U.S. patent application number 10/640502 was filed with the patent office on 2005-04-14 for compositions and methods for modulating serum cholesterol.
This patent application is currently assigned to Johns Hopkins University. Invention is credited to Chatterjee, Subroto.
Application Number | 20050079998 10/640502 |
Document ID | / |
Family ID | 31996528 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050079998 |
Kind Code |
A1 |
Chatterjee, Subroto |
April 14, 2005 |
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.
Inventors: |
Chatterjee, Subroto;
(Columbia, MD) |
Correspondence
Address: |
Peter F. Corless
EDWARDS & ANGELL, LLP
P.O. Box 55874
Boston
MA
02205
US
|
Assignee: |
Johns Hopkins University
Baltimore
MD
|
Family ID: |
31996528 |
Appl. No.: |
10/640502 |
Filed: |
August 13, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10640502 |
Aug 13, 2003 |
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09511532 |
Feb 23, 2000 |
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6713057 |
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60121447 |
Feb 24, 1999 |
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Current U.S.
Class: |
424/94.6 ;
514/423; 514/460; 514/548; 514/7.4; 514/78 |
Current CPC
Class: |
G01N 2800/044 20130101;
G01N 33/92 20130101; A61K 38/1709 20130101; A61K 31/164 20130101;
A61K 31/164 20130101; A61K 38/1709 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/002 ;
514/423; 514/460; 514/548; 514/078 |
International
Class: |
A61K 038/00; A61K
031/685; A61K 031/401; A61K 031/366 |
Goverment Interests
[0002] Funding for the present invention was provided in part by
the Government of the United States by virtue of National Institute
of Health Grants R0-1 DK-31722 and P50-HL4812. Thus, the Government
of the United States has certain rights in and to the invention
claimed herein.
Claims
What is claimed is:
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.sub.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-.alpha.), 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-.alpha.), 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 (.beta.APP) 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.
Description
[0001] This application claims the benefit of U.S. provisional
application No. 60/121,447, filed Feb. 24, 1999, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to compositions and methods
for modulating serum cholesterol. 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 anti-lipemic drug includes at a sphingolipid
or protein modifying same linked to the serum cholesterol
inhibitor. Additionally provided are methods for using the
anti-lipemic drugs to significantly stabilize or reduce serum
cholesterol levels in a subject mammal and particularly a human
patient.
BACKGROUND OF THE INVENTION
[0004] There is nearly universal agreement that cholesterol is a
key lipid constituent of cell membranes. Cholesterol is generally
understood to be essential for normal growth and viability of most
higher organisms. Too much serum cholesterol has been correlated
with life threatening lipid related diseases including
hyperlipoproteinemia, stoke, coronary heart disease, and especially
artherosclerosis and related conditions. See generally Stryer, L.
(1988) in Biochemistry, 3.sup.rd Ed. W.H. Freeman and Co. New York,
pp. 547-574; and Brown, M. S. and Goldstein, J. L. (1993) in The
Pharmacological Basis of Therapuetics (8.sup.th Ed.) Gilman, A. G.
et al. eds. McGraw-Hill/New York, pp. 874-896.
[0005] The regulation of serum cholesterol in mammals and
particularly primates has attracted significant attention. It is
often reported that regulation of cholesterol homeostasis in humans
and other mammals involves regulation of cholesterol production,
bile acid biosynthesis and catabolism of specific serum cholesterol
carriers. Important serum cholesterol carriers are called LDL (low
density lipoprotein) particles. The LDL receptor has been reported
to facilitate internalization of the LDL particle into those cells
in need of cholesterol. See e.g., Brown, M. S. and Goldstein, J. L.
(1986) Science 232: 34-47; and Goldstein, J. L. and Brown, (1986)
Nature, 348: 425; and references cited therein.
[0006] The LDL receptor has been disclosed as impacting serum
cholesterol levels in humans. For example, there has been
recognition that cells with enough cholesterol do not make
sufficient LDL receptors, thereby reducing or even blocking uptake
of cholesterol by the cell. In this instance, serum cholesterol
levels rise substantially which can contribute to the development
or severity of disease. Conversely, cells in need of cholesterol
often have capacity to make more LDL receptors, thereby
facilitating a decrease in serum cholesterol. Accordingly, there
has been specific attention focused on regulating the LDL receptor
as one therapeutic approach for stabilizing or reducing serum
cholesterol levels in human patients.
[0007] In particular, it has been reported that transcription of
the LDL receptor gene is suppressed when sterols accumulate and
induced when sterols are depleted. Sterol sensitivity is thought to
be conferred by a 10 basepair (bp) sequence upstream of the LDLr
gene known as the sterol regulatory element (SRE). It has been
disclosed that the mature form of the sterol regulatory element
binding protein-1 (SREBP-1) binds to the SRE and promotes
transcription.
[0008] There have been additional reports that the activity of
SREBP-1 is influenced by sterol induced proteolysis. There is
recognition that the SREBP-1 proteolysis is impacted in some
settings by a cell receptor termed "cytokine tumor necrosis factor"
(TNF-.alpha.).
[0009] In particular, the TNF-.alpha. receptor has been reported to
influence a wide range of biological effects. However, the
TNF-.alpha. receptor remains incompletely characterized.
Elucidation of TNF-.alpha. pathways is sometimes complicated by
presence of at least two TNF receptors. The receptors share some
common downstream effectors but also signal via receptor specific
pathways. See the references cited below for additional disclosure
relating to the TNF-.alpha. receptor.
[0010] There has been understanding that one consequence of TNF
signaling is the activation of neutral sphingomyelinase (N-SMase).
Neutral sphingomyelinase is a membrane bound enzyme that catalyzes
the hydrolysis of sphingomyelin to ceramide and phosphocholine at a
pH optima of 7.4. The role of neutral sphingomyelinase in signal
transduction has primarily been related to ability to generate the
lipid second messenger ceramide. In addition to TNF-.alpha., Fas
receptor ligand, vitamin D.sub.3, interleukin-1.beta., nerve growth
factor, anti-CD28 antibodies and .gamma.-interferon have all been
shown to increase ceramide levels.
[0011] In particular, sphingomyelinases type-C (E.C. 3.1.4.12) are
a group of phospholipases that catalyze the hydrolytic cleavage of
sphingomyelin via the following reaction (1).
Sphingomyelin---->Ceramide+Phosphocholine (1)
[0012] See S. Chatterjee, Adv. Lipid Res., 26:25-48 (1993); S.
Chatterjee et al., J. Biol. Chem., 264:12,534-12,561 (1989); and S.
Chatterjee et al., Methods in Enzymology, Phospholipase,
197:540-547 (1991).
[0013] In addition to the biological roles of sphingomyelin and
ceramide in signal transduction pathways involving cell regulation,
more recent evidence has emerged suggesting that sphingomyelinases
may be involved in cholesterol homeostasis and particularly
induction of LDL receptor activity. See S. Chatterjee, Advances in
Lipid Research, 26:25-48 (1993). Additional work supports a
possible role of ceramide in programmed cell death and/or
"apoptosis" and activation of the gene for nuclear factor (NF)-kB.
See A. Alessenko and S. Chatterjee, Mol. Cell. Biochem., 143:169
(1995).
[0014] It has been suggested that certain enzymes involved in
making cholesterol exert a significant effect on cholesterol
homeostasis. Accordingly, there has been substantial interest in
identifying drugs with capacity to modulate these enzymes
especially to stabilize or reduce serum cholesterol to tolerable
ranges. Illustrative agents include commercially available serum
cholesterol inhibitors such as fluvastatin, simvastatin,
lovastatin, pravastatin, and atorvastatin. See Brown, M. S. and
Goldstein, J. L. (1993), supra for additional disclosure relating
to these and other agents such as mevinolin (compactin).
[0015] Although some clinical benefit has been reported to follow
use of these and other serum lowering agents, there have been
reports of significant side-effects. See e.g., Brown, M. S. and
Goldstein, J. L. (1993), supra; and Physicians Desk Reference 1997
(51.sup.st ed.) Medical Economics Co. Accordingly, there is a need
to have drugs that exhibit more desirable characteristics such as
enhanced potency and better patient tolerance. There is a specific
need to reduce levels of administered cholesterol lowering agents
for some patients.
[0016] There is also a need to identify drugs that can modulate the
SREBP-1 protein and especially the LDL receptor. Moreover, methods
for identifying pharmacological drugs of interest by automated,
high throughput drug screening have become increasing relied upon
in a variety of pharmaceutical and biotechnology drug development
programs. Unfortunately, requisite drugs for such high throughput
screening assays are not widespread. A significant reason for lack
of progress in this area is insufficient understanding of molecules
(i.e. effectors) that impact SREP-1 and the LDL receptor.
[0017] It thus would be desirable to have anti-lipemic drugs with
dual capacity to modulate the LDL receptor and serum cholesterol
levels. It would be particularly desirable if such anti-lipemic
drugs could be administered to subject mammal at doses near or
below those presently used with many serum cholesterol inhibitors.
It would be further desirable to have effective in vitro and in
vivo assays for identifying drugs with potential to modulate the
LDL receptor particularly involving SREP-1 protein maturation.
SUMMARY OF THE INVENTION
[0018] The present invention generally relates to compositions and
methods 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.
[0019] We have discovered a wide spectrum of compositions and
methods for treating or preventing disorders modulated by
cholesterol. Sometimes the disorders will be referred to herein as
"cholesterol related disorders" or a similar term. More
specifically, we have identified anti-lipemic drugs that include at
least one identified effector of the LDL receptor, and particularly
an effector of SREBP-1 and at least one identified serum
cholesterol inhibitor. Particular anti-lipemic drugs of this
invention usually have one of each component although drugs having
multiple effectors and inhibitors (e.g., between from about 2 to 5
of each) are contemplated. Preferred anti-lipemic drugs feature
specifically defined characteristics such as capacity to stabilize
or reduce serum cholesterol levels in a subject mammal as
determined by in vitro or in vivo assays described below.
[0020] More specifically, the present invention provides a variety
of specific anti-lipemic drugs and methods for using same for the
treatment or prevention of one or more than one cholesterol related
disorder in a subject mammal. Illustrative disorders are known in
the field and include hyperlipoproteinemia including
hypercholesterolemia, stroke, obesity, compulsive eating disorders,
cardiac disease including atherosclerosis, cerebral
atherosclerosis, cholesteryl ester storage disorder, liver disease
including organ transplantation failure and cirrhosis; diseases of
the biliary system, and viral infection, particularly those
infections facilitating encephalitis or related disorders.
[0021] Particular anti-lipemic drugs in accord with this invention
include one SREBP-1 effector and one synthetic or semi-synthetic
inhibitor of an enzyme associated with cholesterol biosynthesis.
Preferred enzymes have been extensively characterized and include
3-hydroxy-3-methylglutaryl (HMG) CoA reductase and HMG CoA
synthetase. Additionally contemplated anti-lipemic drugs feature,
as the effector component, an identified caspase, particularly the
cpp32 protease (caspase-3), neutral sphingomyelinase (N-SMase),
ceramide, SREBP-1 (precursor), or SREBP-1 (mature). Effective
fragments of the N-SMase, cpp32 protease, SREBP-1 (precursor), or
the SREBP-1 (mature) protein are contemplated as effector molecules
within the scope of this invention.
[0022] Additionally specific anti-lipemic drugs include one
effector of SREBP-1 which effector can be a sphingolipid, e.g.,
sphingmyelin or ceramide; or N-SMase or an effective fragment
thereof. In embodiments in which the anti-lipemic drug includes
ceramide, that ceramide molecule is preferably naturally-occurring
(ie. can be isolated in substantially pure form from a biological
source). A more preferred ceramide for use in the drug is any one
of C-2, C-4, C-6 or C-8 ceramide. A preferred N-SMase molecule is
encoded by specific nucleotide sequences disclosed herein including
those encoding enzymatically active forms of that enzyme and
effective fragments thereof. Preferred effectors in accord with
this invention demonstrate substantial capacity to modulate the LDL
receptor and especially maturation of the SREBP-1 protein as
determined by specific assays described below.
