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WO2008156833A2 - Récepteur g2a comme cible thérapeutique - Google Patents

Récepteur g2a comme cible thérapeutique Download PDF

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Publication number
WO2008156833A2
WO2008156833A2 PCT/US2008/007706 US2008007706W WO2008156833A2 WO 2008156833 A2 WO2008156833 A2 WO 2008156833A2 US 2008007706 W US2008007706 W US 2008007706W WO 2008156833 A2 WO2008156833 A2 WO 2008156833A2
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subject
density lipoprotein
hdl
levels
apoe
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WO2008156833A3 (fr
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Janusz H.S. Kabarowski
Brizn W. Parks
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UAB Research Foundation
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UAB Research Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present disclosure is directed to methods of treating and preventing disease states and conditions associated with or characterized by decreased high-density lipoprotein (HDL) particle levels, increased very-low density lipoprotein (VLDL) particle levels and/or increased low-density lipoprotein (LDL) particle levels. Diagnostic methods and screening/assay methods for the identification of compounds useful in the treatment and prevention methods herein described are also disclosed.
  • HDL high-density lipoprotein
  • VLDL very-low density lipoprotein
  • LDL low-density lipoprotein
  • Atherosclerosis is the primary cause of cardiovascular heart disease and as such remains the leading cause of death in Western societies (1).
  • Atherosclerosis is a chronic inflammatory disease of the large arteries characterized by the accumulation of LDL- cholesterol and/or VLDL-cholesterol particles in the arterial wall (2).
  • High cholesterol levels results in elevated circulating concentrations of LDL-cholesterol and is therefore a major risk factor for atherosclerosis.
  • Circulating LDL-cholesterol particles and/or VLDL-cholesterol particles can penetrate vascular endothelium at arterial sites prone to disruption due to disturbed blood flow and become trapped in the arterial wall.
  • Oxidized LDL/VLDL (oxLDL/VLDL)-derived phospholipids also directly attract monocytes (5), which differentiate into macrophages and take up oxidized lipid particles via scavenger receptors as part of a defense mechanism against the accumulation of this harmful moiety.
  • monocytes (5) which differentiate into macrophages and take up oxidized lipid particles via scavenger receptors as part of a defense mechanism against the accumulation of this harmful moiety.
  • These macrophages become engorged with cholesterol and transform into "foam cells" which dominate the early atherosclerotic lesion (6, 7).
  • Macrophage foam cells also produce factors which propagate the oxidative and lipolytic modification of lipid particles (8,9).
  • the presence of oxidized lipids can induce a self-propagating cycle of vascular inflammation and LDL/VLDL modification which unabated will eventually lead to the development of an advanced rupture-prone atherosclerotic plaque.
  • LDL and/or VLDL carries cholesterol to peripheral tissues
  • HDL particles transport excess cholesterol from peripheral tissues to the liver for excretion in the bile and feces in a process called reverse cholesterol transport (RCT) (10) (FIG.s 1-3).
  • RCT reverse cholesterol transport
  • HDL- C High levels of HDL-cholesterol (HDL- C) therefore reduce the risk for a number of disease states and conditions such as but not limited to, atherosclerosis and chronic inflammatory autoimmune diseases as they counteract the accumulation of excessive amounts of cholesterol in the arterial wall and thereby reduce foam cell formation, reduce inflammation and reduce oxidative modification of lipids.
  • atherosclerosis and chronic inflammatory autoimmune diseases as they counteract the accumulation of excessive amounts of cholesterol in the arterial wall and thereby reduce foam cell formation, reduce inflammation and reduce oxidative modification of lipids.
  • other functions of HDL have been described that may contribute to its beneficial effects, including antioxidant and anti-inflammatory activities (11). Results of clinical trials have shown that treating individuals who have low HDL-C with therapeutic agents that raise HDL-C can reduce the occurrence of major cardiovascular events (12).
  • VLDL VLDL
  • LDL LDL
  • HDL hyperlipidemia
  • atherosclerosis a variety of diseases and conditions
  • chronic inflammatory autoimmune disorders such as but not limited to lupus (13).
  • the present disclosure provides methods of treating and preventing disease states and conditions associated with or characterized by decreased HDL levels, increased VLDL levels and/or increased LDL levels, such as by increasing HDL levels, decreasing VLDL levels and/or decreasing LDL levels, altering HDL particle biogenesis, composition and/or size.
  • such treatment and prevention methods involve modulating the expression levels and/or activity of the G2A receptor or the signaling pathway in which the G2A receptor takes part.
  • Such disease states and conditions include, but are not limited to, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus.
  • the present disclosure is also directed to diagnostic methods for identifying subjects suffering from or at risk for such disease states and conditions.
  • the present disclosure is directed to methods for identifying compounds that modulate the expression levels and/or activity of the G2A receptor or the signaling pathway in which the G2A receptor takes part.
  • the present disclosure demonstrates its teachings using a murine model of atherosclerosis.
  • the present disclosure should not be limited in scope to atherosclerosis.
  • the present disclosure provides methods for increasing HDL levels and decreasing VLDL and/or LDL levels; therefore, the present disclosure is applicable to the treatment and prevention of a variety of disease states and conditions characterized by low HDL levels and/or high VLDL and/or LDL levels.
  • the present disclosure shows that G2A provides a robust pro-atherogenic stimulus in LDLR-/-mice. Increased HDL-C levels and decreased VLDL-C levels are associated with atherosclerosis suppression in G2A-/-LDLR-/-mice.
  • the G2A receptor or the signaling pathway in which the G2A receptor takes part by decreasing the expression levels and/or activity of the G2A receptor or the signaling pathway in which the G2A receptor takes part, HDL-C levels are increased and/or VLDL levels are decreased and atherosclerosis is reduced in experimental mice.
  • the present disclosure shows that chemotactic effects of G2A in blood cells do not modulate atherosclerosis, as bone marrow deficiency of G2A has no effect on lipoprotein levels or atherosclerosis in LDLR-/-mice. While not being limited to the following, the teachings of the present disclosure suggest that G2A expressed in resident non- hematopoietic cells, such as but not limited to, liver or the small intestinal enterocytes, are responsible for HDL formation are the target of G2A action in atherosclerosis.
  • FIG. 1 shows a simplified schematic depicting Reverse Cholesterol Transport.
  • VLDL and LDL particles transport cholesterol to peripheral tissues.
  • Hypercholesterolemia results in high circulating concentrations of these lipoprotein particles, which can penetrate the vascular endothelium of large arteries at sites prone to disruption by turbulent blood flow.
  • VLDL and LDL particles trapped in the arterial wall become oxidized and are taken up by macrophages that infiltrate the sub-endothelial space as a response to the generation of pro-inflammatory lipids.
  • Uptake of oxidized VLD/LDL particles by macrophages results in their transformation into cholesterol-engorged "foam cells", which drive the development of an atherosclerotic lesion.
  • FIG. 2 shows a model for HDL-mediated reverse cholesterol transport protecting against atherosclerosis by counteracting the accumulation of excess cholesterol in the vascular wall derived from VLD/LDL.
  • Liver hepatocytes synthesize apolipoprotein Al (apoAl) and apolipoprotein E (apoE). ApoAl is secreted with phospholipids and cholesterol from hepatocytes as "nascent HDL" particles.
  • small intestinal enterocytes also synthesize and secrete apoAl and contribute 20-30% of circulating HDL (see FIG. 1).
  • HDL particles accept cholesterol effluxed from peripheral tissues, including macrophages in the vascular wall, which also synthesize apoE.
  • Cholesterol carried by HDL is esterified by the action of LCAT, an enzyme associated with HDL particles, resulting in the maturation of HDL particles.
  • Mature HDL particles are taken up by hepatocytes via scavenger receptors (SRs), such as the prototypical SRBl .
  • ApoE derived from HDL is recycled by hepatocytes and secreted as nascent HDL.
  • apoE derived from VLDL taken up by hepatocytes is also recycled and secreted as nascent HDL.
  • Esterified cholesterol derived from HDL is processed in the liver and excreted through the bile and feces.
  • FIG. 3 shows an overview of reverse cholesterol transport incorporating processes illustrated in FIGS. 1 and 2.
  • FIG. 4 shows a schematic depicting the principal sources of lysophosphatidylcholine (LPC) associated with hypercholesterolemia and atherosclerosis.
  • LPC is a major product of LDL oxidation (Platelet-Activating Factor Acetyl Hydrolase, PAF-AH) mediated hydrolysis of oxidized phosphatidylcholine [PC]), pro-inflammatory Secretory Phospholipase A 2 (sPLA 2 )- mediated PC hydrolysis, and Lecithin:Cholesterol Acyltransferase (LCAT)-mediated HDL- cholesterol esterification.
  • PAF-AH Platinum-Activating Factor Acetyl Hydrolase
  • sPLA 2 pro-inflammatory Secretory Phospholipase A 2
  • LCAT Lecithin:Cholesterol Acyltransferase
  • FIG. 5 shows the G protein-coupled receptor G2A is expressed in key cell-types relevant to atherosclerosis.
  • FIG. 5 A shows RT-PCR analysis of G2A expression in the indicated primary cells.
  • FIG. 5B shows one potential mechanism of G2A signaling in response to LPG. High extracellular LPG concentrations induce the rapid redistribution of intracellular endosomal receptor pools to the surface where G2A can activate signal transduction pathways through specific membrane-associated G proteins (Gg/1 1 and Gl 3 G proteins).
  • FIG. 6 shows that the loss of G2A inhibits atherosclerosis in LDLR-/-mice.
  • Representative sudan IV-stained aorta from male (upper panel) and female (lower panel) G2A+/+LDLR-/- and G2A-/-LDLR-/-mice maintained on a high cholesterol diet for 20 weeks. Quantification of aortic lesion coverage shown alongside. Symbols indicate statistically significant differences in aortic lesion coverage by Mann Whitney rank sum test.
