Compositions and methods for modulating serum cholesterol

Abstract

Compositions and methods are provided for modulating serum cholesterol in a subject mammal. In one aspect, the invention features novel anti-lipemic drugs that include at least one identified effector of the Low Density Lipoprotein (LDL) receptor and at least one identified serum cholesterol inhibitor. In a particular aspect, the drugs include one identified sphingolipid or protein modifying same linked to one identified serum cholesterol inhibitor. Additionally provided are methods for identifying anti-lipemic drugs capable of modulating the LDL receptor and specifically SREBP-1 maturation, including assays designed to identify pharmacological drugs capable of stabilizing or reducing serum cholesterol levels in a mammal and particularly a human patient.

1 . An anti-lipemic drug comprising a therapeutically effective amount of at least one effect of the sterol regulatory element binding protein-1 (SREBP-1).  
     
     
         2 . The anti-lipemic drug of  claim 1 , wherein the therapeutically effective amount is sufficient to reduce serum cholesterol level in a mammal compared to a suitable control mammal.  
     
     
         3 . The anti-lipemic drug of  claim 1 , wherein the drug further comprises at least one synthetic or semi-synthetic inhibitor of an enzyme associated with cholesterol biosynthesis.  
     
     
         4 . The anti-lipemic drug of  claim 3 , wherein the enzyme is HMG CoA reductase or HMG-CoA-synthetase.  
     
     
         5 . The anti-lipemic drug of  claim 1 , wherein the drug further comprises at least one caspase.  
     
     
         6 . The anti-lipemic drug of  claim 5 , wherein the caspase is cpp32 protease (caspase-3).  
     
     
         7 . The anti-lipemic drug of  claim 3 , wherein the inhibitor is a drug selected from the group consisting of fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin (compactin) atorvastatin or a derivative thereof.  
     
     
         8 . The anti-lipemic drug of  claim 2 , wherein at least one of the SREBP-1 effectors is a sphingolipid, neutral sphingomyelinase (N-SMase) or a therapeutically effective fragment thereof.  
     
     
         9 . The anti-lipemic drug of  claim 8 , wherein the sphingolipid is a ceramide.  
     
     
         10 . The anti-lipemic drug of  claim 9 , wherein the ceramide is naturally occurring ceramide or any one of C-2, 4, 6, or 8 ceramide.  
     
     
         11 . The anti-lipemic drug of  claim 1 , wherein the drug comprises a sphingolipid associated with an inhibitor of HMG CoA reductase or HMG-CoA-synthetase.  
     
     
         12 . The anti-lipemic drug of  claim 11 , wherein the drug comprises the sphingolipid covalently linked to one of fluvastatin, simvastatin, lovastatin, pravastatin mevinolin (compactin) atorvastatin; or a derivative thereof.  
     
     
         13 . The anti-lipemic drug of  claim 12 , wherein the sphingolipid is ceramide and the ceramide is linked to one of the inhibitors through a hydroxyl (—OH) group on the inhibitor.  
     
     
         14 . The anti-lipemic drug of  claim 13 , wherein the hydroxyl (—OH) group on the inhibitor is covalently linked to the C-3 group of the ceramide.  
     
     
         15 . The anti-lipemic drug of  claim 14 , wherein the anti-lipemic drug comprises covalently linked in sequence: 1) ceramide, 2) a heterobifunctional spacer group linked to the C-3 group of the ceramide, and 3) the hydroxyl (—OH) group of the fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin (compactin), artorvastatin; 
 or decivahi thereof linked to a reactive carbon atom on the heterobifunctional spacer.    
     
     
         16 . The anti-lipemic drug of  claim 1 , wherein the drug comprises neutral sphingomyelinase (N-SMase) or a therapeutically effective fragment thereof; the N-SMase or fragment being associated with an inhibitor of HMG CoA reductase or HMG CoA synthetase.  
     
     
         17 . The anti-lipemic drug of  claim 16 , wherein the drug further comprises the neutral sphingomyelinase (N-SMase) or the fragment thereof covalently linked to one of fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin (compactin) atorvastatin; or a derivative thereof.  
     
     
         18 . The anti-lipemic drug of  claim 1 , wherein the drug comprises ceramide, neutral sphingomyelinase (N-SMase) or a therapeutically effective fragment thereof.  
     
