U.S. patent application number 14/711381 was filed with the patent office on 2015-09-24 for use of delta tocopherol for the treatment of lysosomal storage disorders.
The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health & Human Services, The United States of America, as represented by the Secretary, Dept. of Health & Human Services. Invention is credited to Christopher P. AUSTIN, Ke LIU, Juan Jose MARUGAN, Noel Terrence SOUTHALL, Wei ZHENG.
Application Number | 20150265571 14/711381 |
Document ID | / |
Family ID | 44629822 |
Filed Date | 2015-09-24 |
United States Patent
Application |
20150265571 |
Kind Code |
A1 |
ZHENG; Wei ; et al. |
September 24, 2015 |
USE OF DELTA TOCOPHEROL FOR THE TREATMENT OF LYSOSOMAL STORAGE
DISORDERS
Abstract
This disclosure relates generally to the treatment of lysosomal
storage disorders. Specifically, the disclosure relates to a novel
use of .delta. tocopherol in the treatment of diseases and
conditions related to lysosomal storage disorders. Included in the
present disclosure is a method for the modulation of cholesterol
recycling. Further, the disclosure relates to conditions such as
Niemann-Pick type C disease, Farber disease, Niemann-Pick type A
disease, Wolman disease and Tay Sachs disease. Further included in
the present disclosure is a method for treating lysosomal storage
disorders comprising the administration of .delta. tocopherol.
Further included in the present disclosure is a method for treating
lysosomal storage disorders comprising the administration of
.delta. tocopherol in combination with cyclodextrin to a patient in
need thereof.
Inventors: |
ZHENG; Wei; (POTOMAC,
MD) ; MARUGAN; Juan Jose; (GAITHERSBURG, MD) ;
LIU; Ke; (CAMBRIDGE, MD) ; SOUTHALL; Noel
Terrence; (CHEVY CHASE, MD) ; AUSTIN; Christopher
P.; (POTOMAC, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health & Human Services |
ROCKVILLE |
MD |
US |
|
|
Family ID: |
44629822 |
Appl. No.: |
14/711381 |
Filed: |
May 13, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13810774 |
Jan 17, 2013 |
9044451 |
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PCT/US2011/044590 |
Jul 19, 2011 |
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14711381 |
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61365712 |
Jul 19, 2010 |
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Current U.S.
Class: |
514/458 |
Current CPC
Class: |
A61K 31/496 20130101;
A61P 43/00 20180101; A61K 31/724 20130101; A61K 31/496 20130101;
A61K 31/724 20130101; A61K 31/36 20130101; A61K 36/185 20130101;
A61K 45/06 20130101; A61K 31/355 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 31/36 20130101; A61K 2300/00 20130101; A61K 36/185
20130101; A61K 31/355 20130101 |
International
Class: |
A61K 31/355 20060101
A61K031/355 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] The instant application was made with government support,
the government has certain rights in this invention.
Claims
1. A method for treating a lysosomal storage disorder comprising
administering to a subject in need thereof a pharmaceutically
effective composition comprising .delta. tocopherol.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/810,774, filed Jan. 17, 2013, which is a 35 U.S.C. .sctn.371
application of PCT/US2011/044590, filed Jul. 19, 2011, which claims
the benefit of U.S. Provisional Application No. 61/365,712, filed
Jul. 19, 2010, all of which are incorporated herein by reference in
their entirety.
TECHNICAL FIELD
[0003] This disclosure relates generally to the treatment of
lysosomal storage disorders. Specifically, the disclosure relates
to a novel use of delta tocopherol in the treatment of diseases and
conditions related to lysosomal storage disorders. Included in the
present disclosure is a method for the modulation of cholesterol
recycling. Further, the disclosure relates to conditions such as
Niemann-Pick type C disease, Farber disease, Niemann-Pick type A
disease, Wolman disease and Tay Sachs disease. Further included in
the present disclosure is a method for treating lysosomal storage
disorders comprising the use of .delta.-tocopherol. Further
included in the present disclosure is a method for treating
lysosomal storage disorders comprising the use of
.delta.-tocopherol in combination with cyclodextrin.
