, a cell, or a non-human mammal that includes an ABCl nucleic acid molecule of the present invention.
  • the invention features a cell from a non-human mammal having a transgene that includes a nucleic acid molecule of the present invention.
  • the invention features a method for determining whether a candidate compound decreases the inhibition of a dominant-negative ABCl polypeptide.
  • the method includes (a) providing a cell expressing a dominant-negative ABCl polypeptide; (b) contacting the cell with the candidate compound; and (c) measuring ABCl biological activity of the cell, wherein an increase in the ABCl biological activity, relative to a cell not contacted with the compound, indicates that the candidate compound decreases the inhibition of a dominant-negative ABCl polypeptide.
  • a preferred dominant-negative ABCl polypeptide is M1091T ABCl.
  • the invention features a method of determining in a subject a propensity for a disease or condition selected from the group consisting of a lower than normal HDL level, a higher than normal triglyceride level, and a cardiovascular disease.
  • This method involves determining the presence or absence of at least one ABCl polymo ⁇ hism in the polynudeotide sequence of an ABCl regulatory region, promoter, or coding sequence or in the amino acid sequence of an ABCl protein in a sample obtained from the subject, wherein the presence or absence of the ABCl polymo ⁇ hism is indicative of a risk for the disease or condition.
  • the method also includes analyzing at least five ABCl polymorphic sites in the polynudeotide sequence or the amino acid sequence.
  • the invention features a method for determining whether an ABCl polymo ⁇ hism is indicative of a risk in a subject for a disease or condition selected from the group consisting of a lower than normal
  • the method includes (a) determining whether the prevalence of the disease or condition in a first subject or set of subjects differs from the prevalence of the disease or condition in a second subject or set of subjects; (b) analyzing the polynudeotide sequence of an ABCl regulatory region, promoter, or coding sequence or the amino acid sequence of an ABCl protein in a sample obtained from the first subject or set of subjects and the second subject or set of subjects; and (c) determining whether at least one ABCl polymo ⁇ hism differs between the first subject or set of subjects and the second subject or set of subjects, wherein the presence or absence of the ABCl polymo ⁇ hism is correlated with the prevalence of the disease or condition, thereby determining whether the ABCl polymo ⁇ hism is indicative of the risk.
  • the method further includes analyzing at least five ABCl polymo ⁇ hic sites in the polynudeotide sequence of an ABCl regulatory region, promoter,
  • the invention provides an electronic database having a plurality of sequence records of ABCl polymorphisms correlated to records of predisposition to or prevalence of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the invention features a method for selecting a preferred therapy for modulating ABCl activity or expression in a subject. This method includes (a) determining the presence or absence of at least one
  • the method further includes analyzing at least five ABCl polymo ⁇ hic sites in the polynudeotide sequence of an ABCl regulatory region, promoter, or coding sequence or the amino acids sequence of ABCl.
  • the invention provides a method for determining whether a candidate compound is useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • This method includes (a) providing an assay system having a measurable ABCl biological activity; (b) contacting the assay system with the candidate compound; and (c) measuring ABCl biological activity or ABCl phosphorylation. Modulation of ABCl biological activity or ABCl phosphorylation in this assay system, relative to the ABCl biological activity or ABCl phosphorylation in a corresponding control assay system not contacted with the candidate compound, indicates that the candidate compound is useful for the treatment of the disease or condition.
  • the assay system is a cell based system or a cell free system.
  • the candidate compound modulates both ABCl protein phosphorylation and ABCl activity.
  • the invention provides a method for identifying a compound to be tested for an ability to ameliorate a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • This method includes
  • the candidate compound modulates both ABCl protein phosphorylation and the ABCl activity.
  • the invention provides a method for determining whether a candidate compound is useful for modulating a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the method includes (a) providing a cell expressing an ABCl gene or a fragment thereof;
  • the invention provides a method for determining whether a candidate compound is useful for modulating a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • This method includes (a) contacting a cell expressing an ABCl protein with the candidate compound; and (b) measuring the phosphorylation of the ABCl protein. Altered ABCl protein phosphorylation in this cell, relative to the ABCl protein phosphorylation in a corresponding control cell not contacted with the candidate compound, indicates that the is useful for modulating the disease or condition.
  • the invention provides a compound useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the compound modulates ABCl biological activity, and is identified by the steps of (a) providing an assay system having a measurable ABCl biological activity; (b) contacting the assay system with the compound; and (c) measuring ABCl biological activity, wherein modulation of ABCl biological activity, relative to the ABCl biological activity in a corresponding control assay system not contacted with the compound, indicates that the compound is useful for the treatment of the disease or condition.
  • the invention provides a compound useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the compound induces a change in ABCl biological activity that mimics the change in ABCl biological activity induced by the R219K ABCl mutation.
  • the invention provides a compound useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the compound binds or interacts with residue R219 of ABCl, thereby mimicking the change in ABCl activity induced by the R219K ABCl mutation.
  • the invention provides a compound useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the compound induces a change in ABCl biological activity that mimics the change in ABCl biological activity induced by the V339A ABCl mutation.
  • the invention provides a compound useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the compound binds or interacts with residue V399 of ABCl, thereby mimicking the change in ABCl activity induced by the V399A ABCl mutation.
  • the invention provides a compound that modulates ABCl activity and binds or interacts with an amino acid of ABCl, wherein the amino acid is a residue selected from amino acids 119 to 319 of ABCl (SEQ ID NO: 5) or amino acids 299 to 499 of ABCl (SEQ ID NO: 5).
  • the invention provides a method for determining whether a candidate compound is useful for the treatment a disease or condition selected from the group consisting of a lower than normal
  • This method involves (a) providing an assay system having a measurable LXR biological activity; (b) contacting the assay system with the candidate compound; and (c) measuring LXR biological activity, wherein modulation of LXR biological activity, relative to the LXR biological activity in a corresponding control assay system not contacted with the candidate compound, indicates that the candidate compound is useful for the treatment of the disease or condition.
  • the invention provides method for determining whether a candidate compound is useful for modulating ABCl biological activity.
  • This method involves (a) providing an assay system having a measurable LXR biological activity; (b) contacting the assay system with the candidate compound; and (c) measuring LXR biological activity, wherein modulation of LXR biological activity, relative to the LXR biological activity in a corresponding control assay system not contacted with the candidate compound, indicates that the candidate compound is useful for modulating ABCl biological activity.
  • the LXR biological activity is modulation of ABCl expression.
  • the invention provides method for identifying a compound to be tested for an ability to modulate ABCl biological activity.
  • This method involves (a) contacting a subject or cell with a candidate compound; (b) assaying the activity of the LXR gene product in the subject or cell; wherein modulation of the activity, relative to the activity in a corresponding control subject or cell not contacted with the candidate compound, identifies the candidate compound as a compound to be tested for an ability to modulate the biological activity of ABCl.
  • the invention provides the use of an LXR gene product in an assay to identify compounds useful for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the invention features the use of a compound that modulates the activity or expression of an LXR gene product for the treatment of a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease.
  • the invention provides a method for identifying a compound to be tested for an ability to treat a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease. This method involves
  • the invention provides a method for screening an candidate LXR agonist for the ability to treat a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease. This method involves
  • the invention provides a method for screening a candidate LXR modulating compound for the ability to treat a disease or condition selected from the group consisting of a lower than normal HDL cholesterol level, a higher than normal triglyceride level, and a cardiovascular disease. This method involves (a) contacting a cell with the candidate LXR modulating compound; and
  • the invention provides a method for determining whether a candidate compound is useful for modulating triglyceride levels.
  • the method involves (a) providing a cell comprising an ABCl polypeptide comprising amino acids 1 to 60 of SEQ ID NO: 5; (b) contacting the cell with the candidate compound; and (c) measuring the half-life of the ABCl polypeptide, wherein an increase in said half-life, relative to the half-life in a corresponding control cell not contacted with the compound, indicates that the candidate compound is useful for modulating triglyceride levels.
  • the invention features method for determining whether a candidate compound mimics ABCl biological activity.
  • the method includes (a) providing a cell that is not expressing an ABCl polypeptide; (b) contacting the cell with the candidate compound; and (c) measuring ABCl biological activity of the cell, wherein altered ABCl biological activity, relative to a corresponding control cell not contacted with the compound, indicates that the candidate compound modulates ABCl biological activity.
  • the cell has an ABCl null mutation.
  • the cell is in a mouse or a chicken (e.g., a WHAM chicken) in which its ABCl gene has been mutated.
  • the cell is in an animal.
  • the preferred biological activity is transport of cholesterol (e.g., HDL cholesterol or LDL cholesterol) or interleukin-1, or is binding or hydrolysis of ATP by the ABCl polypeptide.
  • the ABCl polypeptide used in the screening methods includes amino acids 1-60 of SEQ ID NO: 5.
  • the ABCl polypeptide can include a region encoded by a nucleotide sequence that hybridizes under high stringency conditions to nudeotides 75 to 254 of SEQ ID NO: 6.
  • the subject is a human.
  • the cell or assay system has an exogenously supplied copy of an
  • LXRE selected from the group consisting of SEQ ID NO: 94, SEQ ID NO: 92, and the LXRE consensus motif at nucleotide -7670 of the 3' end of intron 1.
  • a preferred LXR biological activity is modulation of ABCl expression.
  • a preferred LXR gene product is an ABCl nucleic acid molecule or protein.
  • ABCl regulatory regions may be determined by sequencing the rest of the 418, 31J20, 47019, or 179G21 Research Genetics RPCI-11 BACs using the methods described herein.
  • Substantially pure nucleic acids containing regions substantially identical to at least 50, 100, 150, 300, 500, 750, 1000, 2000, 3000, 4000, 5000 consecutive nudeotides of these regions may be used in the methods of the present invention.
  • polypeptide any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
  • reporter gene any gene which encodes a product whose expression is detectable and/or quantifiable by physical, immunological, chemical, biochemical, or biological assays.
  • a reporter gene product may, for example, have one of the following attributes, without restriction: a specific nucleic acid/chip hybridization pattern, fluorescence (e.g., green fluorescent protein), enzymatic activity (e.g., lacZ/ ⁇ -galactosidase, luciferase, chloramphenicol acetyltransferase), toxicity (e.g., ricin A), or an ability to be specifically bound by a second molecule (e.g., biotin or a detectably labeled antibody). It is understood that any engineered variants of reporter genes, which are readily available to one skilled in the art, are also included, without restriction, in the foregoing definition.
  • operably linked is meant that a gene and a regulatory sequence are connected in such a way as to permit expression of the gene product under the control of the regulatory sequence.
  • a promoter may also be operably linked to a gene such that expression of the gene product is under control of the promoter.
  • regulatory region is meant a region that, when operably linked to a promoter and a gene (e.g., a reporter gene), is capable of modulating the expression of the gene from the promoter.
  • Regulatory regions include, for example, nuclear hormone transcription factor binding sites such as those described herein and may be found in intronic sequence.