     
         19 . The anti-lipemic drug of  claim 16  or  18 , wherein the neutral sphingomyelinase (N-SMase) is encoded by a sequence having at least 70% sequence identity to the sequence represented by SEQ ID NO:1 or the completement thereof.  
     
     
         20 . The anti-lipemic drug of  claim 19 , wherein the effective fragment of the neutral sphingomyelinase (N-SMase) comprises a sequence having at least 70% sequence identity to nucleotides 862 to 1414 of SEQ ID NO:1 or the completement thereof.  
     
     
         21 . The anti-lipemic drug of  claim 20 , wherein the therapeutically effective fragment of the neutral sphingomyelinase (N-SMase) consists of nucleotides 862 to 1414 of SEQ ID NO:1 or the complement thereof.  
     
     
         22 . The anti-lipemic drug of any one of claims  8 - 21 , wherein the neutral sphingomyelinase (N-SMase) or the fragment thereof is linked to one of the inhibitors through an amide bond of the N-SMase or the fragment.  
     
     
         23 . The anti-lipemic drug of any one of claims  8 - 22 , wherein the anti-lipemic drug comprises covalently linked in sequence: 1) the neutral sphingomyelinase (N-SMase) or the therapeutically effective fragment thereof, 2) a heterobifunctional spacer linked to the amide group of the N-Smase or the fragment, and 3) the hydroxyl (—OH) group of the fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin (compactin), artorvastatin or derivative thereof linked to a reactive carbon atom of heterobifunctional spacer.  
     
     
         24 . The anti-lipemic drug of any one of claims  1 - 23 , wherein the drug is specifically formulated for topical or related use.  
     
     
         25 . The anti-lipemic drug of  claim 24 , wherein the drug further comprises components sufficient to provide the drug as a liposome formulation.  
     
     
         26 . A method for modulating serum cholesterol level in a mammal, wherein the method comprises administering to the mammal a therapeutically effective amount of the anti-lipemic drug of any one of  claims 1  to  25 .  
     
     
         27 . A method for modulating SREBP-1 levels in a mammal, wherein the method comprises administering to the mammal a therapeutically effective amount of the anti-lipemic drug of any one of  claims 1  to  25 .  
     
     
         28 . A method for modulating LDL receptor levels in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the anti-lipemic drug of any one of  claims 1  to  25 .  
     
     
         29 . The method of  claim 28 , wherein the mammal is a recognized animal model for atherosclerosis or related disease.  
     
     
         30 . The method of  claim 29 , wherein the mammal is a Watanabe heritable hyperlipidemic rabbit or an apolipoprotein E negative mouse.  
     
     
         31 . The method of any one of claims  26 - 28 , wherein the mammal is a primate.  
     
     
         32 . The method of  claim 31 , wherein the primate is a human patient who has been diagnosed as having, is suspected of having, or is susceptible to a cholesterol related disorder.  
     
     
         33 . The method of  claim 32 , wherein the cholesterol related disorder is at least one of hyperlipoproteinemia including hypercholesterolemia, stroke, obesity, cardiac disease including atherosclerosis, cerebral atherosclerosis, cholesterol ester storage disorder, liver disease including organ transplantation failure and cirrhosis; diseases of the biliary system, and viral infection facilitating encephalitis.  
     
     
         34 . The method of  claim 32  or  33 , wherein the susceptibility of the human patient is related to a genetic or environmental pre-disposition to the cholesterol related disorder.  
     
     
         35 . A method for treating a disorder in a mammal associated with high serum cholesterol levels, the method comprising administering to the mammal a therapeutically effective amount of the anti-lipemic drug of any one of claims  1 - 25 .  
     
     
         36 . The method of  claim 35 , wherein the mammal is a primate.  
     
     
         37 . The method of  claim 36 , wherein the primate is a human patient who has been diagnosed as having, is suspected of having, or is susceptible to a high serum cholesterol levels.  
     
     
         38 . A method for modulating serum cholesterol level in a mammal, wherein the method comprises administering to the mammal a therapeutically effective amount of the anti-lipemic drug of  claim 1 , wherein the SREBP-1 effector is neutral sphingomyelinase (N-SMase) or a therapeutically effective fragment thereof; or a sphingolipid.  
     
     
         39 . The method of  claim 38 , wherein the mammal is a recognized animal model for atherosclerosis or a related disease.  
     