  • FIG. 7 shows that the loss of G2A suppresses lesion progression at the aortic sinus of LDLR- /-mice.
  • FIG. 8 shows increased plasma HDL-cholesterol levels and decreased VLDL-cholesterol levels in G2A-/LDLR-/-mice.
  • FIG. 8A shows plasma concentrations (mg/dl) of total cholesterol (total C), unesterified cholesterol (UC), LDL-cholesterol (LDL-C), and HDL- cholesterol (HDL-C) in male G2A+/+LDLR-/-and G2A-/-LDLR-/-mice maintained on a high cholesterol diet for 20 weeks. Mean (-) with standard deviation shown. *p ⁇ 0.02, statistically significant differences in plasma lipoprotein concentrations by Mann Whitney rank sum test.
  • FIG. 8A shows plasma concentrations (mg/dl) of total cholesterol (total C), unesterified cholesterol (UC), LDL-cholesterol (LDL-C), and HDL- cholesterol (HDL-C) in male G2A+/+LDLR-/-and G2A-/-LDLR-/-mice maintained on a high cholesterol diet for 20 weeks
  • FIG. 8B shows a representative column lipoprotein profile (CLiP)-based FPLC lipoprotein profile in plasma samples (lO ⁇ l) from one male G2A+/+LDLR-/-mouse and one male G2A-/- LDLR-/-mouse maintained on a high cholesterol diet for 20 weeks.
  • Relative cholesterol distribution within VLDL, LDL and HDL fractions are shown with HDL-C levels being increased and VLDL-C levels being decreased in G2A-/-LDLR-/- mice.
  • FIG. 9 shows FPLC analysis of plasma lipoprotein cholesterol distribution and HDL apolipoprotein composition in 300 ⁇ l samples of pooled plasma from 3 male G2A+/+LDLR-/- and 3 male G2A-/-LDLR-/-mice maintained on a high cholesterol diet for 20 weeks.
  • FPLC was performed on a Biologic chromatography system (BioRad; Hercules, CA) using a HiLoad 16/60 Superdex 200 prep grade column (Amersham Biosciences) to obtain optimal separation of HDL, VLDL and LDL particles.
  • Plasma samples were eluted with phosphate- buffered saline (PBS) at a flow rate of ImI per minute.
  • PBS phosphate- buffered saline
  • Cholesterol concentrations were measured in each 0.5ml eluate fractions. Profiles show the amount of cholesterol in each fraction.
  • the inset shows immunoblot of pooled consecutive pairs of HDL fractions (indicated by dashed lines) with antibodies specific for apoE and apoAl (1 indicates LDLR-/- G2A+/+ and 2 indicates LDLR-/-G2A-/-). Data shown is representative of 3 independent experiments.
  • FIG. 10 shows that modulation of HDL by G2A is dependent on apoE. Loss of G2A in apoE knockout mice does not raise circulating HDL-cholesterol concentrations.
  • FIG. 10 shows plasma concentrations (mg/dL) of HDL-C and LDL-C in apoE-/-G2A+/+ and apoE-/-G2A-/- mice fed standard chow or high cholesterol diets for the indicated periods. Differences in lipoprotein concentrations not statistically significant by Mann Whitney rank Sum Test.
  • FIG. 10 shows that modulation of HDL by G2A is dependent on apoE. Loss of G2A in apoE knockout mice does not raise circulating HDL-cholesterol concentrations.
  • FIG. 10 shows plasma concentrations (mg/dL) of HDL-C and LDL-C in apoE-/-G2A+/+ and apoE-/-G2A-/- mice fed standard chow or high cholesterol diets for the indicated periods. Difference
  • FIG. 11 shows FPLC analysis of plasma lipoprotein (300 ⁇ l samples of pooled plasma from 3 male animals) from male high cholesterol diet-fed (20 weeks) apoE-/-G2A+/+ and apoE-/- G2A-/- mice using a HiLoad Superdex column.
  • the inset shows immunoblot of pooled consecutive pairs of HDL fractions indicated by dashed lines with an apoAl -specific and apoE specific antibody (1 indicates apoE-/-G2A+/+ and 2 indicates: apoE-/-G2A-/-). Differences in apoAl lipoprotein concentrations not statistically significant by Mann Whitney rank Sum Test
  • FIG. 12 shows FPLC separation of plasma lipoproteins from male high cholesterol diet-fed (20 weeks) ApoE+/+LDLR+/+G2A+/+ (C57BL6J), ApoE-/-LDLR-/-G2A+/+ and ApoE- ⁇ LDLR-/-G2A-/- mice using a conventional Superose 6 column.
  • Plasma lipoprotein profile from male high cholesterol diet-fed (20 weeks) ApoE+/+LDLR+/+G2A+/+ (C57BL/6J) mice is shown to demonstrate the position of HDL fractions.
  • the inset shows anti-apoAl immunoblot analysis of apoAl content in pooled consecutive pairs of HDL fractions indicated by dashed lines.
  • 1 Control high cholesterol diet-fed (20 weeks) C57BL6J
  • 2 ApoE-/-LDLR- /-G2A+/+
  • 3 ApoE-/-LDLR-/-G2A-/-.
  • FIG. 13 shows that the loss of G2A in apoE-/-mice does not inhibit atherosclerosis.
  • FIG. 14 shows that bone marrow (BM) deficiency of G2A does not affect circulating HDL- cholesterol concentrations in LDLR-/-mice.
  • BM bone marrow
  • FIG. 15 shows that bone marrow (BM) deficiency of G2A does not inhibit atherosclerosis in LDLR-/-mice.
  • BM bone marrow
  • FIG. 16 shows that G2A expressed in resident non-hematopoietic tissue (i.e., not bone marrow-derived blood cells) modulates atherosclerosis in LDLR-/-mice.
  • Representative sudan rV-stained aorta are shown from male G2A+/+LDLR-/-and G2A-/-LDLR-/-mice transplanted with the indicated donor bone marrow cells.
  • FIG. 17 shows one potential role of G2A as a regulator of apoE recycling, increased HDL levels and decreased VLDL levels.
  • ATP -binding cassette transporters including ABCAl, mediate free cholesterol (FC) efflux to apolipoprotein acceptors and HDL, facilitated by scavenger receptor class B type 1 (SR-Bl) binding to HDL.
  • FC mediate free cholesterol
  • SR-Bl scavenger receptor class B type 1
  • Esterif ⁇ cation of FC by HDL- associated lecithinxholesterol acyltransferase (LCAT) generates esterified cholesterol (EC) and LPC.
  • Other sources of LPC include the LPA 2 -like activity of HDL-associated paraoxonase-1 (PON-I).
  • G2A may control the modulatory effect of these local increases in LPC on apoE recycling/secretion and resulting HDI assembly (lighter arrows). Trafficking of G2A, apoE and ABCAl through the same endosomal pathways suggests possible modes of G2A-mediated regulation of apoE recycling (co-localization of G2A and apoE within endosomes not depicted, but may occur).
  • FIG. 18 demonstrates that G2A is expressed by primary mouse hepatocytes.
  • FIG. 18A shows conventional RT-PCR analysis on total RNA from the indicated mouse tissues.
  • FIG. 8B shows indistinguishable morphological characteristics of primary hepatocytes isolated from high cholesterol diet-fed (20 weeks) LDLR-/-G2A+/+ and LDLR-/-G2A-/- mice.
  • FIG 18C shows quantitative real-time PCR analysis demonstrating G2A expression by primary hepatocytes (macrophages shown as positive control). The data shown is the average of 3 independent experiments, each performed in triplicate.
  • FIG. 18D shows ethidium bromide- stained agarose gel illustrating the expected size of G2A-specific amplification products from FIG. 18C.
  • Lane 1 is LDLR-/-G2A+/+ hepatocytes
  • lane 2 is LDLR-/-G2A-/- hepatocytes
  • lane 3 is LDLR-/-G2A+/+ macrophages
  • lane 4 is LDLR-/-G2A-/- macrophages.
  • FIG. 19 shows deletion of G2A in high cholesterol diet-fed LDLR-/- mice increases aApoE- HDL secretion by hepatocytes without altering apoE or apoAl gene expression.
  • FIG. 19 shows deletion of G2A in high cholesterol diet-fed LDLR-/- mice increases aApoE- HDL secretion by hepatocytes without altering apoE or apoAl gene expression.
  • FIG. 19A shows quantitative real-time PCR analysis of the expression of the indicated genes in primary hepatocytes and macrophages from male high cholesterol diet-fed (20 weeks) LDLR-/- G2A+/+ and LDLR-/-G2A-/- mice. The data shown is the average of 3 independent experiments, each performed in triplicate.
  • FIG. 19B shows immunoblot analysis of apoE, apoAl and ABCAl levels in primary hepatocytes from male high cholesterol diet-fed (20 weeks) LDLR-/-G2A+/+ and LDLR-/-G2A-/- mice (lane 1 : LDLR-/-G2A+/+; lane 2: LDLR- /-G2A-/-).
  • FIG. 19C shows FPLC separation of lipoproteins secreted into culture medium over 18 hours by freshly isolated hepatocytes from high cholesterol diet-fed (20 weeks) LDLR-/-G2A+/+ and LDLR-/- G2A-/- mice. Phospholipid profiles of HDL fractions secreted by LDLR-/-G2A+/+ and LDLR-/-G2A-/- hepatocytes shown to the right.
  • the inset shows immunoblot of HDL fractions with antibodies specific for apoE and apoAl showing significant increases in apoE- HDL in conditioned medium from LDLR-/-G2A-/- hepatocytes. Data shown is representative of 3 independent experiments.
  • Lysophosphatidylcholine is a bioactive lysophospholipid generated in the arterial wall by platelet activating factor-acetylhydrolase (PAF-AH) mediated hydrolysis of oxidized phosphatidylcholine (PC) and is therefore a decomposition products of LDL oxidation (14) (FIG. 4).