  • promoter is meant a minimal sequence sufficient to direct transcription of an operably-linked gene.
  • substantially identical is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% identity to a reference amino acid or nucleic acid sequence.
  • the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids.
  • the length of comparison sequences will generally be at least 50 nudeotides, preferably at least 60 nudeotides, more preferably at least 75 nudeotides, and most preferably 110 nudeotides.
  • One sequence may include additions or deletions (i.e., gaps) of 20% or less when compared to the second sequence.
  • Optimal alignment of sequences may be conducted, for example, by the methods of Gish and States (Nature Genet. 3:266-272, 1993), Altshul et ai. (J. Mol. Biol. 215:403-410, 1990), Madden et ai. (Meth. Enzymol. 266: 131-141, 1996), Althsul et al (Nucleic Acids Res. 25:3389-3402, 1997), or Zhang et al (Genome Res. 7:649-656, 1997).
  • Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • substantially pure nucleic acid nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid.
  • the term therefore includes, for example, a recombinant nucleic acid that is inco ⁇ orated into a vector; into an autonomously replicating plasmid or virus; into the genomic nucleic acid of a prokaryote or a eukaryote cell; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant nucleic acid that is part of a hybrid gene encoding additional polypeptide sequence.
  • high stringency conditions hybridization in 2X SSC at 40 °C with a DNA probe length of at least 40 nudeotides.
  • high stringency conditions see F. Ausubel et al., Current Protocols in Molecular Biology, pp. 6.3.1-6.3.6, John Wiley & Sons, New York, NY, 1994, hereby inco ⁇ orated by reference.
  • modulates increase or decrease.
  • a compound that modulates LXR-mediated transcription, RXR-mediated transcription, ABCl gene expression, HDL-C levels, or triglyceride levels does so by at least 5%, more preferably by at least 10%, and most preferably by at least 25% or even at least 50%.
  • purified antibody is meant antibody which is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably 90%, and most preferably at least 99%, by weight, antibody.
  • a purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
  • telomere sequence of Fig. 2A (SEQ ID NO: 5).
  • polymo ⁇ hism is meant that a nucleotide or nucleotide region is characterized as occurring in several different forms.
  • a “mutation” is a form of a polymo ⁇ hism in which the expression level, stability, function, or biological activity of the encoded protein is substantially altered.
  • LXR nuclear receptors LXR ⁇ and LXR ⁇ .
  • Preferred LXRs include human LXR ⁇ (GenBank accession no. Q13133) and human LXR ⁇ (GenBank accession no. P55055)(see Apfel et al., Mol. Cell. Biol.
  • RXR nuclear receptors RXR ⁇ , RXR ⁇ ., and RXR ⁇ .
  • Preferred RXRs include human RXR ⁇ (GenBank accession no. Q13133), human RXR ⁇ (GenBank accession no. S37781), and human RXR ⁇ .(GenBank accession no. Q13133).
  • ABC transporter or “ABC polypeptide” is meant any transporter that hydrolyzes ATP and transports a substance across a membrane.
  • an ABC transporter polypeptide includes an ATP Binding Cassette and a transmembrane region.
  • Examples of ABC transporters include, but are not limited to, ABCl, ABC2, ABCR, and ABC8.
  • ABCl polypeptide is meant a polypeptide having substantial identity to an ABCl polypeptide having the amino acid sequence of SEQ ID NO: 5.
  • ABSC biological activity or “ABCl biological activity” is meant hydrolysis or binding of ATP, transport of a compound (e.g., cholesterol, interleukin-1) or ion across a membrane, or regulation of cholesterol or phospholipid levels (e.g., either by increasing or decreasing HDL-cholesterol or LDL-cholesterol levels).
  • a compound e.g., cholesterol, interleukin-1
  • ion across a membrane
  • regulation of cholesterol or phospholipid levels e.g., either by increasing or decreasing HDL-cholesterol or LDL-cholesterol levels.
  • the invention provides methods for treating patients having low HDL- C and/or higher than normal triglyceride levels by administering compounds that modulate ABCl biological activity or expression.
  • the compounds may modulate the transcriptional activity of LXR/RXR heterodimers.
  • Many compounds that modulate LXR transcriptional activity or RXR transcriptional activity are known in the art.
  • Preferred compounds of the invention are oxysterols; additional compounds are described herein.
  • the invention also provides screening procedures for identifying therapeutic compounds (cholesterol-modulating, triglyceride-modulating, or anti-CVD pharmaceuticals) which can be used in human patients.
  • Screening methods of the invention involve screening any number of compounds for therapeutically active agents by employing any number of in vitro or in vivo experimental systems. Exemplary methods useful for the identification of such compounds are detailed below.
  • the methods of the invention simplify the evaluation, identification and development of active agents for the treatment and prevention of low HDL, higher than normal triglyceride levels, and CVD.
  • the screening methods provide a facile means for selecting natural product extracts or compounds of interest from a large population which are further evaluated and condensed to a few active and selective materials. Constituents of this pool are then purified and evaluated in the methods of the invention to determine their HDL-raising, triglyceride-lowering, anti-CVD activities, or a combination thereof.
  • Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof.
  • Fig. 1 shows the genomic sequence of human ABCl, including exons 1-50 (SEQ ID NO: 1).
  • Capital letters denote exonic sequence, lower case letters denote 5' regulatory sequence or intronic sequence.
  • “Z” denotes any nucleotide or other, including no nucleotide.
  • Fig. 2 A is the amino acid sequence of the human ABCl protein (SEQ ID NO: 5).
  • Fig. 2B is the nucleotide sequence of the human ABCl cDNA (SEQ ID NO: 6).
  • the numbering is based on the first base of the promoter as nucleotide number -1.
  • the numbering is based on the first base of exon 1 as nucleotide number +1.
  • the numbering is based on the first position in intron 1 as +1.
  • the numbering is based on the first base '5 to the start of exon 2 as nucleotide number -1.
  • Fig. 4 is a table summarizing polymorphisms in the genomic ABCl sequences.
  • Figs. 5 A and 5B are bar graphs showing the percent of heterozygotes or unaffected family members with HDL (Fig. 5A) and triglycerides (TG) (Fig 5B) within a given range of percentiles for age and sex, based on the LRC criteria (Heiss et al, Circulation 62:IV-116-IV-136, 1980). A broad distribution of HDL levels was seen in the heterozygotes, extending up to the 31 st percentile for age and sex.
  • Fig. 6 is a table characterizing TD patients, ABCl heterozygotes, and unaffected family members.
  • Fig. 7 is a table summarizing the incidence of CAD in ABCl heterozygotes.
  • Fig. 8 is a graph showing the average HDL levels in heterozygotes for each mutation versus the efflux levels measured in a heterozygous carrier of each mutation.
  • the HDL levels are expressed as the percentage of the mean HDL level in the unaffected members of that family.
  • the efflux levels are highly correlated with the levels of HDL cholesterol and are associated with 82% of the variation in HDL cholesterol levels.
  • Fig. 9 is a table summarizing the HDL levels and presence or absence of
  • the deletion of C6825 in the nucleotide sequence is a frame-shift mutation that results in a STOP codon at the codon corresponding to amino acid 2145 of the encoded protein.
  • CTC6952-4TT->2203X "CTC” is replaced by " ⁇ / ⁇ ” ⁇ n the nucleotide sequence, resulting in the conversion of the codon encoding amino acid 2203 to a stop codon.
  • Fig. 10 is a table comparing the mean lipid levels in unaffected family members and ABCl heterozygotes with either missense or sever mutations.
  • Fig. 11 is a schematic diagram of the ABCl protein, illustrating the location of the mutations and the presence or absence of CAD in carriers of the mutations. The number (n) of heterozygotes who are 40 years or older and may have developed CAD are listed.
  • Figs. 12A and 12B are pedigrees of two FHA kindreds, FHA3 and
  • FHA1 FHA1
  • Males are denoted by square symbols, females by circles.
  • Individuals heterozygous for mutations are given half-shaded symbols, with the probands indicated by arrows.
  • a diagonal line indicates a deceased individual.
  • the youngest individuals have HDL cholesterol at higher percentile ranges than those in the older generations.
  • Fig. 13 is a bar graph showing the percentage of individuals less than 30 years of age and from 30 to less than 70 years of age with HDL cholesterol levels in a given percentile range. Younger individuals have a far broader distribution of HDL cholesterol levels, clearly indicating that the impact of
  • ABCl on HDL levels is influenced by age.
  • Fig. 14 is a table summarizing HDL and TG levels in different age groups for ABCl heterozygotes and unaffected family members.
  • Figs. 15 A and 15B are graphs showing the mean HDL level in heterozygous males (Fig. 15A) and females (Fig. 15B) in 10 year age groups
  • Figs. 16A and 16B are graphs showing the mean HDL (Fig. 16 A) and triglyceride levels (Fig. 16B) in heterozygotes and unaffected family members falling within each tertile of BMI.
  • Fig. 17 is a table showing the oligonucleotides and reaction conditions used for RFLP screening of ABCl polymo ⁇ hisms.
  • Fig. 18 is a picture of a gel showing RFLP genotyping of the R219K variant.
  • the 177 base pair PCR product is not digested for the A allele, whereas the B allele is digested producing fragments of 107 and 70 base pairs.
  • Fig. 19 is a table showing the allele frequencies of polymo ⁇ hisms in the ABCl gene.
  • Fig. 20 is a table comparing MSD, MOD, and frequency of coronary events in R219K ABCl variant carriers compared to controls.
  • Fig. 21 is a graph showing the event-free survival curves for carriers (AB+BB) and non-carriers (AA) of the R219K ABCl variant. Carriers of the variant have a 29% increased event-free survival over the two years of the trial, compared with non-carriers.
  • Fig. 22 is a table showing the baseline demographics and lipid levels in the Regression Growth Evaluation Statin Study (REGRESS) cohort by R219K ABCl genotype.
  • Fig. 23 is a table showing the lipid levels and CAD above and below the median age in R219K ABCl carriers and controls.
  • Fig. 24 is a bar graph showing the percent difference in HDL cholesterol levels between those greater and less than the median age (56.7 years) for each R219K genotype.
  • Figs. 25 A and 25B are graphs showing the correlations of HDL cholesterol (Fig. 25 A) and efflux (Fig. 25B) with age, by R219K genotype.
  • Figs. 26A and 26B are graphs showing the change in MSD (Fig. 26A) and MOD (Fig. 26B) by median age in carriers (AB+BB) and non-carriers (AA) of the R219K ABCl variant.
  • Fig. 27 is a table showing the ethnic distribution of the R219K ABCl variant.
  • the human ABCl also known as ABCA1 genomic region contains consensus binding sites for transcription factors such as LXRs, RXRs, PPARs, SREBPs, and RORs.
  • the sequence of additional regions of the ABCl regulatory region which also contain consensus binding sites for transcription factors.
  • heterozygotes for ABCl mutations have age- modulated decreases in HDL, increases in triglyceride levels, and significantly increased risk for CAD.