[0023] As discussed, particular anti-lipemic drugs of this
invention include a suitable SREBP-1 effector such as sphingolipid,
particularly a sphingomyelin or ceramide, N-SMase or effective
fragment thereof, although other drugs may include other effectors
as needed. In this embodiment, the anti-lipemic drug further
includes the inhibitor of HMG CoA reductase. It is generally
preferred that the effector and the inhibitor are be combined in a
way to facilitate function for which the drug was intended. A
preferred function is to stabilize or reduce serum cholesterol as
determined by a conventional in vivo assays defined below. In most
instances, covalent attachment between the effector and the
inhibitor will be preferred although other associations will be
suitable for some applications. Preferred cholesterol inhibitors
have recognized capacity to inhibit the reductase, thereby lowering
serum cholesterol. Illustrative inhibitors include commercially
available serum cholesterol inhibitors acceptable for human use,
e.g., fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin
(compactin), atorvastatin; or a clinically acceptable derivative
thereof.
[0024] In a particular embodiment, the anti-lipemic drugs include
one effector of the SREBP-1 protein, e.g., the N-SMase or effective
fragment; or a sphingolipid. In this example, the effector is also
preferably associated with the inhibitor of HMG CoA reductase. By
the term "associated" or related term is meant that the SREBP-1
effector and the inhibitor are attached by at least one bond
preferably at least on covalent bond. Particular examples of
bonding are described below. In some instances, the association can
also be provided by a suitable combination of covalent and
non-covalent chemical bonds. Alternatively, association between the
SREBP-1 effector and the inhibitor can be provided by essential
co-administration of the effector and the inhibitor to a desired
subject mammal. More specific methods for making and using the
drugs of this invention are provided in the discussion and examples
which follow.
[0025] In one embodiment, the anti-lipemic drug includes the
sphingolipid attached to the inhibitor by at least one covalent
bond. As noted, preferred are recognized cholesterol inhibitors
such as fluvastatin, simvastatin, lovastatin, pravastatin,
mevinolin (compactin), atorvastatin. In this illustration, the
sphingolipid is preferably ceramide or a related molecule,
particularly any one of the preferred ceramides described
previously, which ceramide is covalently linked to a reactive
hydroxyl group on the inhibitor molecule. Also in this example, the
hydroxyl group of the inhibitor is usually covalently linked to a
reactive carbon atom on the ceramide such as the C-3 carbon.
[0026] Additional anti-lipemic drugs of this invention include at
least one bifunctional spacer group, typically a heterobifunctional
spacer group, which group spaces the SREBP-1 effector from the
inhibitor or other drug moiety. A particular example of this type
of anti-lipemic drug includes one SREBP-1 effector covalently
linked to one heterobifunctional spacer group. That spacer group is
preferably covalently linked to the serum cholesterol inhibitor.
Typically, the bifunctional spacer is linked to suitably reactive
chemical group on the effector and the inhibitor, usually
specifically reactive carbon atoms and hydroxyl groups,
respectively.
[0027] Further anti-lipemic drugs in accord with the present
invention include one effector of SREBP-1 such as the neutral
sphingomyelinase (N-SMase) or an effective fragment thereof. A
preferred drug includes the N-SMase or the fragment in association
with an inhibitor of HMG CoA reductase or HMG CoA synthetase as
described previously. Preferred examples of the N-SMase and
fragment are provided in the examples and discussion which
follow.
[0028] Further contemplated anti-lipemic drugs include the effector
of SREBP-1, preferably the neutral sphingomyelinase (N-SMase) or
the fragment thereof; which effector is covalently linked to one
inhibitor of the HMG CoA reductase. Preferred inhibitors of the
reductase have already been discussed. Preferably, the covalent
linkage is made by binding a chemically reactive group on the
enzyme or fragment, preferably an amide bond. More particular
anti-lipemic drugs are disclosed below featuring an amide linkage
between the enzyme or fragment and the serum cholesterol
inhibitor.
[0029] Preferred anti-lipemic drugs of this invention are generally
formulated to suit intended use and specifically include those
drugs formatted for topical or related use. Additionally, the
invention includes anti-lipemic drugs that include components
sufficient to provide the drug as a liposome formulation suitable
for in vitro or in vivo use. Methods for making and using such
preferred drugs are described below.
[0030] In general, therapeutic methods in accord with this
invention include administering to a subject, particularly a mammal
such as a primate, especially a human, a therapeutically effective
amount of at least one anti-lipemic drug of interest. That drug can
be administered as a sole active agent. Alternatively, the
anti-lipemic drug can be administered in combination with other
drugs or agents exhibiting a desired pharmacological activity. In
most cases, the amount of anti-lipemic drug use will be one which
exhibits good activity in a standard in vitro or in vivo assay
described below.
[0031] As discussed, the anti-lipemic drugs of this invention
advantageously provide dual "anti-cholesterol" activity, ie, by
increasing LDL receptor activity, particularly by enhancing LDL
receptor levels; and by reducing serum cholesterol levels.
Particular in vitro and in vivo assays to detect and quantitate
these activities are provided below and in the discussion and
examples which follow.
[0032] As an illustration, preferred anti-lipemic drugs of this
invention are capable of stimulating production of the mature form
of SREBP-1 (maturation) by at least about 2 fold, as determined by
a standard SREBP-1 proteolysis (maturation) assay. That assay is
provided below and generally involves monitoring in a time and dose
dependent manner, the maturation of the SREBP-1 protein. Mature
SREBP-1 protein is believed to move to the nucleus and stimulate
production of LDL receptor.
[0033] Additionally preferred anti-lipemic drugs of this invention
are capable of increasing LDL receptor mRNA levels by at least
about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, or 90% as determined
by Northern blot or related mRNA detection assay. An exemplary
Northern blot assay for detecting and optionally quantitating LDL
receptor mRNA levels are provided below.
[0034] Also preferred anti-lipemic drugs of this invention exhibit
an ID.sub.50 of between from about 20%, 30%, 40%, 50%, 60%, or 70%
to about 90% as determined in a standard HMG CoA reductase assay.
In this assay, the activity of the reductase enzyme is monitored in
the presence and absence (control) of the anti-lipemic agent. An
example of the standard HMG CoA reductase assay is provided
below.
[0035] Further preferred anti-lipemic drugs are capable of
significantly reducing serum cholesterol as determined by a
standard serum cholesterol assay. Preferably, an administered
anti-lipemic drug is capable of reducing serum cholesterol in a
subject mammal by at least about 5%, 10% to 20% or 30%, 40%, 50%,
60% or 70%. An example of the assay is described below. Typically,
the reduction in serum cholesterol is monitored with respect to a
suitable control subject. The serum cholesterol assays are
optimally performed in vivo and preferably include use of a
recognized animal model such as specific rabbit and mouse strains
provided below.
[0036] Preferred animal models for use in the serum cholesterol
assay or other suitable assay disclosed herein are generally
recognized test systems for an identified cholesterol related
disease. Typically such animal models include commercially
available in-bred strains of rabbits or mice, e.g., the Watanabe
heritable hyperlipidemic rabbit and the apolipoprotein E negative
mouse. In this example, the reduction in serum cholesterol can be
evaluated using well-known testing strategies adopted for use with
the specific animal model. However for some applications it may be
useful to test a desired anti-lipemic drug on a normal
("wild-type") animal such as those genetically defined (e.g.,
isogenic) wild-type animal strains known in the field.
[0037] The anti-lipemic drugs of this invention are preferably
tested by at least one and preferably all of the standard assays
summarized above. Preferred are anti-lipemic drugs that demonstrate
about the stated activity ranges in one or more of the assays.
[0038] Significantly, use of multiple testing strategies (e.g., a
combination of one in vitro and/or in vivo assays) with a single
anti-lipemic drug can extend the selectivity and effectiveness of
the testing as needed. That is, the testing strategy can be
tailored for treatment or prevention of a particular cholesterol
related disease or group of patients if required.
[0039] Such broad spectrum testing provides additional advantages.
For example, preferred anti-lipemic drugs have capacity to enhance
LDL receptor activity (typically by enhancing production of the LDL
receptor) and provide for a reduction in serum cholesterol level.
Thus by providing such dual "anti-cholesterol" activity, the
invention is a significant advance over prior therapies and agents
that have been reported to reduce serum cholesterol in one way,
usually by targeting cholesterol biosynthesis. Accordingly,
preferred anti-lipemic drugs of this invention feature better
activity, can be administered at lower dosages then prior agents.
Patient tolerance of the anti-lipemic drugs will also be positively
impacted.
[0040] In another aspect, the invention includes methods for
modulating and particularly reducing serum cholesterol level in a
mammal. In this embodiment, the methods generally include
administering to the mammal a therapeutically effective amount of
at least one and typically one of the anti-lipemic drugs disclosed
herein.
[0041] Also provided are methods for modulating LDL receptor levels
in a mammal in which the method includes administering to the
mammal a therapeutically effective amount of at least one and
typically one of the anti-lipemic drugs disclosed herein.
[0042] The present invention also provides methods treating a
disorder in a mammal having or suspected of having high serum
cholesterol levels. In this embodiment, the method includes
administering to the mammal a therapeutically effective amount of
at least one of the anti-lipemic drugs disclosed herein. A
preferred mammal is a primate and especially a human patient, e.g.,
those susceptible to coronary heart disease, obesity, eating
disorders or other cholesterol related disorders described herein.
Accordingly, the methods are especially applicable to a subject
mammal such as a human patient who has been diagnosed as having, is
suspected of having, or is susceptible to a high serum cholesterol
level, e.g., through adverse genetic or dietary influences.
[0043] Also provided by this invention are methods for modulating
serum cholesterol level in a mammal in which the method includes
administering to the mammal a therapeutically effective amount of
at least one of the anti-lipemic drugs disclosed herein. In this
embodiment, the SREBP-1 effector is preferably neutral
sphingomyelinase (N-SMase) or an effective fragment thereof; or a
sphingolipid such as ceramide. Preferred methods employ a primate
such as a human patient. Preferred anti-lipemic agents for use in
the methods are typically tested for activity using a recognized
animal model for a cholesterol related disorder and especially
atherosclerosis, e.g., the Watanabe heritable hyperlipidemic rabbit
or an apolipoprotein E negative mouse discussed previously.
[0044] Additionally contemplated are methods for modulating LDL
receptor in a mammal in which the methods include administering to
the mammal a therapeutically effective amount of at least one of
the anti-lipemic drugs disclosed herein. The modulation is
preferably an increase in the synthesis (or sometimes decrease in
the degradation of) the LDL receptor. In this example, the SREBP-1
effector is neutral sphingomyelinase (N-SMase) or an effective
fragment thereof; or a sphingolipid such as ceramide. Methods for
evaluating an increase or decrease in LDL receptor levels are known
in the field and involve, e.g., molecular and immunological
approaches using anti-LDL antibodies capable of detecting and
quantitating LDL receptor in vitro or in vivo.
[0045] Particular methods of this invention involve use of at least
one suitable anti-lipemic drug which includes one effector of
SREBP-1 associated with an identified inhibitor of serum
cholesterol as discussed herein. In this example, that effector is
preferably a sphingolipid such as ceramide. Preferred examples of
ceramide include naturally occurring ceramide and other ceramide
forms as discussed previously. As discussed, preferred methods are
conducted using a mammalian subject such as a primate and
especially a human patient who has been diagnosed as having, is
suspected of having, or is susceptible to a cholesterol related
disorder as disclosed.
[0046] In an embodiment of the methods disclosed herein, the
anti-lipemic drug is preferably disposed as a liposome formulation.
In this example, the liposome formation can be compatible for
hepatic administration in accordance with standard practice. Also
in this example, the liposome formulation can be administered to
the liver or associated organ in a human patient according to
standard medical techniques involving, e.g., oral, intramuscular,
intraperitoneal, administration via a stent or related
implementation. Particular routes of administration are provided
below.
[0047] Other aspects of the invention are discussed below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 is graph showing effect of TNF-.alpha. on neutral
sphingomyelinase (N-SMase) activity.
[0049] FIGS. 2A and 2B are graphs illustrating effects of
TNF-.alpha., sphinogmyelinase, and C.sub.2-ceramide on the kinetics
of SREBP-1 maturation. 2A) kinetics of SREBP-1 maturation, 2B)
ratio of immature/mature SREBP-1 versus time.
[0050] FIG. 2C is a representation of a Western immunoblot showing
expression of TNF-.alpha., sphingomyelinase and C.sub.2
ceramide.
[0051] FIGS. 3A-C are graphs showing effects of TNF-.alpha. (3A),
sphinogmyelinase (3B), and C.sub.2-ceramide (3C) on SREBP-1
maturation.
[0052] FIG. 4 is a representation of a Western immunoblot showing
effect of anti-N-SMase antibodies on TNF-.alpha.-induced SREBP-1
maturation.
[0053] FIGS. 5A-D are representations of indirect
immunofluorescence micrographs showing SREBP-1 expression in
cells.