BACKGROUND
[0004] Tocopherols are natural products widely used as vitamin E
for human health, cosmetic ingredients and food antioxidants. The
main source of tocopherols is the deodorized distillate produced by
the manufacture process of edible oil. The main constituents of the
tocopherols extracted from deodorized distillate are .alpha.,
.beta., .gamma., and .delta.-tocopherols. Their molecular
structures have a common core ring, differing only in the number of
attached methyl groups. Although the structures are quite similar,
it is known in the art that .alpha.-tocopherol has the highest
biopotency among all the isomeric forms. Current research involving
tocopherols is focused on .alpha.-tocopherol, emphasizing
.alpha.-tocopherol's use as an antioxidant.
[0005] In normal physiological cell function, lysosomal enzymes
break down macromolecules in cells. Normal metabolism allows the
cells to remove expended metabolites. Defects or deficiencies of
lysosomal enzymes or other lysosomal components can result in the
accumulation of undegraded metabolites. These defects or
deficiencies result in Lysosomal Storage Disorders (LSDs).
[0006] Cholesterol is an essential component of the cell membrane
and plays an important role in maintaining integrity and fluidity
of the cell membrane with additional lipids. The appropriate
proportion and makeup of cholesterol in the cell membrane is
important for regulation of cell signaling, receptor function, ion
permeability and endocytosis. In addition, cholesterol is the
precursor molecule for synthesis of bile acids, Vitamin D and
steroid hormones. Cholesterol concentration is regulated on two
levels; in the body as a whole and intracellularly. Cholesterol is
primarily synthesized in cells and transported in blood by lipid
binding proteins. While the mechanisms of cholesterol transport in
blood and cholesterol synthesis in cells are well characterized,
the many details of free cholesterol trafficking in cells remain
unclear. Cholesterol enters cells through low-density lipoprotein
(LDL) receptors via endocytosis or cholesterol is de novo
synthesized inside cells. The internalized cholesterol esters are
hydrolyzed by acid lipase to form free cholesterols in late
endosomes/lysosomes that are delivered to NPC1 and NPC2 proteins
for further processing. Cholesterol may be transported to other
proteins and/or to membrane microdomains such as lipid rafts and
other vesicles. It is known that most of these free cholesterols
recycle back to cell membrane; some of them efflux out of cells,
and some of them move to ER for storage after esterification.
[0007] Lysosomal storage diseases (lysosomal storage disorders) are
a group of .about.50 diseases with a common feature of lysosomal
accumulation of macromolecules such as lipids or glycoproteins in
patient cells. The diseases are caused by inherited genetic
mutations that result in deficiency of lysosomal enzyme or protein.
Once the hydrolysis or transport of macromolecules such as lipids
and glycoproteins is reduced by a mutation of an enzyme or protein
in lysosome, they are accumulated which causes the enlargement of
lysosomes as well as malfunction of lipids recycling and
utilization in cells. Degeneration or death of affected cells
occurs in certain tissues which are varied in different lysosomal
storage diseases. The hepatosplenomegaly and neuronal degeneration
are common features of many lysosomal storage diseases.
[0008] One identified LSD is Niemann-Pick type C disease (NPCD), an
autosomal recessive disease with an estimated incidence of
1:150,000. This disease is characterized by a lysosomal
accumulation of unesterified cholesterol and other lipids in many
cell types, probably due to impairment of the retrograde transport
of lipids from the late endosomes and/or lysosomes to the plasma
membrane or endoplasmic reticulum (ER). The clinical manifestation
includes hepatosplenomegaly, vertical gaze palsy, and progressive
neurodegeneration characterized by cerebellar ataxia, bulbar
dysfunction, and variable degrees of cognitive decline. Most often,
the onset of symptoms occurs in early childhood, leading to death
within a decade. Two human genes have been identified for NPCD:
mutations in NPC1 are causative in nearly 95% of all NPC cases,
while mutations in NPC2 account for the rest. Over 230 mutations
have been identified in the NPC1 gene, nearly 2/3 of which are
missense mutations. NPC1 is a highly-conserved integral membrane
protein with 13 transmembrane domains, while the NPC2 gene encodes
a small soluble protein. Both proteins are located in late
endosomes and lysosomes. Recent structure and biochemical studies
have revealed that NPC1 and NPC2 bind to cholesterol in opposite
orientations, leading to a working model of these two proteins:
NPC2 captures the cholesterol liberated from LDL, and then
transfers it to the NPC1 on the membrane for subsequent trafficking
out of the lysosomes. In NPCD patients, the cholesterol trafficking
out of late endosomes and lysosomes is blocked resulting in an
accumulation of free cholesterols in late endosomes and lysosomes.