  • PAF-AH platelet activating factor-acetylhydrolase
  • PC oxidized phosphatidylcholine
  • LPC LDL oxidation
  • sPLA2 macrophage-expressed secretory phospholipase A 2
  • LPC is also a major by-product of cholesterol esterification by HDL- associated lecithin:cholesterol acyltransferase (LCAT) (16, 17).
  • LPC circulating levels of LPC are also significantly elevated under hypercholesterolemic conditions (18).
  • Chemotactic action of LPC has been shown to be mediated via G2A, a G protein-coupled receptor (GPCR) expressed in monocytes/macrophages, lymphocytes and aortic endothelial cells (ECs) (18-22).
  • GPCR G protein-coupled receptor
  • ECs aortic endothelial cells
  • 9-S-HODE 9-S-hydroxyeicosadienoic acid
  • Other lysophospholipids structurally related to LPC have also recently been reported to activate G2A in neutrophils (26).
  • GPCR G protein-coupled receptor
  • Activation of cellular responses by LPC via G2A are associated with mobilization of intracellular endosomal G2A pools to the plasma membrane resulting in an increased number of cell-surface receptors (22, 26).
  • cell-surface receptors 22, 26
  • global inhibition of intracellular recycling by monensin inhibited G2A mobilization to the cell surface and suppressed LPC-stimulated cellular chemotaxis (22).
  • LPC-stimulated cellular chemotaxis 22).
  • intracellular sequestration and surface expression control G2A-mediated responses to LPC.
  • This mode of G2A regulation while distinct from those utilized by most GPCRs, is reminiscent of intracellular recycling mechanisms utilized by other receptors whose activities are controlled by lipid molecules.
  • apolipoprotein and receptor recycling in macrophages and hepatocytes has been shown to constitute a major regulatory mechanism by which cells control cholesterol mobilization and HDL assembly.
  • internalization and trafficking of the ATP -binding cassette transporter 1 (ABCA 1) is functionally important in mediating apoAl -dependent cholesterol efflux from macrophages (27-29).
  • the present disclosure demonstrates that the G2A receptor suppresses atherosclerosis in LDLR deficient mice, a model of atherosclerosis.
  • the suppression of atherosclerosis was demonstrated by statistically significant decreases in atherosclerotic lesion surface areas in G2A deficient mice on the LDLR-/- background.
  • the present disclosure demonstrates that G2A deficiency on the LDLR-/- background causes an increased level of circulating HDL particles.
  • the increased level of circulating HDL particles was concurrent with an increase in apoAl and apoE content in circulating HDL particles, increased volume/size of circulating HDL particles, and a decrease in circulating VLDL levels.
  • the present disclosure demonstrates that G2A modulating effects on circulating HDL, LDL and/or VLDL levels are dependent upon apoE.
  • concurrent G2A deficiency does not increase HDL levels.
  • the present disclosure shows that resident non-hematopoietic cells expressing G2A mediate the effects on modulation of circulating levels of HDL particles.
  • modulating the expression levels and/or activity of the G2A receptor or the signaling pathway in which the G2A receptor takes part has significant potential for the treatment and prevention of disease states and conditions characterized by or associated with by decreased HDL levels, increased VLDL levels and/or increased LDL levels.
  • disease states and conditions include, but are limited to, cardiovascular disease, stroke, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus.
  • methods for diagnosing patients with elevated G2A receptor activity are needed to identify patients suffering from or at risk for disease states and conditions characterized by or associated with atherosclerosis.
  • the present disclosure demonstrates that the G2A receptor and its associated signaling pathways modulate the levels of HDL, LDL and/or VLDL in the circulation.
  • the present disclosure also demonstrates that loss of G2A function in vivo results in athero-protection by modulating HDL, LDL and/or VLDL levels and/or metabolism.
  • the present disclosure shows that apoE is required for G2A to modulate HDL metabolism. Therefore, the present disclosure provides new therapeutic targets and diagnostic markers for the treatment and/or prevention of disease states and conditions characterized by and/or associated with decreased HDL levels, increased VLDL levels and/or increased LDL levels and the diagnosis of such disease states and conditions.
  • the teachings of the present disclosure are applicable to all disease states and conditions characterized by decreased HDL levels, increased VLDL levels and/or increased LDL levels.
  • Such disease states and conditions include, but are limited to, cardiovascular disease, stroke, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus. Definitions
  • prevention refers to a course of action (such as administering a compound or pharmaceutical composition of the present disclosure) initiated prior to the onset of a clinical manifestation of a disease state or condition so as to prevent or reduce such clinical manifestation of the disease state or condition. Such preventing and suppressing need not be absolute to be useful.
  • treatment refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a clinical manifestation of a disease state or condition so as to eliminate or reduce such clinical manifestation of the disease state or condition.
  • Such treating need not be absolute to be useful.
  • in need of treatment refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a condition that is treatable by a method, compound or pharmaceutical composition of the disclosure.
  • in need of prevention refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a condition that is preventable by a method, compound or pharmaceutical composition of the disclosure.
  • the term "individual”, “subject” or “patient” as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans.
  • mammals such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans.
  • the term may specify male or female or both, or exclude male or female.
  • terapéuticaally effective amount refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease state or condition. Such effect need not be absolute to be beneficial.
  • the present disclosure provides for compounds that inhibit G2A activity (either directly or through inhibition of expression).
  • the present disclosure also provides for compounds that modulate the activity of a polypeptide regulated by G2A.
  • Modulating the activity of a polypeptide regulated by G2A as used herein refers to modulating the function of such polypeptide in a manner opposite of is regulation by G2A. For example, if G2A stimulates the activity or induces translocation a given polypeptide, then modulating the activity of such polypeptide refers to inhibiting the activity of such polypeptide or inhibiting translocation.
  • G2A inhibits the activity or inhibits translocation of a given polypeptide
  • modulating the activity of such polypeptide refers to stimulating the activity of such polypeptide or inducing translocation.
  • Such compounds may be used to treat and/or prevent a disease state or condition associated with or characterized by increased VLDL or LDL levels or decreased HDL levels, as well as by increased G2A activity.
  • disease states and conditions include, but are limited to, cardiovascular disease, stroke, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus.
  • the compounds disclosed can inhibit G2A activity or modulate the activity of a polypeptide regulated by G2A in vivo and/or in vitro.
  • a compounds of the present disclosure can be an antagonist of a G2A or a polypeptide regulated by G2A. Such compounds can exert their effect on the G2 A activity or the activity of a polypeptide regulated by G2A via changes in expression, post-translational modifications or by other means.
  • Suitable compounds include, but are not limited to, polypeptides, functional nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of G2A. Such compounds may be identified in the methods of screening discussed herein. Nucleic acid inhibitors
  • the compounds of the present disclosure that inhibit G2 A activity or modulate the activity of a polypeptide regulated by G2A are functional nucleic acids.
  • Functional nucleic acids are nucleic acid molecules that carry out a specific function in a cell, such as binding a target molecule or catalyzing a specific reaction.
  • Such functional nucleic acids may inhibit the activity of G2 A or modulate the activity of a polypeptide regulated by G2A.
  • Functional nucleic acids include but are not limited to antisense molecules, aptamers, ribozymes, triplex forming molecules, small interfering RNA (siRNA), RNA interference (RNAi), and external guide sequences (EGS).
  • siRNA small interfering RNA
  • RNAi RNA interference
  • EGS external guide sequences
  • a siRNA could be used to reduce or eliminate expression of G2A or a polypeptide regulated by G2A.
  • Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing.
  • the interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation.
  • the antisense molecule is designed to interrupt a processing function that normally would take place on the target nucleic acid molecule, such as transcription or replication.
  • Antisense molecules can be designed based on the sequence of the target nucleic acid molecule (such as a nucleic acid encoding G2A or a polypeptide regulated by G2A). Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target nucleic acid molecule exist. Exemplary methods include, but are not limited to, in vitro selection experiments and DNA modification studies using DMS and DEPC.
  • Aptamers are molecules that interact with a target nucleic acid molecule, preferably in a specific way.
  • aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in, for example, U.S. Patent Nos. 5,476,766 and 6,051,698 (which are hereby incorporated by reference).
  • the secondary structure inhibits expression of the polypeptide encoded by the gene or inhibits a processing function as discussed above.
  • Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly.
  • ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as, but not limited to, hammerhead ribozymes, hairpin ribozymes and tetrahymena ribozymes.
  • ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (including, but not limited to, those described in U.S. Patent Nos.
  • Ribozymes may cleave RNA or DNA substrates. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in U.S. Patent Nos. 5,837,855; 5,877,022; 5,972,704; 5,989,906; and 6,017,756 (which are hereby incorproated by reference).
  • Triplex forming functional nucleic acid molecules are nucleic acid molecules that can interact with either double-stranded or single-stranded nucleic acid.
  • triplex forming nucleic acids interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing.
  • Triplex molecules can bind target regions with high affinity and specificity. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in U.S. Patent Nos. 5,650,316; 5,683,874; 5,693,773; 5,834,185; 5,869,246; 5,874,566; and 5,962,426 (which are hereby incorporated by reference).
  • EGSs are molecules that bind a target nucleic acid molecule forming a complex, which is recognized by RNase P. RNaseP then cleaves the target nucleic acid molecule. EGSs can be designed to specifically target a RNA molecule of choice. Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in U.S. Patent Nos. 5,168,053; 5,624,824; 5,683,873; 5,728,521; 5,869,248; and 5,877,162 (which are hereby incorproated by reference).
  • siRNA is a double-stranded RNA that can induce sequence-specific post- transcriptional gene silencing, thereby decreasing or even inhibiting gene expression from a target nucleic acid.
  • an siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA.
  • Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer.
  • siRNA can be chemically or in Wtro-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell.
  • shRNAs short double-stranded hairpin-like RNAs
  • Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer.
  • siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit (Ambion, Austin, TX).