  • this phenotype was highly correlated with efflux, clearly demonstrating that impairment of reverse cholesterol transport is associated with decreased plasma HDL cholesterol, increased triglyceride levels, and increased atherogenesis.
  • the present invention features screening methods to identify therapies that increase ABCl function, resulting in increased plasma HDL cholesterol, decreased triglyceride levels, protection against atherosclerosis, or a combination of these effects.
  • Cholesterol is normally assembled with intracellular lipids and secreted, but in TD the process is diverted and cholesterol is degraded in lysosomes. This disturbance in intracellular trafficking of cholesterol results in an increase in intracellular cholesterol ester accumulation associated with mo ⁇ hological changes of lysosomes and the Golgi apparatus and cholesteryl ester storage in histiocytes, Schwann cells, smooth muscle cells, mast cells and fibroblasts.
  • FHA is much more common than TD, although its precise frequency is not known. While TD has been described to date in only 40 families, we have identified more than 40 FHA families in the Netherlands and Quebec alone. After initial suggestions of linkage to 9q31, thirteen polymo ⁇ hic markers spanning approximately 10 cM in this region were typed and demonstrated the highest LOD score at D9S277. Analysis of the homozygosity of markers in the TD-2 proband, who was expected to be homozygous for markers close to TD due to his parents' consanguinity, placed the TD gene distal to D95127. Combined genetic data from TD and FHA families pointed to the same genomic segment spanning approximately 1,000 kb between D9S127 and D9S1866.
  • the ABCl transporter gene was contained within the minimal genomic region. RT-PCR analysis in one family demonstrated a deletion of leucine at residue 693 ( ⁇ 693) in the first transmembrane domain of ABCl, which segregated with the phenotype of HDL deficiency in this family.
  • ABCl is part of the ATP-binding cassette (ABC transporter) superfamily, which is involved in energy-dependent transport of a wide variety of substrates across membranes (Dean et ai, Curr. Opin. Gen. Dev. 5:779-785, 1995). These proteins have characteristic motifs conserved throughout evolution which distinguish this class of proteins from other ATP binding proteins. In humans these genes essentially encode two ATP binding segments and two transmembrane domains (Dean et al, Curr. Opin. Gen. Dev. 5:779- 785, 1995). We have now shown that the ABCl transporter is crucial for intracellular cholesterol transport.
  • TD and FHA now join the growing list of genetic diseases due to defects in the ABC group of proteins including cystic fibrosis (Zielenski, et al,
  • heterozygotes for TD do have reduced HDL levels and that the same mechanisms underlie the HDL deficiency and cholesterol efflux defects seen in heterozygotes for TD as well as FHA. Furthermore, the more severe phenotype in TD represents loss of function from both alleles of the ABCl gene.
  • ABCl is activated by protein kinases, presumably via phosphorylation, which also provides one explanation for the essential role of activation of protein kinase C in promoting cholesterol efflux (Drobnick et al, Arterioscler. Thromb. Vase. Biol. 15: 1369-1377, 1995).
  • Brefeldin which inhibits trafficking between the endoplasmic reticulum and the Golgi, significantly inhibits cholesterol efflux, essentially reproducing the effect of mutations in ABCl, presumably through the inhibition of ABCl biological activity. This finding has significance for the understanding of mechanisms leading to premature atherosclerosis.
  • TD homozygotes develop premature coronary artery disease, as seen in the proband of TD-1 (111-01) who had evidence for coronary artery disease at 38 years. This is particular noteworthy as TD patients, in addition to exhibiting significantly reduced HDL, also have low LDL cholesterol, and yet they develop atherosclerosis despite this. This highlights the importance of HDL intracellular transport as an important mechanism in atherogenesis. There is significant evidence that heterozygotes for TD are also at increased risk for premature vascular disease (Schaefer et al, Ann. Int. Med. 93:261-266, 1980; Serfaty-Lacrosniere et ai, Atherosclerosis 107:85-98, 1994).
  • the ABCl gene plays a crucial role in cholesterol transport and, in particular, intracellular cholesterol trafficking in monocytes and fibroblasts. It also appears to play a significant role in other tissues such as the nervous system, GI tract, and the comea. Completely defective intracellular cholesterol transport results in peripheral neuropathy, comeal opacities, and deposition of cholesterol esters in the rectal mucosa.
  • HDL deficiency is heterogeneous in nature.
  • the delineation of the genetic basis of TD and FHA underlies the importance of this particular pathway in intracellular cholesterol transport, and its role in the pathogenesis of atherosclerosis.
  • Unraveling of the molecular basis for TD and FHA defines a key step in a poorly defined pathway of cholesterol efflux from cells and could lead to new approaches to treatment of patients with HDL deficiency in the general population.
  • HDL has been implicated in numerous other biological processes, including but not limited to: prevention of lipoprotein oxidation; abso ⁇ tion of endotoxins; protection against Trypanosoma brucei infection; modulation of endothelial cells; and prevention of platelet aggregation (see Genest et al, J. Invest. Med. 47: 31-42, 1999, hereby incorporated by reference). Any compound that modulates HDL levels may be useful in modulating one or more of the foregoing processes.
  • Our previous discovery that ABCl functions to regulate HDL levels links, for the first time, ABCl with the foregoing processes.
  • the phenotype of the heterozygotes was compared to that of unaffected family members, enabling the results to be controlled, at least in part, for other genetic and environmental influences.
  • prior studies in obligate heterozygotes have been limited to small numbers, often within a single family, and thus restricted in the ability to analyze the phenotypic expression with multiple mutations over a range of ages.
  • ABCl heterozygotes have an approximate 50% decrease in HDL cholesterol and apoAI, and a mild but significant decrease in apoAII.
  • ABCl heterozygotes have increased triglycerides, but in contrast to TD patients, have no significant change in total or LDL cholesterol.
  • the changes in HDL, apoAI, and triglycerides were gene-dose dependent, suggesting that they are directly related to ABCl function.
  • heterozygotes have an over three-fold increased risk of developing CAD, and younger average age-of-onset compared to unaffected individuals.
  • the heterozygotes with the most severe deficiency in efflux had a higher frequency and greater severity of CAD.
  • the severity of the phenotype observed in the heterozygotes appeared to be mutation-dependent, but there was no obvious relationship between the site of mutation and the phenotype.
  • One notable exception is the Ml 09 IT missense mutation which had the most severe phenotype, with marked reductions in HDL cholesterol and efflux in affected family members, suggesting that this mutation may act in a dominant-negative fashion, down- regulating the function of the wild-type allele.
  • Another interesting finding is the small cluster of mutations at the very C-terminal region of the protein, which suggests that this region is critical for ABCl function.
  • the phenotype in ABCl heterozygotes is also age-modulated. From 20 years of age in members of the control cohort, there is a small but definite increase in HDL with advancing age that is obviously absent in the heterozygotes. One explanation for this finding is that there is normally an age-related increase in ABCl function, which is not seen in heterozygotes, perhaps because the remaining functioning allele has already been maximally up-regulated secondary to an increase in intracellular cholesterol. This lack of age-related increase in ABCl function in heterozygotes would exaggerate the difference in HDL levels between heterozygotes and control individuals in older age groups. There is some evidence for an age-modulated increase in expression of ABC transporters (Gupta, Drugs Aging 7:19-29, 1995).
  • TD-1 is of Dutch descent while TD-2 is of British descent.
  • D9S1690 Recombination with the most proximal marker, D9S1690 was seen in 11-09 in Family TD-1, providing a centromeric boundary for the disease gene.
  • a physical map spanning approximately 10 cM in this region was established with the development of a YAC contig.
  • 22 other polymo ⁇ hic multi-allelic markers which spanned this particular region were mapped to the contig, and a subset of these were used in construction of a haplotype for further analysis.
  • D9S127 but was heterozygous at D9S127 and DNA markers centromeric to it.
  • the ABCl transporter gene had previously been mapped to 9q31, but its precise physical location had not been determined (Luciani et al, Genomics 21 : 150-159, 1994).
  • the ABCl gene is a member of the ATP binding cassette transporters which represents a super family of highly conserved proteins involved in membrane transport of diverse substrates including amino acids, peptides, vitamins and steroid hormones (Luciani et al, Genomics 21 : 150-159, 1994; Dean et al, Curr. Opin. Gen.
  • the ABCl gene was revealed encompassing 49 exons and a minimum of 75 Kb of genomic sequence.
  • ABCl its expression in fibroblasts and localization to the minimal genomic segment underlying TD, we formally assessed ABCl as a candidate.
  • RNA was used in Northern blot analysis and RT-PCR and sequence analyses.
  • RT-PCR and sequence analysis of TD-1 revealed a heterozygous T to C substitution in the TD-1 proband, which would result in a substitution of arginine for cysteine at a conserved residue between mouse and man.
  • This mutation confirmed by sequencing exon 31 of the ABCl gene, exhibited complete segregation with the phenotype on one side of this family. This substitution creates a Hgal site, allowing for RFLP analysis of amplified genomic DNA and confirmation of the mutation.
  • RT-PCR analysis of fibroblast RNA encoding the ABCl gene from the proband in TD-2 revealed a homozygous nucleotide change of A to G at nucleotide 1864 in exon 14, resulting in a substitution of arginine for glutamine at residue 597, occurring just proximal to the first predicted transmembrane domain of ABCl at a residue conserved in mouse and as well as a C. elegans homolog.
  • This mutation creates a second Act! site within exon 14.
  • Segregation analysis of the mutation in this family revealed complete concordance between the mutation and the low HDL phenotype as predicted.
  • the proband in TD-2 is homozygous for this mutation, consistent with our expectation of a disease causing mutation in this consanguineous family.
  • TD and FHA have thus far been deemed distinct with separate clinical and biochemical characteristics. Even though the genes for these disorders mapped to the same region, it was uncertain whether FHA and TD were due to mutations in the same gene or, alternatively, due to mutations in genes in a similar region. Refinement of the region containing the gene for FHA was possible by examining haplotype sharing and identification of critical recombination events. Seven separate meiotic recombination events were seen in these families, clearly indicating that the minimal genomic region containing the potential disease gene was a region of approximately 4.4 cM genomic DNA spanned by marker D9S1690 and D9S1866.
  • This region is consistent with the results of two point linkage analysis which revealed maximal LOD scores with markers D9S277 and D9S306 and essentially excluded the region centromeric to D9S1690 or telomeric to D9S1866.
  • An 8 th meiotic recombination event further refined the FHA region to distal to D9S277.
  • the ABCl gene mapped within this interval.
  • the overlapping genetic data strongly suggested that FHA may in fact be allelic to TD.
  • Utilization of sets of genetic data from FHA and TD provided a telomeric boundary at D9S1866 (meiotic recombinant) and a centromeric marker at D9S127 based on the homozygosity data of TD-2. This refined the locus to approximately 1 mb between D9S127 and D9S1866.