This accumulation of cholesterol eventually affects the lysosomal
homeostasis, triggering also the accumulation of other lysosomal
substrates such us glycosphingolipids. Moreover, glycosphingolipids
accumulation in sphingolipid storage diseases (SLSD) as result of a
defective lysosomal hydrolase or activator protein also affects
cholesterol homeostasis elevating cellular cholesterol levels.
[0009] Currently, there is no cure for NPCD and all established
therapies are for relief of symptoms with limited efficacy.
Clinical trials with Miglustat (N-butyldeoxynojirimycin,
Zavesca.RTM.) are in progress with favorable preliminary results.
Miglustat is an iminosugar that inhibits glucosylceramide synthase,
an enzyme responsible for a series of reactions that lead to the
synthesis of most glycosphingolipids (GSL). This drug crosses the
blood-brain barrier, reduces the substrate availability for
synthesizing GSL, and thus reduces GSL accumulation in the brain.
This substrate reduction effect seems to relieve the symptoms in
NPC patients. Recently, several other compounds including
allopregnanolone, T0901317, curcumin and cyclodextrin have also
been reported to have beneficial effects on NPC cell or animal
models. The full therapeutic benefits of these drugs still need to
be evaluated. Despite these studies, an effective treatment for NPC
patients is still an unmet medical need.
[0010] Recent advances in technologies for high-throughput
screening (HTS) have made it possible to screen the cell-based NPC
disease model against compound libraries to identify lead compounds
for the new drug development. Two such attempts have been made
which applied the filipin cholesterol staining assay in NPC
patient-derived skin fibroblasts. Though some active compounds were
reported from these screens, none of them are useful for the
further drug development. In addition, the drug development process
usually takes an average of 8-12 years costing on the order of
hundreds of millions of dollars. The failure rate of drug
development is very high due to unpredictable compound toxicity in
human and disproportional efficacy between human and model systems.
The present invention is directed toward overcoming the problems
discussed above.
SUMMARY OF THE EMBODIMENTS
[0011] We identify, in one embodiment, the use of
.delta.-tocopherol for the treatment of LSDs. Further, we identify
the use of .delta.-tocopherol in reducing the accumulation of free
cholesterols. Additionally we teach the use of .delta.-tocopherol
for the reduction of the size of enlarged lysosomes.
[0012] In one embodiment, we disclose a method for treating
lysosomal storage disorder comprising administering to a patient in
need thereof a pharmaceutically effective amount of a .delta.
tocopherol. In one embodiment, we disclose a pharmaceutically
effective amount of .delta. tocopherol of 50 uM plasma and/or
tissue concentration. In one embodiment, we disclose a
pharmaceutically effective amount of .delta. tocopherol of less
than 50 uM. In one embodiment, we disclose a pharmaceutically
effective amount of .delta. tocopherol of less than 40 uM. In one
embodiment, we disclose a pharmaceutically effective amount of
.delta. tocopherol of less than 30 uM. In one embodiment, we
disclose a pharmaceutically effective amount of .delta. tocopherol
of less than 20 uM. In one embodiment, we disclose a
pharmaceutically effective amount of .delta. tocopherol of less
than 15 uM. In one embodiment, we disclose a pharmaceutically
effective amount of .delta. tocopherol of less than 10 uM. In one
embodiment, we disclose a pharmaceutically effective amount of
.delta. tocopherol of less than 7.8 uM. In one embodiment, we
disclose a pharmaceutically effective amount of .delta. tocopherol
without significant cytotoxicity.
