US20040006129A1

US20040006129A1 - Prevention or treatment of abnormal lipoprotein, atherosclerosis and cholestasis

Info

Publication number: US20040006129A1
Application number: US10/430,770
Authority: US
Inventors: Monty Krieger; Anne Braun-Egles; Helena Miettinen
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 2002-05-16
Filing date: 2003-05-05
Publication date: 2004-01-08
Also published as: US7361684B2; EP1511379A1; WO2003096808A1; AU2003241374A1; JP2005526128A; US20030046718A1

Abstract

Using an animal model, transgenic animals that do not express functional SR-BI and apoE which develop severe atherosclerosis, by age four weeks in transgenic mice, a class of drugs, PROBUCOL (4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol)) and monoesters of PROBUCOL, and BO 653, 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran, has been discovered which is useful in normalizing abnormal lipoprotein levels and/or characteristics, such as those found in lipoprotein X-associated disease. These animals are good models for screening of drugs useful in the treatment and/or prevention of cardiac fibrosis, myocardial infarction, defects in electrical conductance, atherosclerosis, unstable plaque, and stroke, as well as for lipoprotein disorders such as cholestasis and lipoprotein X associated disorders. Studies demonstrate normalization of lipoprotein levels and structure, as well as significant decreases in atherosclerosis and prevention of heart attack, even when administered after disease onset.

Description

This application claims priority to U.S. Ser. No. 10/147,651 filed May 16, 2002, entitled “Screening of Compounds for Treatment of Atherosclerosis and Heart Attack” by Monty Krieger, Anne Braun-Egles and Helena Miettinen.[0001]

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. government has certain rights to this invention by virtue of Grants HL41484, HI-52212, and HL20948 from the National Institutes of Health-National Heart, Lung and Blood Institute to Monty Kreiger and HL63609 and HL53793 to M. Simons and M. J. P. from the National Institutes of Health.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of methods of prevention or treatment of abnormal lipoprotein disorders, atherosclerosis, and heart attack.

Atherosclerosis is the leading cause of death in western industrialized countries. Atherosclerosis is the process in which deposits of fatty substances, cholesterol, cellular waste products, calcium and other substances build up in the inner lining of an artery. This buildup is called plaque. It usually affects large and medium-sized arteries. Some hardening of arteries often occurs when people grow older. Plaques can grow large enough to significantly reduce the blood's flow through an artery, and it is thought that much damage occurs when they become fragile and rupture. Plaques that rupture cause blood clots to form that can block blood flow or break off and travel to another part of the body. If either happens (occlusive plaques with or without occlusive blood clots) and blocks a blood vessel that feeds the heart, it causes a heart attack. If it blocks a blood vessel that feeds the brain, it causes a stroke.

Atherosclerosis is a slow, complex disease that typically starts in childhood and often progresses when people grow older. In some people it progresses rapidly, even in their third decade. Many scientists think it begins with inflammation or damage to the artery, including its innermost layer of cells called the endothelium. Causes of damage to the arterial wall include elevated levels of cholesterol and triglyceride in the blood, high blood pressure, tobacco smoke, and diabetes. The risk of developing atherosclerosis is directly related to plasma levels of LDL cholesterol and inversely related to HDL cholesterol levels. Because of the inflammation and/or damage to the endothelium, fats, cholesterol, platelets, cellular waste products, calcium and other substances are deposited in the artery wall. These may stimulate artery wall cells to produce other substances that result in further buildup of cells. These cells and surrounding material thicken the artery wall significantly. The artery's luminal diameter shrinks and blood flow decreases, reducing the oxygen and other nutrients supply to downstream tissue. A blood clot can form near this plaque and blocks the artery, stopping the blood flow. Alternatively, the plaque can grow large enough to effectively occlude the vessel. Research also suggests that inflammation may play an important role in triggering heart attacks and strokes. Inflammation is the body's response to infection and injury, and blood clotting is often part of that response.

Atherosclerosis often shows no symptoms until flow within a blood vessel has become seriously compromised. Typical symptoms of atherosclerosis include chest pain when a coronary artery is involved, or leg pain when a leg artery is involved. Sometimes symptoms occur only with exertion. In some people, however, they may occur at rest.

Cholestasis is any condition in which bile excretion from the liver is blocked, which can occur either in the liver or in the bile ducts. There are many causes of cholestasis. Extrahepatic cholestasis (which occurs outside the liver) can be caused by bile duct tumors, strictures, cysts, diverticula, and other damage. Other potential causes for this type include stones in the common bile duct, pancreatitis, pancreatic tumor or pseudocyst, primary sclerosing cholangitis, and compression due to a mass or tumor on a nearby organ. Intrahepatic cholestasis (which occurs inside the liver) can be caused by sepsis (generalized infection), cacterial abscess, drugs, total parenteral nutrition (being fed intravenously), lymphoma, tuberculosis, sarcoidosis and amyloidosis. Other causes of this form of the disorder include primary biliary cirrhosis, primary sclerosing cholangitis, viral hepatitis (A, B, C, etc.), alcoholic liver disease, pregnancy, Sjogren's syndrome and others.

The cellular mechanisms of cholestasis have as yet to be completely understood. Diagnosis can be made using history, physical examination, a few laboratory parameters, serology and also ERCP. Treatment depends largely on the cause of the cholestasis. Normally, bile is produced in the liver, moved to the gallbladder and excreted into the gut through the biliary tract, to aid in the digestion of fats and the excretion of small molecules from the body. Flow from the liver to the gallbladder and ultimately to the gut can be slowed or stopped by certain drugs. When the flow of bile is inhibited, an individual may become jaundiced. Drugs such as ursodesoxycholic acid and immunosuppressants are causes of the leading disorders such as PBC and PSB. Many drugs can cause cholestasis. Some more common culprits include: gold salts, nitrofurantoin, anabolic steroids, oral contraceptives, chlorpromazine, prochlorperazine, sulindac, cimetidine, erythromycin, tobutamide, imipramine, ampicillin and other penicillin-based antibiotics. This list is not comprehensive, as other medications can also unexpectedly cause cholestasis in some individuals.

The disturbance of lipid metabolism is seen in some inherited diseases and also in patients with some kinds of underlying diseases. The presence of its disturbance can be detected by measuring the concentrations of cholesterol and triglyceride in serum. Although hyperlipidemia or hypolipidemia can be the result of abnormal lipid metabolism, hyperlipidemia is of more concern to physicians because of the close association with atherosclerosis and kidney disease. Responsible genes for some primary (or hereditary) hyperlipidemic diseases have been confirmed as follows; LPL or apo C-II for primary chylomicronemia, LDL receptor for familial hypercholesterolemia and apo B-100 for familial defective apo B-100. However, the responsible gene remains controversial for familial combined hyperlipidemia, though AI/CIII/AIV cluster is one of the possible candidate genes. Secondary hyperlipidemia is caused by various diseases such as diabetes mellitus, renal diseases and cholestasis. This type of hyperlipidemia is improved by therapy for the underlying diseases. Hyperlipidemia with a marked increase of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels is a common feature in patients with chronic cholestatic liver disease. Excess morbidity and mortality from cardiovascular disease has not been reported in these patients. This may be due to the particular lipoprotein pattern observed during chronic cholestasis, characterized by elevated serum HDL cholesterol, which may have a cardioprotective effect. However, in a subgroup of patients with chronic cholestasis, hyperlipidemia is characterized by markedly elevated LDL levels with normal or low HDL levels, probably reflecting hypercholesterolemia with coexisting familial and nutritional origins.