  • the ABCl gene mapped within this minimal region.
  • Antisense oligonucleotide AN-6 is directed to the novel start codon 5' to the one indicated in AJ012376.1; this antisense oligonucleotide effectively suppresses efflux. Polymorphisms in ABCl 5' regulatory region and 5' UTR
  • Several polymo ⁇ hisms in the 5' regulatory region of human ABCl have been identified (Fig. 4). Because of their location, it is likely that ABCl gene expression will differ among humans having different promoter polymo ⁇ hisms, and these individuals may also respond differently to the same dmg treatment. Thus, using these newly-identified polymorphisms, one can tailor dmg treatment depending on which polymo ⁇ hism(s) is/are present in a patient. The presence or absence of particular ABCl polymo ⁇ hisms may also be used in determining an individual's predisposition to developing CVD.
  • the methods of the invention may be performed using the following materials and methods.
  • Lipoprotein measurement is performed on fresh plasma as described elsewhere (Rogler et al, Arterioscler. Thromb. Vase. Biol. 15:683-690, 1995). Lipids, cholesterol and triglyceride levels are determined in total plasma and plasma at density d ⁇ 1.006 g/mL (obtained after preparative ultracentrifugation) before and after precipitation with dextran manganese. Apolipoprotein measurement is performed by nephelometry for ApoB and ApoAI.
  • the genetic markers of interest at 9q31 were identified within YAC con tigs. Additional markers that mapped to the approximate 9q31 interval from public databases and the literature were then assayed against the YAC clones by PCR and hybridization analysis. The order of markers was based on their presence or absence in the anchored YAC contigs and later in the B AC contig. Based on the haplotype analysis, the region between D9S277 and D9S306 was targeted for higher resolution physical mapping studies using bacterial artificial chromosomes (BACs). BACs within the region of interest were isolated by hybridization of DNA marker probes and whole YACs to high-density filters containing clones from the RPCI-11 human BAC library.
  • BACs bacterial artificial chromosomes
  • This may represent a leader sequence, or another transmembrane or membrane- associated region of the ABCl protein.
  • antibodies directed to the region of amino acids 1-60 are raised against and used to determine the physical relationship of amino acids 1-60 in relation to the cell membrane.
  • Other standard methods can also be employed, including, for example, expression of fusion proteins and cell fractionation.
  • the mouse ABCl sequence used has accession number X75926. It is very likely that this mouse sequence is incomplete, as it lacks the additional 60 amino acids described herein for human ABCl. Version 1.7 of ClustalW was used for multiple sequence alignments with BOXSHADE for graphical enhancement (http://www.isrec.isb- sib.ch:8080/ software/BOX_form.html) with the default parameter.
  • a Caenorhabditis elegans ABCl orthologue was identified with BLAST (version 2.08) using CAA1005.1 (see above) as a query, with the default parameter except for doing an organism filter for C. elegans.
  • the selected protein sequence has accession version number AAC69223.1 with a score of 375, and an E value of 103.
  • BAC DNA was extracted from bacterial cultures using NucleoBond Plasmid Maxi Kits (Clontech, Palo Alto, CA). For DNA sequencing, a sublibrary was first constmcted from each of the BAC DNAs (Rowen et al, Automated DNA Sequencing and Analysis, eds. Adams, M.D., Fields, C. & Venter, J.C., 1994). In brief, the BAC DNA was isolated and randomly sheared by nebulization.
  • the sheared DNA was then size fractionated by agarose gel electrophoresis and fragments above 2 kb were collected, treated with Mung Bean nuclease followed by T4 DNA polymerase and klenow enzyme to ensure blunt-ends, and cloned into Sr ⁇ l-cut M13mpl9. Random clones were sequenced with an ABI373 or 377 sequencer and fluorescently labeled primers (Applied BioSystems, Foster City, CA). DNAStar software was used for gel trace analysis and contig assembly. All DNA sequences were examined against available public databases primarily using BLASTn with RepeatMasker (University of Washington). The sequence of each of the assembled contigs is shown in Figs. 1 A-D.
  • RT-PCR products were purified by Qiagen spin columns. Sequencing was carried out in a Model 373A Automated DNA sequencer (Applied Biosystems) using Taq di-deoxy terminator cycle sequencing and Big Dye Kits according to the manufacturer's protocol.
  • Skin fibroblast cultures are established from 3.0 mm punch biopsies of the forearm of FHD patients and healthy control subjects as described (Marcil et ai, Arterioscler. Thromb. Vase. Biol. 19: 159-169, 1999).
  • the protocol for cellular cholesterol efflux experiments has been described in detail elsewhere (Marcil et al, Arterioscler. Thromb. Vase. Biol. 19: 159-169, 1999).
  • the cells are 3 H-cholesterol labeled during growth and free cholesterol loaded in growth arrest.
  • Efflux studies are carried out from 0 to 24 hours in the presence of purified ApoAI (10 ⁇ g protein/mL medium). Efflux is determined as a percent of free cholesterol in the medium after the cells were incubated for specified periods of time. All experiments are preferably performed in triplicate, in the presence of cells from one control subject and the cells from the study subjects to be examined.
  • genomic structure ofthe ABCl gene Most splice junction sequences were determined from genomic sequence generated from BAC clones spanning the ABCl gene. More than 160 kb of genomic sequence were generated. Genomic sequences were aligned with cDNA sequences to identify intron/exon boundaries. In some cases, long distance PCR between adjacent exons was used to amplify intron/exon boundary sequences using amplification primers designed according to the cDNA sequence. The genomic sequence of human ABCl is shown in Figs.
  • the presence or absence of mutations identified by genomic sequencing of probands from each family was subsequently confirmed by restriction fragment length polymo ⁇ hism (RFLP) assays, to define heterozygous and unaffected individuals, respectively.
  • the control cohort consisted of unaffected members of the 11 families.
  • Lipid levels in ABCA1 heterozygotes were measured as previously described (Brooks-Wilson et al, supra; Marcil et al, supra), at standardized lipid clinics in Vancouver, Montreal and Amsterdam. LDL was calculated by the method of Friedewald et ai (Clin. Chem. 18:499-502, 1972), modified to account for lipid measurements in mmol/L.
  • the analyzed cohort comprised 77 individuals from 11 families identified as heterozygous for mutations in the ABCl gene.
  • Heterozygotes have an approximately 40-45% decrease in HDL and apoA-I and a mild (approximately 10%) decrease in apoA-II compared to unaffected family members.
  • Mean triglycerides (TG) were increased by approximately 40% in heterozygotes compared to unaffected family members, and were further increased in TD patients.
  • the heterozygote phenotype was further examined by calculating the percentage of individuals falling within a given range of age and sex specific percentiles (based on LRC criteria (Heiss et al, supra; Heiss et al, Circulation 61 :302-315, 1980). Much variability in the heterozygote phenotype was evident. As illustrated in Fig. 5A, although a significantly higher percentage of heterozygotes had HDL cholesterol less than the 5 th percentile for age and sex compared to unaffected controls (65% vs. 5%, p ⁇ 0.0001), 5% of the heterozygotes had HDL greater than the 20 th percentile, with HDL ranging up to the 31 st percentile for age and sex. Thus, in some individuals clearly the low
  • HDL phenotype is less severe.
  • a broad distribution of triglyceride (TG) levels was also evident (Fig. 5B).
  • TG triglyceride
  • CAD coronary artery disease
  • vascular disease was generally more severe in the heterozygotes than in their unaffected family members (Fig. 7). Heterozygotes had myocardial infarctions (five, one fatal) and severe vascular disease requiring multiple interventions, whereas in unaffected individuals, CAD was manifest as angina in two cases and as a transient ischemic attack at the age of 80 in another. Furthermore, the mean age-of-onset was on average a decade younger in heterozygotes compared to unaffected controls (Fig. 6)
  • Relative cholesterol efflux levels are also related to CAD within the family. Families with clearest evidence for premature CAD had individuals with the lowest cholesterol efflux (bold on Fig. 8 and Fig. 9). These data suggest that the level of residual ABCl function is a critical determinant of both HDL cholesterol levels and susceptibility to CAD. Comparison of mutation type and location to the severity of phenotype in individuals heterozygous for ABCl mutations
  • Missense mutations result in the change of only a single amino acid and may result in a protein product that still retains partial activity. Lipid levels were compared in heterozygous carriers of severe and missense mutations. While there was a trend to decreased HDL levels in carriers of severe compared to missense mutations, this trend did not reach significance (Fig. 10). A range of HDL levels in individual missense and severe mutations were observed (Fig. 9). The Ml 09 IT missense mutation is the most severe mutation in terms of effects on efflux and HDL levels, with a more severe phenotype than even early tmncations of the protein (e.g. R909X).
  • the site of mutation (e.g. N-terminal or C-terminal) within the ABCl protein did not influence the phenotype (Fig. 11).
  • the presence of CAD is seen in carriers of mutations in several domains of the protein. Patients with mutations on both alleles manifest with splenomegaly alone or in association with CAD (TDI).
  • TDI splenomegaly alone or in association with CAD
  • the phenotype appears to be mutation specific, and most likely dependent on remaining ABCl function of the wild-type allele and residual function of the mutant allele, similar to what has been shown for mutations in ABCR, a close homologue of ABCl (van Driel et al, Ophthalmic Genet. 19: 117-122, 1998).
  • BMI Another factor known to influence HDL and triglyceride levels is BMI.
  • the entire cohort was divided into tertiles of BMI, and the mean HDL and triglyceride levels of heterozygotes and unaffected individuals by BMI tertile are shown in Figs. 16A and 16B.
  • BMI had a significant effect on both HDL and triglycerides in both heterozygotes and controls (p ⁇ 0.0001).
  • the effect of BMI on HDL-C and triglyceride levels was more severe in heterozygotes for ABCl than in controls, being evident at lower BMIs (mid-tertile) in heterozygotes.
  • a raised BMI was more obviously associated with changes in HDL and triglyceride levels in heterozygotes compared to controls.
  • SNPs in the ABCl gene were identified during the complete genomic sequencing of 14 unrelated probands with low HDL-C (Brooks- Wilson et al, supra 1999; Marcil et al, supra 1999). Variants that were identified within the low HDL families that did not co-segregate with the low HDL phenotype or that were observed in unaffected individuals were assumed to be SNPs. Based on the sequencing of BAC clones spanning the entire ABCl region (described above), sites identified as heterozygous or different from that found in sequenced individuals were also identified as polympo ⁇ hisms. Sequence data was available from at least one control individual at all variant coding sites.
  • the SNPs are numbered from the nucleotide described as position 1 (Pullinger et al, Biochemical and Biophysical Research Communications 271:451-455, 2000), naming the first exon number 1. As a standardized nomenclature for all variants, the "wild-type" allele (more frequent in the
  • A the variant (less frequent) allele is designated B.