  • Antibody inhibitors are generally designed using algorithms and a conventional DNA/RNA synthesizer. siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit (Ambion, Austin, TX).
  • Polypeptides that inhibit G2A activity of the activity of a polypeptide regulated by G2A include antibodies with antagonistic or inhibitory properties.
  • fragments, chimeras, or polymers of immunoglobulin molecules are also useful in the methods taught herein, as long as they are chosen for their ability to inhibit G2A activity of the activity of a polypeptide regulated by G2A.
  • the antibodies can be tested for their desired activity using in vitro assays, or by analogous methods, after which their in vivo therapeutic or prophylactic activities are tested according to known clinical testing methods.
  • the term antibody is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. Monoclonal antibodies can be made using any known procedure.
  • monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (which is hereby incorproated by reference).
  • DNA encoding the disclosed monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, as described in U.S. Patent No. 5,804,440 and U.S. Patent No. 6,096,441 (which are hereby incorproated by reference).
  • Antibody fragments include Fv, Fab, Fab' or other antigen binding portion of an antibody. Digestion of antibodies to produce fragments thereof can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published and U.S. Pat. No. 4,342,566 (which are hereby incorproated by reference). Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross linking antigen.
  • the antibodies or antibody fragments may also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues. These modifications can provide additional or improved function. For example, the removal or addition of acids capable of disulfide bonding may increase the bio-longevity of the antibody. In any case, the modified antibody or antibody fragment retains a desired bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide. Such methods are readily apparent to a skilled practitioner in the art and can include site-specific mutagenesis of the nucleic acid encoding the antibody or antibody fragment. (Zoller, M.J. Curr.
  • antibody or antibodies can also refer to a human antibody and/or a humanized antibody.
  • examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol., 147(1):86 95, 1991).
  • Human antibodies (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. MoI. Biol., 227:381, 1991; Marks et al., J. MoI. Biol., 222:581, 1991).
  • the disclosed human antibodies can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 255 (1993); Jakobovits et al., Nature, 362:255 258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).
  • Antibody humanization techniques generally involve the use of recombinant DNA technology to manipulate the DNA sequence encoding one or more polypeptide chains of an antibody molecule.
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain that contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody. Fragments of humanized antibodies are also useful in the methods taught herein. Methods for humanizing non human antibodies are well known in the art.
  • humanized antibodies can be generated according to the methods of Winter and co workers (Jones et al., Nature, 321 :522 525 (1986), Riechmann et al., Nature, 332:323 327 (1988), Verhoeyen et al., Science, 239:1534 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Methods that can be used to produce humanized antibodies are also described in U.S. Patent Nos. 4,816,567, 5,565,332, 5,721,367, 5,837,243, 5, 939,598, 6,130,364, and 6,180,377.
  • the teachings of the present disclosure provide for the treatment and/or prevention of disease states and conditions associated with or characterized by decreased HDL levels, increased VLDL levels and/or increased LDL levels in a subject in need of such treatment.
  • disease states and conditions include, but are limited to, cardiovascular disease, stroke, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus.
  • the present disclosure describes a role for the G2A receptor and its signal transduction pathway which is involved, at least in part, in decreasing the levels of HDL and increasing the levels of LDL and VLDL; the composition, cholesterol content and biogenesis of HDL particles is also modulated.
  • HDL levels can be increased, LDL and/or VLDL levels can be decreased and the composition, cholesterol content and biogenesis of HDL particles can be favorable altered.
  • the method of treatment and/or prevention comprises the steps of identifying a subject in need of such treatment or prevention and administering to the subject a compound or composition that inhibits the activity of the G2A receptor and/or modulates the activity of a polypeptide regulated by the G2A receptor.
  • a compound or composition that inhibits the activity of the G2A receptor and/or modulates the activity of a polypeptide regulated by the G2A receptor.
  • said inhibition or modulation is accomplished by decreasing the expression, in whole or in part, of the G2A gene, to reduce the levels of such polypeptides in the subject.
  • Such decreased expression is accomplished by administering a compound or pharmaceutical composition containing at least one agent capable of decreasing the expression of such genes, such as a functional nucleic acid which may be delivered via gene- therapy or other techniques known in the art.
  • said modulation is accomplished by decreasing the activity, in whole or in part, of G2A receptor and/or a polypeptide regulated by the G2A receptor, so as to reduce the activity of the G2A receptor and/or downstream signaling pathways of the G2A receptor in the subject.
  • Such decreased activity is accomplished by administering a compound or pharmaceutical composition containing at least one agent capable of decreasing the activity of the G2A receptor or one or more of the polypeptides involved in G2A signaling such as but not limited to, a specific or non-specific inhibitor of such polypeptides, agents that reduce the stability or half-life of the such polypeptides, or agents that promote the intracellular sequestration of the such polypeptides. Any inhibitor known or subsequently determined to have activity against G2A or a polypeptide regulated by G2A may be used.
  • said modulation is accomplished by decreasing the activity, number or distribution, in whole or in part, of cells expressing G2A, thereby decreasing the activation of these cells in the presence of endogenous activators of G2A receptor, such as but not limited to lysophosphotidylcholine (LPC).
  • the cells are resident non-hematopoietic cells of the liver.
  • Such decreased activation is accomplished by administering a an agent capable of decreasing the expression of G2A or decreasing the activation of such cells, such as but not limited to, factors that decrease the activation, number or distribution of resident non-hematopoietic cells expressing G2A, agents, such as, but not limited to, antibodies, that sequester factors that activate resident non- hematopoietic cells expressing G2A, increasing the expression of factors that decrease the activation of resident non-hematopoietic cells expressing G2A or decreasing the gene expression of factors that activated resident non-hematopoietic cells expressing G2A.
  • Such modulation would thereby reduce G2A receptor mediated activation of cells expressing G2 A receptor in a subject and treat the disease states or conditions discussed herein.
  • the results of inhibiting the activity and/or expression of G2A or a polypeptide regulated by G2 A include, but are not limited to, (1) reducing/regressing atherosclerotic plaques in a subject, (2) increasing the levels of circulating HDL particles in the vasculature of a subject, (3) increasing the volume/cholesterol content of circulating HDL particles in the vasculature of a subject, (4) increasing the apoE content and/or concentration of circulating HDL particles in the vasculature of a subject, (5) increasing the apoAl content and/or concentration in circulating HDL particles in the vasculature of a subject, (6) decreasing cardiovascular disease, plaque formation, and/or atherosclerotic lesions in a subject, (7) decreasing the content of oxidized LDL particles in a subject and/or atherosclerotic lesions in a subject, (8) reducing the inflammation at chronic or nascent atherosclerotic plaques in a subject, (2) increasing the levels of
  • the compounds/agents used in the above methods may funcitonal nucleic acids, polypeptides, such as those antibodies described herein, or compounds identified in the screening assays disclosed herein. Any known inhibitor of G2A or a polypeptide regulated by G2A may be used.
  • the present disclosure also provides methods for diagnosis for determining if a subject is suffering from or at risk for a disease states and conditions associated with or characterized increased G2A activity and/or by decreased HDL particle levels, increased VLDL particle levels and/or increased LDL particle levels.
  • disease states and conditions include, but are limited to, cardiovascular disease, stroke, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus.
  • the methods for diagnosis involve determining the status of a subject with respect to the activity and/or expression G2A or the activity and/or expression of a polypeptide regulated by G2A.
  • a biological sample which is subjected to testing is a sample derived from a subject and includes, but is not limited to, any biological fluid, preferably a bodily fluid.
  • bodily fluids include, but are not limited to, whole blood, serum, saliva, tissue infiltrate, pleural effusions, lung lavage fluid, bronchoalveolar lavage fluid, and the like.
  • the biological fluid may be a cell culture medium or supernatant of cultured cells.
  • the sample can be a blood sample or a serum sample.
  • Those subjects in which G2A activity and/or expression is increased above a control value or the activity of a polypeptide regulated by G2A is modulated (increased or decreased) as compared to a control value are determined to be suffering from or at risk for a disease states and conditions associated with or characterized by increased G2A activity and/or decreased HDL particle levels, increased VLDL particle levels and/or increased LDL particle levels.
  • such methods determine in the subject 1) the levels of circulating HDL particles in the vasculature of a subject, 2) the volume/cholesterol content of circulating HDL particles in the vasculature of a subject, 3) the apoE content and/or concentration in circulating HDL particles in the vasculature of a subject, 4) the apoAl content and/or concentration in circulating HDL particles in the vasculature of a subject, 5) the extent of plaque formation, and/or atherosclerotic lesions in a subject, 6) the content of oxidized LDL particles in a subject 7) the levels of inflammatory mediators, such as, but not limited to, LPC, in a subject, 8) the numbers of monocytes, macrophages and/or foam cells at atherosclerotic plaques/lesions/foci in a subject and/or 9) the levels of circulating VLDL particles in the vasculature of the subject.
  • In vivo imaging techniques such as
  • An increase or decrease in expression or activity as compared to a control means that the level of expression or activity is higher or lower, respectively, in the biological sample from a subject being tested than in a control sample. In one embodiment, the difference is at least 1.25-fold, 1.5-fold, 2-fold, 5-fold or higher.
  • a control means a sample obtained from a subject that does not exhibit a symptom or characteristic of the disease state or condition to be determined. The control value may be determined empirically from a subject or group of subjects or may be an average value from a selected population.
  • Assay techniques that can be used to determine levels of expression or activity in a sample are known. Such assay methods include, but are not limited to, radioimmunoassays, reverse transcriptase PCR (RT-PCR) assays, immunohistochemistry assays, in situ hybridization assays, competitive-binding assays, Western Blot analyses, ELISA assays and proteomic approaches, two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling.
  • RT-PCR reverse transcriptase PCR
  • immunohistochemistry assays immunohistochemistry assays
  • in situ hybridization assays in situ hybridization assays
  • competitive-binding assays Western Blot analyses
  • ELISA assays and proteomic approaches two-dimensional gel electrophoresis (2D electrophoresis) and non-gel based approaches such as mass spectrometry or protein interaction profiling.