[0013] The disclosure further provides a method for treating a
mammal having a lysosomal storage disorder by administering a
pharmaceutical composition comprising a dosage of a
.delta.-tocopherol effective to treat the lysosomal storage
disorder; and a beta-cyclodextrin compound, in which the dosage of
the .delta.-tocopherol compound is less than about 1000 IU/kg per
day. In particular embodiments, the dosage of .delta.-tocopherol is
less than 500 IU/kg per day, less than 50 IU/kg per day, less than
5 IU/kg per day, or less than 0.5 IU/kg per day. In one embodiment
of the methods of treatment, the disclosure provides a method for
treating a mammal having a lysosomal storage disorder by
administering a pharmaceutical composition comprising a dosage of a
.delta.-tocopherol compound, wherein the dosage of the
.delta.-tocopherol compound is less than an accepted dose of
.alpha.-tocopherol. In one embodiment of the methods of treatment,
the disclosure provides a method for treating a mammal having a
lysosomal storage disorder by administering a pharmaceutical
composition comprising a dosage of a .delta.-tocopherol compound,
wherein the dosage of the .delta.-tocopherol is less than about
1000 IU/kg per day. In particular embodiments, the dosage of
.delta.-tocopherol is less than 500 IU/kg per day, less than 50
IU/kg per day, less than 5 IU/kg per day, or less than 0.5 IU/kg
per day.
[0014] We further disclose the treatment with .delta.-tocopherol,
wherein the lysosomal storage disorder is selected from the group
consisting of Niemann-Pick Disease, Mucopolysaccharidoses disorder,
and Neuronal Ceroid Lipofuscinoses. We further disclose the
treatment with .delta.-tocopherol, wherein the lysosomal storage
disorder is selected from the group consisting of Niemann-Pick type
C disease, Farber disease, Niemann-Pick type A disease, Wolman
disease and Tay Sachs disease. In one embodiment, the composition
of .delta. tocopherol excludes .alpha., .beta., and .gamma.
tocopherol for the treatment of LDS. In one embodiment, the
composition of .delta.-tocopherol excludes .alpha., .beta., and
.gamma. tocopherol for the treatment of NPCD. In one embodiment the
composition of .delta. tocopherol excludes any one of .alpha.,
.beta., and .gamma. tocopherols, and/or any combination thereof,
for the treatment of LDS. In one embodiment, the composition of
.delta. tocopherol comprises cyclodextrin for the treatment of LDS.
In one embodiment the composition of .delta. tocopherol excludes
any one of .alpha., .beta., and .gamma. tocopherols, and/or any
combination thereof, for the treatment of LDS, and comprises
cyclodextrin for the treatment of LDS. In one embodiment, the
composition of .delta.-tocopherol excludes .alpha., .beta., and
.gamma. tocopherol for the treatment of NPCD. We disclose a method
for reducing cholesterol accumulation in cells comprising
administering a composition of .delta. tocopherol. We further
disclose a method for reducing cholesterol accumulation in cells
comprising administering a composition of .delta. tocopherol which
excludes any one of .alpha., .beta., and .gamma. tocopherol or
combinations thereof. We further disclose a method for treatment of
a disease characterized by increased cholesterol accumulation
wherein the cholesterol accumulation in cells is reduced by a
method comprising administering a composition of .delta. tocopherol
which excludes any one of .alpha., .beta., and .gamma. tocopherol
or combinations thereof to a patient in need.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1A) The influence of delta-tocopherol on free
cholesterol (green lines, right y-axis) and ATP level (red lines,
left y-axis) in WT and NPC fibroblasts. On day 0 cells were seeded
in 1536-well plates. On day 1, 3, or 5, delta-tocopherol dissolved
in DMSO was added to the cells at various final concentrations as
indicated in the figure. On day 7, the cells were examined for free
cholesterol and ATP levels by Amplex Red cholesterol assay and
ATP-lite assay, respectively. Free cholesterol and ATP levels of
.delta.-tocopherol treated cells were normalized against those of
DMSO treated cells of the same type and expressed as the percentage
of DMSO treated cells. Error bars represented standard error of n=8
values. FIG. 1B) Filipin staining of the NPC cells treated with
.delta.-tocopherol. On day 0, cells were seeded on black
clear-bottom 96-well plates at 500 cells/well. On day 1, culture
medium was replaced with medium containing different concentrations
of .delta.-tocopherol or matching DMSO. On day 4, media were
replaced again with ones containing fresh compounds. On day 6,
filipin staining (blue) as well as CellMask staining (red) was
performed as described in the method section. FIG. 1C) Lysotracker
staining of the NPC cells treated with .delta.-tocopherol. Cells
were treated similarly as in FIG. 1B and stained by Lysotracker Red
and Hoechst as described in the method session.