  • Phenotypic effects of the cSNPs were examined in relationship to baseline lipid parameters. Patients were randomly assigned to treatment with pravastatin (Pravachol, Bristol-Myers Squibb, Princeton, N.J.) or placebo for a period of two years. Computer-assisted quantitative coronary angiography was carried out at the start and at the end of the study as previously described (Jukema et al, supra (1995)).
  • the baseline values and changes in the average mean segment diameter (MSD), which is a measurement of the average unobstructed diameter along the vessel, and in the minimal obstructive diameter (MOD), which is a measurement of the smallest unobstmcted segment, were used as the primary measures of CAD.
  • the MSD reflects diffuse changes of atherosclerosis, and the MOD reflects focal atherosclerotic changes. Larger
  • MSD and MOD measurements reflect less occlusion of the vessel, and a decrease in these parameters reflects progression of coronary atherosclerosis.
  • the prevalence of coronary events defined as death, myocardial infarction, unscheduled coronary angioplasty or bypass surgery (PTCA, CABG), or stroke/transient ischemic attack; was examined.
  • a restriction enzyme whose cleavage pattern was altered by the variant was identified for development of an RFLP assay. If no suitable enzyme was found, a mismatch strategy was employed, whereby a single nucleotide mismatch was inco ⁇ orated into the PCR primer, creating a restriction site in combination with either the wild-type or variant allele.
  • the specific conditions of all assays are described in Fig. 17. All PCR reactions were carried out in 50 ⁇ L volumes, in the presence of lx PCR buffer and 1.5 ⁇ M MgCl 2 (Life Technologies).
  • Thermocycling parameters for all assays were as follows: 95 °C for 3 min; 35 cycles of denaturation at 95 °C for 10 seconds, annealing for 30 seconds at the temperature specified in Fig. 17, and elongation for 30 seconds at 72°C; and a final elongation at 72°C for 10 min. All digestions (15-20 ⁇ L PCR product) were carried out in manufacturer's buffer (New England Biolabs) for 2 hours at the temperature specified by the manufacturer. As an example, the digest results for the R219K are shown in
  • Fig. 18 A 177 base pair fragment with the A allele is not cut by EcoNI, whereas the B allele is digested to produce fragments of 107 and 70 base pairs. Heterozygous individuals thus display all three bands (177, 107, and 70 base pairs). Geno typing with the TaqMan(r) assay
  • PCR polymerase chain reaction
  • two fluorogenic hybridization probes are labeled with different fluorescent reporter dyes (FAM or TET) at their 5' terminus and a common quencher dye (TAMRA) at their 3' terminus.
  • FAM or TET fluorescent reporter dyes
  • TAMRA quencher dye
  • PCR amplifications with flanking sets of primers (300 nM) in the presence of two TaqMan probes (25 nM each) and 4.5 mM MgCl 2 were performed using the following thermocycling protocol: initial denaturation at 96°C for 10 min, followed by 39 cycles of 96°C for 30 sec, 63 °C for 1 min and 72 °C for 15 sec, followed by a final extension at 72 °C for 10 min. Each plate included controls (no DNA template) as well as standards of each known genotype. Fluorescence quantification and genotype determination were performed on a Perkin Elmer LS50B or ABI Prism 7700 Sequence Detector. The fluorescence from each reaction was normalized to the signal from the no-template controls.
  • the baseline characteristics of the patients in the three genotypes was compared using one way analysis of variance and the chi-square test, where appropriate.
  • the AA group was compared to the combined group
  • triglyceride levels generally decrease with age, a finding seen in all R219K genotypes.
  • Differences between the genotypes are also observed in the MSD and MOD.
  • MSD and MOD measurements decrease significantly with age, reflecting increased atherosclerosis in the older individuals (Figs. 23 and 26).
  • carriers of the R219K variant these measurements do not significantly change with age.
  • vascular disease progresses much more slowly with age in carriers of the R219K variant compared to non-carriers.
  • polympo ⁇ hisms have been examined for their effect on cholesterol regulation and the predisposition for the development of cardiovascular disease.
  • the polympo ⁇ hisms are numbered from the nucleotide described as position 1 (Pullinger et al. , supra), naming the first exon number 1.
  • homozygous carriers of this variant have significantly increased CAD progression compared to non-carriers. Substitution of Afar G at nucleotide 2868 (V825I). Carriers of this variant had significantly more CAD events than individuals who do not have this variant.
  • each of the tested potential LXRE binding sites seem to bind an in vitro LXR-RXR heterodimer.
  • the LXRE binding site at +4 in exon 1 appears to have the highest affinity, closely followed by the LXRE binding site at -7670 in 3' intron 1.
  • Useful therapeutic compounds include those which modulate the expression, activity, or stability of ABCl.
  • ABCl expression, biological activity, or regulated catabolism is measured following the addition of candidate compounds to a culture medium of ABCl -expressing cells.
  • the candidate compounds may be directly administered to animals (for example mice, pigs, or chickens) and used to screen for their effects on ABCl expression.
  • ABCl In addition its role in the regulation of cholesterol, ABCl also participates in other biological processes for which the development of ABCl modulators would be useful.
  • ABCl transports interleukin-1 ⁇ (IL-l ⁇ ) across the cell membrane and out of cells.
  • IL-l ⁇ is a precursor of the inflammatory response and, as such, inhibitors or antagonists of ABCl expression or biological activity may be useful in the treatment of any inflammatory disorders, including but not limited to rheumatoid arthritis, systemic lupus erythematosis (SLE), hypo- or hyper- thyroidism, inflammatory bowel disease, and diabetes mellitus.
  • SLE systemic lupus erythematosis
  • ABCl expressed in macrophages has been shown to be engaged in the engulfment and clearance of dead cells. The ability of macrophages to ingest these apoptotic bodies is impaired after antibody-mediated blockade of ABCl. Accordingly, compounds that modulate ABCl expression, stability, or biological activity
  • ABCl expression is measured, for example, by standard Northern blot analysis using an ABCl nucleic acid sequence (or fragment thereof) as a hybridization probe, or by Western blot using an anti- ABCl antibody and standard techniques.
  • the level of ABCl expression in the presence of the candidate molecule is compared to the level measured for the same cells, in the same culture medium, or in a parallel set of test animals, but in the absence of the candidate molecule.
  • ABCl activity can also be measured using the cholesterol efflux assay.
  • ABCl mRNA is increased approximately 8-fold upon cholesterol loading. This increase is likely controlled at the transcriptional level.
  • genomic sequence described herein, one can identify transcription factors that bind to the 5' regulatory sequence by performing, for example, gel shift assays, DNAse protection assays, or in vitro or in vivo reporter gene-based assays. The identified transcription factors are themselves drug targets.
  • ABCl drug compounds that act through modulation of transcription of
  • ABCl could be used for HDL modulation, triglyceride modulation, atherosclerosis prevention, and the treatment of cardiovascular disease.
  • using a compound to inhibit a transcription factor that represses ABCl would be expected to result in up-regulation of ABCl and, therefore, up-regulation of HDL cholesterol levels and down-regulation of triglyceride levels.
  • a compound that increases transcription factor expression or activity would also increase ABCl expression, increase HDL levels, and decrease triglyceride levels.
  • Transcription factors known to regulate other genes in the regulation of apolipoprotein genes or other cholesterol- or lipid-regulating genes are of particular relevance.
  • Such factors include, but are not limited to, the steroid response element binding proteins (SREBP-1 and SREBP-2), and the PPAR (peroxisomal proliferation-activated receptor), RXR, and LXR transcription factors.
  • SREBP-1 and SREBP-2 steroid response element binding proteins
  • PPAR peroxisomal proliferation-activated receptor
  • RXR peroxisomal proliferation-activated receptor
  • LXR transcription factors include, but are not limited to, the steroid response element binding proteins (SREBP-1 and SREBP-2), and the PPAR (peroxisomal proliferation-activated receptor), RXR, and LXR transcription factors.
  • Oxygens at more than one carbon on the side chain of cholesterol diminished LXR binding and activation as compared to monoxygenated analogs.
  • LXR ligands were found to require a single stereoselective oxygen on the sterol side chain that functioned as a hydrogen acceptor.
  • Introduction of dimethylamide exhibited the greatest binding and activation compared to an ester or carbonyl group.
  • Compounds known to modulate LXR activity include, without limitation, 24-(S),25-epoxycholesterol; 24(S)-hydroxycholesterol; 22-(R)-hydroxycholesterol; 24(R),25-epoxycholesterol; 22(R)-hydroxy-24(S),25-epoxycholesterol; 5 22(S)-hydroxy-24(R),25-epoxycholesterol; 24-(S),25-iminocholesterol; methyl-38-hydroxycholonate; N,N-dimethyl-3 ⁇ -hydroxycholonamide; 24(R)-hy droxy cholesterol; 22(S)-hy droxy cholesterol; 22(R),24(S)-dihydroxycholesterol; 25-hydroxycholesterol; 22(R)-hydroxycholesterol; 22(S)-hydroxycholesterol; l o 24(S ),25 -dihydroxycholesterol ; 24(R) ,25 -dihy droxycholesterol ;
  • Non-steroidal agonists such as RIP140 protein, antibodies (monoclonal 5 or polyclonal) specific for LXR ⁇ or LXR ⁇ ; tetradecycloxy-furnacarboxylic acid (TOFA;); tetradecylthioacetic acid; as well as other fatty acids (see, for example, Tobin et al. Molec. Endocrin. 14: 741-752, 2000) are also useful LXR-modulating agents.
  • Additional transcription factors which may also have an effect in modulating ABCl gene expression and thereby HDL levels, triglyceride levels, atherosclerosis, and CAD risk include REV-ERB ⁇ , SREBP-1 & 2, ADD-1, EBP ⁇ , CREB binding protein, P300, HNF 4, RAR, and ROR ⁇ .
  • Exemplary binding sites are depicted in Fig. 3. Additional binding sites for these factors can be found, for example, through examination of the sequence in SEQ ID NO: 1.
  • RXR heterodimerizes with many nuclear receptors, including LXR, and aids in transactivating the target gene.
  • compounds that modulate RXR- mediated transcriptional activity will also modulate ABCl expression.
  • RXR-modulating compounds include, for example, hetero ethylene derivatives; tricyclic retinoids; trienoic retinoids; benzocycloalkenyl-alka:di- or trienoic acid derivatives; bicyclic-aromatic compounds and their derivatives; bicyclylmethyl-aryl acid derivatives; phenyl-methyl heterocyclic compounds; tetrahydro-napthyl compounds; arylthio-tetrahydro-naphthalene derivatives and heterocyclic analogues; 2,4-pentadienoic acid derivatives; tetralin-based compounds; nonatetraenoic acid derivatives; SRI 1237; dexamethasone; hydroxy, epoxy, and carboxy derivatives of methoprene; bicyclic benzyl, pyridinyl, thiophene, furanyl, and pynole derivatives; benzofuran-acrylic
  • Additional compounds include BRL 49653; troglitazone; pioglitazone; ciglitazone; WAY- 120; englitazone; AD 5075; and darglitazone.