  • Assays also include, but are not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, enzyme immunoassays (EIA), enzyme linked immunosorbent assay (ELISA), sandwich immunoassays, precipitin reactions, gel diffusion reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and Immunoelectrophoresis assays.
  • EIA enzyme immunoassays
  • ELISA enzyme linked immunosorbent assay
  • sandwich immunoassays precipitin reactions
  • gel diffusion reactions immunodiffusion assays
  • immunodiffusion assays agglutination assays
  • complement-fixation assays immunoradiometric assays
  • fluorescent immunoassays fluorescent immunoassays
  • protein A immunoassays protein A immunoassays
  • an antibody is prepared, if not readily available from a commercial source, specific to an antigen, such as, for example, G2A or a polypeptide regulated by G2A.
  • a reporter antibody generally is prepared.
  • the reporter antibody is attached to a detectable reagent such as a radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.
  • a detectable reagent such as a radioactive, fluorescent or enzymatic reagent, for example horseradish peroxidase enzyme or alkaline phosphatase.
  • the sample to be analyzed is incubated with the solid support, during which time the antigen binds to the specific antibody. Unbound sample is washed out with buffer. A reporter antibody specifically directed to the antigen and linked to a detectable reagent is introduced resulting in binding of the reporter antibody to any antibody bound to the antigen. Unattached reporter antibody is then washed out. Reagents for detecting the presence of the reporter antibody are then added. The detectable reagent is then determined in order to determine the amount of antigen present.
  • the antigen is incubated with the solid support, followed by incubation with one ore more antibodies, wherein at least one of the antibodies comprises a detectable reagent. Quantitative results may be obtained by reference to a standard curve.
  • a genetic sample from the biological sample can be obtained.
  • the genetic sample comprises a nucleic acid, preferably RNA and/or DNA.
  • mRNA can be obtained from the biological sample, and the mRNA may be reverse transcribed into cDNA for further analysis.
  • the mRNA itself is used in determining the expression of genes.
  • a genetic sample may be obtained from the biological sample using any techniques known in the art (Ausubel et al. Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: A Laboratory Manual, 2nd Ed., ed.
  • the nucleic acid may be purified from whole cells using DNA or RNA purification techniques.
  • the genetic sample may also be amplified using PCR or in vivo techniques requiring subcloning.
  • the genetic sample can be obtained by isolating mRNA from the cells of the biological sample and reverse transcribing the RNA into DNA in order to create cDNA (Khan et al. Biochem. Biophys. Acta 1423:17 28, 1999).
  • a genetic sample Once a genetic sample has been obtained, it can be analyzed. The analysis may be performed using any techniques known in the art including, but not limited to, sequencing, PCR, RT-PCR, quantitative PCR, restriction fragment length polymorphism, hybridization techniques, Northern blot, microarray technology, and similar techniques.
  • the level of expression may be normalized by comparison to the expression of another gene such as a well known, well characterized gene or a housekeeping gene (for example, actin).
  • RT-PCR reverse- transcriptase PCR
  • RT-PCR can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species.
  • Hybridization to clones or oligonucleotides arrayed on a solid support can be used to both detect the expression of and quantitate the level of expression of that gene.
  • a cDNA encoding an antigen is fixed to a substrate.
  • the substrate may be of any suitable type including but not limited to glass, nitrocellulose, nylon or plastic.
  • At least a portion of the DNA encoding the antigen is attached to the substrate and then incubated with the analyte, which may be RNA or a complementary DNA (cDNA) copy of the RNA, isolated from the sample of interest.
  • Hybridization between the substrate bound DNA and the analyte can be detected and quantitated by several means including but not limited to radioactive labeling or fluorescence labeling of the analyte or a secondary molecule designed to detect the hybrid. Quantitation of the level of gene expression can be done by comparison of the intensity of the signal from the analyte compared with that determined from known standards. The standards can be obtained by in vitro transcription of the target gene, quantitating the yield, and then using that material to generate a standard curve.
  • the present disclosure also provides for methods of screening for identifying compounds that inhibit G2A activity or that modulate the activity of a polypeptide regulated by G2A.
  • Such compounds may be useful as active ingredients included in pharmaceutical compositions.
  • Such compounds may be used to treat and/or prevent a disease state or condition associated with or characterized by increased VLDL or LDL levels or decreased HDL levels, as well as by increased G2A activity.
  • disease states and conditions include, but are limited to, cardiovascular disease, stroke, hyperlipidemia, hypercholesterolemia, atherosclerosis and chronic inflammatory autoimmune disorders, such as but not limited to lupus.
  • such screening methods comprises the steps of providing an assay system (as described in more detail below) that expresses G2A or a polypeptide regulated by G2A, introducing into the assay system a test compound to be tested and determining whether the test compound inhibits the activity of G2A or modulates the activity of a polypeptide regulated by G2A.
  • Such inhibition or modulation may directly on the activity of G2A or a polypeptide regulated by G2 A or may be an inhibition or modulation of expression.
  • the methods involve the identification of candidate or test compounds or agents (polypeptides, functional nucleic acids, carbohydrates, antibodies, small molecules or other molecules) which bind to G2A or a polypeptide regulated by G2A and/or have an inhibitory effect on the biological activity and/or expression of G2A or modulate the biological activity and/or expression a polypeptide regulated by G2A.
  • candidate or test compounds or agents polypeptides, functional nucleic acids, carbohydrates, antibodies, small molecules or other molecules
  • Such compounds may then be further tested in appropriate systems (such as, but not limited to, the animal models systems described herein) to determine the activity of the identified compounds.
  • Candidate compounds are identified using a variety of assays, such as, but not limited to, assays that employ cells which express G2A on the cell surface or a polypeptide regulated by G2A (cell-based assays) or in assays with isolated G2A or isolated polypeptides regulated by G2A (cell-free assays).
  • the various assays can employ a variety of variants of G2A and the polypeptide regulated by G2A (e. g., full-length, a biologically active fragment, or a fusion protein which includes all or a portion of the desired polypeptide).
  • G2A or a desired polypeptide regulated by G2A can be derived from any suitable mammalian species (e. g., human, rat or murine).
  • the cell may either naturally express G2A or a polypeptide regulated by G2A or may be modified to express the same.
  • cells can be modified to express G2A or a polypeptide regulated by G2A through conventional molecular biology techniques, such as by infecting the cell with a virus comprising G2A or a polypeptide regulated by G2A wherein G2A or a polypeptide regulated by G2A is expressed in the cell following infection.
  • the cell can also be a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding G2A or a polypeptide regulated by G2A.
  • full length polypeptides, fragments or fusion proteins containing at least a part of such polypeptide may be used.
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound to G2A or a polypeptide regulated by G2A.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of G2 A or the activity of a polypeptide regulated by G2A.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of mRNA or protein.
  • the various screening assays may be combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms the disease states and conditions discussed herein.
  • the compounds may be evaluated to determine if the impact a parameter associated with the action of G2A or polypeptides regulated by G2A.
  • Such parameters include, but are not limited to, determining 1) the levels of circulating HDL particles in the vasculature of a subject, 2) the volume/cholesterol content of circulating HDL particles in the vasculature of a subject, 3) the apoE content and/or concentration in circulating HDL particles in the vasculature of a subject, 4) the apoAl content and/or concentration in circulating HDL particles in the vasculature of a subject, 5) the extent of plaque formation, and/or atherosclerotic lesions in a subject, 6) the content of oxidized LDL particles in a subject 7) the levels of inflammatory mediators, such as, but not limited to, LPC, in a subject, 8) the numbers of monocytes, macrophages and/or foam cells at atherosclerotic plaques/lesions/foci in a subject and/or 9) the levels of circulating VLDL particles in the vasculature of the subject.
  • the present disclosure provides assays for screening candidate or test compounds which bind to or modulate the activity of a membrane-bound (cell surface expressed) form of G2A.
  • Such assays can employ full-length G2A, a biologically active fragment of G2A, or a fusion protein which includes all or a portion of G2A.
  • the test compound can be obtained by any suitable means (such as from conventional compound libraries).
  • the G2A may be expressed in a whole cell or in a liposome, micelle or similar lipid containing structure.
  • Determining the ability of the test compound to bind to a membrane-bound form of G2A can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the G2A- expressing cell can be measured by detecting the labeled compound in a complex.
  • the test compound can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radio-emission or by scintillation counting.
  • the test compound can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phos-phatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting G2A expressing cell or liposome with a known compound which binds to G2A to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the G2A expressing cell, wherein determining the ability of the test compound to interact with the G2 A expressing cell comprises determining the ability of the test compound to preferentially bind the G2A expressing cell as compared to the known compound. Inhibition of signaling
  • the assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of G2A (a full-length G2A, a biologically active fragment of G2 A, or a fusion protein which includes all or a portion of G2 A) expressed on the cell surface with a test compound and determining the ability of the test compound to inhibit the activity of the membrane- bound form of G2A. Determining the ability of the test compound to inhibit the activity of the membrane-bound form of G2A can be accomplished by any method suitable for measuring the activity of G2A or the activity of a G-protein coupled receptor or other seven-transmembrane receptor.
  • the activity of a seven- transmembrane receptor can be measured in a number of ways, not all of which are suitable for any given receptor. Among the measures of activity are: alteration in intracellular Ca2+ concentration, activation of phospholipase C, alteration in intracellular inositol triphosphate (IP3) concentration, alteration in intracellular diacylglycerol (DAG) concentration, and alteration in intracellular adenosine cyclic 3', 5'-monophosphate (cAMP) concentration. The activation/inhibition of polypeptides regulated by G2A may also be measured.