Lipoprotein-X (Lp-X) is an abnormal low-density lipoprotein frequently found in liver disease and in patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency syndromes. It is regarded as the most sensitive and specific biochemical parameter for the diagnosis of intra- and extrahepatic cholestasis. Moreover, Lp-X is supposed to contribute to the development of hypercholesterolemia in cholestatic liver disease, because it fails to inhibit de novo cholesterol synthesis. Compared to other lipoproteins, Lp-X contains a high content of unesterified cholesterol (30%, w/w) to phosphatidylcholine (60%, w/w). Lp-X isolated from sera of patients with obstructive jaundice has a high content of unesterified cholesterol and phosphatidylcholine and contained apolipoprotein E, apoCs, and albumin.

It is an object of the present invention to provide methods and drugs for treating or preventing abnormal lipoproteins, for use in disorders such as cholestasis and lipoprotein X and for treating diseases due to abnormalities in cholesterol metabolism (e.g., Niemann-Pick Type C and Tangier diseases).

It is another object of the present invention to provide methods and compounds for the treatment of atherosclerosis.

SUMMARY OF THE INVENTION

Using an animal model, transgenic animals that do not express functional SR-BI and apoE which develop severe atherosclerosis by age four-five weeks i, a class of drugs, PROBUCOL (4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol)) and monoesters of PROBUCOL, and BO 653, 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran, has been discovered which is useful in normalizing abnormal lipoprotein levels and/or characteristics. These animals exhibit progressive heart dysfunction starting by age four-six weeks, and die by age eight weeks (between 5 and eight weeks). Pathology shows extensive fibrosis of the heart and occlusion of coronary arteries. The occlusion appears to be due to atherosclerosis, since fat deposition is in the walls. These animals are good models for the following diseases, and for screening of drugs useful in the treatment and/or prevention of these disorders: cardiac ischemia, cardiac fibrosis, myocardial infarction, defects in EKGs, including electrical conductance, heart failure, cardiac dysfunction (e.g., reduced ejection fraction), cardiac hypertrophy/dialation, and occlusive coronary artery disease (e.g., atherosclerosis). These animals can also be used to screen for drugs for use in treating lipoprotein disorders such as cholestasis and lipoprotein X.

Animals (apoE −/− SR-BI +/−) were fed PROBUCOL beginning at the time of mating. Offspring were weaned at three weeks and fed PROBUCOL. Survival is shown in FIG. 1A. In contrast to animals not fed PROBUCOL, 50% of whom are dead at six weeks of age, all animals on PROBUCOL have a normal phenotype (MRI of heart function, ECG, echocardiogram, histology) at six weeks. At seven to eight months, there is evidence of atherosclerosis and some myocardial infarction in the treated animals. This demonstrates that the compound has a preventative action. Animals who are taken off of the PROBUCOL at weaning all die within ten to twelve weeks.

In another study, the majority of animals whose parents were not fed PROBUCOL, but who received the PROBUCOL beginning before or after five weeks of age, survived for a few months (See FIG. 1B), demonstrating that the compound also has a therapeutic benefit. The earlier the treatment with PROBUCOL, the longer the survival of the animals. These studies showed that PROBUCOL treatment could extend the lives of these animals even when it is administered after the development of a significant level of heart disease. Further studies demonstrate that these animals have abnormal lipid structures similar to those present in cases resulting in the appearance of lipoprotein X, which are normalized by treatment with PROBUCOL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1[0016] a and 1 b are graphs of percent survival versus age for double knockout dKO mice compared to mice fed PROBUCOL.
FIG. 1[0017] a, dKO mice, n=13, fed normal chow, dashed line (death by eight weeks); dKO mice, n=10, 0.5% PROBUCOL diet from conception solid line (latest death at 60 weeks); and dKO-Pww mice, n=9, PROBUCOL diet from conception until weaning (death at about 18 weeks). FIG. 1b, dKO-P<5 mice (PROBUCOL diet administered only after weaning but prior to 5 weeks of age, n=9; and dKO-P>5, PROBUCOL diet administered immediately after 5 weeks of age, n=7.
FIG. 2 is a graph of the cholesterol lipoprotein profile (lipoproteins fractionated by size using FPLC) showing total cholesterol (mg/dl) for VLDL, IDL/LDL, and HDL for dKO mice untreated (black circles) or fed 0.5% PROBUCOL from conception (open circles).[0018]

DETAILED DESCRIPTION OF THE INVENTION

I. Pharmaceutical Compositions [0019]
Pharmacologically Active Compounds [0020]
The double knockout (dKO) mouse for apolipoprotein E and the HDL receptor (SR-BI) was used to identify compounds that can normalize abnormal lipoprotein levels and/or structure. SR-BI controls HDL structure and metabolism and appears to be important for reverse cholesterol transport. These mice display characteristics of coronary artery disease such as high serum cholesterol levels, atherosclerosis, patchy myocardial infarct, cardiac enlargement and premature death. They also are characterized by abnormal lipid levels and structure. [0021]
As demonstrated by the examples, PROBUCOL dramatically increases the life expectancy of the dKO mice. Untreated mice have a median lifespan of 6 weeks while PROBUCOL-treated mice can live up to 419 days (mean of 36 weeks). Histological study of the cardiac tissue at 6 weeks of age demonstrates that PROBUCOL also eliminated most of the functional and morphological indicators of occlusive atherosclerotic coronary artery disease, myocardial infarct and cardiac dysfunction. No fibrosis due to myocardial infarct, atherosclerotic plaques or atherosclerosis in the root of the aortic arch is seen in treated mice at this age. Serum cholesterol levels are decreased two fold in PROBUCOL-treated mice and PROBUCOL also corrects the defective red blood cell maturation seen in dKO mice. [0022]
Based on these studies, a number of compounds are useful in altering lipid levels and cholesterol metabolism. A preferred class of compounds are PROBUCOL (4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol)) and monoesters of PROBUCOL, for example, as described in U.S. Pat. No. 6,121,319 to Somers and other derivatives as described by FR 2168137, FR 2140771, FR 2140769, FR 2134810, FR 2133024, and FR 2130975, including the derivatives developed by Atherogenics Corporation. These compounds have potent antioxidant properties and block oxidative modification of LDL. Another useful compound available from Chugai of Japan is BO 653, 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran, an antioxident. Noguchi, et al., Arch. Biochem. Biophys. 1:347 (1997). [0023]
Based on the PROBUCOL data, other compounds that will be effective include vitamin E and vitamin C, both hypocholesterolemic and antioxident compounds, both as fertility enhancing agents as well as for treatment and/or prevention of cardiovascular disease or atherosclerosis. The preferred compounds have both activities. [0024]
Pharmaceutical Carriers [0025]
Compounds are preferably administered in a pharmaceutically acceptable vehicle for oral administration. Suitable pharmaceutical vehicles are known to those skilled in the art. For parenteral administration, the compound will usually be dissolved or suspended in sterile water or saline. For enteral administration, the compound will be incorporated into an inert carrier in tablet, liquid, or capsular form. Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature. [0026]
Alternatively, the compound may be administered in liposomes or microspheres (or microparticles). Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art. U.S. Pat. No. 4,789,734 describe methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. A review of known methods is by G. Gregoriadis, Chapter 14. “Liposomes”, [0027] Drug Carriers in Biology and Medicine pp. 287-341 (Academic Press, 1979). Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Pat. Nos. 4,906,474, 4,925,673, and 3,625,214.
II. Treatment of Disorders or Diseases [0028]
Disorders which may be treated include cholestasis, lipoprotein x, and disorders caused by LCAT deficiencies. Other disorders include atherosclerosis and unstable plaque, where the formulations are administered in an amount effective to normalize the lipoproteins levels and/or structure, reduce plaque deposition, or reduce the levels of other indicators. The pharmaceutical compositions can also be administered in an effective amount to modify or prevent the development of the disorder. Reduction in symptoms are readily determined by measuring blood, urine and/or tissue samples using clinically available tests, as demonstrated below. [0029]
The present invention will be further understood by reference to the following non-limiting examples. [0030]