  • PPARs may alter transcription of ABCl by mechanisms including heterodimerization with retinoid X receptors (RXRs) and then binding to specific proliferator response elements (PPREs). Examples of such PPARs include PPAR ⁇ , ⁇ , ⁇ and ⁇ . These distinct PPARs have been shown to have transcriptional regulatory effects on different genes. PPAR ⁇ is expressed mainly in liver, whereas PPAR ⁇ is expressed in predominantly in adipocytes.
  • PPAR ⁇ and PPAR ⁇ are found in coronary and carotid artery atherosclerotic plaques and in endothelial cells, smooth muscle cells, monocytes and monocyte-derived macrophages.
  • Activation of PPAR ⁇ results in altered lipoprotein metabolism through PPAR ⁇ 's effect on genes such as lipoprotein lipase (LPL), apolipoprotein CIII (apo CIII) and apolipoprotein Al (apo Al) and All (apo All).
  • LPL lipoprotein lipase
  • apo CIII apolipoprotein CIII
  • apo Al apolipoprotein Al
  • All All
  • PPAR ⁇ activation results in overexpression of LPL and apoA-I and apoA-II, but inhibits the expression of apo CIII.
  • PPAR ⁇ activation also inhibits inflammation, stimulates lipid oxidation and increases the hepatic uptake and esterification of free fatty acids (FFA's).
  • PPAR ⁇ activation may inhibit nitric oxide (NO) synthase in macrophages and prevent interleukin-1 (IL-1) induced expression of IL-6 and cyclo-oxygenase-2 (COX-2) and thrombin induced endothelin-1 expression secondary to negative transcriptional regulation of NF-KB and activation of protein- 1 signaling pathway. It has also been shown that PPAR ⁇ induces apoptosis in monocyte-derived macrophages through the inhibition of NF-KB activity.
  • NO nitric oxide
  • Activation of PPAR ⁇ can be achieved by compounds such as fibrates, ⁇ -estradiol, arachidonic acid derivatives, WY- 14,643 and LTB4 or 8(s)HETE.
  • PPAR ⁇ activation can be achieved through compounds such as thiozolidinedione antidiabetic dmgs, 9-HODE and 13-HODE. Additional compounds such as nicotinic acid or HMG CoA reductase inhibitors may also alter the activity of PPARs.
  • PPARs may have an effect on ABCl expression and thereby could affect HDL levels, triglyceride levels, atherosclerosis, and risk of CAD.
  • PPARs are also regulated by fatty acids (including modified fatty acids such as 3 thia fatty acids), leukotrienes such as leukotriene B4 and prostaglandin J2, which is a natural activator/ligand for PPAR ⁇ .
  • Drugs that modulate PPARs may therefore have an important effect on modulating lipid levels (including HDL and triglyceride levels) and altering CAD risk. This effect could be achieved through the modulation of ABCl gene expression.
  • Drugs may also effect ABCl gene expression and thereby HDL and triglyceride levels, by an indirect effect on PPARs via other transcriptional factors such as adipocyte differentiation and determination factor- 1 (ADD-1) and sterol regulatory element binding protein- 1 and 2 (SREBP-1 and 2).
  • Dmgs with combined PPAR ⁇ and PPAR ⁇ agonist activity or PPAR ⁇ and PPAR ⁇ agonists given in combination for example, may increase HDL levels or decrease triglyceride levels even more.
  • a PPAR binding site (PPRE element) is found 5' to the ABCl gene (Fig. 3). Like the PPRE elements found in the C-ACS, HD, CYP4A6 and ApoA-I genes, this PPRE site is a trimer related to the PPRE consensus sequence. Partly because of its similarity in the number and anangement of repeats in this PPAR binding site, this element in particular is very likely to be of physiological relevance to the regulation of the ABCl gene.
  • ABCl polypeptides, nucleic acids, and modulators ABCl may act as a transporter of toxic proteins or protein fragments
  • ABCl agonists/upregulators may be useful in the treatment of other disease areas, including Alzheimer's disease, Niemann- Pick disease, and Huntington's disease.
  • ABC transporters have been shown to increase the uptake of long chain fatty acids from the cytosol to peroxisomes and, moreover, to play a role in ⁇ - oxidation of very long chain fatty acids.
  • ALD x-linked adrenoleukodystrophy
  • Any agent that upregulates ABC transporter expression or biological activity may therefore be useful for the treatment of ALD or any other lipid disorder.
  • ABCl is expressed in macrophages and is required for engulfment of cells undergoing programmed cell death.
  • the apoptotic process itself, and its regulation, have important implications for disorders such as cancer, one mechanism of which is failure of cells to undergo cell death appropriately.
  • ABCl may facilitate apoptosis, and as such may represent an intervention point for cancer treatment.
  • Increasing ABCl expression or activity or otherwise up-regulating ABCl by any method may constitute a treatment for cancer by increasing apoptosis and thus potentially decreasing the abenant cellular proliferation characterized by this disease.
  • down-regulation of ABCl by any method may provide opportunity for decreasing apoptosis and allowing increased proliferation of cells in conditions where cell growth is limited.
  • disorders include but are not limited to neurodeficiencies and neurodegeneration, and growth disorders.
  • ABCl could, therefore, be used as a method for identification of compounds for use in the treatment of cancer, or in the treatment of degenerative disorders.
  • Agents that have been shown to inhibit ABCl include, for example, the anti-diabetic agents glibenclamide and glyburide, flufenamic acid, diphenylamine-2-carbonic acid, sulfobromophthalein, and DIDS.
  • Agents that upregulate ABCl expression or biological activity include but are not limited to protein kinase A, protein kinase C, vanadate, okadaic acid, and IBMX1. Those in the art will recognize that other compounds can also modulate ABCl biological activity, and these compounds are also in the spirit of the invention.
  • the ABCl protein and gene can be used in screening assays for identification of compounds which modulate its activity and may be potential dmgs to regulate cholesterol or triglyceride levels.
  • the ABCl 5' regulatory sequence and other regulatory regions e.g., exon 1 and exon 2 can be used in screening assays for identification of compounds which modulate ABCl expression and may be potential drugs to regulate lipid levels, including, for example, HDL-C, LDL-C, and triglycerides.
  • Dmg screens to identify compounds that modulate ABCl expression may employ an ABCl regulatory region operably linked to ABCl.
  • the regulatory region is operably linked to a reporter gene (e.g., a gene encoding GFP, chloramphenicol acetyltransf erase, or beta-galactosidase).
  • Useful ABCl proteins include wild-type and mutant ABCl proteins or protein fragments, in a recombinant form or endogenously expressed. Dmg screens to identify compounds acting on the ABCl expression product may employ any functional feature of the protein. In one example, the phosphorylation state or other post-translational modification is monitored as a measure of ABCl biological activity. ABCl has ATP binding sites, and thus assays may wholly or in part test the ability of ABCl to bind ATP or to exhibit ATPase activity.
  • ABCl by analogy to similar proteins, is thought to be able to form a channel-like stmcture; drug screening assays could be based upon assaying for the ability of the protein to form a channel, or upon the ability to transport cholesterol or another molecule, or based upon the ability of other proteins bound by or regulated by ABCl to form a channel.
  • drug screening assays could be based upon assaying for the ability of the protein to form a channel, or upon the ability to transport cholesterol or another molecule, or based upon the ability of other proteins bound by or regulated by ABCl to form a channel.
  • phospholipid or lipid transport can also be used as measures of ABCl biological activity.
  • ABCl in addition to its role as a regulator of cholesterol levels, ABCl also transports anions.
  • Functional assays could be based upon this property, and could employ dmg screening technology such as (but not limited to) the ability of various dyes to change color in response to changes in specific ion concentrations in such assays can be performed in vesicles such as liposomes, or adapted to use whole cells.
  • Drug screening assays can also be based upon the ability of ABCl or other ABC transporters to interact with other proteins.
  • interacting proteins can be identified by a variety of methods known in the art, including, for example, radioimmunoprecipitation, co-immunoprecipitation, co- purification, and yeast two-hybrid screening. Such interactions can be further assayed by means including but not limited to fluorescence polarization or scintillation proximity methods.
  • Dmg screens can also be based upon functions of the ABCl protein deduced upon X-ray crystallography of the protein and comparison of its 3-D stmcture to that of proteins with known functions.
  • Dmg screens can be based upon a function or feature apparent upon creation of a transgenic or knockout mouse, or upon overexpression of the protein or protein fragment in mammalian cells in vitro. Moreover, expression of mammalian (e.g., human) ABCl in yeast or C. elegans allows for screening of candidate compounds in wild- type and mutant backgrounds, as well as screens for mutations that enhance or suppress an ABCl -dependent phenotype. Modifier screens can also be performed in ABCl transgenic or knock-out mice.
  • dmg screening assays can also be based upon ABCl functions deduced upon antisense interference with the gene function.
  • Intracellular localization of ABCl, or effects which occur upon a change in intracellular localization of the protein, can also be used as an assay for dmg screening. Immunocytochemical methods will be used to determine the exact location of the ABCl protein.
  • Human and rodent ABCl protein can be used as an antigen to raise antibodies, including monoclonal antibodies. Such antibodies will be useful for a wide variety of pu ⁇ oses, including but not limited to functional studies and the development of drug screening assays and diagnostics. Monitoring the influence of agents (e.g., drugs, compounds) on the expression or biological activity of ABCl can be applied not only in basic dmg screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase ABCl gene expression, protein levels, or biological activity can be monitored in clinical trails of subjects exhibiting altered ABCl gene expression, protein levels, or biological activity.
  • agents e.g., drugs, compounds
  • the effectiveness of an agent determined by a screening assay to modulate ABCl gene expression, protein levels, or biological activity can be monitored in clinical trails of subjects exhibiting decreased altered gene expression, protein levels, or biological activity.
  • the expression or activity of ABCl and, preferably, other genes that have been implicated in, for example, cardiovascular disease can be used to ascertain the effectiveness of a particular dmg.
  • genes, including ABCl that are modulated in cells by treatment with an agent (e.g., compound, dmg or small molecule) that modulates ABCl biological activity (e.g., identified in a screening assay as described herein) can be identified.
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of ABCl and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, or, alternatively, by measuring the amount of protein produced, by one of a number of methods known in the art, or by measuring the levels of biological activity of ABCl or other genes.
  • the gene expression can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other dmg candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of an ABCl protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the ABCl protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the ABCl protein, mRNA, or genomic DNA in the pre-administration sample with the ABCl protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g.,
  • increased administration of the agent may be desirable to increase the expression or activity of ABCl to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of ABCl to lower levels than detected.
  • the ABCl gene or a fragment thereof can be used as a tool to express the protein in an appropriate cell in vitro or in vivo (gene therapy), or can be cloned into expression vectors which can be used to produce large enough amounts of ABCl protein to use in in vitro assays for dmg screening.