[0054] FIGS. 6A-6D are representations of gels showing results of
electrophoretic mobility shift assays.
[0055] FIG. 7 is a model showing how TNF-.alpha. induces SREBP-1
proteolysis(maturation) and mobilizes membrane cholesterol in human
hepatocytes. Effectors of the LDL receptor and particularly SREBP-1
are shown schematically.
[0056] FIG. 8 is a representation of a Western immunoblot showing
N-SMase protein in cells expressing increasing amounts of a
recombinant vector encoding the N-SMase (PHH1 lanes 3-6; PHH11 lane
9).
[0057] FIG. 9 is a representation of a Northern blot showing
expression of the vectors encoding the N-SMase protein (lane 2
PHH1; lane 3 PHH11).
[0058] FIG. 10 is a representation of a Western immunoblot
illustrating SREBP-I expression and maturation in cells.
[0059] FIG. 11 is a drawing showing a nucleotide sequence (SEQ ID
NO:1) of isolated cDNA encoding human N-SMase.
[0060] FIG. 12 is a drawing illustrating the deduced amino acid
sequence (SEQ ID NO:2) of human N-SMase.
[0061] FIG. 13 is a drawing showing examples of particular
anti-lipemic drugs, target organs and particular actions of the
drugs.
[0062] FIG. 14 is a drawing showing chemical structures for
specific serum cholesterol inhibitors mevastatin, fluvastatin,
pravastatin, lovastatin and simvastatin. The inhibitors are HMG-CoA
reductase inhibitors. Fluvastatin is an entirely synthetic
mevalonolactone derivative. Remaining reductase inhibitors are
fungal compactin derivatives based on a hydronapthalene ring.
[0063] FIG. 15A-B are drawings showing (15A) sphingmyelin and (15B)
C-2 ceramide and dihydro-C-2 ceramide. The 3-hydroxyl group and 4,
5 trans carbon-carbon double bond in the sphingosine backbone are
indicated by arrows.
DETAILED DESCRIPTION OF THE INVENTION
[0064] As discussed, the invention relates to anti-lipemic drugs
and methods for using same to stabilize or reduce serum cholesterol
level in a human patient or other subject mammal. Preferred
anti-lipemic drugs generally include one identified effector of the
SREBP-1 protein associated with one identified serum cholesterol
inhibitor. More preferred are anti-lipemic drugs in which the
effector and inhibitor components are specifically covalently
linked together as a single formulation.
[0065] The term "anti-lipemic drug" is used herein to refer
generically to a composition of this invention, preferably a
specific synthetic or semi-synthetic drug, which has dual capacity
to modulate serum cholesterol levels, ie, by modulating the LDL
receptor and stabilizing or reducing serum cholesterol levels in
the subject mammal. Preferred is an anti-lipemic drug with
demonstrated capacity to increase LDL receptor levels and to reduce
serum cholesterol levels as determined by specific in vitro and in
vivo assays described below. As discussed below, capacity to reduce
serum cholesterol levels by the inhibitor component is generally
mediated by modulation of HMG CoA reductase, typically by
inhibiting that enzyme sufficient to reduce serum cholesterol. As
also discussed, the effector portion preferably increases
production of the LDL receptor.
[0066] The anti-lipemic drugs disclosed herein can be made by
recognized methods known in the field. For example, methods for
making specific sphingolipids and especially ceramide and
ceramide-related compounds have been disclosed in co-pending U.S.
patent application Ser. No. 08/998,262 entitled "Methods for
Treatment of Conditions Associated with Lactosylceramide" filed on
Dec. 24, 1997, now issued as U.S. Pat. No. 5,972,928 on Oct. 26,
1999, the disclosure of which is incorporated herein by reference.
See also Abe, A. et al., (1992) J. Biochem. 111:191-196; Inokuchi,
J. et al. (1987) J. Lipid Res. 28:565-571; Shukla, A. et al. (1991)
J. Lipid Res. 32:73; Vunnam, R. R. et al., (1980) Chem. and Physics
of Lipids 26:265; Carson, K. et al., (1994) Tetrahedron Lets.
35:2659; and Akira, A. et al., (1995) J. Lipid Research 36:611.
[0067] More specific anti-lipemic drugs of this invention include
as covalently linked components the effector and the serum
cholesterol inhibitor. However for some applications other
anti-lipemic drugs can be appropriate such as those including
non-covalently linked components. Examples include those drugs
provided as essentially co-administered formulations.
[0068] The molecular weight of a particular anti-lipemic drug will
vary depending, e.g., on the specific SREBP-1 effector and serum
cholesterol inhibitor chosen and the number of effectors and
inhibitors making up the drug. However in most cases the
anti-lipemic drug will have a molecular weight of less than about
10,000 kD to 35,000 kD particularly when the effector molecule is a
protein or polypeptide sequence such as the N-SMase sequences or
fragments thereof disclosed herein. Molecular weights will
generally be significantly lower, e.g., between from about 100 kD
to 1000 kD, preferably between from about 200 kD to 500 kD when the
effector is a sphingomyelin or related molecule. Methods for
determining the molecular weight are known and include standard
molecular sizing methods such as SDS polyacrylamide gel
electrophoresis.
[0069] Illustrative examples of specific anti-lipemic drugs in
accord with this invention are shown in FIG. 13. FIG. 13
particularly shows use of combinations of SREBP-1 maturation
upregulators (effectors) ceramide, N-SMase, and various lipid
lowering molecules; HMG CoA-reductase inhibitors (statins) in
various human pathologies.
[0070] An "effector" of the LDL receptor and particularly the
SREBP-1 protein is a molecule, usually an amino acid sequence,
lipoprotein, lipid or like molecule with demonstrated capacity to
modulate the LDL receptor and specifically maturation of the
SREBP-1 protein as determined by the standard SREBP-1 maturation
assay described below. Illustrative effectors are provided in the
Examples and FIG. 7.
[0071] A "serum cholesterol inhibitor" as that term is used herein
generally refers to a recognized compound capable of reducing serum
cholesterol levels in a subject mammal and especially a human
patient. Preferred serum cholesterol inhibitors particularly
interfere with cholesterol biosynthesis and especially HMG
CoA-reductase activity, e.g., in the liver. More preferred serum
cholesterol inhibitors are readily available commercially and
include mevastatin, fluvastatin, pravastatin, lovastatin and
simvastatin. See FIG. 14 and the discussion below.
[0072] It has been unexpectedly found that TNF-.alpha.
significantly stimulates maturation of SREBP-1 in cells through
action of the N-SMase. That is, we have found that TNF-.alpha. is
capable of inducing SREBP-1 maturation in a time and dose dependent
manner. This induction was consistent with the kinetics of
TNF-.alpha. mediated activation of neutral sphingomyelinase
(N-SMase). Antibodies to N-SMase inhibited TNF-.alpha. induced
SREBP-1 maturation suggesting that N-SMase is a necessary component
of this signal transduction pathway. Ceramide, a product of
sphingomyelin hydrolysis, was also found to be capable of inducing
SREBP-1 maturation. Without wishing to be bound to theory, it
appears that the mature form of SREBP-1 generated by TNF-.alpha.,
sphingomyelinase or ceramide treatment translocates to the nucleus
and binds the sterol regulatory element (SRE). This is believed to
promote transcription of the gene upstream of the SRE. See FIG. 7
for a schematic outline of these findings. It further appears that
effectors of the SREBP-1 stimulate the LDL receptor, particularly
by enhancing SREBP-1 maturation, thereby stabilizing or reducing
serum cholesterol in the subject mammal.
[0073] Therapeutic methods of the invention generally comprise
administration of a therapeutically effective amount of at least
one and typically one anti-lipemic drug as disclosed herein to a
subject mammal such as a primate and especially a human patient in
such treatment. The therapeutic treatment methods more specifically
include administration of an effective amount of the anti-lipemic
drug to a subject, particularly a mammal such as a human in need of
such treatment for an indication disclosed herein.
[0074] Typical subjects of interest include those suffering from,
suspected of suffering from, or susceptible to the conditions,
disorders or diseases disclosed herein, e.g., hyperlipoproteinemia
including hypercholesterolemia, stroke, obesity including
compulsive eating disorders, cardiac disease including
atherosclerosis, cerebral atherosclerosis, cholesteryl ester
storage disorder, liver disease including organ transplantation
failure and cirrhosis; diseases of the biliary system, and viral
infection particularly those infections facilitating encephalitis
or related disorders. More specific disclosure relating to these
and other cholesterol related diseases including accepted methods
for screening and diagnosing these disorders have been reported.
See e.g., Brown, M. S. and Goldstein, J. L. (1993), supra and
references cited therein.
[0075] A variety of specific anti-lipemic drugs can be employed in
the present invention and particularly in the treatment methods
described. Routine testing, e.g., in a standard in vitro assay
optionally combined with another in vitro and/or in vivo assay, can
in most instances readily identify suitable anti-lipemic drugs
exhibiting desired selectivity and activity with respect to the
target disorder or disease. As noted, preferred anti-lipemic drugs
feature a specific effector of the SBREP-1 protein such as those
effectors identified in the Examples including N-SMase or an
effective fragment thereof; a sphingolipid and especially ceramide,
a caspase, e.g., cpp32 protein (caspase-3), or an effective
fragment thereof; as well as other specific effectors discussed
herein.
[0076] Additionally specific effectors are disclosed in the
Examples and discussion which follows. For example, one
anti-lipemic drug of this invention includes covalently linked in
sequence: 1) an SREBP-1 effector comprising a chemically reactive
group; and 2) a serum cholesterol inhibitor such as those disclosed
herein including another chemically reactive group capable of
specifically binding generally by covalent linkage to the reactive
group of the effector. Optionally, the anti-lipemic drug further
includes a bifunctional spacer, e.g., a heterobifunctional spacer,
covalently linked between 1) and 2).
[0077] A more preferred anti-lipemic drug includes covalently
linked in sequence: 1) a sphingolipid and especially sphingomyelin
or ceramide; and 2) a specific serum cholesterol inhibitor as
disclosed herein. In this embodiment, the ceramide is preferably
naturally-occurring and can be any one of C-2, C-4, C-6 or C-8
ceramide. In embodiments in which the SREBP-1 effector is ceramide,
the reactive group will typically be the C-3 group of ceramide.
Preferred are serum cholesterol inhibitors that include a suitably
chemically reactive hydroxyl (--OH) group, e.g., fluvastatin,
simvastatin, lovastatin, pravastatin, mevinolin (compactin), or
artorvastatin. Optionally, the anti-lipemic drug may include a
bifunctional spacer covalently linked between 1) and 2), ie.,
providing a covalent bond between the C-3 group and the hydroxyl
group.
[0078] Chemical structures for sphlingomyelin and specific
ceramides (C-2 ceramide, dihydro-C-2-ceramide) are shown in FIGS.
15A and 15B.
[0079] Also preferred is an anti-lipemic drug that includes
covalently linked in sequence: 1) the neutral sphingomyelinase
(N-SMase) or the effective fragment thereof, and 2) a specific
serum cholesterol inhibitor as disclosed herein. In this embodiment
in which the SREBP-1 effector is N-SMase or the fragment, the
chemically reactive group will be a suitable amide bond. Preferred
are serum cholesterol inhibitors that include a suitably chemically
reactive hydroxyl (--OH) group, e.g., fluvastatin, simvastatin,
lovastatin, pravastatin, mevinolin (compactin), or artorvastatin.
Optionally, the anti-lipemic drug may include a bifunctional spacer
and particularly a heterobifunctional spacer covalently linked
between 1) and 2). Suitable linker sequences are known in the field
and generally include chemically reactive groups on each end of a
suitable polymeric sequence such as an amino acid sequence.
[0080] Illustrative N-SMase and fragments thereof for use in accord
with this invention are provided in the examples and discussion
which follow as well as the co-pending U.S. application Ser. No.
08/774,104 entitled "Recombinant N-SMases and Nucleic Acids
Encoding Same" filed on Dec. 24, 1996, now issued as U.S. Pat. No.
5,919,687 on Jul. 6, 1999, the disclosure of which is incorporated
herein by reference.
[0081] In particular, a preferred neutral sphingomyelinase
(N-SMase) is encoded by a sequence having at least 70%, 80%, 90% or
95% sequence identity to the sequence shown in FIG. 12 (SEQ ID NO:
1) or the complement thereof. A preferred fragment of the N-SMase
includes a sequence having at least 70%, 80% or 90% sequence
identity to nucleotides 862 to 1414 of SEQ ID NO:1 or the
complement thereof. More specifically preferred is an N-SMase
fragment that consists of nucleotides 862 to 1414 of SEQ ID NO: 1
or the complement thereof. Methods for determining nucleotide
sequence identity are known in the field and include use of
well-known computer assisted programs such as FASTA and BLAST. See
S. Altschul et al. J. Mol. Biol., 215:403 (1990); and S. Altschul
et al. Nuc. Acids Res., 25: 3389-3402 (1997) for disclosure
relating to the BLAST and related programs.