EXAMPLE 1

Analysis of CHD and Atherosclerosis in SR-BI/apoE Double Knockout Mice

Mice with homozygous null mutations in the genes for both the high density lipoprotein receptor SR-BI and apolipoprotein E (SR-BI/apoE double KO, ‘dKO’, mice) express many features of CHD. dKO mice fed a low-fat, low cholesterol diet were examined using a variety of histologic, angiographic and functional (electrocardiography, hemodynamics, MRI) methods. They spontaneously exhibited the following characteristics: hypercholesterolemia, atherosclerosis, extensive lipid-rich coronary occlusions, multiple myocardial infarctions, cardiac dysfunction (e.g., cardiomegaly, markedly reduced ejection fraction (˜50%) and contractility (˜3-fold)) and death at ˜6 weeks of age. Their coronary lesions were strikingly similar to human atherosclerotic plaques. Many of their fibroatheromatous plaques contained structures reminiscent of cholesterol clefts and extensive deposition of fibrin, suggesting the possibility of plaque rupture/bleeding into the plaque. [0031]
Materials and Methods [0032]
Animals: [0033]
Mice (mixed with C57BL/6×129 background) were housed and fed a normal chow diet and genotypes were determined by PCR analysis as described in the patent application. All the analyses described were performed on 4-6 weeks old, male and/or female mice. No significant differences were observed between male and female animals. The study complied with all institutional and NIH guidelines for the use of laboratory animals. [0034]
Histology: [0035]
Mice were euthanized and tissues were prepared for cryosectioning. Tissue for paraffin sections were first fixed in 10% buffered formalin (J. T. Baker, NJ). In some instances, heparin was administered (450 U/20 g, i.v.) prior to euthanasia to prevent systemic coagulation. Tissues were sectioned and stained with Masson's trichrome (Sigma), hematoxylin and eosin (reference to protocol) or oil red O (lipid) and Mayer's hematoxylin. Immunohistochemistry was performed with a mouse monoclonal anti-fibrin antibody (NYB-T2G1, Accurate Chemical & Scientific Corp., NY, diluted to 1:g/ml), using the M.O.M.™ staining procedure (as described in the Vector M.O.M.™ Immunodetection kit; Vector, CA) and peroxidase activity was revealed with 3-amino-9-ethylcarbazole (AEC) substrate (as described, Vector, CA). Immunodetection of macrophages was performed using the monoclonal rat anti-mouse F4/80 antibody (MCA 497, Serotec, NC, diluted 1:10) and a biotinylated rabbit anti-rat, mouse adsorbed, secondary antibody (Vector, CA). Staining was revealed using avidin-biotinylated peroxidase complex (Vectastain Elite ABC kit, Vector, CA) and diaminobenzidine substrate (Vector, CA) as suggested by the manufacturer. Sections were counterstained with Mayer's hematoxylin. [0036]
Gravimetric Analysis: [0037]
Mice were euthanized with a lethal dose of anesthetic (avertin 2.5%; 0.8 ml/20 g) and immediately weighed. After perfusion, hearts were excised, dissected free of connective tissue and major systemic vessels, and weighed. The atria were then removed, the right ventricular free wall was removed and weighed, and the left ventricle+septum was opened, blotted to remove internal liquid, and weighed. [0038]
Magnetic Resonance Imaging: [0039]
Mice were anesthetized with chloral hydrate (i.p. 200-320 mg/kg; Sigma, St. Louis, Mo.) and placed in a 2T small bore magnet (Bruker) on a custom designed body coil that also contains ECG electrode patches. Anesthesia was adjusted by 1-2% isoflurane to keep the heart rates constant during the experiment. Image acquisition was ECG gated. After a set of scout images, 6-7 images corresponding to 1 mm thick cross-sectional slices (perpendicular to the long axis of the heart as shown in FIG. 5, and spanning the entire heart) were collected for each heart, both at end systole and end diastole. For each slice, dimensions (cross-sectional areas) were calculated for the left ventricular (LV) wall+septum and the LV chamber. These were multiplied by the thickness of each slice and summed for all of the appropriate slices to obtain total LV tissue volumes as well as LV end diastolic and end systolic luminal volumes (LVEDV and LVESV respectively). These values were used to determine ejection fractions (EF (%)=((LVEDV−LVESV)/LVEDV)×100). [0040]
Hemodynamic Evaluation: [0041]
Heparin (1 U/10 g i.p.) was administered and mice were anesthetized with chloral hydrate as described above. Mice were intubated and ventilated (model 687, Harvard Apparatus Inc, Holliston, Mass.) with room air at a rate of 130/min and a tidal volume of 0.14 μl/g. Local anesthesia (0.05 ml of 0.5% Lidocaine HC1; Abbott Laboratories, North Chicago, Ill.) was administered at the neck. The right carotid artery was exposed via a midline neck incision through blunt dissection and cannulated with a 1.4 Fr micromanometer catheter (SPR-671, Millar Instruments, Houston, Tex.). The catheter was advanced into the aorta for aortic pressure measurements, and then into the LV for direct measurement of ventricular pressure. Measurements were performed before and after cutting both right and left vagal nerves to determine the effects of the vagal reflexes. Pressure data from the micromanometer were recorded using a Windaq DI 220 converter and Windaq Pro software (Dataq Instrument, Akron, Ohio). This software was also used to calculate systolic and diastolic left ventricular pressures, heart rate and maximum positive and negative dP/dt values. [0042]
Ex vivo Angiography: [0043]
After measurement of hemodynamic, each heart was exposed by median sternotomy and the ascending aorta was cannulated with polyethylene tubing (PE50: Becton Dickinson and Company, Sparks, Md.). The right atrium was opened for drainage. The heart was flushed with PBS (retrograde direction) until the effluent was clear, harvested and then a barium sulfate suspension (E-Z-EM, Inc., Westbury, N.Y.) was injected manually at a maximum pressure of 80 mmHg. Coronary angiograms were obtained with a [0044] Micro 50 X-ray (Micro 50, General Electric, Milwaukee, Wis., 20 kV, 20 sec exposure).
Electrocardiography: [0045]
Electrocardiograms (ECG's) were recorded using two methods. For mice anesthetized with avertin (reference) measurements were made using standard limb leads. For conscious, non-anesthetized mice, ECG's were recorded using AnonyMOUSE® ECG Screening Tools (Mouse Specifics, Inc., Boston). The apparatus includes paw-sized conductive pads embedded in a platform, and electronics with solid-state gating programmed to record ECGs when three single pads contact three paws of the unrestrained mouse. The signals were acquired digitally (DI-220, DATAQ Instruments, Inc., Akron, Ohio) at a sampling rate of 2500 samples/second. Data were recorded for 2-3 sec to provide a continuous record of 20-30 ECB signals. From these signals, the shapes, QRS widths and heart rates were determined. Average heart rates were 646±80 for SR-BI heterozygous null (n-4), 698±70 for SR-BI JI (n=6), 761±54 for apoE KO (n=9) and 652±82 for dKO (n=12) mice (p=0.01). As a control, ECGs of 3 avertin-anesthetized double KO mice were recorded using the AnonyMOUSE® method. In 2 of the 3 animals, patterns of abnormal conductance similar to those frequently observed with the standard limb-leads method in similarly anesthetized mice were observed. [0046]
Statistical Analysis: [0047]
Data were analyzed with StatView software, using either a two-tailed, unpaired Student's t test, for comparison of two groups, or an ANOVA test, for comparison of three or four groups. [0048]
Results [0049]
When fed a normal low fat/low cholesterol standard chow diet, dKO mice develop extensive, accelerated atherosclerosis in their aortic sinuses by approximately 5 weeks of age, and die at an early age. None of the control mice died during this period from weaning (three weeks of age) to eight weeks of age. The controls included homozygous apoE deficient (apoE KO) mice and apoE KO mice with a heterozygous SR-BI null mutation as well as wild type and homozygous SR-BI deficient (SR-BI KO) mice. In contrast, the dKO mice died between 5 and 8 weeks of age, with 50% of the deaths occurring by 6 weeks of age. Before they died, the dKO mice exhibited a 1-2 day period of progressively reduced general activity associated with a change in outward appearance (ruffled fur, abnormal gait). A series of studies of the cardiac morphology, physiology, and histology of 4-6 week old dKO mice were conducted using wild type, SR-BI KO and apoE KO mice as controls. [0050]
Macroscopic Lesions and Extensive Cardiac Fibrosis in the Myocardium of dKO Mice [0051]