     
         40 . The method of  claim 39 , wherein the mammal is a Watanabe heritable hyperlipidemic rabbit or an apolipoprotein E negative mouse.  
     
     
         41 . A method for modulating SREBP-1 levels in a mammal, wherein the method comprises administering to the mammal a therapeutically effective amount of the anti-lipemic drug of  claim 1 , wherein the SREBP-1 effector is neutral sphingomyelinase (N-SMase) or a therapeutically effective fragment thereof; or a sphingolipid.  
     
     
         42 . A method for modulating LDL receptor levels in a mammal, the method comprising administering to the mammal a therapeutically effective amount of the anti-lipemic drug of  claim 1 , wherein the SREBP-1 effector is neutral sphingomyelinase (N-SMase) or a therapeutically effective fragment thereof; or a sphingolipid.  
     
     
         43 . The method of any one of claims  38 - 42 , wherein the neutral sphingomyelinase (N-SMase) is encoded by a sequence having at least 70% sequence identity to the sequence represented by SEQ ID NO:1 or completement thereof.  
     
     
         44 . The method of any one of claims  38 - 42 , wherein the effective fragment of the neutral sphingomyelinase (N-SMase) comprises a sequence having at least 70% sequence identity to nucleotides 862 to 1414 of SEQ ID NO:1 or completement thereof.  
     
     
         45 . The method of any one of claims  38 - 42 , wherein the sphingolipid is ceramide.  
     
     
         46 . The method of any one of claims  38 - 42 , wherein the mammal is a primate.  
     
     
         47 . The method of  claim 46 , wherein the primate is a human patient who has been diagnosed as having, is suspected of having, or is susceptible to a cholesterol related disorder.  
     
     
         48 . The method of  claim 47 , wherein the cholesterol related disorder is at least one of hyperlipoproteinemia including hypercholesterolemia, stroke, obesity, cardiac disease including atherosclerosis, cholesterol ester storage disorder, liver disease including organ transplantation failure and cirrhosis; and diseases of the biliary system.  
     
     
         49 . The method of  claim 47  or  48 , wherein the susceptibility of the human patient is a related to a genetic or environmental pre-disposition to the cholesterol related disorder.  
     
     
         50 . The method of any one of claims  26 - 49 , wherein the anti-lipemic drug is provided as a liposome formulation.  
     
     
         51 . The method of  claim 50 , wherein the liposome formulation is specifically adapted for hepatic administration.  
     
     
         52 . The method of  claim 50  or  51 , wherein the liposome formulation is administered to liver orally, intramuscularly, intraperitoneally, or via a stent or related implementation.  
     
     
         53 . The method of any one of claims  26 - 52 , wherein each of the methods reduces serum cholesterol levels in the mammal by at least 20% when compared to a suitable control mammal as determined by a standard serum cholesterol assay.  
     
     
         54 . The anti-lipemic drug of any one of claims  1 - 25 , wherein the drug exhibits an ID 50  of between from about 20% to 90% as determined in a standard in vitro HMG CoA reductase assay.  
     
     
         55 . The anti-lipemic drug of  claim 54 , wherein the drug is capable of reducing serum cholesterol in the mammal by at least about 20% when compared to a suitable control mammal as determined by a standard serum cholesterol binding assay.  
     
     
         56 . A method for detecting an effector of the sterol regulatory element binding protein-1 (SREBP-1), the method comprising: 
 a) providing a population of cells capable of expressing SREBP-1,    b) contacting the cells with a candidate effector in an amount sufficient to induce maturation of the SREBP-1,    c) culturing the cells in medium; and    d) detecting maturation of the SREBP-1 as indicative of the effector of the SREBP-1.    
     
     
         57 . The method of  claim 56 , wherein the effector of SREBP-1 is tumor necrosis factor (TNF-α), neutral sphingomyelinase (N-SMase) or an effective fragment thereof, sphinogmyelin, ceramide, cpp32, or cholesterol.  
     
     
         58 . The method of  claim 56  or  57  further comprising monitoring LDL receptor activity as being indicative of the effector of the SREBP-1.  
     
     
         59 . A method for detecting an effector of LDL receptor biosynthesis, the method comprising: 
 a) providing a population of cells responsive to ceramide and capable of expressing SREBP-1,    b) contacting the cells with a candidate effector in an amount sufficient to induce maturation of the SREBP-1,    c) culturing the cells in medium; and    d) detecting biosynthesis of the LDL receptor as being indicative of the effector of the LDL receptor.    
     