[0082] The term "effective fragments" as it relates to preferred
N-SMase nucleotide fragments is used herein to refer to a specific
nucleotides having significant activity in the standard in vitro
SREBP-1 maturation assay described below. A specifically preferred
example of an effective fragment of the N-SMase is nucleotides 862
to 1414 of SEQ ID NO: 1.
[0083] As discussed, preferred anti-lipemic drugs of this invention
exhibit significant activity in a standard SREBP-1 maturation
assay. Preferably, the drug exhibits at least about 2 fold,
preferably between about 2 to 10 fold, and more preferably from
about 2 to 5 fold as determined by the assay. A preferred assay
generally involves:
[0084] a) culturing suitable cells, e.g., HH-25 cells, in medium
and adding the anti-lipemic drug for between from about 2 to 60
minutes, preferably between from about 10 to 30 minutes with about
15 minutes being generally preferred, typically followed by
washing; and
[0085] b) detecting mature SREBP-1 (ie. proteolyically cleaved) and
precursor SREBP-1 by performing Western immunoblotting with an
anti-SREP-1 antibody such as those described below. In general,
mass of the mature form of SREBP-1 can quantitatively determined
vs. the precursor form. Presence of that mature form is indicative
of SREBP-1 maturation and proteolysis. More specific methods for
performing the assay are provided in the Examples which follow.
Typically suitable control cells are included as a reference which
cells are not exposed to the drug.
[0086] As also discussed, preferred anti-lipemic drugs of this
invention exhibit good activity in a Northern blot assay for
detecting and preferably quantifying LDL receptor mRNA.
Additionally preferred anti-lipemic drugs are capable of increasing
LDL receptor mRNA levels by at least about 10% and preferably at
least from between about 20% to 50% as determined by the Northern
blot assay or related mRNA detection assay. Methods for performing
Northern blot assays are generally known and have been described,
e.g., in Sambrook et al. in Molecular Cloning: A Laboratory Manual
(2d ed. 1989); and Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, New York, 1989.
[0087] Suitable probes for detecting LDL mRNA are generally
available and include cloned sequences of the human LDL receptor or
related mammalian sequence available from Genbank. Information
about Genbank can be obtain from the National Library of Medicine,
38A, 8N05, Rockville Pike, Bethesda, Md. 20894. Genbank is also
available on the internet at http://www. ncbi .nlm. nih.gov. See
generally Benson, D. A. et al. (1997) Nucl. Acids. Res. 25: 1 for a
more complete description of Genbank.
[0088] An exemplary Northern blot assay for detecting and
optionally quantitating LDL receptor mRNA levels is discussed
below.
[0089] Preferred inhibitors of the HMG CoA reductase generally
reduce or block synthesis of cholesterol in the liver, thereby
facilitating compensatory reactions that can lead to a reduction in
plasma LDL. A preferred assay for measuring this phenomenon is the
standard HMG CoA reductase assay. As mentioned previously preferred
anti-lipemic drugs of this invention exhibit an ID.sub.50 of
between from about 20%, 30%, 40%, 50%, 60%, 70%, or 80% to about
90%, preferably between from about 30% to 50% as determined in the
HMG CoA reductase assay. The standard HMG CoA reductase assay has
been disclosed by Brown et al. (1978) J. Biol. Chem. 253: 1121. In
this assay cultured human fibroblasts respond to an inhibition of
the reductase by accumulating increased amounts of the enzyme when
compared to a suitable control.
[0090] As also discussed additionally preferred anti-lipemic drugs
are capable of reducing serum cholesterol as determined by a
standard cholesterol assay. The drug preferably registers at least
from about 5% or 10% to 20%, 30%, 40% or 50% decrease, preferably
at least about 30% to 50% decrease as determined by the assay. A
preferred assay for measuring LDL cholesterol is commercially
available from Sigma (St. Louis, Mo.) and involves immunological
separations. See also the National Cholesterol Education Program
(NCEP) for information relating to acceptable cholesterol levels in
humans.
[0091] A "high" or "high risk" cholesterol level or related term is
defined herein as from between about 200 to 240 mg/dl (mM)
cholesterol with a level greater than or equal to 240 mg/dl (mM)
cholesterol being more generally understood to be indicative of
high serum cholesterol. A normal serum cholesterol level is defined
herein as being less than about 200 mg/dl (mM). For specific
disclosure relating to conducting cholesterol tests see Brown, M.
S. and Goldstein, J. L. supra, discussing the Guidelines of the
NCEP Report of 1988.
[0092] Accordingly, "stabilization" or "reduction" of serum
cholesterol as those terms are used herein will be understood to
mean manifestation of a normal or near normal serum cholesterol
level in the subject mammal. Also, a suitable control mammal in
accord with this invention will preferably have a normal or near
normal serum cholesterol level as determined by standard serum
cholesterol tests.
[0093] Additional methods of this invention include modulating
SREBP-1 levels in a mammal in which the method includes
administering to the mammal a therapeutically effective amount of
at least one and typically one of the anti-lipemic drugs disclosed
herein. Typically, modulation of the SREBP-1 is evaluated by
determining maturation of the protein as determined by the SREBP-1
maturation tests described in the Examples below.
[0094] The present invention also provides methods for modulating
SREBP-1 levels in a mammal in which the method includes
administering to the mammal a therapeutically effective amount of
at least one and preferably one of the anti-lipemic drugs disclosed
herein. In this embodiment, the SREBP-1 effector is neutral
sphingomyelinase (N-SMase) or a therapeutically effective fragment
thereof, or a sphingolipid. As discussed, modulation of the SREBP-1
is typically evaluated by determining maturation of the protein as
determined by the SREBP-1 maturation tests described in the
Examples below. A preferred assay is the SREBP-1 proteolysis assay
described below in the Examples.
[0095] Methods of this invention can be performed in vitro or in
vivo using acceptable primary, cultured or immortalized cells such
as those disclosed herein. Generally, these cells will be capable
of exhibiting SPEBP-1 maturation as defined herein including the
HH-25 human hepatocytes described below. Methods for testing
anti-lipemic drugs of interest and especially for use in human
patient will preferably be conducted in vivo and may involve use of
a suitable animal model depending on the method used. In this
example, the model can be a suitable animal model such as those
discussed previously. Alternatively, the methods can be performed
on a suitable primate such as a human patient. Preferred is a human
patient has been diagnosed as having, is suspected of having, or is
susceptible to a cholesterol related disorder as defined above.
[0096] In embodiments in which the human patient is susceptible to
one or more cholesterol related disorders, that susceptibility can
be related to a genetic or environmental predisposition to the
cholesterol related disorder. Methods for determining such
pre-disposition are known in the field and include genetic testing.
See Brown, M. S. and Goldstein, J. L. (1993) supra.
[0097] The invention thus provides methods for treating
inappropriate (i.e. high) serum cholesterol levels as well as a
disorder or condition associated therewith. In general, the methods
include administration of a therapeutically effective amount of one
or more anti-lipemic compounds of this invention to a subject
mammal, particularly a human, suffering from or susceptible to the
high serum cholesterol levels. Additionally contemplated is use of
the present anti-lipemic compounds as prophylactic drugs to prevent
development of or reduce the severity of inappropriate serum
cholesterol levels.
[0098] Compounds of the invention will be especially useful to a
human patient who has or is suspected of having a cholesterol
related disease, disorder or condition as defined herein. Compounds
of the invention will be particularly useful in lowering serum
cholesterol to normal or near normal levels in human patients.
Specific examples of diseases which may be treated in accordance
with the invention include hyperlipoporteinemia, stroke,
cardiovascular disease and especially atherosclerosis as well as
other specific disorders of conditions mentioned herein.
[0099] Without wishing to be bound by theory, it is believed the
multiple and distinct covalently linked compounds of this invention
(i.e. at least one identified anti-lipemic drug in combination with
at least one identified SREP-1 effector) can significantly enhance
efficacy of the anti-lipemic drug, e.g., by increasing synthesis of
LDL receptor in subject cells.
[0100] Moreover, by virtue of the covalent linkage, the conjugates
of the invention present the anti-lipemic drug and the SREP-1
effector to the subject cell essentially simultaneously, an effect
that may not be readily achieved by administering the same
compounds in a drug "cocktail" formulation without covalently
linking the compounds.
[0101] It also has been reported that treatment with treatment with
one drug can in turn sensitize a patient to another drug.
Accordingly, the essentially simultaneous presentation to the
subject cell of an anti-lipemic drug and SREP-1 effector via a
conjugate of the invention may enhance drug activity, e.g., by
providing synergistic results and/or by enhancing production of LDL
receptors. Particular SREP-1 effectors of interest include
sphingomyelin and especially ceramide and related compounds. Also
preferred is N-SMase as well as therapeutically effective fragments
of that enzyme.
[0102] Administration of compounds of the invention may be made by
a variety of suitable routes including oral, topical (including
transdermal, buccal or sublingal), nasal and parenteral (including
intraperitoneal, subcutaneous, intravenous, intradermal or
intramuscular injection) with oral or parenteral being generally
preferred. It also will be appreciated that the preferred method of
administration and dosage amount may vary with, for example, the
condition and age of the recipient.
[0103] Compounds of the invention may be used in therapy in
conjunction with other medicaments such those with recognized
pharmacological activity to lower concentrations of plasma
lipoproteins. See Brown, M. S. and Goldstein, J. L. supra.
Exemplary medicaments are recognized serum cholesterol inhibitors
(i.e. reported to inhibit HMG CoA reductase) such as Lescol.TM.
(fluvastatin from Sandoz Pharmaceuticals), Mevacor.TM. and
Zocor.TM. (simvastatin and lovastatin, respectively, from Merck
& Co.), Pravachol.TM. (pravastatin from Bristol-Myers Squibb
Co.) and mevinolin (compactin).
[0104] The compounds of this invention may be used alone or in
combination with other accepted anti-lipemic therapies including
those implementing use of fibric acids, e.g., gembibrozil,
clofibrate, fenofibrate, ciprofibrate or bezafibrate; bile
acid-binding resins such as cholestyramine or colestipol; and
probucol. The compounds of this invention can be administered
before, during or after such therapies as needed.
[0105] While one or more compounds of the invention may be
administered alone, they also may be present as part of a
pharmaceutical composition in mixture with conventional excipient,
i.e., pharmaceutically acceptable organic or inorganic carrier
substances suitable for parenteral, oral or other desired
administration and which do not deleteriously react with the active
compounds and are not deleterious to the recipient thereof.
Suitable pharmaceutically acceptable carriers include but are not
limited to water, salt solutions, alcohol, vegetable oils,
polyethylene glycols, gelatin, lactose, amylose, magnesium
stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty
acid monoglycerides and diglycerides, petroethral fatty acid
esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc. The
pharmaceutical preparations can be sterilized and if desired mixed
with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, colorings, flavorings and/or aromatic
substances and the like which do not deleteriously react with the
active compounds.
[0106] For parenteral application, particularly suitable are
solutions, preferably oily or aqueous solutions as well as
suspensions, emulsions, or implants, including suppositories.
Ampules are convenient unit dosages.
[0107] For enteral application, particularly suitable are tablets,
dragees or capsules having talc and/or carbohydrate carrier binder
or the like, the carrier preferably being lactose and/or corn
starch and/or potato starch. A syrup, elixir or the like can be
used wherein a sweetened vehicle is employed. Sustained release
compositions can be formulated including those wherein the active
component is protected with differentially degradable coatings,
e.g., by microencapsulation, multiple coatings, etc.
[0108] Therapeutic compounds of the invention also may be
incorporated into liposomes. The incorporation can be carried out
according to known liposome preparation procedures, e.g. sonication
and extrusion. Suitable conventional methods of liposome
preparation are also disclosed in e.g. A. D. Bangham et al., J.
Mol. Biol., 23:238-252 (1965); F. Olson et al., Biochim. Biophys.
Acta, 557:9-23 (1979); F. Szoka et al., Proc. Nat. Acad. Sci.,
75:4194-4198 (1978); S. Kim et al., Biochim. Biophys. Acta,
728:339-348 (1983); and Mayer et al., Biochim. Biophys. Acta,
858:161-168 (1986).