Macroscopic examination of the hearts from control and dKO mice revealed two striking abnormalities in the dKO hearts. First, dKO hearts appeared enlarged relative to those from control wild type or single KO mice. Furthermore, hearts from dKO mice exhibited pale, discolored patched, which were not observed in hearts from any of the control mice. These lesions suggested the presence of extensive myocardial infarction and scarring. These lesions were always present in the AV groove of the left ventricle and frequently, but not always, present at various locations on the right ventricular wall, the left ventricular wall and/or the apex. [0052]
Histologic analysis of heart sections was performed to characterize the nature of these lesions. A longitudinal section of a dKO mouse heart was trichrome stained. Trichrome stains healthy mycardium red and fibrotic tissue blue. Fibrotic areas were invariably seen in the regions surrounding the mitral valves and left ventricular outflow tract. The mitral valves themselves appeared unaffected. Higher magnification of trichrome and H&E stained sections show that these lesions were composed of fibrotic connective tissue, very few remaining myocytes and large, dilated cells, many of which appeared to be mononuclear inflammatory cells. These were characterized by extensive fibrosis, inflammation, and in some cases, diffuse necrosis and myocardial scarring, typical of a healed infarct. These lesions appeared more well-organized and contained fewer dilated cells than those in the outflow tract area. Numerous macrophages were also detected in these lesions, as well as in lesions in the papillary muscle. Thus, macroscopic and microscopic observations indicate that multiple myocardial infarcts developed in the dKO mice. [0053]
In hypercholesterolemic animals, macrophages can accumulate extensive cytosolic lipid deposits (foam cells). Because the dKO mice are hypercholesterolemic, outflow tract and papillary muscle sections were stained with oil red O, which stains neutral lipid droplets red. Lipid staining was particularly intense in the macophage-rich regions exhibiting extensive fibrosis. In these regions, oil red O staining appeared both in a concentrated, intense, globular pattern, reminiscent of intracellular lipid and in a puntate pattern, reminiscent of extracellular lipid. Some oil red O staining was also detected in non-fibrotic tissue throughout the heart, between, but not within, the myocardial fibers. This staining was substantially more diffuse than that in fibrotic regions. The coincident distribution of lipid and macrophages suggested that at least some of the apparently intracellular lipid was present in macrophage foam cells within the myocardium. [0054]
Heart Function in dKO Mice [0055]
To determine if the extensive lesions present in the double KO hearts were associated with altered heart function, a series of morphologic and functional analyses were performed using control and dKO mice. These included an assessment of heart size (gravimetry and MRI), measurements of physiological parameters (hemodynamics and left ventricular ejection fractions) and electrocardiographic (ECG) analysis. [0056]
Cardiomegaly [0057]
Macroscopically, hearts from dKO mice appeared larger than those from age-matched control mice. Gravimetric and MRI analyses were conducted to quantitatively characterize the sizes of the intact hearts and their chambers. Gravimetric analysis revealed a strong linear correlation (r[0058] ²=0.74, p<0.0001) between heart and body weights for all control mice. The hearts of dKO mice, however, were disproportionately large, both because the dKO mice were smaller than age-matched controls and because their absolute heart sizes were larger. The mean heart-to-body weight ratio for dKO mice (9.4 mg/g±2.3, n=9) was 1.6-fold greater than that for apoE KO mice (5.9±0.5 mg/g, n=9, P=0.002) and 1.8-fold greater than those for SR-BI KO and wild-type mice (5.3 mg/g±0.3, n=8 for each, P=0.001). The increase in the heart weights of dKO mice included an increase in left ventricular plus septum and right ventricular tissue mass (normalized to body weight). This was confirmed by MRI analysis of LV+S tissue volume to body weight ratio. In contrast, the body weight corrected LV end diastolic chamber volumes were only slightly higher for the dKO hearts, suggesting only minor dilation. Thus, the increased size of dKO hearts was due primarily to increased ventricular tissue mass, a finding consistent with compensating cardiomegaly due to an underlying abnormality in heart activity.
Hemodynamic and MRI Analysis [0059]
Cardiac function was evaluated by hemodynamic analysis and MRI. Average aortic diastolic and systolic blood pressures and heart rates (HR) were significantly lower in dKO than in wild type or single KO control mice. A substantial decrease in left ventricular systolic pressure (LVSP) and contractility (positive dP/dt) was observed in the dKO mice, indicating left ventricular systolic dysfunction. There was a similarly significant (3-fold) reduction in negative dP/dt, suggesting that left ventricular relaxation was also impaired. The moderately elevated LVEDP was not statistically significant. Average HR of dKO mice remained low relative to controls after bilateral disruption of the vagal nerves, indicating that this difference in HR was not due to extracardiac neuronal influences. Although reduced HR might have contributed to low blood pressure and low contractility (dP/dt), and small differences in LVEDP and HR complicate interpretation of dP/dt differences between the strains, it is unlikely that these relatively small baseline differences lead to the large changes in +dP/dt and −dP/dt. Therefore, the results are consistent with a primary cardiac dysfunction, including decreased aortic blood pressures and abnormalities in both contractility and relaxation. [0060]
MRI was used to determine cardiac ejection fractions (EF), a critical measure of heart function, of control apoE KO and dKO mice. MRI images at, end-diastole or -systole show that, while the LVEDVs were similar, the LV end systolic volumes (LVESVs) were higher in dKO hearts than in the controls. Consequently, the EFs of the dKO hearts were substantially lower (˜50%) than those of the controls. [0061]
Electrocardiograms (ECG) were performed on unanesthetized, conscious mice. While the ECGs of the controls were normal, striking abnormalities were observed in the ECGs of six of 12 unanesthethized dKO mice. One exhibited an ST elevation of unclear etiology and five showed severe ST depression, indicating subendocardial ischemia. When ECGs were performed on avertin-anesthetized mice, in 5 of 8 dKO mice, but not in any controls, that anesthesia induced or uncovered cardiac conductance defects, which in some cases included escaped QRSs and progressed to complete AV blocks and bradycardic death. These results, together with the gravimetric, hemodynamic and MRI findings, unequivocally demonstrate impaired heart function in dKO mice, possibly because of extensive myocardial fibrosis. [0062]
Coronary Artery Disease: Angiography and Histology [0063]
To determine if occlusive coronary disease may have contributed to cardiac dysfunction, ex vivo angiography was performed. No obvious defects were apparent in control hearts. In contrast, out of 7 dKO hearts examined, 5 clearly showed stenoses and occlusions of branches of the left coronary arteries. Two instances of apparent stenoses were also observed in the main coronary arteries. [0064]
Histologic analyses of hearts revealed extensive coronary artery disease (CAD) in dKO mice. There were complex occlusions of major arterial branches in the LV free wall (9 of 10 mice analyzed), the septum (10 of 11) and/or the RV wall (11 of 12). No occlusions were seen in age-matched controls. A partially cellular, lipid-rich lesion almost completely occluding the lumen of a left coronary branching artery was observed. Fibrosis and inflammatory cells surrounding an occluded artery in the RV wall of another dKO mouse was also observed. Proximal lesions in coronary ostia were also seen in 7 of 10 dKO mice. These lesions are probably responsible for the patchy MIs in the LV and RV. Serial cross-sections through an occluded coronary artery from a different dKO mouse were stained with trichrome and lipid staining, which revealed numerous structures reminiscent of cholesterol clefts within a lipid-rich core. Immunostaining showed fibrin deposits in the core regions of eight of ten lesions observed in 3 of 3 dKO mice but not in any age-matched apoE KO control mice (n=3). The presence of fibrin within the plaques occluding coronary arteries in dKO mice indicates thrombosis, probably resulting from rupture of or bleeding into these complex lesions. [0065]