  • Expression systems which may be employed include baculovims, he ⁇ es vims, adenovirus, adeno-associated vims, bacterial systems, and eucaryotic systems such as CHO cells. Naked DNA and DNA-liposome complexes can also be used.
  • Assays of ABCl activity includes binding to intracellular interacting proteins; interaction with a protein that up-regulates ABCl activity; interaction with HDL particles or constituents; interaction with other proteins which facilitate interaction with HDL or its constituents; and measurement of cholesterol efflux.
  • assays may be based upon the molecular dynamics of macromolecules, metabolites and ions by means of fluorescent- protein biosensors.
  • the effect of candidate modulators on expression or activity may be measured at the level of ABCl protein production using the same general approach in combination with standard immunological detection techniques, such as Western blotting or immunoprecipitation with an ABCl-specific antibody.
  • useful cholesterol- or triglyceride-regulating or anti-CVD therapeutic modulators are identified as those which produce an change in ABCl polypeptide production.
  • Agonists may also affect ABCl activity without any effect on expression level.
  • Candidate modulators may be purified (or substantially purified) molecules or may be one component of a mixture of compounds (e.g., an extract or supernatant obtained from cells).
  • ABCl expression is tested against progressively smaller subsets of the candidate compound pool (e.g., produced by standard purification techniques, e.g., HPLC or FPLC; Ausubel et al.) until a single compound or minimal compound mixture is demonstrated to modulate ABCl expression.
  • Agonists, antagonists, or mimetics found to be effective at modulating the level of cellular ABCl expression or activity may be confirmed as useful in animal models (for example, mice, pigs, rabbits, or chickens).
  • the compound may ameliorate the low HDL levels of mouse or chicken hypoalphalipoproteinemias or may lower the triglyceride levels in animal models.
  • a compound that promotes an increase in ABCl expression or activity is considered particularly useful in the invention; such a molecule may be used, for example, as a therapeutic to increase the level or activity of native, cellular ABCl and thereby treat a low HDL or high triglyceride condition in an animal (for example, a human).
  • treatment with an agonist of the invention may be combined with any other HDL-raising, triglyceride-lowering, or anti- CVD therapies.
  • ABC biological activity is to increase the stabilization of the ABC protein or to prevent its degradation.
  • compounds that increase the stability of a wild-type ABC polypeptide or decrease its catabolism may also be useful for the treatment of low HDL-C or any other condition resulting from loss of ABCl biological activity.
  • Such mutations and compounds can be identified using the methods described herein.
  • cells expressing an ABC polypeptide having a mutation are transiently metabolically labeled during translation and the half-life of the
  • ABC polypeptide is determined using standard techniques. Mutations that increase the half-life of an ABC polypeptide are ones that increase ABC protein stability. These mutations can then be assessed for ABC biological activity. They can also be used to identify proteins that affect the stability of ABCl mRNA or protein. One can then assay for compounds that act on these factors or on the ability of these factors to bind ABCl.
  • cells expressing wild-type ABC polypeptide are transiently metabolically labeled during translation, contacted with a candidate compounds, and the half-life of the ABC polypeptide is determined using standard techniques.
  • Compounds that increase the half-life of an ABC polypeptide are useful compounds in the present invention.
  • ABCl is the prefened ABC transporter for the dmg screens described herein, other ABC transporters can also be used.
  • the replacement of ABCl with another ABC transporter is possible because it is likely that ABC transporter family members, such as ABC2, ABCR, or ABC8 will have a similar mechanism of regulation.
  • ABCl polypeptide purified or unpurified can be used in an assay to determine its ability to bind another protein (including, but not limited to, proteins found to specifically interact with ABCl). The effect of a compound on that binding is then determined.
  • ABCl protein (or a polypeptide fragment thereof or an epitope-tagged form or fragment thereof) is harvested from a suitable source (e.g., from a prokaryotic expression system, eukaryotic cells, a cell-free system, or by immunoprecipitation from ABCl -expressing cells).
  • the ABCl polypeptide is then bound to a suitable support (e.g., nitrocellulose or an antibody or a metal agarose column in the case of, for example, a his-tagged form of ABCl). Binding to the support is preferably done under conditions that allow proteins associated with ABCl polypeptide to remain associated with it. Such conditions may include use of buffers that minimize interference with protein-protein interactions.
  • the binding step can be done in the presence and absence of compounds being tested for their ability to interfere with interactions between ABCl and other molecules.
  • other proteins e.g., a cell lysate
  • the immobilized ABCl polypeptide is then washed to remove proteins or other cell constituents that may be non-specifically associated with it the polypeptide or the support.
  • the immobilized ABCl polypeptide is then dissociated from its support, and so that proteins bound to it are released (for example, by heating), or, alternatively, associated proteins are released from
  • ABCl without releasing the ABCl polypeptide from the support.
  • the released proteins and other cell constituents can be analyzed, for example, by SDS- PAGE gel electrophoresis, Western blotting and detection with specific antibodies, phosphoamino acid analysis, protease digestion, protein sequencing, or isoelectric focusing.
  • Normal and mutant forms of ABCl can be employed in these assays to gain additional information about which part of ABCl a given factor is binding to.
  • comparison of the normal and mutant forms of the protein can be used to help distinguish tme binding proteins.
  • the foregoing assay can be performed using a purified or semipurified protein or other molecule that is known to interact with ABCl. This assay may include the following steps.
  • FRET Fluorescent Resonance Energy Transfer
  • This assay can be performed as follows. 1. Provide ABCl protein or a suitable polypeptide fragment thereof and couple a suitable FRET donor (e.g.,. nitro-benzoxadiazole (NBD)) to it;
  • a suitable FRET donor e.g.,. nitro-benzoxadiazole (NBD)
  • a FRET acceptor e.g., rhodamine
  • the ABCl protein can also be tested for its effects on membrane permeability. For example, beyond its putative ability to translocate lipids, ABCl might affect the permeability of membranes to ions.
  • Other related membrane proteins most notably the cystic fibrosis transmembrane conductance regulator and the sulfonylurea receptor, are associated with and regulate ion channels.
  • ABCl or a fragment of ABCl is inco ⁇ orated into a synthetic vesicle, or, alternatively, is expressed in a cell and vesicles or other cell sub-stmctures containing ABCl are isolated.
  • the ABCl -containing vesicles or cells are loaded with a reporter molecule (such as a fluorescent ion indicator whose fluorescent properties change when it binds a particular ion) that can detect ions (to observe outward movement), or alternatively, the external medium is loaded with such a molecule (to observe inward movement).
  • a reporter molecule such as a fluorescent ion indicator whose fluorescent properties change when it binds a particular ion
  • the external medium is loaded with such a molecule (to observe inward movement).
  • a molecule which exhibits differential properties when it is inside the vesicle compared to when it is outside the vesicle is prefened.
  • a molecule that has quenching properties when it is at high concentration but not when it is at another low concentration would be suitable.
  • the movement of the charged molecule (either its ability to move or the kinetics of its movement) in the presence or absence of a compound being tested for its ability to affect this process can be determined.
  • membrane permeability is determined electro- physiologically by measuring ionic influx or efflux mediated by or modulated by ABCl by standard electrophysiological techniques.
  • a suitable control e.g., TD cells or a cell line with very low endogenous ABCl expression
  • uptake of radioactive isotopes into or out of a vesicle can be measured. The vesicles are separated from the extravesicular medium and the radioactivity in the vesicles and in the medium is quantitated and compared.
  • ABCl nucleic acid may be used in an assay based on the binding of factors necessary for ABCl gene transcription.
  • the association between the ABCl DNA and the binding factor may be assessed by means of any system that discriminates between protein-bound and non-protein-bound DNA (e.g., a gel retardation assay).
  • a gel retardation assay The effect of a compound on the binding of a factor to
  • ABCl DNA is assessed by means of such an assay.
  • in vitro binding assays in which the regulatory regions of the ABCl gene are linked to reporter genes can also be performed.
  • a cell-based or cell-free system can be used to screen for compounds based on their effect on the half-life of ABCl mRNA or ABCl protein.
  • the assay may employ labeled mRNA or protein.
  • ABCl mRNA may be detected by means of specifically hybridizing probes or a quantitative PCR assay. Protein can be quantitated, for example, by fluorescent antibody-based methods.
  • Mutant ABCl polypeptides are likely to have dominant negative activity (i.e., activity that interferes with wild-type ABCl function).
  • An assay for a compound that can interfere with such a mutant may be based on any method of quantitating normal ABCl activity in the presence of the mutant. For example, normal ABCl facilitates cholesterol efflux, and a dominant negative mutant would interfere with this effect.
  • the ability of a compound to counteract the effect of a dominant negative mutant may be based on cellular cholesterol efflux, or on any other normal activity of the wild-type ABCl that was inhibitable by the mutant.
  • the effect of a compound on ABCl phosphorylation can be assayed by methods that quantitate phosphates on proteins or that assess the phosphorylation state of a specific residue of a ABCl .
  • methods include but are not limited to 32 P labeling and immunoprecipitation, detection with antiphosphoamino acid antibodies (e.g., antiphosphoserine antibodies), phosphoamino acid analysis on 2-dimensional TLC plates, and protease digestion finge ⁇ rinting of proteins followed by detection of 32 P-labeled fragments.
  • the effect of a compound on the post-translational modification of ABCl is based on any method capable of quantitating that particular modification.
  • effects of compounds on glycosylation may be assayed by treating ABCl with glycosylase and quantitating the amount and nature of carbohydrate released.
  • azido-ATP analogs can be used to allow covalent attachment of the azido-ATP to protein (by means of U. V. light), and permit easier quantitation of the amount of ATP bound to the protein;
  • Quantitation of the ATPase activity of ABCl can also be assayed for the effect of compounds on ABCl. This is preferably performed in a cell-free assay so as to separate ABCl from the many other ATPases in the cell.
  • An ATPase assay may be performed in the presence or absence of membranes, and with or without integration of ABCl protein into a membrane. If performed in a vesicle-based assay, the ATP hydrolysis products produced or the ATP hydrolyzed may be measured within or outside of the vesicles, or both. Such an assay may be based on disappearance of ATP or appearance of ATP hydrolysis products.
  • a coupled ATPase assay is preferable.
  • a reaction mixture containing pymvate kinase and lactate dehydrogenase can be used.
  • the mixture includes phosphoenolpymvate (PEP), nicotinamide adenine dinucleotide (NAD + ), and ATP.
  • PEP phosphoenolpymvate
  • NAD + nicotinamide adenine dinucleotide
  • ATPase activity of ABCl generates ADP from ATP.
  • the ADP is then converted back to ATP as part of the pymvate kinase reaction.
  • the product, pymvate is then converted to lactate.
  • NADH a colored quinone
  • NAD+ colorless substrate
  • ADP is limiting for the pymvate kinase reaction
  • this coupled system precisely monitors the ATPase activity of ABCl.
  • a transport-based assay can be performed in vivo or in vitro.