[0016] FIG. 2A) Structures of vitamin E analogs. FIG. 2B) The
influence of different forms of vitamin E on free cholesterol level
of NPC cells. FIG. 2C) The influence of other vitamins and
anti-oxidants on the free cholesterol level of NPC cells. FIG. 2D)
The influence of various compounds in cellular ATP level of NPC
cells. FIG. 2E) Delta-tocopherol treatment did not change the
expression level of NPC1 and HMG-CoA reductase in NPC
fibroblasts.
[0017] FIG. 3. Lysotracker staining of lysosomal storage disease
cells treated with .delta.-tocopherol. Cells were treated similarly
as in FIG. 1 and stained by Lysotracker Red and Hoechst as
described in the method session.
[0018] Effects of .delta.-tocopherol and .alpha.-tocopherols on
(FIG. 4A) free cholesterol, (FIG. 4B) cholesterol ester and (FIG.
4C) sphingomyelin in NPC cells. NPC or WT cells were treated with
DMSO, 80 .mu.M .alpha.-tocopherol or 40 uM .delta.-tocopherol for 5
days. Cell pellets were collected and extracted for lipids. FC EC
and sphingomyelin levels were measured by GC/MS. (FIG. 4D) Effects
of .delta.-tocopherols and .alpha.-tocopherols on cholesterol
efflux.
[0019] FIG. 5A) Filipin staining of the WT or NPC MSC cells. FIG.
5B) NPC cells treated with .delta.-tocopherol or DMSO were
co-stained with filipin and the antibody against neuronal beta III
tubulin. Cells were treated and stained as described in the method
section.
[0020] FIG. 6. A model for mechanism of action for tocopherols.
[0021] FIG. 7. A model for mechanism of action for tocopherols.
[0022] FIG. 8. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Farber disease
fibroblasts are in the bottom panel. Drugs (80 .mu.M
alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 40X objectives.
[0023] FIG. 9. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Niemann Pick Type A
disease (NPA) fibroblasts are in the bottom panel. Drugs (80 .mu.M
alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 40X objectives.
[0024] FIG. 10. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Wolman6144 disease
fibroblasts (from two patients) are in the bottom panel. Drugs (80
.mu.M alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 40X objectives.
[0025] FIG. 11. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Wolman11851 disease
fibroblasts (from two patients) are in the bottom panel. Drugs (80
.mu.M alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 40X objectives.
[0026] FIG. 12. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Tay Sachs disease
fibroblasts are in the bottom panel. Drugs (80 .mu.M
alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 40X objectives.
[0027] FIG. 13. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Farber disease
fibroblasts are in the bottom panel. Drugs (80 .mu.M
alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 20.times. objectives.
[0028] FIG. 14. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Niemann Pick Type A
disease (NPA) fibroblasts are in the bottom panel. Drugs (80 .mu.M
alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 20.times. objectives.
[0029] FIG. 15. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Wolman6144 disease
fibroblasts (from one patient) are in the bottom panel. Drugs (80
.mu.M alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 20.times. objectives.
[0030] FIG. 16. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Wolman11851 disease
fibroblasts (from one patient) are in the bottom panel. Drugs (80
.mu.M alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 20.times. objectives.
[0031] FIG. 17. Lysotracker staining images from WT (wild type)
control cells are in the top panel and from Tay Sachs disease
fibroblasts are in the bottom panel. Drugs (80 .mu.M
alpha-tocopherol, 40 .mu.M delta-tocopherol and 300 .mu.M
methyl-beta-cyclodextrin) were treated for four days in cell
culture. 20.times. objectives.
[0032] FIG. 18. Control WT cells treated with 80 .mu.M
alpha-tocopherol and 40 .mu.M delta-tocopherol for four days. Top
panel with red emission is for total lipids including the polar
lipids such as phospholipids. Bottom panel is with emission for
non-polar lipids including cholesteryl ester and triglycerides.
[0033] FIG. 19. Images of Nile red staining in Farber disease cells
treated with 80 .mu.M alpha-tocopherol and 40 .mu.M
delta-tocopherol for four days. Top panel with red emission is for
total lipids including the polar lipids such as phospholipids.
Bottom panel is with emission for non-polar lipids including
cholesteryl ester and triglycerides.