EXAMPLE 2

Analysis of the Abnormal Lipoproteins in SR-BI/ApoE Double Knockout Mice

Materials and Methods [0066]
Animals are the same as analyzed in Example 1 for atherosclerosis and heart disease. [0067]
Plasma and Bile Analysis [0068]
Blood was collected in a heparinized syringe by cardiac puncture from mice fasted overnight. Plasma was subjected to FPLC analysis, either immediately after isolation or after storage at 4° C. Total cholesterol was assayed. Cholesterol from non-apoB containing lipoproteins was determined either using the EZ HDL kit (Sigma, based on an antibody which blocks detection of cholesterol in non-HDL lipoproteins, and validated by us using human or mouse lipoproteins, not shown) or after precipitation with magnesium/dextran sulfate (Sigma). Plasma (0.4 μl) and FPLC fractions or pools were analyzed by SDS-polyacrylamide or agarose gel electrophoresis and immunoblotting with chemiluminescence detection using primary anti-apolipoprotein antibodies (Sigma, or gifts from J. Herz and H. Hobbs) and corresponding horseradish peroxidase coupled secondary antibodies (Jackson Immuno Research or Amersham). The Attophos chemifluorescence kit (Amersham) and an alkaline phosphatase coupled goat anti-rabbit secondary antibody (gift from D. Housman) were used with a Storm Fluorimager (Molecular Dynamics) for quantitative analysis. Plasma progesterone concentrations were determined by radioimmunoassay (Diagnostics Products Corp, Los Angeles, Calif.). Cholesterol was extracted from gallbladder bile and assayed. [0069]
Histology and Immunofluorescence Microscopy: [0070]
Mice anesthetized with 2.5% avertin were perfused through the left ventricle with 20 ml of ice cold PBS containing 5 mM EDTA. Hearts were collected directly, or the mice were perfused (5 ml) with paraformaldehyde and the hearts collected and treated. Hearts and ovaries were frozen in Tissue Tek OCT (Sakura, Torrance, Calif.). Serial cross sections (10 μm thickness through aortic sinuses, 5 μm for ovaries, Reichert-Jung cryostat) were stained with oil red O and Meyer's hematoxylin. Images were captured for morphometric analysis using a computer assisted microscopy imaging system and lesion size was quantified as the sum of the cross-sectional areas of each oil red O staining atherosclerotic plaque in a section using NIH Image software. Immunohistochemistry with a monoclonal anti-α smooth muscle actin antibody (Sigma, gift from R. Hynes) was performed). Cumulus/oocyte complexes, isolated from the oviducts of superovulated females or denuded oocytes (zona pellucida removed) were immunostained with polyclonal rabbit anti-murine SR-BI antibodies gift from K. Kozarsky) and Cy3-labeled donkey anti-rabbit secondary antibodies (gift from R. Rosenberg). [0071]
Statistical Analysis [0072]
Data were analyzed using either a two-tailed, unpaired Student t-test (total or EZ HDL cholesterol from plasma, bile or FPLC fractions, progesterone and apoA-I levels) or an unpaired nonparametric Kruskall-Wallis test (atherosclerotic plaque lesion sizes) (Statview and Microsoft Excel). Values are presented as means±standard deviations. [0073]
Results and Discussion [0074]
To analyze the effects of SR-BI on atherosclerosis, SR-BI KO and apoE KO (spontaneously atherosclerotic) mice were crossed and the lipoprotein profiles and development of atherosclerosis in the single and double homozygous KO females at 4-7 weeks of age compared. Results for males were similar, except as noted. The results are shown in FIG. 2. Plasma total cholesterol in the single SR-BI KOs was increased relative to controls, because of an increase in large, apoE-enriched HDL particles, while the even greater relative plasma cholesterol increase in the single apoE KOs was a consequence of a dramatic increase in cholesterol in VLDL and IDL/LDL size particles. There was increased plasma cholesterol in the double KOs relative to the single apoE KOs, mainly in VLDL size particles. This might have occurred if SR-BI, which can bind apoB containing lipoproteins, directly or indirectly contributes to the clearance of the cholesterol in VLDL size particles in single apoE KO mice (reduced clearance in its absence). [0075]
The normal size HDL cholesterol peak seen in the single apoE KOs virtually disappeared in the double KOs. However, no statistically significant differences (P=0.1) in plasma levels of HDL's major apolipoprotein, apoA-I, were detected. Based on the analysis of lipoproteins in the single SR-BI KO mice, abnormally large HDL-like particles were expected to appear in the double KOs. Indeed, the loss of normal sized HDL cholesterol and apoA-I in the double KOs was accompanied by a shift of the apoA-I into the VLDL and IDL/LDL size fractions. Furthermore, analysis of HDL-like cholesterol in the FPLC fractions using the EZ HDL assay provides evidence for the presence of abnormally large HDL-like particles in the double KO mice. In the single apoE KO males, most of this cholesterol was in particles with the size of normal HDL, while in their double KO counterparts almost all of this cholesterol was in abnormally large particles. In addition, there was approximately 3.7-fold more of this HDL-like cholesterol in the double (133±24 mg/dl) than in the single (36±16 mg/dl, P=0.005) KO mice. These increases in the amounts and sizes of HDL-like cholesterol by inactivation of the SR-BI gene in an apoE KO background were reminiscent of those seen in a wild-type background (approximately 2.2-fold increase in cholesterol, although the HDL-like particles in the double KO mice were much larger and more heterogeneous than those in the SR-BI single KO mice. A similar trend was seen for female mice, except that there were increased levels of abnormally large HDL-like cholesterol in the single apoE KO females relative to males. Preliminary cholesterol measurements using magnesium/dextran sulfate precipitation of lipoproteins support the EZ HDL findings of large HDL in the double KO animals. [0076]