  • the assay may be based on any part of the reverse cholesterol transport process that is readily re-created in culture, such as cholesterol or phospholipid efflux.
  • the assay may be based on net cholesterol transport in a whole organism, as assessed by means of a labeled substance (such as cholesterol).
  • fluorescent lipids can be used to measure ABCl -catalyzed lipid efflux.
  • a fluorescent precursor, C6-NBD-phosphatidic acid can be used. This lipid is taken up by cells and dephosphorylated by phosphatidic acid phosphohydrolase.
  • the product, NBD-diglyceride is then a precursor for synthesis of glycerophospholipids like phosphatidylcholine.
  • the efflux of NBD-phosphatidylcholine can be monitored by detecting fluorescence resonance energy transfer (FRET) of the NBD to a suitable acceptor in the cell culture medium.
  • FRET fluorescence resonance energy transfer
  • This acceptor can be rhodamine-labeled phosphatidylethanolamine, a phospholipid that is not readily taken up by cells.
  • the use of short-chain precursors obviates the requirement for the phospholipid transfer protein in the media.
  • NBD-cholesterol ester can be reconstituted into LDL.
  • the LDL can efficiently deliver this lipid to cells via the LDL receptor pathway.
  • the NBD-cholesterol esters are hydrolyzed in the lysosomes, resulting in NBD-cholesterol that can now be transported back to the plasma membrane and efflux from the cell.
  • the efflux can be monitored by the aforementioned FRET assay in which NBD transfers its fluorescence resonance energy to the rhodamine-phosphatidylethanoline acceptor.
  • Test compounds identified as having activity in any of the above-described assays are subsequently screened in any available animal model system, including, but not limited to, pigs, rabbits, and WHAM chickens. Test compounds are administered to these animals according to standard methods. Test compounds may also be tested in mice bearing mutations in the ABCl gene. Additionally, compounds may be screened for their ability to enhance an interaction between ABCl and any HDL particle constituent such as ApoAI,
  • the cholesterol efflux assay measures the ability of cells to transfer cholesterol to an extracellular acceptor molecule and is dependent on ABCl function.
  • cells are loaded with radiolabeled cholesterol by any of several biochemical pathways (Marcil et al, Arterioscler. Thromb. Vase. Biol. 19:159-169, 1999).
  • Cholesterol efflux is then measured after incubation for various times (typically 0 to 24 hours) in the presence of HDL3 or purified ApoAI. Cholesterol efflux is determined as the percentage of total cholesterol in the culture medium after various times of incubation. ABCl expression levels and/or biological activity are associated with increased efflux while decreased levels of ABCl are associated with decreased cholesterol efflux.
  • This assay can be readily adapted to the format used for dmg screening, which may consist of a multi-well (e.g., 96-well) format. Modification of the assay to optimize it for dmg screening would include scaling down and streamlining the procedure, modifying the labeling method, using a different cholesterol acceptor, altering the incubation time, and changing the method of calculating cholesterol efflux. In all these cases, the cholesterol efflux assay remains conceptually the same, though experimental modifications may be made. A transgenic mouse overexpressing ABCl would be expected to have higher than normal HDL levels.
  • An animal such as a mouse, that has had one or both ABCl alleles inactivated (e.g., by homologous recombination) is likely to have low HDL-C levels and higher than normal triglyceride levels, and thus is a prefened animal model for screening for compounds that raise HDL-C levels or lower triglyceride levels.
  • Such an animal can be produced using standard techniques.
  • the animals having mutant ABCl genes are useful for further testing of efficacy and safety of drugs or agents first identified using one of the other screening methods described herein.
  • Cells taken from the animal and placed in culture can also be exposed to test compounds. HDL-C and triglyceride levels can be measured using standard techniques, such as those described herein.
  • WHAM chickens an animal model for low HDL cholesterol
  • This chicken low HDL locus is Z-linked, or sex-linked. (In birds, females are ZW and males are ZZ). Genetic mapping placed the Y locus on the long arm of the Z chromosome (Bitgood, 1985), proximal to the ID locus (Bitgood, 1988). Examination of cunent public mapping data for the chicken genome mapping project, ChickMap (maintained by the Roslin Institute; http://www.ri.bbsrc.ac.uk/ chickmap/ChickMapHomePage.html) showed that a region of synteny with human chromosome 9 lies on the long arm of the chicken Z chromosome (Zq) proximal to the ID locus.
  • ALDOB aldolase B locus
  • the human ALDOB locus maps to chromosome 9q22.3 (The Genome Database, http://gdbwww.gdb.org/), not far from the location of human ABCl. This comparison of maps showed that the chicken Zq region near chicken ALDOB and the human 9q region near human ALDOB represent a region of synteny between human and chicken.
  • It may be an important regulatory region (there is a phosphorylation site for casein kinase near the mutated residue), or it may help to dictate a precise topological relationship with cellular membranes (the N-terminal 60 amino acid region contains a putative membrane-spanning or membrane-associated segment).
  • amino-terminal region of the protein (up to the first 6-TM region at approximately amino acid 639) is an ideal tool for screening factors that affect
  • ABCl activity It can be expressed as a tmncated protein in ABCl wild-type cells in order to test for interference of the normal ABCl function by the tmncated protein. If the fragment acts in a dominant negative way, it could be used in immunoprecipitations to identify proteins that it may be competing away from the normal endogenous protein.
  • the C-terminus also lends itself to such experiments, as do the intracellular portions of the molecule, expressed as fragments or tagged or fusion proteins, in the absence of transmembrane regions.
  • any animal model to be used for a human genetic disease represents the homologous locus in that animal, and not a different locus with a similar function.
  • the evidence above establishes that the chicken Y locus and the human chromosome 9 low HDL locus are homologous. WHAM chickens are therefore an important animal model for the identification and testing of drugs that modulate cholesterol efflux.
  • the WHAM chickens' HDL deficiency syndrome is not, however, known to be associated with an increased susceptibility to atherosclerosis in chickens. This may reflect the shorter lifespan or, more likely, the impaired abso ⁇ tion of dietary cholesterol in these chickens.
  • Compounds of the invention including but not limited to, ABCl polypeptides, ABCl nucleic acids, other ABC transporters, LXR-modulating compounds, RXR-modulating compounds, and any therapeutic agent that modulates biological activity or expression of ABCl identified using any of the methods disclosed herein, may be administered with a pharmaceutically- acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients.
  • Any appropriate route of administration may be employed, for example, intravenous, perenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, or oral administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for agonists of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, or example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • novel dmgs for the treatment of abenant lipid levels and/or CVD are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art.
  • test extracts or compounds are not critical to the screening procedure(s) of the invention.
  • chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
  • Synthetic compound libraries are commercially available from Brandon Associates (Menimack, NH) and Aldrich Chemical (Milwaukee, WI).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the cmde extract having HDL-raising, triglyceride - lowering, or anti-CVD activities, ability to modulate ABCl gene expression, or a combination thereof.
  • the same in vivo and in vitro assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogeneous extracts are known in the art.
  • compounds shown to be useful agents for the treatment of pathogenicity are chemically modified according to methods known in the art.
  • Compounds identified as being of therapeutic value are subsequently analyzed using any standard animal model of diabetes or obesity known in the art. It is understood that compounds that modulate activity of proteins that modulate ABCl gene expression or activity are useful compounds for modulating HDL-C levels and triglyceride levels. Exemplary compounds are provided herein; others are known in the art. Compounds that are structurally related to cholesterol, or that mimic
  • ApoAI or a related apolipoprotein, and increase ABCl biological activity are particularly useful compounds in the invention.
  • Other compounds, known to act on the MDR protein, can also be used or derivatized and assayed for their ability to increase ABCl biological activity.
  • Exemplary MDR modulators are PSC833, bromocriptine, and cyclosporin A.
  • Other examples of compounds that may be assayed for the ability to increase ABCl biological activity include oxysterols and their derivatives.
  • Screening patients having low HDL-C or high triglyceride levels ABCl expression, biological activity, and mutational analysis can each serve as a diagnostic tool for low HDL or higher than normal triglyceride levels; thus determination of the genetic subtyping of the ABCl gene sequence can be used to subtype low HDL or higher than normal triglyceride individuals or families to determine whether the low HDL or higher than normal triglyceride phenotype is related to ABCl function.
  • This diagnostic process can lead to the tailoring of drug treatments according to patient genotype, including prediction of side-effects upon administration of HDL increasing or triglyceride lowering dmgs (refened to herein as pharmacogenomics).
  • Pharmacogenomics allows for the selection of agents (e.g., dmgs) for therapeutic or prophylactic treatment of an individual based on the genotype of the individual (e.g., the genotype of the individual is examined to determine the ability of the individual to respond to a particular agent).
  • Agents, or modulators which have a stimulatory or inhibitory effect on ABCl biological activity or gene expression can be administered to individuals to treat disorders (e.g., cardiovascular disease, low HDL cholesterol, or a higher than normal triglyceride level) associated with abenant ABCl activity.
  • disorders e.g., cardiovascular disease, low HDL cholesterol, or a higher than normal triglyceride level
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • the individual may be considered.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., dmgs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of ABCl protein, expression of ABCl nucleic acid, or mutation content of ABCl genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to dmgs due to altered drug disposition and abnormal action in affected persons (Eichelbaum, M., Clin. Exp. Pharmacol. Physiol., 23:983-985,
  • dmg action a single factor altering the way dmgs act on the body
  • genetic conditions transmitted as single factors altering the way the body acts on dmgs altered drug metabolism
  • Altered drug action may occur in a patient having a polymo ⁇ hism (e.g., an single nucleotide polymo ⁇ hism or SNP) in promoter, intronic, or exonic sequences of ABCl.
  • polymo ⁇ hisms in the promoter region may be critical in determining the risk of HDL deficiency, higher than normal triglyceride level, and CVD.
  • polymo ⁇ hisms in the human ABCl gene Fig. 4
  • These polymo ⁇ hisms are located in promoter, intronic, and exonic sequence of ABCl.
  • standard methods such as direct sequencing, PCR, SSCP, or any other polymo ⁇ hism-detection system, one could easily ascertain whether these polymo ⁇ hisms are present in a patient prior to the establishment of a dmg treatment regimen for a patient having low HDL, a higher than normal triglyceride level, cardiovascular disease, or any other ABCl -mediated condition. It is possible that some these polymo ⁇ hisms are, in fact, weak mutations. Individuals harboring such mutations may have an increased risk for cardiovascular disease; thus, these polymo ⁇ hisms may also be useful in diagnostic assays.

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Abstract

L'invention concerne des méthodes permettant de traiter des patients ayant un faible taux de HDL cholestérol, un taux de triglycérides supérieur à la normale ou une maladie cardio-vasculaire, en leur administrant des composés qui modulent l'expression ou l'activité de ABC1.
EP00974705A 1999-09-01 2000-09-01 Compositions et methodes permettant de moduler le taux de hdl cholesterol et de triglycerides Withdrawn EP1239848A2 (fr)

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