[0109] The liposome may be made from one or more of the conjugates
discussed above alone, or more preferably, in combination with any
of the conventional synthetic or natural phospholipid liposome
materials including phospholipids from natural sources such as egg,
plant or animal sources such as phosphatidylcholine,
phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin,
phosphatidylserine or phosphatidylinositol. Synthetic phospholipids
also may be used e.g., dimyristoylphosphatidylcholine,
dioleoylphosphatidylcholine, dioleoylphosphatidycholine and
corresponding synthetic phosphatidylethanolamines and
phosphatidylglycerols. Cholesterol or other sterols, cholesterol
hemisuccinate, glycolipids, 1,2-bis(oleoyloxy)-3-(tr- imethyl
ammonio)propane (DOTAP), N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethy-
lammonium chloride (DOTMA), and other cationic lipids may be
incorporated into the liposomes. The relative amounts of the one or
more compounds and additives used in the liposomes may vary
relatively widely. Liposomes of the invention suitably contain
about 60 to 90 mole percent of natural or synthetic phospholipid;
cholesterol, cholesterol hemisuccinate, fatty acids or cationic
lipids may be used in amounts ranging from 0 to 50 mole percent;
and the one or more therapeutic compounds of the invention may be
suitably present in amounts of from about 0.01 to about 50 mole
percent.
[0110] It will be appreciated that the actual preferred amounts of
active compounds used in a given therapy will vary according to the
specific compound being utilized, the particular compositions
formulated, the mode of application, the particular site of
administration, etc. Optimal administration rates for a given
protocol of administration can be readily ascertained by those
skilled in the art using conventional dosage determination tests
conducted with regard to the foregoing guidelines.
[0111] In general, for treatment of a lipid related disease as
disclosed herein and particularly hyperlipoproteinemia, stroke,
coronary heart disease and especially atherosclerosis, a suitable
effective dose of one or more compounds of this invention will be
in the range of from 0.01 to 100 milligrams per kilogram of
bodyweight of recipient per day, preferably in the range of from
0.1 to 50 milligrams per kilogram bodyweight of recipient per day,
more preferably in the range of 1 to 20 milligrams per kilogram
bodyweight of recipient per day. The desired dose is suitably
administered once daily, or several sub-doses, e.g. 2 to 5
sub-doses, are administered at appropriate intervals through the
day, or other appropriate schedule.
[0112] A preferred dose for many compounds of this invention will
be in the range of those dosages accepted for identified HMG CoA
reductase inhibitors with lower than that range being preferred for
many patients. See the Physicians Desk Reference, supra for more
specific information relating to recommended doses for HMG CoA
reductase inhibitors with anti-lipemic activity.
[0113] In another aspect, the invention also provides methods for
detecting an effector of the sterol regulatory element binding
protein-1 (SREBP-1). In one embodiment, the method includes the
steps of:
[0114] a) providing a population of cells capable of expressing
SREBP-1,
[0115] b) contacting the cells with a candidate effector in an
amount sufficient to induce maturation of the SREBP-1,
[0116] c) culturing the cells in medium; and
[0117] d) detecting maturation of the SREBP-1 as indicative of the
effector of the SREBP-1.
[0118] Illustrative effectors for use in the method are include
those specifically described in the Examples and FIG. 7, e.g.,
tumor necrosis factor (TNF-.alpha.), neutral sphingomyelinase
(N-SMase) or an effective fragment thereof, sphinogmyelin,
ceramide, cpp32, or cholesterol. See also FIG. 13.
[0119] The invention also includes a method for detecting an
effector of LDL receptor biosynthesis. In one embodiment, the
method includes:
[0120] a) providing a population of cells responsive to ceramide
and capable of expressing SREBP-1,
[0121] b) contacting the cells with a candidate effector in an
amount sufficient to induce maturation of the SREBP-1,
[0122] c) culturing the cells in medium; and
[0123] d) detecting biosynthesis of the LDL receptor as being
indicative of the effector of the LDL receptor.
[0124] In one embodiment of the method, illustrative candidate
effectors of the LDL receptor is tumor necrosis factor
(TNF-.alpha.), neutral sphingomyelinase (N-SMase) or an effective
fragment thereof; sphinogmyelin, ceramide, cpp32, or
cholesterol.
[0125] Also provided by the present invention is a method for
determining therapeutic capacity of an effector of SREBP-1 for
treating a cholesterol related disease in a mammal. In one
embodiment, the method includes:
[0126] a) providing a population of cells capable of expressing
SREBP-1,
[0127] b) contacting the cells with a candidate compound in an
amount sufficient to induce maturation of the SREBP-1,
[0128] c) culturing the cells in medium; and
[0129] d) detecting maturation of the SREBP-1 as indicative of the
therapeutic capacity of the effector in treating the disease.
[0130] The present invention also provides methods for determining
therapeutic capacity of any one of the anti-lipemic drugs disclosed
herein for treating a cholesterol related disease in a mammal. In
one embodiment, the method includes:
[0131] a) providing a population of cells capable of expressing
SREBP-1,
[0132] b) contacting the cells with the anti-lipemic drug in an
amount sufficient to induce maturation of the SREBP-1,
[0133] c) culturing the cells in medium; and
[0134] d) detecting maturation of the SREBP-1 as indicative of the
therapeutic capacity of the anti-lipemic drug in treating the
disease.
[0135] Also provided herein are methods for determining therapeutic
capacity of one or more of the anti-lipemic drugs disclosed herein
using a Watanabe heritable hyperlipidemic rabbit or apolipoprotein
and negative mouse as an animal model. In one embodiment, the
method includes:
[0136] 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
[0137] 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.
[0138] Methods of this invention can optionally include monitoring
LDL receptor activity as being indicative of the effector of the
SREBP-1. In this embodiment, the receptor activity can be suitably
monitored and quantified if desired by one or a combination of
standard strategies. For example, a variety of specific methods
have been reported to monitor LDL receptor activity and
particularly to detect increases or decreases in the level of LDL
receptors. See Brown, M. S. and Goldstein, J. L. (1993), supra and
references cited therein for several immunological and molecular
approaches. A preferred method is the standard LDL receptor
Northern blot assay disclosed herein.
[0139] Suitable cells for use in the methods of this invention are
described in the Examples which follow.
[0140] Preferred are cells which include SREBP-1 and are capable of
SREBP-1 maturation as determined by the standard assay described
herein. More preferred are cells responsive to an increase or
decrease in intracellular sphingolipid and especially ceramide or a
related compound such as human hepatocytes as provided in the
Examples below.
[0141] Suitable effectors or candidate compounds for use with the
methods can be those specific compounds described herein neutral
sphingomyelinase (N-SMase) or an effective fragment thereof;
sphinogmyelin, ceramide, cpp32, or cholesterol. An illustrative
neutral sphingomyelinase (N-SMase) is encoded by a sequence having
at least 70%, 80%, or 90% sequence identity to the sequence
represented by SEQ ID NO: 1 or complement thereof. Alternatively,
the effective fragment of the neutral sphingomyelinase (N-SMase)
can include a sequence having at least 70%, 80% or 90% sequence
identity to nucleotides 862 to 1414 of SEQ ID NO: 1 or complement
thereof.
[0142] It is preferred that the anti-lipemic drugs as well as
components thereof (e.g., ceramide) be substantially pure. That is,
the drugs will be present in at least 90 to 95% homogeneity (w/w).
Anti-lipemic drugs having at least 98 to 99% homogeneity (w/w) are
most preferred for many pharmaceutical, clinical and research
applications. Once substantially purified the drug should be
substantially free of contaminants for therapeutic applications.
Once purified partially or to substantial purity, the drugs can be
used therapeutically, or in performing preferred in vitro or in
vivo assays as disclosed herein. Substantial purity can be
determined by a variety of standard techniques such as
chromatography and gel electrophoresis.
[0143] The Examples 1-8 below illustrate that TNF-.alpha. is
capable of inducing SREBP-1 proteolysis independent of the presence
of sterols. Exogenously supplied sphingomyelinase and ceramide are
also capable of inducing SREBP-1 proteolysis in a time and dose
dependent manner. The kinetics of SREBP-1 maturation is consistent
with those of neutral sphingomyelinase activation by TNF-.alpha..
Further, SREBP-1 maturation can be blocked with anti-N-SMase
antibodies indicating that neutral sphingomyelinase is necessary
for TNF-.alpha. induced, sterol independent SREBP-1 cleavage. The
product of sterol independent SREBP-1 proteolysis is capable of
nuclear translocation and binds to the sterol regulatory
element.
[0144] All documents mentioned herein are incorporated herein by
reference.
[0145] The following abbreviations are used throughout this
disclosure including the following examples as needed: N-SMase,
neutral sphingomyelinase; LDLr, Low Density Lipoprotein receptor;
SREBP-1, Sterol Regulatory Element Binding Protein-1. Numbered
citations are listed in numerical order below.
EXAMPLE 1
The Effect of TNF-.alpha. on Neutral Sphingomyelinase Activity
[0146] Neutral sphingomyelinase activity increased rapidly with the
addition of TNF-a. See FIG. 1. A maximal 2.5 fold increase in
activity was observed 15 minutes after TNF-.alpha. was added to the
cells. The gradual return of N-SMase activity to control levels
within 1 hour contrasted the rapid onset of activation and is
reflected in the asymmetric kinetic profile observed.
[0147] FIG. 1 illustrates the effect of TNF-.alpha. on neutral
sphingomyelinase activity and is explained in more detail as
follows: Confluent cultures of HH-25 cells were washed once with
PBS and incubated in serum free media for 30 minutes prior to the
addition of TNF-.alpha. (10 ng/ml). At the indicated time, cells
were harvested in PBS, pelleted and frozen. Cells were subsequently
lysed as described in materials and methods. N-SMase assays were
performed in duplicate as described. Error bars represent.+-.one
standard deviation from the mean.
EXAMPLE 2
Kinetics of SREBP-1 Proteolysis
[0148] Sterol independent SREBP-1 maturation in response to
TNF-.alpha. closely paralleled the kinetics of TNF-.alpha. induced
N-SMase activation. The mass of the mature form of SREBP-1 was
found to increase 2 fold after 5 minutes and 3 fold after 15
minutes of incubation with TNF-.alpha.. See FIG. 2A. The amount of
mature SREBP-1 returned to control levels within one hour. This
effect could not be recapitulated with EGF or PDGF treatment. The
increase in mature SREBP-1 levels was accompanied by a concomitant
decrease in the intensity of the band corresponding to the
precursor form of SREBP-1. See FIG. 2B. After 60 minutes of
treatment significantly less precursor SREBP-1 was visible.
[0149] To incorporate the observed increase in mature SREBP-1 and
the concomitant decrease in precursor SREBP-1 into a single
variable, the ratio of precursor SREBP-1 to mature was plotted. See
FIG. 2B. A maximal 1.5 fold decrease in the precursor to mature
ratio occurred 45 minutes after TNF-.alpha. was added to the media.
The decrease in precursor to mature ratio was more pronounced in
the initial 30 minutes of treatment. This is also consistent with
the kinetics of TNF-.alpha. induced N-SMase activation.
[0150] To explore the possibility that plasma membrane
sphingomyelinase was involved in the signal transduction pathway
leading to SREBP-1 proteolysis, cells were treated with exogenously
supplied bacterial sphingomyelinase. Sphingomyelinase induced a
dramatic change in the relative amounts of precursor and mature
SREBP-1. As seen in FIGS. 2A-2B, a 2.5 fold increase in mature
SREBP-1 levels was observed after 15 minutes treatment. Unlike
TNF-.alpha., the increase in mature SREBP-1 induced by
sphingomyelinase persisted after 60 minutes. Sphingomyelinase was
also capable of reducing the level of the precursor form of
SREBP-1. See FIGS. 2A-B. Treatment with purified recombinant human
sphingomyelinase produced similar results.
[0151] Much of the signal transducing ability of N-SMase has been
ascribed to its ability to generate the lipid second messenger
ceramide. Accordingly, the ability of a cell permeable ceramide
analog C.sub.2-ceramide (N-acetylsphingosine) was tested to induce
SREBP-1 maturation. C.sub.2-ceramide also induces SREBP-1
maturation in a sterol independent manner with greater magnitude
than what was observed with either TNF-.alpha. or sphingomyelinase.