Additional evidence for abnormally large HDL-like particles in the IDL/LDL size range from both males and females was obtained using agarose gel electrophoresis and immunoblotting. There was a significant reduction in the amount of immunodetectable apoB present in the IDL/LDL-sized particles from the double KOs relative to the single apoE KOs, even though there was as much or more total cholesterol in these fractions in the double KOs. In addition, there was significantly greater heterogeneity in the electrophoretic mobilities of apoA-I containing IDL/LDL-sized particles. This was in part due to the presence of novel apoA-I containing, apoB-free, HDL-like particles. In contrast, most of the apoA-I in the single apoE KOs appeared to comigrate with apoB. Thus, it appears that normal size HDL in the single apoE KO animals was replaced by very large (VLDL/IDL/LDL-size) HDL-like particles in the double KO animals. It is possible that normal size HDL is converted into these large HDL-like particles in the absence of both apoE and SR-BI because of substantially reduced selective (SR-BI, mediated) and apoE-mediated uptake or transfer of cholesterol from HDL particles. [0077]
In addition to examining plasma cholesterol, biliary cholesterol was measured in the mice. Cholesterol levels in gallbladder bile were significantly reduced in SR-BI single KO (30%, P<0.005) and SR-BI/apoE double KO (47%, P<0.0005) mice relative to their SR-BI[0078] ^+/+ controls. This is consistent with the previous finding that hepatic overexpression of SR-BI increases biliary cholesterol levels and indicates that SR-BI may normally play an important role in the last stage of reverse cholesterol transport—transfer of plasma HDL cholesterol into bile. The data also suggest that apoE expression can regulate biliary cholesterol content in a SR-BI KO, but not SR-BI^+/+, background.
Atherosclerosis in the animals was further assessed by analyzing plaque areas in aortic sinuses and the effects of SR-BI gene disruption on plasma lipoproteins in apoE KO mice. Mice were 4-7 weeks old. Plasma apoA-I levels (mean±SD, expressed as relative units) were determined by SDS-polyacrylamide (15%) gel electrophoresis followed by quantitative immunoblotting for apoE[0079] ^−/− (n=7) and SR-BI^−/−apoE^−/− females (n=5) (P=0.1). Lipoprotein cholesterol profiles: Plasma lipoproteins from individual apoE^−/− or SR-BI^−/− apoE^−/− females were separated based on size (SUPEROSE 6-FPLC) and total cholesterol in each fraction (expressed as mg/dl of plasma) was measured. Pooled Superose 6-FPLC fractions (approximately 21 μl per pool) from females in an independent experiment were analyzed by SDS-polyacrylamide gradient (3-15%) gel electrophoresis and immunoblotting with an anti-apoA-I antibody. Each pool contained 3 fractions and lanes are labeled with the number of the middle fraction in each pool. Average EZ HDL cholesterol FPLC profiles for apoE^−/− or SR-BI^−/− apoE^−/− males (n=3) or females (n=3). Agarose gel electrophoresis and immunoblotting: Pooled fractions (11-21, 3.5 μl) from the IDL/LDL region of the lipoprotein profile from individual apoE^−/− or SR-BI^−/− apoE^−/− females were analyzed using either anti-apoA-I or anti-apoB antibodies. Migration was upward from negative to positive. Gallbladder biliary cholesterol (mean±SD): Total gallbladder biliary cholesterol from both male and female mice of the indicated genotypes (n=10 or 11 per genotype) was measured. Except for the wild-type and apoE^−/−values, all pairwise differences were statistically significant (P<0.025-0.0005).
To determine the effects of SR-BI gene disruption on atherosclerosis in apoE KO mice, atherosclerosis in SR-BI[0080] ^−/− (n=8, 4-6 weeks old), apoE^−/− (n=8, 5-7 weeks old), or SR-BI^−/−apoE^−/− (n=7, 5-6 weeks old) female mice was analyzed in cryosections of aortic sinuses stained with oil red O and Meyer's hematoxylin as described in Methods. Representative sections through the aortic root region and cross-sectional areas of oil red O stained lesions in the aortic root region, showed average lesion areas (mm²±SD) for SR-BI^−/−apoE^−/−, apoE^−/− or SR-BI^−/− mice, respectively, were as follows 0.10±0.07, 0.002±0.002, and 0.001±0.002 (P=0.0005). There were virtually no detectable lesions in the single KO animals at this relatively young age (4-7 weeks). However, there was substantial, statistically significant, lesion development in the double KOs in the aortic root region, elsewhere in the aortic sinus, and in coronary arteries. The lipid-rich lesions were cellular (hematoxylin stained nuclei were seen at high magnification) and in some cases had a cellular cap which stained with antibodies to smooth muscle actin. Thus, the atherosclerotic plaques were relatively advanced.
While most did not exhibit overt signs of illness at that time, they all died suddenly around 6 weeks of age. Electrocardiographic studies indicated that premature death of the double KOs was due to progressive heart block (cardiac conduction defects) and histology revealed extensive cardiac fibrosis and narrowing or occlusion of the coronary arteries, suggesting myocardial infarction (MI) due to advanced atherosclerotic disease. The anti-atherosclerotic effect of SR-BI expression in apoE KO mice is consistent with the reports that adenovirus- or transgene-mediated hepatic overexpression of SR-BI in the cholesterol and fat-fed LDLR KO mouse reduces atherosclerosis. Thus, pharmacologic stimulation of endogenous SR-BI activity may be anti-atherogenic, possibly because of its importance for RCT. The accelerated atherogenesis and loss of normal size HDL cholesterol in the double KOs resembles that reported for high-fat diet fed single apoE KO mice; although those mice have far higher total plasma cholesterol levels (1800-4000 vs. approximately 600 mg/dl). It is thought the similarities arise in part because the very high levels of large lipoproteins in the fat-fed single apoE KO might block the ability of SR-BI to interact with HDL and other ligands (functional SR-BI deficiency due to competition), or because of dietary suppression of hepatic SR-BI expression. [0081]
Results [0082]
The endogenous substrate assay showed that LCAT activity in SR-BI KO plasma has 20% of wild-type activity. This is consistent with the possibility that reduced substrate activity of the abnormal lipoproteins in SR-BI KO mice or reduced activity of the LCAT enzyme is responsible for the increased fraction of the cholesterol in the plasma that is in the unesterified form (see Example 3). [0083]