  • IP3 inositol triphosphate
  • DAG diacylglycerol
  • cAMP adenosine cyclic 3', 5'-monophosphate
  • Determining the ability of the test compound to modulate the activity of G2A can be accomplished, for example, by determining the ability of G2A to bind to or interact with a target molecule, such as a polypeptide regulated by the G2A receptor.
  • the target molecule can be a molecule with which G2A binds or interacts with in nature, for example, a molecule on the surface of a cell which expresses G2A, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • the target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (a signal generated by binding of a G2A ligand) through the cell membrane and into the cell.
  • the target G2A molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with G2A.
  • Determining the ability of G2A to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding.
  • determining the ability of a compound of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (intracellular Ca2+, diacyl glycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (such as a regulatory element that is responsive to a compound operably linked to a nucleic acid encoding a detectable marker, e. g., luciferase), or detecting a cellular response.
  • a reporter gene such as a regulatory element that is responsive to a compound operably linked to a nucleic acid encoding a detectable marker, e. g.,
  • the present disclosure also includes cell-free assays.
  • Such assays involve contacting a form of G2A or a polypeptide regulated by G2A (full-length, a biologically active fragment, or a fusion protein comprising all or a portion of a desired polypeptide) with a test compound and determining the ability of the test compound to bind to G2A or a polypeptide regulated by G2A or to inhibit G2A or a polypeptide regulated by G2A G2A. Binding of the test compound to G2A can be determined either directly or indirectly as described above. Regulation of G2A activity or of a polypeptide regulated by G2A can be determined as discussed above.
  • the assay includes contacting a cell free system containing G2A or a polypeptide regulated by G2A with a known compound to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with G2A, wherein determining the ability of the test compound to interact with G2A comprises determining the ability of the test compound to preferentially bind to G2A as compared to the known compound.
  • the cell-free assays of the present disclsoure are amenable to use of either a membrane-bound form of G2A or a soluble fragment thereof.
  • non-ionic detergents such as n-octy
  • G2A or a polypeptide regulated by G2A
  • binding of a test compound to G2A or a polypeptide regulated by G2A, or interaction of G2A with a polypeptide regulated by G2A in the presence and absence of a test compound can be accomplished in any vessel suitable for containing the reactants.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST glutathione-S-transferase
  • glutathione-S-transferase fusion proteins or glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads or glutathione derivatized microtitre plates, which are then combined with the test compound and the mixture incubated under conditions conducive to complex formation (for example at physiological conditions for salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of binding or activity of G2A or a polypeptide regulated by G2A can be determined using standard techniques.
  • the screening assay can also involve monitoring the expression of G2A or a polypeptide regulated by G2A.
  • regulators of expression of G2A or a polypeptide regulated by G2A can be identified in a method in which a cell is contacted with a test compound and the expression of G2A polypeptide or a polypeptide regulated by G2A or mRNA encoding the foregoing in the cell is determined. The level of expression of polypeptide or mRNA the presence of the test compound is compared to the level of expression of in the absence of the test compound. The test compound can then be identified as a regulator of expression of G2A or a polypeptide regulated by G2A based on this comparison.
  • test compound when expression of polypeptide or mRNA protein is decreased in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of polypeptide or mRNA expression.
  • the level of polypeptide or mRNA expression in the cells can be determined by methods described below.
  • the level of mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used.
  • the presence of polypeptide products of G2A polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, Northern blots, Southern blots, microarray testing, PCR techniques, including but not limited to, real-time PCR and immunohistochemistry.
  • polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into G2A.
  • Such screening can be carried out either in a cell-free assay system or in an intact cell.
  • Any cell which expresses G2A polynucleotide or polynucleotide encoding a polypeptide regulated by G2A can be used in a cell-based assay system.
  • the polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line can be used.
  • test compounds for use in the screening assays can be obtained from any suitable source, such as conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one- compound” library method and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage. Modeling compounds
  • Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can inhibit G2A or modulate the activity of a polypeptide regulated by G2A (either through expression or activity). Having identified such a compound, the active sites or regions are identified. Such active sites might typically be ligand binding sites. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand.
  • Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry.
  • the incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
  • test compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. Alternatively, these methods can be used to identify improved compounds known in the art or identified in one of the screening assays above. These compounds found may be used to inhibit G2A activity or modulate the activity of a peptide regulated by G2A.
  • kits for carrying out any method of the present disclosure which can contain any of the compounds and/or compositions disclosed herein or otherwise useful for practicing a method of the disclosure.
  • compositions of the present disclosure may comprise one or more compounds useful in the treatment and prevention methods of the present disclosure, such as, but not limited to, those compounds that modulate the expression or activity of G2A or of the signaling pathway in which G2A is involved; such compounds may be identified by a screening method of the present disclosure.
  • such compounds decrease the expression, in whole or in part, of the G2A gene, thereby reducing the levels of such proteins in the subject.
  • such compounds decrease the activity, in whole or in part, of G2A receptor, so as to reduce the activity/activation of the G2A receptor and/or downstream signaling pathways of the G2A receptor in the subject.
  • such compounds decrease the activity, number or distribution, in whole or in part, of resident non-hematopoietic cells expressing G2A, thereby decreasing the activation of these cells in the presence of endogenous activators of G2 A receptor, such as but not limited to lysophosphotidylcholine (LPC).
  • endogenous activators of G2 A receptor such as but not limited to lysophosphotidylcholine (LPC).
  • such compounds are in the form of compositions, such as but not limited to, pharmaceutical compositions.
  • the compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington: The Science and Practice of Pharmacy (20 th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor). To form a pharmaceutically acceptable composition suitable for administration, such compositions will contain a therapeutically effective amount of a compound(s).
  • compositions of the disclosure may be used in the treatment and prevention methods of the present disclosure. Such compositions are administered to a subject in amounts sufficient to deliver a therapeutically effective amount of the compound(s) so as to be effective in the treatment and prevention methods disclosed herein.
  • the therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject's condition, weight, sex and age. Other factors include the mode and site of administration.
  • the pharmaceutical compositions may be provided to the subject in any method known in the art. Exemplary routes of administration include, but are not limited to, subcutaneous, intravenous, topical, epicutaneous, oral, intraosseous, intramuscular, intranasal and pulmonary.
  • compositions of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week, once per month or once per year. The compositions may also be administered to the subject more than one time per day.
  • the therapeutically effective amount of the nucleic acid molecules and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects.
  • co-administration or sequential administration of other agents may be desirable.
  • the compositions of the present disclosure may be administered systemically, such as by intravenous administration, or locally such as by subcutaneous injection or by application of a paste or cream.
  • compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and/or or decrease the toxicity of the compounds(s). Examples of such agents are described in a variety of texts, such a, but not limited to, Remington: The Science and Practice of Pharmacy (20 th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor).
  • compositions of the present disclosure can be administered in a wide variety of dosage forms for administration.
  • the compositions can be administered in forms, such as, but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous administration or injection.
  • Other dosage forms include administration transdermally, via patch mechanism or ointment.
  • Further dosage forms include formulations suitable for delivery by nebulizers or metered dose inhalers. Any of the foregoing may be modified to provide for timed release and/or sustained release formulations.
  • the pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrating agents and carriers, as well as accessory agents, such as, but not limited to, coloring agents and flavoring agents (collectively referred to herein as a carrier).
  • the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use.
  • the pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.
  • the compound(s) may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier, such as, but not limited to, inert fillers, suitable binders, lubricants, disintegrating agents and accessory agents.
  • suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthum gum and the like.
  • Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid as well as the other carriers described herein.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • the nucleic acid molecules of the present disclosure can be dissolved in diluents, such as water, saline, or alcohols.
  • the oral liquid forms may comprise suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like.
  • suitable and coloring agents or other accessory agents can also be incorporated into the mixture.
  • Other dispersing agents include glycerin and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compound(s) may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4- methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxy
  • Oils which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.
  • Suitable fatty acids for use in parenteral formulations include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkylbeta-aminopropionates, and 2- alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.
  • HLB hydrophile-lipophile balance
  • Topical dosage forms such as, but not limited to, ointments, creams, pastes, emulsions, containing the nucleic acid molecule of the present disclosure, can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. Inclusion of a skin exfoliant or dermal abrasive preparation may also be used. Such topical preparations may be applied to a patch, bandage or dressing for transdermal delivery or may be applied to a bandage or dressing for delivery directly to the site of a wound or cutaneous injury.
  • carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, all
  • the compound(s) of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Such liposomes may also contain monoclonal antibodies to direct delivery of the liposome to a particular cell type or group of cell types.
  • the compound(s) of the present disclosure may also be coupled with soluble polymers as targetable drug carriers.
  • Such polymers can include, but are not limited to, polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • G2A knockout mice backcrossed 10 generations onto the C57BL/6J strain with C57BL/6J LDL receptor knockout (LDLR-/-) mice were bred.
  • G2A+/+LDLR-/- and G2A-/- LDLR-/- mice were subsequently derived by intercrossing compound heterozygotes.
  • G2A+/+LDLR-/- and G2A-/-LDLR-/- mice were maintained on a high cholesterol diet (1.25% cholesterol) for 20 weeks to induce atherosclerosis.
  • G2A+/+LDLR-/- mice developed extensive atherosclerosis throughout the descending aorta (average % lesion coverage $G2A+/+LDLR-/- 19.9% ⁇ 20.6) while that in G2A-/-LDLR-/- mice was almost completely suppressed in comparison (average % lesion coverage cJ ⁇ A-ALDLR-/- 0.9% ⁇ 0.6).
  • Atherosclerotic lesion areas in serial aortic sinus sections from G2A+/+LDLR-/- and G2A-/-LDLR-/- mice maintained on a high cholesterol diet for 20 weeks were also measured.
  • G2A deficiency resulted in significantly reduced aortic sinus lesion size (average lesion area c ⁇ G2A+/+LDLR-/- 922,000mm 2 ⁇ 198,091, c?G2A-/-LDLR-/- 529,769mm 2 ⁇ 125,221, $G2A+/+LDLR-/- 786,900mm 2 ⁇ 74,301, $G2A-/-LDLR- ⁇ 457,438mm 2 ⁇ 87,757) (FIG. 7).