C.sub.2-ceramide increased the level of mature SREBP-14 fold after
30 minutes of treatment. See FIGS. 2A-2B. The persistent elevation
of mature SREBP-1 levels observed with sphingomyelinase treatment
also accompanied C.sub.2-ceramide treatment. The increase in mature
SREBP-1 is recapitulated with the addition of bovine brain
ceramides but could not be induced with C.sub.2-dihydroceramide,
PL-A.sub.2, or Phospholipid D treatment.
[0152] The kinetics of SREBP-1 maturation presented in this example
would suggest that SREBP-1 proteolysis is a sufficiently early
event to be involved in providing cholesterol to apoptotic cells.
However, there was no evidence of apoptosis in the HH-25 human
hepatocyte cell line used in this study. Without wishing to be
bound to theory, it is conceivable that the sterol independent
induction of SREBP-1 maturation in hepatocytes is a physiologic
process that does not require that apoptosis be induced.
Alternatively, the two pathways may diverge before the cell has
been committed to apoptosis suggesting a manner in which sterol
independent SREBP-1 proteolysis could be employed independent of
the induction of apoptosis.
[0153] The sterol-independent cleavage of SREBP-1 observed with
human hepatocytes could also occur by ceramide generated by the
TNF-.alpha. induced N-SMase activation. This phenomenon may be
reconstituted by the exogenous addition of N-SMase and/or C.sub.2
ceramide to the hepatocytes.
[0154] FIGS. 2A-2C illustrate effects of TNF-.alpha.
sphingomyelinase and C.sub.2-ceramide on the kinetics of SREBP-1
maturation--FIGS. 2A-2C is explained in more detail as follows:
Cells were maintained in media supplemented with 1 .mu.g/ml
25-hydroxy cholesterol and 15 .mu.g/ml cholesterol for 24 hours
before the experiment. The cells were treated for the indicated
time as described in materials and methods. The cells were then
harvested in PBS, pelleted and frozen. Lysis and nuclear
fractionation were performed as described. Nuclear fractions (50
.mu.g of protein) were electrophoresed on a 7.5% polyacrylamide gel
and transferred to a PVDF membrane. Western blotting was performed
as described. Band intensity was quantified via densitometry. Error
bars represent.+-.one standard deviation from the mean. 2A) The
kinetics of SREBP maturation as measured by the increase in mature
SREBP-1 are plotted. Fold increase was calculated by comparing each
time point to the corresponding control value (TNF-.alpha. is
represented by stippled bars, bacterial sphingomyelinase is
represented by light gray bars and C.sub.2-ceramide by the dark
bars) 2B) Cells were treated with TNF-.alpha. (10 ng/ml) and
prepared as described above. The bands corresponding to the
precursor and mature forms of SREBP-1 were quantified and their
ratio plotted. 2C) Representative Western blots from which
numerical data was derived. Incubation time is indicated above and
applies to all conditions. The membranes were exposed to film for
15 seconds. P and M denote the precursor and mature forms of
SREBP-1 respectively.
EXAMPLE 3
Effects of TNF-.alpha., Sphingomyelinase and C.sub.2-ceramide on
Apoptosis in Hepatocytes
[0155] To demonstrate that the observed maturation of SREBP-1 was
not an artifact of the more general phenomenon of apoptosis induced
proteolysis we performed DNA laddering assays. The 160 bp DNA
ladder characteristic of cells undergoing apoptosis was not
observed in any of the samples.
[0156] TNF-.alpha., C.sub.2-ceramide and sphingomyelinase did not
induce apoptosis demonstrating that in hepatocytes, SREBP-1
maturation is not part of the more general phenomenon of apoptotic
protein hydrolysis.
EXAMPLE 4
Effects of TNF-.alpha., Sphingomyelinase and C.sub.2-ceramide
Concentration on SREBP-1 Maturation
[0157] The extent of TNF-.alpha. induced SREBP-1 maturation did not
vary appreciably with concentration. A maximal effect was observed
with 10 ng/ml of TNF-.alpha.. See FIGS. 3A-C. 250 milliunits of
sphingomyelinase activity induced an 80% decrease in the precursor
to mature ratio. As little as 1 .mu.M of C.sub.2-ceramide was
effective in producing an 81% maximal effect. The maximal effect
however, was obtained with a C.sub.2-ceramide concentration of 50
M. See FIGS. 3A-C.
[0158] FIGS. 3A-3C show effects of TNF-.alpha., sphingomyelinase
and C.sub.2-ceramide concentration on SREBP-1 maturation--The FIGS.
3A-3C are explained in more detail as follows. Cells were treated
with either TNF-.alpha., sphingomyelinase or C.sub.2-ceramide at
the indicated concentrations. Nuclear pellets were prepared and
electrophoresed (50 .mu.g of protein). The bands corresponding to
the precursor and mature froms of SREBP-1 were quantified. The
precursor to mature ratios were normalized to a single control to
facilitate comparison. The control ratio was arbitrarily assigned a
value of 1. A Unit of sphingomyelinase activity hydrolyzes 1.0
.mu.mol of sphingomyelin per minute at 37.degree. C. FIG. 3A (ng/ml
TNF-.alpha.); FIG. 3B (mUnits of sphingomyelinase); FIG. 3C
(micromolar C.sub.2-ceramide).
EXAMPLE 5
The Effect of Anti-N-SMase Antibodies on TNF-.alpha. Mediated
SREBP-1 Maturation
[0159] The availability of anti-N-SMase antibodies allowed us to
examine the effects of TNF-.alpha. on this pathway independent of
N-SMase activation (10). Polyclonal anti-N-SMase antibodies at a
dilution of 1:200 completely block TNF-.alpha. induced SREBP-1
maturation. See FIG. 4. The suppression of TNF-.alpha. mediated
SREBP-1 maturation was relieved with increasing antibody dilution.
Preincubation with preimmune serum at the same dilution had no
appreciable effect.
[0160] This example shows that pre incubation with anti-N-SMase
antibody effectively blocked TNF-.alpha. induced SREBP-1
maturation. Inhibition was not observed with pre-immune serum
treatment and was relieved with increasing antibody dilution. Such
findings are confirmed by other studies such as those showing the
ability of the antibody to inhibit TNF-.alpha. induced increases in
cholesterol ester synthesis and N-SMase induced increases in
[.sup.125]I-LDL binding, internalization and degradation in human
fibroblasts (15, 16).
[0161] FIG. 4 shows effect of anti-N-SMase antibodies on
TNF-.alpha. induced SREBP-1 maturation. The FIG. 4 is explained in
more detail as follows. Cells were maintained in media supplemented
with 1 .mu.g/ml 25-hydroxycholesterol and 15 g/ml cholesterol for
24 hours before the experiment. The cells were switched to serum
free media for 15 minutes and then incubated with anti-N-SMase
antibodies or rabbit preimmune serum at the indicated dilution for
30 minutes prior to TNF-.alpha. addition (10 ng/ml). The cells were
then harvested, pelleted and lysed as described. The samples were
electrophoresed on a 7.5% polyacrylamide gel and transferred to a
PVDF membrane. Bands were visualized as described. Film was exposed
for 15 seconds.
EXAMPLE 6
Effects of TNF-.alpha., C.sub.2-ceramide and Sphingomyelinase on
the Subcellular Localization of SREBP-1
[0162] To determine if the SREBP-1 fragment generated by
TNF-.alpha., C.sub.2-ceramide or sphingomyelinase treatment was
capable of nuclear translocation, immunofluorescence studies were
persued. Previous immunofluorescence studies have relied on the
overexpression of precursor and mature forms of SREBP-1 (14). We
were able to visualize endogenous SREBP-1 in treated and untreated
cells with polyclonal antibodies directed against the DNA binding
domain of SREBP-1. Since the DNA binding domain is common to both
the precursor and mature forms, examination of the total
distribution of endogenous SREBP-1 was possible.
[0163] TNF-.alpha., C.sub.2-ceramide and sphingomyelinase all are
capable of inducing changes in the subcellular localization of
SREBP-1. See FIG. 5A. Untreated cells display an even staining
pattern throughout their cell bodies. This is consistent with the
localization of precursor SREBP-1 to intracellular membranes (14).
However, cells treated with TNF-.alpha., C.sub.2-ceramide or
sphingomyelinase all exhibit intense nuclear staining and little
extra-nuclear staining. See FIGS. 5B-5D. The rapid change in the
subcellular localization of SREBP-1 is consistent with a
precursor/product relationship between the two forms and provides
additional evidence that the mature SREBP-1 fragment generated by
treatment is capable of nuclear translocation.
[0164] FIGS. 5A-5D show indirect immunofluorescence of SREBP-1.
FIGS. 5A-5D are discussed in more detail as follows. SREBP-1 was
visualized with rabbit polyclonal antibodies directed towards the
N-terminal DNA binding domain which is common to both the precursor
and mature forms. Cells were maintained in media supplemented with
1 .mu.g/ml 25-hydroxycholesterol and 15 .mu.g/ml cholesterol for 24
hours before the experiment. Immunofluorescence was performed as
described. All magnifications are 40.times. and all photographs
were taken of samples that were fixed 30 minutes after initiating
treatment. FIG. 5A) Control cells, FIG. 5B) Cells treated with
TNF-.alpha. (10 ng/ml), FIG. 5C) Cells treated with
sphingomyelinase (100 m Units), FIG. 5D) Cells treated with
C.sub.2-ceramide (10 .mu.M).
EXAMPLE 7
Electrophoretic Mobility Shift Assays
[0165] Electrophoretic mobility shift assays were pursued to
demonstrate that the mature SREBP-1 fragment is additionally
capable of binding to its consensus sequence. The amount of
electorphoretically retarded probe increases with time following
TNF-.alpha. treatment. See FIG. 6A. The kinetics of this process is
consistent with the activation of N-SMase. The amount of probe
bound increases with sphingomyelinase and ceramide treatment. As
expected, C.sub.2-ceramide induces a more rapid accumulation of
active, nuclear SREBP-1 than either TNF-.alpha. or
sphingomyelinase. See FIGS. 6A-6C. Antibodies directed towards the
DNA binding domain of SREBP successfully compete with the
oligonucleotide probe for binding. See FIG. 6D. Binding of the
probe is not titrated by an unrelated oligonucleotide but is
decreased with the addition of a non-radioactive competing
probe.
[0166] FIGS. 6A-6D show electrophoretic mobility shift assays.
FIGS. 6A-D are explained in more detail as follows. Cells were
maintained in sterol supplemented media. Nuclear pellets were
prepared and assayed as described in materials and methods. Probe
that has been bound by mature SREBP-1 is indicated as "Bound".
Unbound probe is indicated as "Free". The kinetics (in minutes) of
SREBP-1 binding to the probe in response to treatment with (FIG.
6A). TNF-.alpha. (10 ng/ml), (FIG. 6B) sphingomyelinase (100
mUnits) and (FIG. 6C) C.sub.2-ceramide (10 M). (FIG. 6D). The cells
were treated with either TNF-.alpha. (10 ng/ml), sphingomyelinase
(100 mUnits) or C.sub.2-ceramide (10 .mu.M) for 15 minutes.
Supershift assays were then performed with antibodies raised
against the DNA binding domain of SREBP-1. The presence or absence
of antibody is indicated by (+) and (-) respectively. Pre-immune
IgG was used as a control.
[0167] The gel mobility shift experiments in FIGS. 6A-D clearly
indicate that TNF-.alpha., N-SMase and C.sub.2 ceramide all induce
SREBP-1 levels in hepatocytes. It is known that TNF-.alpha. induces
sterol metabolism in cultured human fibroblasts (15) and LDL
receptors (16, 17). The present data indicate that indeed
TNF-.alpha. induces LDL receptor mRNA level in human hepatocytes.
One result is that TNF-.alpha. induced increase in mature SREBP-1
level is accompanied by increased LDL receptors and sterol
metabolism.
EXAMPLE 8
Effects of Overexpression of Neutral Sphingomyelinase (N-SMase) and
Recombinant N-SMase on the Maturation of Sterol-regulatory Element
Binding Protein-1 and Low Density Lipoprotein Receptor Expression
in Cultured Human Hepatocytes
[0168] The present example was conducted to address whether the
overexpression of N-SMase employing two separate N-SMase plasmid
DNA(PHH-1, representing the entire nucleotide sequence in N-SMase
cDNA and PHH-11, representing nucleotide sequence 862-1414) would
increase the maturation of SREBP-1 and LDL receptor mRNA expression
in a human hepatocyte cell line HH-11. Cells transfected with mock
plasmid cDNA(PSV-SPOT) served as a control and cells incubated with
C-2 ceramide previously shown to induce SREBP-1 maturation served
as a positive control.