EXAMPLE 3

Prevention or Treatment of Atherosclerosis and Normalization of Lipid Levels and Structure in SR-BI Knockout Mice

The animals described in the preceeding examples are useful to screen for compounds that are effective for the prevention or treatment of atherosclerosis and heart disease and treatment of diseases that lead to the accumulation of lipoprotein X in the plasma (liver diseases (cholestasis), LCAT dificiency) and other diseases that influence cholesterol metabolism (NPC1, Tangier disease). Several studies were conducted to demonstrate this. [0084]
Materials and Methods [0085]
The same animals were used as in the preceding examples. [0086]
Animals. [0087]
Mice were housed and fed a normal chow diet or chow (Teklad 7001) supplemented with 0.5% (wt/wt) 4,4′-(isopropylidene-dithio)-bis-(2,6-di-tertbutylphenol (PROBUCOL; Sigma Chemical Co., St. Louis, Mo., USA). Mouse strains (genetic backgrounds) were: wild-type and SR-BI KO (both 1:1 mixed C57BL/6×129 backgrounds), apoA-I KO (C57BL/6; The Jackson Laboratory, Bar Harbor, Me., USA). Double SR-BI/apoA-I KO mice were produced by (a) mating SR-BI KO males with apoA-I KO females, (b) transferring the resulting embryos into Swiss Webster recipients, and (c) intercrossing the double heterozygous offspring. Colonies were maintained by crossing double-KO males with females heterozygous for the SR-BI null mutation and homozygous for the apoA-I mutation to optimize the low yield of SR-BI homozygotes. [0088]
These animals were fed PROBUCOL at various times prior to or after birth, as described in results. [0089]
Results [0090]
Effects on Survival of Genetic Disruption of the ApoA-I Gene or PROBUCOL Treatment on the Fertility of Female SR-BI KO Mice. [0091]
Wild-type males were mated with female SR-BI KO (n=13, average litter size=1, 2- to 6-month mating), SR-BI/apoA-I KO (n=17, dark gray bars, average litter size=2.2, 4-month mating), PROBUCOL-fed SR-BI KO (n=14s, average litter size=5.7, 1- to 2-month mating), and PROBUCOL-fed wild-type (n=9, white bars, average litter size=5.3, 1- to 2-month mating) mice. [0092]
In the first study, PROBUCOL was fed to a mating pair (ApoE −/− and SR-BI +/−). The offspring, who have had PROBUCOL since conception, were weaned at three weeks of age. They continued to receive PROBUCOL in the chow. At 6 weeks, when typically 50% of the animals are dead in the absence of treatment, there is no abnormal phenotype, as measured by MRI of heart function, ECG, echocardiogram, and histology. There is no evidence of atherosclerosis. [0093]
At 7-8 months, many of the animals receiving PROBUCOL are still alive. However, they do have substantial atherosclerosis and some myocardial infarctions. In contrast, normal wild-type mice show no evidence of heart disease or atherosclerosis. [0094]
In a second study, animals receiving PROBUCOL were taken off of the treatment at weaning (approximately three weeks of age). All of the animals die within 10-12 weeks. [0095]
In a third study, the animals were not treated with PROBUCOL until after weaning, either prior to 5 weeks of age (dKO-P<5; mean age, 4.1 weeks, range 3.9-4.6 weeks) or later (dKO-P>5; mean age, 5.2 weeks, range 5.1-5.3 weeks). The majority of those fed PROBUCOL before 5 weeks of age survive a few months. [0096]
The survival of animals receiving PROBUCOL from conception is shown in FIG. 1A; the survival of animals receiving PROBUCOL from prior to or after five weeks of age is shown in FIG. 1B. [0097]
These results demonstrate that drugs can be used as preventatives as well as therapeutics. [0098]
Effects of PROBUCOL Treatment on Lipoproteins and Red Blood Cell Maturation [0099]
PROBUCOL treatment lowered by about two-fold the levels of plasma total cholesterol (Table 1) and phospholipids (Table 2) in dKO mice compared to those in untreated apoE KO mice. Treatment did not lower the plasma triglyceride level of dKO mice that was somewhat lower than that of apoE KO mice (Table 2). The abnormally large HDL particles in dKO mice are found in the VDL/IDL/LDL, but not the HDL, size ranges, because of the loss of SR-BI-mediated selective lipid uptake. Unexpectedly, PROBUCOL treatment (open circles) induced the appearance of a peak of cholesterol at the position of normal sized HDL (fractions 29-34) that contained apoA-I that is also seen in untreated apoE KO mice. In contrast, PROBUCOL decreases the normal size HDL peak in apoE KO mice. The shapes of the lipoprotein unesterified cholesterol and phospholipid FPLC profiles were similar to that of total cholesterol. The ratios of the relative amounts of unesterified cholesterol (UC) to total cholesterol (TC) and phospholipid (PL) to TC in the major FPLC lipoprotein fractions are shown in Tables 1 and/or 2. [0100]
There was a surprisingly high ratio of unesterified to total cholesterol in both dKO and SR-BI KO mice compared to SR-BI positive controls (Table 1). As a consequence in dKO mice the ratio of surface/polar (UC and PL) to core/nonpolar (esterified cholesterol and triglycerides) lipids was about 6-fold larger than in apoE KO controls (Table 2). This high ratio suggested that the structures of the lipoproteins in dKO mice would be abnormal. Electron micrographs of negatively stained total plasma and major lipoprotein fractions from dKO mice showed the presence of numerous lamellar/vesicular, and stacked discoidal particles with abnormal morphologies, reminiscent of those (e.g., lipoprotein-X) seen in LCAT deficiency, cholestasis and other cases of abnormally high UC. The large PROBUCOL-induced decrease (˜4.8-fold) in plasma unesterified cholesterol lowered the abnormally high ratios of UC:TC in both dKO and SR-BI KO mice to the normal values in SR-BI-positive controls (Table 1) and lowered the surface to core lipid ratio in dKO mice to that in apoE KO controls (Table 2). As a consequence, the morphologies of the major lipoprotein fractions in dKO-P mice were restored to those in apoE KO controls. [0101]
There is a profound, reversible defect in RBC maturation in dKO mice due to excessive accumulation in precursor reticulocytes of unesterified cholesterol (visualized by staining with the cholesterol-binding, fluorescent dye filipin). See Table 3. Compared to wild-type and apoE KO mice, the untreated dKO mice were anemic and exhibited profound reticulocytosis (irregularly shaped, precursor reticulocytes, no biconcave mature erythrocytes). Their large, macrocytic red cells (elevated mean copuscular volume) differed morphologically from classic abnormal ‘target’ cells seen in LCAT-deficient patients and contained big, cholesterol-rich autophagolysosomal inclusion bodies. Almost all of these abnormalities, except the somewhat irregular shape, were corrected by PROBUCOL treatment. Correction of the reticulocytosis in dKO mice by PROBUCOL may contribute to its suppression of their CHD, e.g., by improving tissue oxygenation. [0102]
Mice with homozygous null mutations in the HDL receptor SR-BI and apoE genes (dKO mice) on a low fat diet rapidly develop many cardinal features of human CHD, including hypercholesterolemia, atherosclerosis in the aortic sinus, occlusive coronary plaques, patchy MIs, cardiac enlargement and dysfunction (reduced systolic function and ejection fraction, ECG abnormalities), and premature death (50% mortality at ˜6 weeks) (1,16). They also exhibit a block in RBC maturation. Their abnormally high ratio of lipoprotein surface (unesterified cholesterol (UC), phospholipids)-to-core (esterified cholesterol, triglycerides) lipids led to abnormal lipoprotein morphology (lamellar/vesicular and stacked discoidal particles reminiscent of those in other cases of high UC, e.g. LCAT deficiency, cholestasis and apoE/hepatic lipase (HL) deficiency. The mechanism causing excess UC in SR-BI null (dKO, SR-BI KO) mice is unknown, but may involve the activities of LCAT, HL or other lipoprotein-metabolizing proteins. [0103]