     
         60 . The method of  claim 58  or  59 , wherein the effector of the LDL receptor is tumor necrosis factor (TNF-α), neutral sphingomyelinase (N-SMase) or an effective fragment thereof; sphinogmyelin, ceramide, cpp32, or cholesterol.  
     
     
         61 . A method for determining therapeutic capacity of an effector of SREBP-1 for treating a cholesterol related disease in a mammal, the method comprising: 
 a) providing a population of cells capable of expressing SREBP-1,    b) contacting the cells with a candidate compound in an amount sufficient to induce maturation of the SREBP-1,    c) culturing the cells in medium; and    d) detecting maturation of the SREBP-1 as indicative of the therapeutic capacity of the effector in treating the disease.    
     
     
         62 . The method of  claim 61 , wherein the cells are further capable of responding to an increase or decrease in intracellular ceramide levels.  
     
     
         63 . The method of  claim 61  or  62  further comprising monitoring LDL receptor activity as being indicative of the therapeutic activity of the candidate compound.  
     
     
         64 . The method of  claim 63 , wherein the candidate compound is a neutral sphingomyelinase (N-SMase) or an effective fragment thereof; sphinogmyelin, ceramide, cpp32, or cholesterol.  
     
     
         65 . The method of any one of claims  57 - 64 , wherein the neutral sphingomyelinase (N-SMase) is encoded by a sequence having at least 70% sequence identity to the sequence represented by SEQ ID NO:1 or completement thereof.  
     
     
         66 . The method of any one of claims  43 - 51 , wherein the effective fragment of the neutral sphingomyelinase (N-SMase) comprises a sequence having at least 70% sequence identity to nucleotides 862 to 1414 of SEQ ID NO:1 or completement thereof.  
     
     
         67 . A method for determining therapeutic capacity of any one of the anti-lipemic drugs of claims  1 - 25  for treating a cholesterol related disease in a mammal, the method comprising: 
 a) providing a population of cells capable of expressing SREBP-1,    b) contacting the cells with the anti-lipemic drug in an amount sufficient to induce maturation of the SREBP-1,    c) culturing the cells in medium; and    d) detecting maturation of the SREBP-1 as indicative of the therapeutic capacity of the anti-lipemic drug in treating the disease.    
     
     
         68 . The method of  claim 67 , wherein the cells are further capable of responding to an increase or decrease in intracellular ceramide levels.  
     
     
         69 . The method of  claim 67  or  68  further comprising monitoring LDL receptor activity as being indicative of the therapeutic activity of the anti-lipemic agent.  
     
     
         70 . The method of  claim 69 , wherein the anti-lipemic drug is a neutral sphingomyelinase (N-SMase) or an effective fragment thereof; sphinogmyelin, ceramide, cpp32, or cholesterol.  
     
     
         71 . The method of  claim 70 , wherein the neutral sphingomyelinase (N-SMase) is encoded by a sequence having at least 70% sequence identity to the sequence represented by SEQ ID NO:1 or completement thereof.  
     
     
         72 . The method of any one of claims  56 - 57 , wherein the effective fragment of the neutral sphingomyelinase (N-SMase) comprises a sequence having at least 70% sequence identity to nucleotides 862 to 1414 of SEQ ID NO:1 or completement thereof.  
     
     
         73 . A method for determining therapeutic capacity of any one of the anti-lipemic drugs of claims  1 - 25  in a Watanabe heritable hyperlipidemic rabbit or apolipoprotein and negative mouse, the method comprising: 
 a) administering at least one of the anti-lipemic drugs to the rabbit or mouse in an amount sufficient to reduce serum cholesterol levels by at least from about 10 to 20% as determined by a standard cholesterol assay; and    b) detecting the serum cholesterol reduction in the rabbit or mouse as being indicative    of the therapeutic capacity of the anti-lipemic drug to treat the cholesterol related disease.    
     
     
         74 . A method for modulation production of the amyloid precursor protein (βAPP) comprising administering to a subject mammal a therapeutically effective amount of any one of the anti-lipemic drugs of claims  1 - 25 .  
     
     
         75 . A method for modulating fatty acid synthesis, the method comprising administering to a subject mammal a therapeutically effective amount of any one of the anti-lipemic drugs of claims  1 - 25 .