[0169] Briefly, human hepatocytes(1.times.10.sup.4) were seeded in
sterile 100 mm.sup.2 in medium containing 10% dialyzed, heat
inactivated fetal bovine serum without antibiotics. Twenty four
hours later medium was replaced with 9 ml of fresh serum free
medium. After incubation for 30 min at 37.degree. C. 5-40 .mu.g of
the plasmid DNA in 1 ml of a CaCl.sub.2 solution (mixed with equal
volume of 0.25-2.5 M CaCl.sub.2 solution in HEPES buffer and HEPES
buffer pH 6.95). Following gentle mixing incubation of cells was
continued for 5-24 hr at 37.degree. C. The transfection reaction
was terminated by removing the medium and washing the cells with
serum free medium. Next, fresh serum supplemented medium was added
and incubation was continued for an additional 24 hr and cells were
harvested in appropriate buffer centrifuged and stored frozen until
further analysis. Cell pellets were homogenized and suitable
aliquots subjected to Western immunoblot analysis as described
below and in Examples 1-7 above. Total RNA was isolated from
another batch of cells transfected as above and subjected to
Northern analysis employing a .sup.32P labeled LDL receptor
consensus sequence. The autoradiographs were developed and
photographed.
[0170] Cells transfected with 0.2 .mu.g/ml of PHH1 or PHH11 showed
a 2-fold increase in N-SMase activity compared to mock cDNA
transfected cells. This was accompanied with a PHH1 and PHH11
concentration dependent increase in the maturation of SREBP-1 in
human hepatocytes. See FIG. 7. As shown in lanes 3-6 transfection
of cells with 5,10,20,40 .mu.g of PHH1 plasmid DNA/dish resulted in
a gradual but marked increase in the maturation of SREBP-1 as
compared to mock cDNA transfected cells (lane 1, FIG. 7). In
contrast, a marked increase in the maturation of SREBP-1 was noted
in cells transfected with 20 .mu.g/dish of PHH11 plasmid DNA (lane
9 FIG. 7) but subsided at a higher concentration. As expected form
the Examples 1-7 above, cells incubated with C-2 ceramide (.mu.M)
markedly increased the maturation of SREBP-1 (lane 2 FIG. 7). In
additional experiments we observed that increasing the time of
transfection from 8 hr to 24 hr decreased the maturation of SREBP-1
in hepatocytes. Moreover, decreasing the concentration of
CaCl.sub.2 from 2.5 M to 0.25 M was ineffective.
[0171] Northern gel analysis revealed that transfection with PHH1
and PHH11(lanes 2, 3, respectively in FIG. 8) significantly
increased the level of LDL receptor mRNA as compared to cells
transfected with mock cDNA (lane 1 FIG. 8).
[0172] In another experiment hepatocytes were incubated with
purified bacterial recombinant N-SMase. Preferred methods of making
and using the recombinant N-SMase are described in the co-pending
U.S. patent application Ser. No. 08/774,104, now issued as U.S.
Pat. No. 5,919,687. That N-SMase was subjected to western
immunoblot analysis employing antibody against SREBP-1. As shown in
FIG. 9, cells incubated with C-2 ceramide markedly increased the
maturation of SREBP-1 (lane 1). In comparison the r-N-SMase exerted
a concentration-dependent increase in the maturation of SREBP-1
(lane 2, 3, 4, 5 representing 0.4, 0.8, 2, and 4 .mu.g/ml of
r-N-SMase, respectively).
[0173] This example shows that overexpression of N-SMase or feeding
r-N-SMase to hepatocytes stimulates the maturation of SREBP-1 and
consequently an increase in the LDL receptor mRNA levels.
[0174] The Examples 1-8 above highlight a novel pathway by which
SREBP-1 maturation could be effected in a sterol independent
manner. It was found that TNF-.alpha. is capable of inducing
SREBP-1 maturation in a sterol independent manner in human
hepatocytes. These findings are not a general response to growth
factors, as they could not be recapitulated with EGF or PDGF. The
maturation, nuclear translocation, and SRE binding activity of
SREBP-1 in response to TNF-.alpha. closely paralleled the kinetics
of N-SMase activation. The effect of TNF-.alpha. on SREBP-1
maturation could be reconstituted with exogenously supplied
bacterial or human sphingomyelinase C.sub.2-ceramide but could not
be recapitulated with dihydroceramide, PL-A.sub.2, or PL-D.
[0175] In particular, Examples 1-7 show that addition of
C.sub.2-ceramide, a water soluble ceramide analog, or bacterial
sphingomyelinase mimicked the effect of TNF-.alpha. on SREBP-1
maturation. C.sub.2-ceramide and sphingomyelinase induced more
extensive SREBP-1 maturation than TNF-.alpha.. Without wishing to
be bound to theory, this observation may reflect the presence of a
regulatory event upstream of ceramide generation that is
effectively bypassed with exogenous ceramide or sphingomyelinase.
Also, the lack of apparent dose dependence observed with
TNF-.alpha. treatment might be attributable to saturable binding of
the TNF-.alpha. receptors or an internal regulatory event that
reduces the signaling capacity of the TNF-.alpha. receptors.
[0176] The present data and discussion indicate a model in which
TNF-.alpha. initiates SREBP-1 proteolysis. The model (FIG. 7) in
which there is shown TNF-.alpha. binding to one or more of its cell
surface receptors and in so doing promotes the activation of
N-SMase. N-SMase hydrolyzes membrane sphingomyelin into ceramide
and phosphocholine. Ceramide, in turn, activates a protease perhaps
CPP32 that mediates SREBP-1 maturation. According to the model, the
mature SREBP-1 then migrates into the nucleus as shown and drives
the transcription of genes with an upstream sterol regulatory
element.
[0177] The model illustrated in FIG. 7 clarifies how sterol
homeostasis can occur in the presence of increased cytosolic
sterols, which would be predicted to suppress SREBP-1 maturation.
One advantage conferred by the participation of neutral
sphingomyelinase in cholesterol homeostasis is that it is capable
of providing a short term solution to cholesterol starvation
through mobilization of plasma membrane cholesterol and can
facilitate long term compensatory mechanisms by promoting the
maturation of SREBP-1.
[0178] The model shown in FIG. 7 also shows that TNF-.alpha. is
capable of inducing SREBP-1 proteolysis independent of the presence
of sterols. Exogenously supplied sphingomyelinase and ceramide are
also capable of inducing SREBP-1 proteolysis in a time and dose
dependent manner. The kinetics of SREBP-1 maturation is consistent
with the activation of neutral sphingomyelinase by TNF-.alpha..
Furthermore, recombinant human N-SMase can also exert a time and
concentration dependent induction of SREBP-1 maturation. In
addition, anti-N-SMase antibodies block SREBP-1 maturation. These
findings indicate that neutral sphingomyelinase is necessary for
TNF-.alpha. induced, sterol independent SREBP-1 cleavage.
[0179] The present examples and discussion identify N-SMase in the
TNF-.alpha. initiated signal transduction pathway leading to
SREBP-1 maturation and provide evidence that ceramide is the second
messenger employed. Also shown is an important role for TNF-.alpha.
in the regulation of cholesterol homeostasis.
[0180] The present findings are summarized as follows. The role of
TNF-.alpha. as a mediator of SREBP-1 maturation was investigated in
human hepatocytes.
[0181] One significant aspect of the above Examples and discussion
is that ceramide stimulated SREBP-1 maturation even in the presence
of cholesterol and 25-hydroxycholesterol both of which are known
suppressers of SREBP-1 maturation. This indicates that ceramide
mediated maturation of SREBP-1 maturation is a novel, sterol
independent mechanism by which cholesterol homeostasis may be
regulated.
[0182] The following materials and methods were used as needed in
the above Examples 1-8.
[0183] 1. Materials--A continuous line of human hepatocytes
designated HH-25 were prepared from normal human tissue (18). Alpha
modified minimal essential medium was purchased from Mediatech
(Herndon, VA). Fetal bovine serum was purchased from Hyclone, Salt
Lake City, Utah. F10 media and the insulin-transferrin-selenium
supplement were purchased from Gibco-BRL (Gaithersburg, Md.). Human
recombinant EGF, PDGF and TNF-.alpha. were from Upstate
Biotechnology (Lake Placid, N.Y.). C.sub.2-ceramide
(N-acetylsphingosine) was obtained from Matreya (Pleasant Gap,
Pa.). [.sup.14C]-sphingomyclin (specific activity 50 mCi/mrnol) was
from American Radiolabeled Chemicals (St. Louis, Mo.). Anti-SREBP-1
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Sphingomyelinase from Streptomyces species and all other
redrugs were obtained from Sigma.
[0184] 2. Cell Culture--HH-25 cells were grown in alpha-minimal
essential media supplemented with 100 units/ml penicillin, 100 g/ml
streptomycin, 10 g/ml insulin, 0.1 .mu.M selenium, 5.5 .mu.g/ml
transferrin, 0.5 .mu.g/ml linoleic acid and 10% fetal bovine serum
(media A). The cells were incubated in serum free F10 media for 30
to 60 minutes prior to initiating treatment with TNF-.alpha.,
C.sub.2-ceramide or sphingomyelinase.
[0185] 3. Cellfractionation--Following treatment, the cells were
washed with 5 ml of PBS and pelleted at 1500.times.g for 10 minutes
at 4.degree. C. The pellet was stored at -70.degree. C. and lysed
in 0.5 ml buffer A (10 mM HEPES pH 7.4, 5 mM EDTA, 0.25 mM EGTA, 50
mM NaF, 7 mM .beta.-mercaptoethanol, 0.35M sucrose, 0.1% NP-40 and
protease inhibitors 1 mM PMSF, 2 .mu.g/ml aprotinin, 10 .mu.g/ml
leupeptin and 5 .mu.g/ml pepstatin). The lysate was centrifuged at
12,000.times.g for 15 minutes at 4.degree. C. to prepare a nuclear
fraction. The protein concentration of these samples was determined
by the method of Lowry et al. al. (19).
[0186] 4. Neutral Sphingomyelinase Assay-After stimulation with
TNF-.alpha. for the indicated time intervals, the cells were washed
once with 5 ml PBS and harvested. The pellet was stored frozen at
-70.degree. C. and resuspended in 0.5 ml buffer B (100 mM Tris HCl
pH 7.4, 0.1% triton X-100, 1 mM EDTA and protease inhibitors). The
cell suspension was sonicated 3 times (3 second bursts) using a
probe sonicator and centrifuged at 500.times.g at 4.degree. C. for
5 minutes. The supernatant was used as the enzyme source.
[0187] 100 .mu.g of protein was assayed for neutral
sphingomyelinase activity in a buffer consisting of 50 mM Tris HCl
pH 7.4, 0.1% triton X-100, 0.1 mg BSA, 5 mM MgCl.sub.2, and 50
moles [.sup.14C] sphingomyelin (12,000 dpm). The assay was
incubated at 37.degree. C. for 1.5 hours and terminated with the
addition of 1 ml of 10% TCA. The precipitate was pelleted
(1000.times.g at 4.degree. C. for 20 minutes) and 1 ml of the
supernatant was extracted with 1 ml anhydrous diethyl ether. 0.5 ml
of the aqueous phase was removed for liquid scintillation
counting.
[0188] 5. Immunoblotting--50 .mu.g of nuclear protein was separated
by gel electrophoresis on a 7.5% polyacrylamide gel. Gels were
calibrated by high range molecular weight markers (Bio-Rad product
#161-0303, New York, N.Y.) which were transferred to a polyvinyl
diflouride (PVDF) membrane and visualized with coomassie staining.
Rabbit polyclonal antibodies against SREBP-1 were used at 0.5
.mu.g/ml according to the instructions of the manufacturer. The
antibody was visualized with horseradish peroxidase conjugated
anti-rabbit IgG made in donkey (Amersham) using the Enhanced
Chemiluminescence (ECL) Western Blotting Detection System Kit
(Amersham). PVDF membranes were exposed to Hyperfilm ECL (Amersham)
for the indicated time. Immunoblots were quantified via
densitometry performed on a PDI densitometer scanner (model 20J7)
coupled to a SPARC IRC workstation.
[0189] 5. Indirect Immunofluorescence-Cultured HH-25 cells were
grown on coverslips and treated as described. After treatment, the
cells were washed 3.times.5 minutes with PBS containing 1 mM
MgCl.sub.2 and 0.1 mM CaCl.sub.2 (solution A). The cells were fixed
with 3% paraformaldehyde in solution A for 10 minutes and
permeabilized with 0.5% Triton X-100 in solution A for 6 minutes at
room temperature. The coverslips were then washed 3.times.5 minutes
with solution A.