Treatment of dKO mice with the lipid-lowering- and anti-oxidation-drug PROBUCOL extended their lives (from 6 to 36 weeks) and reversed essentially all early onset CHD-associated anatomical, functional, cellular and biochemical pathology. A substantial portion of the PROBUCOL-sensitive pathology apparently occurred between 3 and 5 weeks of age.

TABLE 1


Effects of Probucol on Plasma Unesterified
and Total Cholesterol

	Unesterified	Total
Genotype	Cholesterol	Cholesterol	Ratio (Unesterified/
drug treatment	(mg/dL)	(mg/dL)	Total Cholesterol)

DKO
none (n = 11)	781 ± 65*†	970 ± 83*†	0.806 ± 0.007*†
Probucol (n = 10)	151 ± 21	456 ± 45	0.318 ± 0.013
SR-BI^+/−apoE KO
none (n = 5)	124 ± 11¶	421 ± 31**	0.296 ± 0.012§
Probucol (n = 5)	57 ± 3	244 ± 20	0.238 ± 0.015
apoE KO
none (n = 5)	126 ± 14¶,	433 ± 66**	0.291 ± 0.02¥
Probucol (n = 3)	69 ± 6	249 ± 8	0.277 ± 0.02
SR-BI KO#
none (n = 13)	108 ± 5	211 ± 6	0.515 ± .027
Probucol (n = 13)	24 ± 2	110 ± 6	0.218 ± .012
Wild-type control#
none (n = 7)	33 ± 2	103 ± 4	0.315 ± .011
Probucol (n '2 7)	8 ± 2	34 ± 4	0.222 ± .033

[0105]

TABLE 2

Effects of Probucol on Plasma Phospholipid,

Triglyceride and lipid ratios

dKO dKO + probucol apoE KO

Plasma Phospholipid 678 ± 96 246 ± 95 (n = 3) 307 ± 51 (n '2 9)

(mg/dL) (n = 8)

Plasma Triglyceride 53 ± 16 60 ± 44 (n = 3) 89 ± 28 (n = 6)

(mg/dL) (n = 4)

UC*100 Plasma§ 86:60 43:61 30:68

TC VLDL§ 83:55 41:52 35:56

to IDL/LDL 79:60 37:51 36:62

PL*100 §

TC HDL§ — 49:70 nd

ratio

Plasma Surface:Core 6.0 1.1 1.1

Lipid Ratio†

TABLE 3


Effects of Probucol on Red Blood Cell Parameters in apoE
KO and dKO mice

Genotype	Hematocrit	Mean corpuscular	Retinculocytes (%
Drug treatment	(%)	volume (fL)	of total RHCs)

apoE KO none*
(n = 8)	52.5 ± 2.1§	57.6 ± 0.5	2.8 ± 1.2
Probucol (n = 3)	49.8 ± 1.5	54.2 ± 2.1	4.1 ± 0.8
dKO
none*† (n = 6)	34.2 ± 1.7	84.1 ± 4.1	100 ± 10
Probucol (n = 3)	50.8 ± 2.4	54.2 ± 2.3	4.1 ± 0.5

EXAMPLE 4

Measurement of LCAT in SR-BI KO Mice

Decreased levels of the enzyme LCAT are found in certain disorders, including lipoprotein X. Therefore, LCAT levels in the DKO animals were measured. [0107]
Materials and Methods [0108]
To assay LCAT activity with endogenous substrate, tracer [[0109] ³H]cholesterol is added to the plasma overnight at 4° C., then the plasma warmed up and ester formation measured.
This assay was performed on plasma from control (wt) and SR-BI KO mice. [0110]
Results [0111]
The endogenous substrate assay showed that LCAT activity in SR-BI KO plasma has 20% of wild-type activity. This is consistent with the possibility that reduced substrate activity of the abnormal lipoproteins in SR-BI KO mice or reduced activity of the LCAT enzyme is responsible for the increased fraction of the cholesterol in the plasma that is in the unesterified form (see Example 3 above). [0112]
Modifications and variations of the methods and materials described herein will be obvious to those skilled in the art and are intended to be encompassed by the following claims. The teachings of the references cited herein are specifically incorporated herein. [0113]

Claims

We claim:

1. A method for treating or preventing a disorder or disease characterized by abnormal lipoprotein or cholesterol metabolism comprising administering to an individual in need thereof a compound selected from the group consisting of 4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol), monoesters and other derivatives thereof, 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran or a derivative thereof, vitamin E and vitamin C, wherein the compound is administered in an amount effective to decrease lipoprotein levels or normalize lipoprotein structure or reduce abnormal cholesterol metabolism.

2. The method of claim 1 wherein the compound is 4,4′-(isopropylidenedithio) bis(2,6-di-tert-butylphenol), monoesters and other derivatives thereof.

3. The method of claim 1 wherein the compound is 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di-tert-butyl-benzofuran or a derivative thereof.

4. The method of claim 1 wherein the compounds are selected from the group consisting of vitamin E and vitamin C.

5. The method of claim 1 wherein the compound is administered orally.

6. The method of claim 1 wherein the compound is administered to an individual with cholestasis.

7. The method of claim 1 wherein the compound is administered to an individual with atherosclerosis.

8. The method of claim 1 wherein the compound is administered to an individual with lipoprotein X.

9. The method of claim 1 wherein the compound is administered to an individual with a disease due to abnormalities in cholesterol metabolism.

10. The method of claim 9 wherein the disease is Niemann-Pick Type C or Tangier diseases.

11. A formulation for use in any of the methods of claims 1-10.