  • plasma samples (10ml) from one randomly selected male G2A+/+LDLR-/- mouse and one male G2A-/- LDLR-/- mouse maintained on a high cholesterol diet for 20 weeks were examined by Column Lipoprotein Profile (CLiP)-based FPLC (FIG. 8B).
  • CLiP Column Lipoprotein Profile
  • the CLiP method is a simple and convenient procedure for determining plasma lipoprotein cholesterol profiles in small sample volumes (35).
  • FPLC analysis of plasma lipoprotein cholesterol distribution and HDL apolipoprotein composition in 300ml samples of pooled plasma from 3 male G2A+/+LDLR-/- and 3 male G2A-/-LDLR-/- mice maintained on a high cholesterol diet for 20 weeks was carried out (FIG. 9).
  • FPLC FPLC was performed using a HiLoad 16/60 Superdex 200 prep grade column to obtain optimal separation of lipid particles. Both methods confirmed significant elevations in HDL-C and revealed an increase in HDL particle size in hypercholesterolemic G2A-/-LDLR-/- mice (note leftward shift of HDL "peak" in G2A- ⁇ LDLR-/- samples). Furthermore, VLDL-C was decreased. In addition, plasma apoB- containing lipoprotein (VLDL+LDL)-cholesterol concentrations were moderately reduced, but not to a statistically significant extent, in the same mice (data not shown).
  • plasma lipoproteins from high cholesterol diet-fed LDLR-/-G2A+/+ and LDLR-/-G2A-/- mice were fractionated by FPLC and immunoblot analysis for apolipoprotein Al (apoAl) and apolipoprotein E (apoE) was performed.
  • ApoAl is a principal HDL component that is required for HDL formation and is present at stoichiometric levels on virtually all HDL particles (36).
  • ApoE is an apolipoprotein present on a sub-fraction of HDL particles. The presence of apoE on HDL particles is known to significantly facilitate their capacity to carry cholesterol by promoting expansion of the cholesterol ester-rich core.
  • the increased apoE content indicates the cholesterol content of the HDL particles was increased in LDLR-/-G2A-/- mice.
  • HDL-C particles were slightly larger in size in LDLR-/-G2A-/- mice as indicated by a leftward shift of the HDL peak (FIGS. 8A and 9), consistent with an increased cholesterol content of a sub-fraction of HDL particles in these mice.
  • lipoproteins were separated through a high capacity Superdex column in order to maximize separation of HDL particles and thereby minimize their contamination with nonHDL-derived ApoE.
  • ApoE knockout mice are a widely used murine model of atherosclerosis.
  • ApoE is a key component of HDL, VLDL and other triglyceride rich lipoproteins (TRLs) required for their clearance from the circulation through interaction with hepatic receptors.
  • TRLs triglyceride rich lipoproteins
  • a failure to efficiently clear VLDL and TRLs therefore contributes to the predisposition of apoE-/- mice to atherosclerosis.
  • the absence of apoE whose recycling is important for maintaining optimal levels of circulating HDL, results in reduced HDL levels and thereby attenuates cholesterol removal from vascular macrophages to promote foam cell formation.
  • the present disclosure shows that levels of apoE-containing HDL are increased in G2A-/-LDLR-/- mice (FIG. 9) and suggests that increased apoE biosynthesis and/or recycling may contribute, at least in part to, the ability of G2A to modulate HDL particle levels, HDL particle size and HDL particle composition (increased cholesterol content) and the resulting atheroprotection. If so, one would expect that effects of G2A deficiency on HDL-C and atherosclerosis would be attenuated or absent in apoE-/- mice.
  • G2A-/- and apoE-/- mice were bred to generate genetically matched G2A+/+apoE-/- and G2A-/-apoE-/- mice.
  • G2A+/+apoE-/- and G2A-/-apoE-/- mice were maintained on a high cholesterol diet identical to that fed to LDLR-/- mice as described above for 20 weeks.
  • LDL-C 6 v ApoE-/-G2A+/+ 489.7 ⁇ 45.9 mg/dL, $ApoE-/-G2A-/- 540.7 ⁇ 108.3 mg/dL) (FIG. 10).
  • G2A deficiency did not result in statistically significant alterations in plasma HDL-C and LDL-C concentrations (FIG. 10).Total cholesterol and unesterified cholesterol were also not altered in a statistically significant manner (data not shown).
  • FPLC analysis of plasma from G2A+/+apoE-/- and G2A-/-apoE-/- mice confirmed that G2A had no effect on HDL-C levels in the absence of apoE (FIG. 11).
  • FPLC profiles revealed mild increases in VLDL-C in apoE-/-G2A-/- mice as compared to apoE-/- G2A+/+ (FIG. 11) which were consistent with the moderately raised concentrations of apoB- containing lipoproteins (VLDL+LDL) in the same mice (FIG. 10, LDL-C graph).
  • G2A deletion in LDLR-/- mice resulted in increased immunoreactivity of FPLC- separated HDL fractions with an apoAl -specific antibody (FIG. 9), consistent with the presence of an increased number of circulating HDL particles in LDLR-/-G2A-/- mice.
  • FIG. 9 To address the possibility that a similar effect of G2A deletion may have occurred in apoE-/- mice but was obscured by a concomitant reduction in the cholesterol content of individual HDL particles to produce equivalent measurements of HDL-C in apoE-/-G2 A+/+ and apoE-/- G2A-/- mice (FIG.
  • LDLR-/-G2A-/- mice we crossed LDLR-/-G2A-/- mice with apoE-/- mice to generate genetically matched (C57BL/6 NlO) LDLR-/-apoE-/-G2A+/+ and LDLR-/-apoE-/-G2A-/- mice.
  • Plasma lipoproteins from regular chow diet-fed or high cholesterol diet-fed LDLR-/- apoE-/-G2A+/+ and LDLR-/-apoE-/-G2A-/- mice were analyzed by FPLC.
  • G2A provides a robust pro-atherogenic stimulus in LDLR-/- mice.
  • Increased HDL-C levels are associated with atherosclerosis suppression in G2A-/-LDLR-/- mice. Therefore, blocking G2A receptor function results in beneficial effects by raising HDL-C levels, lowering VLDL-C levels and thereby reducing atherosclerosis in experimental mice.
  • Reported chemotactic effects of G2A in blood cells do not modulate atherosclerosis, as demonstrated by the finding that bone marrow deficiency of G2A has no effect on lipoprotein levels or atherosclerosis in LDLR-/- mice.
  • results of bone marrow transplantation experiments further support the notion that G2A expressed in liver, or perhaps small intestinal enterocytes responsible for HDL formation, are the target of G2A action in atherosclerosis.
  • G2A deficiency raises HDL-C levels is provided by data showing increases in apoAl and apoE in HDL, a requirement for apoE expression to manifest HDL-C elevation and atheroprotection in the absence of G2A, and a requirement for G2A expression in resident non-hematopoietic tissue, not bone marrow- derived blood cells, for its effects on HDL metabolism and atherosclerosis.
  • G2A is expressed in primary hepatocvtes.
  • the cumulative data in LDLR-/- and apoE-/- mice show a direct association between the pro-atherogenic action of the G2A receptor and its ability to modulate plasma HDL levels.
  • the increase in the number of plasma HDL particles (as demonstrated by increased apoAl levels) observed in LDLR-/-G2A-/- mice (FIG. 9) suggest that HDL biosynthesis or HDL clearance/catabolism is affected by G2A deficiency. Reduced hepatic clearance or catabolism of HDL is unlikely to be responsible for HDL elevation in these mice because this would have resulted in the accumulation of mature dysfunctional HDL which would be expected to promote, rather than suppress, atherosclerosis (28, 29).
  • hepatocytes express G2A
  • isolated primary mouse hepatocytes from LDLR-/-G2A+/+ and LDLR-/-G2A-/- mice were isolated by hepatic collagenase perfusion (FIG. 18B) and quantitative real-time PCR analysis was performed to determine G2A expression.
  • Primary hepatocytes from LDR-/-G2A+/+ and LDR-/-G2A-/- mice were morphologically indistinguishable (FIG. 18B).
  • Quantitative real-time PCR showed primary hepatocytes from high cholesterol diet-fed LDLR-/-G2A+/+ mice were found to express G2A (FIGS.
  • genes encoding other ATP -binding cassette transporters (ABCG5, ABCG8), SRBl, LDLR-related protein- 1 (LRPl) and lipases capable of modifying plasma lipoprotein-cholesterol levels by affecting lipoprotein clearance (hepatic lipase, lipoprotein lipase) (31) were also unaffected by G2A deletion in hepatocytes from high cholesterol diet-fed LDLR-/- mice (FIG. 19A). This data shows that the elevations in plasma apoE-HDL in LDLR-/-G2A-/- mice are not the result of increased hepatic expression of apoE, apoAl or cholesterol transporters.
  • G2A may modulate the secretion of apoE-HDL at a post-transcriptional level in hepatocytes.
  • lipoprotein secretion by freshly isolated hepatocytes from high cholesterol diet-fed LDLR-/-G2A+/+ and LDLR-/-G2A-/- mice was examined to determine if plasma HDL elevation in the latter group of mice was associated with an increased capacity of their hepatocytes to secrete HDL particles.
  • Conditioned medium from LDLR-/-G2 A+/+ and LDLR-/-G2 A-/- hepatocytes cultured for 18 hours was concentrated and fractionated by FPLC.
  • Contamination of these HDL fractions with VLDL-derived apoE can be excluded as a contributing factor because little apoB48 could be detected by immunoblotting in these fractions (data not shown). Therefore, it is possible that hepatocyte secretion of a minor population of particles containing apoE only may be increased in LDLR-/-G2A-/- mice. Although hepatocyte-specific overexpression of apoE in apoAl -deficient mice can result in the appearance of such particles in the plasma (26), mechanisms regulating their hepatic biogenesis in normal mice are poorly understood.