[0190] Primary antibody (anti-SREBP1) was used at a dilution of 0.5
g/ml in PBS and applied for 1 hour with gentle shaking. The cells
were washed as above and a FITC conjugated anti-rabbit IgG
secondary antibody, was applied for {fraction (1/2)} hour according
to the instructions of the manufacturer. The coverslips were
washed, mounted on microscope slides and were viewed and
photographed at the indicated magnification on a Zeiss Axiovert 25
fluorescence microscope.
[0191] 6. DNA laddering assay--Cells were treated with either
TNF-.alpha., sphingomyelinase or C.sub.2-ceramide for 1 hour at
concentrations identical to those used in the SREBP-1 maturation
studies. The cells were then washed twice with minimal essential
medium and refed with media A for 6 hours. The cells were harvested
and genomic DNA was prepared as described (22). Genomic DNA was
electrophoresed and stained with ethidium bromide.
[0192] 7. Electrophoretic Mobility Shift Assays--Gel mobility shift
assays were performed as follows. Each 20 .mu.l reaction mixture
contained 8-10 .mu.g of nuclear protein plus a
.alpha.-[.sup.32P]-labeled 25-base pair oligonucleotide probe
containing the SREBP-binding site (14) in binding buffer (10 mM
Hepes, pH 7.5, 0.5 mM spermidine, 0.15 mM EDTA, 10 mM
dithiothreitol, 0.35 mM sucrose). The reaction mixture was
incubated at room temperature for 15 min and loaded directly onto a
6.5% polyacrylamide (49:0.6 acrylamide/bisacrylamide) gel in a
buffer of 25 mM Tris borate (pH 8.0), 0.25 mM EDTA. In some
experiments, antisera specific for SREBP or preimmune sera were
added to reaction mixtures to determine the composition of
protein-probe complexes. For these "supershift" assays, extracts
were incubated with 1 .mu.l of preimmune sera or an equal volume of
anti-SREBP antisera at 4.degree. C. for 30 min prior to addition of
.alpha.-[.sup.32P]-labeled probe. In all experiments, proteins were
separated by electrophoresis at 200 V for 2 h at room temperature.
Gels were dried and exposed to Kodak XAR film with intensifying
screens. Assays were repeated with nuclear extracts obtained from
three unique experiments and evaluated by phosphoimage analysis to
ensure reproducibility of results.
REFERENCES
[0193] 1) Goeddel, D. V., Aggarwal, B. B., Gray, P. W., Leung, D.
W., Nedwin, G. E., Palladino, M. A., Patton, J. S., Pennica, D.,
Shepard, H. M., Sugarman, B. J. and Wong, G. H. W. (1986) Cold
Spring Harbor Symp. Quant. Biol. 51, 597-609.
[0194] 2) Baringa, M., (1996) Science 273, 735-737. Bazzoni, F. and
Beutler, B. (1996) NEJM334, 1717-1725.
[0195] 3) Chatterjee, S., (1993) Adv. Lipid Res. 26, 25-48.
[0196] 4) Tepper, C. G., Jayadev, S., Liu, B., Bielawska, A.,
Wolff, R., Yonehara, S., Hannun, Y. A., and Seldin, M. F. (1995)
Proc. Natl. Acad. Sci. U.S.A. 92, 8443-8447.
[0197] 5) Cifone, M. G., DeMaria, R., Roncaioli, P., Rippo, M. R.
Azuma, M. Lanier, L. L., Santoni, A., and Testi, R. (1994) J. Exp.
Med. 180, 1547-1552.
[0198] 6) Okazaki, T., Bell, R. M., and Hannun, Y. A. (1995) J.
Biol. Chem. 264, 19076-19080.
[0199] 7) Mathias, S., Younes, A., Kan, C. C., Orlow, I., Joseph,
C., and Kolesnick, R. N., (1993) Science 259, 519-522.
[0200] 8) Dobrowsky, R. T., Werner, M. H., Castellino, A. M., Chao,
M. V. and Hannun, Y. A. (1994) Science 265, 1596-1599.
[0201] 9) Chan, C. G., and Ochi, A. (1995) Eur. J. Immunol. 25,
1999-2004.
[0202] 10) Kim, M. Y., Linardic, C., and Hannun, Y. A. (1991) J.
Biol. Chem. 266, 484-489.
[0203] 11) Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G.,
Coso, O. A., Gutkind, J. S., and Spiegel, S., Nature 381,
800-803.
[0204] 12) Goldstein, J. L., and Brown, M. S. (1986) Nature 343,
425-430.
[0205] 13) Dawson, P. A., Hofmann, S. L., van der Westhhuyzen, D.
R., Brown, M. S., and Goldstein, J. L. (1988) J. Biol. Chem. 263,
3372-3379.
[0206] 14) Wang, X., Sato, R., Brown, M. S., and Goldstein, J. L.
(1994) Cell 77, 53-62.
[0207] 15) Chatterjee, S., (1994) J. Biol. Chem. 269, 879-882.
[0208] 16) Chatterjee, S. (1993) J. Biol. Chem. 268, 3401-3406.
[0209] 17) Hamanaka, R., Kohno, K., Seguchi, T., Okamura, K.,
Morimoto, A., Ono, M., Ogata, J., and Kuwano, M. (1992) J. Biol.
Chem. 267, 13160-13165.
[0210] 18) Wang, X., Zelenski, N. G., Yang, J., Sakai, J., Brown,
M. S., and Goldstein, J. L. (1996) EMBO 15, 1012-1020.
[0211] 19) Mizushima, N., Koike, R., Kohsaka, H., Kushi, Y., Handa,
S., Yagita, H., Miyasaka N. (1996) FEBS Lett. 395, 267-271.
[0212] 20) Bittman, R., Kasireddy, C. R., Mattjus, P., and Slotte,
J. P. (1994) Biochemistry 33, 11776-11781.
[0213] 21) Kan, C., Ruan, Z., and Bittman, R., (1991) Biochemistry
30, 10746-10754.
[0214] 22) Clejan, S., and Bittman, R., (1984) J. Biol. Chem. 259,
10823-10826.
[0215] 23) Adam-Klages, S., Adam, D., Weigmann, K., Struve, S.,
Kolanus, W., Schneider-Mergener, J., and Kronke, M. (1996) Cell,
86, 937-947.
[0216] 24) Lawler, F. J. et al. (1998) J. Biol. Chem. 273:
5058.
[0217] 25) Shimomura, I., et al. (1998) J. Biol. Chem. 273:
35299.
[0218] 26) Brown, M. S. and J. L. Goldstein (1997) Cell 89:
331.
[0219] The invention has been described with reference to preferred
embodiments thereof. However, it will be appreciated that those
skilled in the art, upon consideration of this disclosure, may make
modifications and improvements within the spirit and scope of the
invention.
Sequence CWU 1
1
2 1 1197 DNA Homo sapiens 1 atgatgacat atcacgaaac gcgcgcgttg
gctcaaagcg acttacagca actctatgcg 60 gcacttgaaa caactgaatt
tggcgcttac tttgcgacac ccgctgatga tactttacgt 120 tttggcattg
gcgcaatcgc tacggcaaaa acggctcagg cattacaagg tgcggttgtt 180
tttggtgcgc agtcatttga tgaacaagag tacccgcagt ctgaattgat ggcgggtttt
240 tggtttgtcc ccgaagtgat ggtgaccatc gcggcagata aaatcacgtt
cggatcagat 300 accgtatctg attttacgac gtggctggcg cagttcgtgc
caaaacagcc aaatacggtg 360 accactagtc atgtgacaga tgaagtggat
tggatcgaac ggacagagaa tttgattgat 420 accttagcca tcgatcaaac
cttagccaaa gtcgtttttg gtcggcaaca gaccctgcag 480 ttatccgaca
cgttacgact ggcacaaatt attcgtgcgt tagctgagca ggcgaatacg 540
tatcatgtgg ttttaaagcg acatgatgaa ttgtttattt cagcaacacc ggaacggtta
600 gtggctatgt caggtggtca gatcgctacg gcggcggtcg ctgggacaag
ccggcgcggg 660 acggatggcg ctgacgatat cgcgttaggc gaagcgttgt
tagccagtca gaaaaaccgc 720 attgaacatc aatatgtcgt ggcaagtatc
acgacacgct tgcaagacgt gacgacgtcg 780 ctaaaggtgc cggccatgcc
aagtttactc aaaaataagc aagttcagca tttgtacaca 840 ccaattacag
gggacattgc ggcacattta agtgtgaccg cgattgttga ccgcttgcat 900
ccaacaccag cactgggtgg cgtcccacgt gaagcggccc tgtattacat tgcgacccat
960 gagaagacac ctcgtggctt gtttgcaggt cctattggct attttaccgc
agataatagt 1020 ggggaatttg tggttggcat ccgttccatg tatgtgaatc
aaacgcagcg acgagcaact 1080 ttatttgctg gtgccgggat tgtggctgac
tccgatgcgc aacaagaata tgaagaaact 1140 gggttgaaat ttgaacccat
gcggcaattg ttaaaggact acaatcatgt cgaatga 1197 2 397 PRT Homo
sapiens 2 Met Met Thr Tyr His Glu Thr Arg Ala Leu Ala Gln Ser Asp
Leu Gln 1 5 10 15 Gln Leu Tyr Ala Ala Leu Glu Thr Thr Glu Phe Gly
Ala Tyr Phe Ala 20 25 30 Thr Pro Ala Asp Asp Thr Leu Arg Phe Gly
Ile Gly Ala Ile Ala Thr 35 40 45 Ala Lys Thr Ala Gln Ala Leu Gln
Gly Ala Val Phe Gly Ala Gln Ser 50 55 60 Phe Asp Glu Gln Glu Tyr
Pro Gln Ser Glu Leu Met Ala Gly Phe Trp 65 70 75 80 Phe Val Pro Glu
Val Met Val Thr Ile Ala Ala Asp Lys Ile Thr Phe 85 90 95 Gly Ser
Asp Thr Val Ser Asp Phe Thr Thr Trp Leu Ala Gln Phe Val 100 105 110
Pro Lys Gln Pro Asn Thr Val Thr Thr Ser His Val Thr Asp Glu Val 115
120 125 Asp Trp Ile Glu Arg Thr Glu Asn Leu Ile Asp Thr Leu Ala Ile
Asp 130 135 140 Gln Thr Leu Ala Lys Val Val Phe Gly Arg Gln Gln Thr
Leu Gln Leu 145 150 155 160 Ser Asp Thr Leu Arg Leu Ala Gln Ile Ile
Arg Ala Leu Ala Glu Gln 165 170 175 Ala Asn Thr Tyr His Val Val Leu
Lys Arg His Asp Glu Leu Phe Ile 180 185 190 Ser Ala Thr Pro Glu Arg
Leu Val Ala Met Ser Gly Gly Gln Ile Ala 195 200 205 Thr Ala Ala Val
Ala Gly Thr Ser Arg Arg Gly Thr Asp Gly Ala Asp 210 215 220 Asp Ile
Ala Leu Gly Glu Ala Leu Leu Ala Ser Gln Lys Asn Arg Ile 225 230 235
240 Glu His Gln Tyr Val Val Ala Ser Ile Thr Thr Arg Leu Gln Asp Val
245 250 255 Thr Thr Ser Leu Lys Val Pro Ala Met Pro Ser Leu Leu Lys
Asn Lys 260 265 270 Gln Val Gln His Leu Tyr Thr Pro Ile Thr Gly Asp
Ile Ala Ala His 275 280 285 Leu Ser Val Thr Ala Ile Val Asp Arg Leu
His Pro Thr Pro Ala Leu 290 295 300 Gly Gly Val Pro Arg Glu Ala Ala
Leu Tyr Tyr Ile Ala Thr His Glu 305 310 315 320 Lys Thr Pro Arg Gly
Leu Phe Ala Gly Pro Ile Gly Tyr Phe Thr Ala 325 330 335 Asp Asn Ser
Gly Glu Phe Val Val Gly Ile Arg Ser Met Tyr Val Asn 340 345 350 Gln
Thr Gln Arg Arg Ala Thr Leu Phe Ala Gly Ala Gly Ile Val Ala 355 360
365 Asp Ser Asp Ala Gln Gln Glu Tyr Glu Glu Thr Gly Leu Lys Phe Glu
370 375 380 Pro Met Arg Gln Leu Leu Lys Asp Tyr Asn His Val Glu 385
390 395
* * * * *
References