  • G2A-/- mice (10 generations backcrossed onto the C57BL/6J background: NlO) were bred with C57BL/6J LDLR-/- or with C57BL/6J ApoE-/- mice and resulting compound heterozygotes were intercrossed to obtain genetically matched LDLR-/-G2A+/+, LDLR-/-G2A-/-, ApoE-/-G2A+/+ and ApoE-/-G2A-/- mice.
  • ApoE-/-LDLR-/-G2A+/+ and apoE-/-LDLR-/-G2A-/- mice were generated by intercrossing compound heterozygotes derived from breeding LDLR-/-G2A-/- with apoE-/-G2A+/+ mice.
  • Mice were weaned at 4 weeks of age and maintained on a standard rodent chow diet (Diet #5015; Harlan Teklad, Madison, WI). At 8 weeks of age, mice were fasted for 12 hours, weighed, bled by retro- orbital puncture and transferred onto a high cholesterol diet (15.8% fat, 1.25% cholesterol, 7.5% casein without sodium cholate: Diet #94059; Harlan Teklad, Madison, WI). Following diet intervention, mice were fasted for 12 hours, weighed and bled by retro-orbital puncture for lipid analyses. All experimental procedures were performed with approval from the Animal Care Committee of the University of Alabama.
  • Atherosclerotic lesion quantification En face atherosclerotic lesion quantification throughout the aorta and atherosclerotic lesion quantification at the aortic root was performed as previously described (19).
  • Plasma samples were processed for measurement of total cholesterol, unesterified cholesterol, HDL cholesterol, LDL-cholesterol, triglycerides and free fatty acids by enzymatic procedures as previously described (19, 39). LDL-cholesterol concentrations obtained using this method includes both VLDL and LDL fractions.
  • Online cholesterol analysis of plasma lipoproteins by FPLC (CLIP) was performed as described by Garber and colleagues (25). Briefly, 10 ⁇ l of plasma was separated with a Superose 6 10/300 GL column (Amersham Biosciences) using PBS/0.02% Sodium Azide running buffer at a flow rate of 0.6 ml/min.
  • Plasma HDL fractions for subsequent immunoblot analysis of apolipoprotein composition
  • plasma was fractionated using a HiLoad 16/60 Superdex 200 prep grade column (Amersham Biosciences) on a Biologic DuoFlow FPLC system (Bio-Rad) to obtain maximum separation of VLDL/LDL and HDL fractions and thereby minimize the detection of VLDL-derived ApoE in immunoblots of HDL fractions.
  • Plasma 400 ⁇ l was injected onto the column and separated with PBS containing 0.02% sodium azide at a flow rate of 1 ml/minute. 120 0.5ml fractions were collected after a 42ml delay. Fractions were subsequently analyzed for cholesterol content utilizing an enzymatic cholesterol reagent (Thermo Electron Corporation).
  • HDL-C fractions were identified and processed for SDS-polyacrylamide gel electrophoresis and immunoblotting as described below.
  • Immunoreactive proteins were visualized using enhanced chemiluminescence (Immun-star; Bio-Rad).
  • Antibodies used were as follows: Rabbit anti-ApoE (Biodesign), rabbit anti-ApoAl (Biodesign) and rabbit anti- ABCAl (Novus Biologicals).
  • mice were anesthetized using a ketamine/xylazine mixture and the superior vena cava was clamped with a micro vascular clamp.
  • the inferior vena cava was immediately cannulated with a 25 gauge winged infusion needle (Terumo) and the hepatic portal vein was severed.
  • the liver was perfused with 30 ml of Ca 24 VMg 2+ - free Hank's Balanced Salt Solution (HBSS) containing 0.2 mM EDTA and 20 mM glucose at a constant rate of 4 ml/min using a syringe pump.
  • HBSS Hank's Balanced Salt Solution
  • the liver was then perfused with 30ml of Ca 2 VMg 2+ - containing HBSS with 20 mM glucose and 100 U/ml collagenase type I (Sigma) at a rate of 4ml/min.
  • the liver was removed and placed into a culture dish containing 20 ml of ice-cold William's medium E (Invitrogen) containing 0.1% BSA, L-glutamine (2mM), penicillin (100 U/ml), streptomycin sulfate (100 ⁇ g/ml), and fungizone (250 ng/ml; Omega Scientific).
  • the liver was gently teased apart and subsequently passed through a 1 OO ⁇ m strainer.
  • Hepatocytes were obtained by gravity sedimentation (15 minutes) followed by 2 washes in Williams medium (50g for 3 minutes at 4°C). Hepatocytes were resuspended in 20ml of Williams medium, counted and assessed for viability using trypan blue exclusion. The yield of hepatocytes averaged from 20-4OxIO 6 from each liver and viability was consistently greater than 90%.
  • RNA concentrations and RNA quality were measured using the Experion automated microfluidics electrophoresis system (BioRad) with preformed RNA standards for single-platform RNA detection and data analysis. lOOng of each RNA sample was used to synthesize cDNA using the iScript cDNA synthesis kit (BioRad).
  • PCR conditions were: 95°C 3 minutes, 95°C 10 seconds, followed by 40 cycles at 95°C for 15 seconds, 58°C for 1 minute, 72°C for 30 seconds, and finally 95°C for 1 minute.
  • Data showing G2A expression (FIG. 17C) is presented as expression relative to G3PDH in G2A+/+ and G2A-/- cells (average CT G3PDH ⁇ average CT G2A).
  • Hepatocvte culture For analysis of hepatocyte apolipoprotein secretion in high cholesterol diet-fed LDLR-/-G2A+/+ and LDLR-/-G2A-/- mice, 8x10 6 hepatocytes were plated at 10 6 /ml in serum-free Williams medium onto fibronectin-coated (lO ⁇ g/well) 10cm tissue culture plates. 18 hours later, conditioned medium (16ml each from LDLR-/-G2A+/+ and LDLR-/- G2A-/- hepatocyte cultures) was collected and concentrated to ⁇ 500 ⁇ l using centrifugal filter devices (Amicon Ultra- 15, 10K).
  • Lipoproteins in concentrated conditioned media were subsequently separated by FPLC using a Superose-6 column as described above. Because we anticipated that HDL secreted by primary mouse hepatocytes may have very low cholesterol/lipid content, we also fractionated plasma from a control C57BL/6J mouse to delineate HDL fractions in subsequent cholesterol profiles. Total cholesterol and phospholipid (Phospholipids C Kit, Wako, VA) content was measured in FPLC fractions from concentrated conditioned media and control plasma. HDL fractions were subjected to SDS- PAGE and immunoblotting for analysis of apoE and apoAl levels.

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Abstract

La présente invention porte sur des procédés de traitement et de prévention d'états et de conditions de maladie associés à ou caractérisés par des taux diminués de particules de lipoprotéine haute densité (HDL), des taux accrus de particules de lipoprotéine très faible densité (VLDL) et/ou des taux accrus de particules de lipoprotéine faible densité (LDL). Dans un mode de réalisation, de tels procédés de traitement et de prévention mettent en jeu la modulation des taux d'expression et/ou de l'activité du récepteur G2A ou d'un polypeptide régulé par le récepteur G2A. La présente invention porte également sur des procédés de diagnostic pour identifier des sujets souffrant ou étant à risque de présenter de tels états et conditions de maladie. De plus, la présente invention porte sur des procédés pour identifier des composés qui modulent les taux d'expression et/ou l'activité du récepteur G2A ou la voie de signalisation à laquelle le récepteur G2A participe.
PCT/US2008/007706 2007-06-19 2008-06-19 Récepteur g2a comme cible thérapeutique Ceased WO2008156833A2 (fr)

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US94507707P 2007-06-19 2007-06-19
US60/945,077 2007-06-19

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3299032A1 (fr) 2016-09-23 2018-03-28 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Inhibiteurs de gpr132 destinés à être utilisés dans la prévention et/ou le traitement de la douleur neuropathique induite par chimiothérapie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PARKS B.W. ET AL.: 'Loss of G2A promotes macrophage accumulation in atherosclerotic lesions of low density lipoprotein receptor-deficient mice' JOURNAL OF LIPID RESEARCH vol. 46, no. 7, July 2005, pages 1405 - 1415 *
PARKS B.W. ET AL.: 'Loss of the lysophosphatidylcholine effector, G2A, ameliorates aortic atherosclerosis in low-density lipoprotein receptor knockout mice' ARTERIOSCLEROSIS, THROMBOSIS AND VASCULAR BIOLOGY vol. 26, no. 12, December 2006, pages 2703 - 2709 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3299032A1 (fr) 2016-09-23 2018-03-28 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Inhibiteurs de gpr132 destinés à être utilisés dans la prévention et/ou le traitement de la douleur neuropathique induite par chimiothérapie
WO2018055082A1 (fr) 2016-09-23 2018-03-29 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Inhibiteurs de gpr132 utilisables dans la prévention et/ou le traitement de la douleur neuropathique induite par la chimiothérapie
CN109715212A (zh) * 2016-09-23 2019-05-03 弗劳恩霍夫应用研究促进协会 用于预防和/或治疗化疗引起的神经性疼痛的gpr132抑制剂
JP2019529461A (ja) * 2016-09-23 2019-10-17 フラウンホファー ゲセルシャフト ツール フェールデルンク ダー アンゲヴァンテン フォルシュンク エー．ファオ． 化学療法誘発性神経障害性疼痛の予防及び／又は治療における使用のためのｇｐｒ１３２阻害剤
EP3515498B1 (fr) * 2016-09-23 2021-03-03 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung E.V. Inhibiteurs de gpr132 destinés à être utilisés dans la prévention et/ou le traitement de la douleur neuropathique induite par chimiothérapie

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