[0034] FIG. 20. Images of Nile red staining in NPA disease cells
treated with 80 .mu.M alpha-tocopherol and 40 .mu.M
delta-tocopherol for four days. Top panel with red emission is for
total lipids including the polar lipids such as phospholipids.
Bottom panel is with emission for non-polar lipids including
cholesteryl ester and triglycerides.
[0035] FIG. 21. Images of Nile red staining in Wolman6144 disease
cells (from one patient) treated with 80 .mu.M alpha-tocopherol and
40 .mu.M delta-tocopherol for four days. Top panel with red
emission is for total lipids including the polar lipids such as
phospholipids. Bottom panel is with emission for non-polar lipids
including cholesteryl ester and triglycerides.
[0036] FIG. 22. Images of Nile red staining in Wolman11851 disease
cells (from one patient) treated with 80 .mu.M alpha-tocopherol and
40 .mu.M delta-tocopherol for four days. Top panel with red
emission is for total lipids including the polar lipids such as
phospholipids. Bottom panel is with emission for non-polar lipids
including cholesteryl ester and triglycerides.
[0037] FIG. 23. Images of Nile red staining in Tay Sachs disease
cells treated with 80 .mu.M alpha-tocopherol and 40 .mu.M
delta-tocopherol for four days. Top panel with red emission is for
total lipids including the polar lipids such as phospholipids.
Bottom panel is with emission for non-polar lipids including
cholesteryl ester and triglycerides.
[0038] FIG. 24. Combination therapy of vitamin E and cyclodextrin.
In Wolman disease cells, the combination sub-effective
concentrations of alpha-tocopherol (20 uM) or delta-tocopherol (10
.mu.M) with 10 .mu.M MBCD (methyl-beta-cyclodextrin) showed
cholesterol reduction effect as the sole therapy with 40 .mu.M
delta-tocopherol and 80 .mu.M alpha-tocopherol. MBCD at 0.3 mM
showed the time dependent reduction on cholesterol level in these
cells. These results indicated the synergetic or additive effect
for delta-tocopherol and cyclodextrin for the treatment of
lysosomal storage diseases. This drug combination therapy has
advantages including reduced dosages for both drugs (to reduce
potential adverse effects) and enhanced therapeutic effect (because
two drugs have different mechanism of action).
[0039] Unless otherwise indicated, all numbers expressing
quantities of ingredients, dimensions reaction conditions and so
forth used in the specification and claims are to be understood as
being modified in all instances by the term "about".
[0040] In this application and the claims, the use of the singular
includes the plural unless specifically stated otherwise. In
addition, use of "or" means "and/or" unless stated otherwise.
Moreover, the use of the term "including", as well as other forms,
such as "includes" and "included", is not limiting. Also, terms
such as "element" or "component" encompass both elements and
components comprising one unit and elements and components that
comprise more than one unit unless specifically stated
otherwise.
DETAILED DESCRIPTION
[0041] We disclose .delta.-tocopherol for the treatment of LSDs.
Surprisingly, .delta.-tocopherol is the most potent of the eight
isoforms of vitamin E for the reduction of cholesterol accumulation
in NPCD fibroblasts. We disclose a potency with delta tocopherol 6
to 8 times higher than with .alpha.-, .beta.- and
.gamma.-tocopherols (EC.sub.50 values between 60-80 .mu.M). It has
been reported that in human plasma .alpha.-tocopherol is the most
abundant vitamin E vitamin with a concentration in the range of 26
.mu.M. The concentrations for .beta.- and .gamma.-tocopherols are
much less (.about.1-3 .mu.M) whereas the plasma concentration for
.delta.-tocopherol is the lowest at the tracer value (.about.0.1
.mu.M). We disclose .delta.-tocopherol for the treatment of LSDs.
.delta.-tocopherol has a low physiological concentration in the
body and is significantly potent in reduction of cholesterol
accumulation.
[0042] Vitamin E, as disclosed in the art, generally refers to a
tocopherol; however, other vitamers or isoforms exist, which
include .alpha.-, .beta.-, .gamma.- and .delta.-isoforms. The
differences in .alpha.-, .beta.-, .gamma.- and .delta.-isoforms
relies on the position and number of methyl groups on the phenol
ring, in addition to the corresponding tocotrienols having three
double bonds in the side chain. The function of vitamin E is
thought to be through its antioxidant effects in cell membrane by
reducing free radicals critical for the structural integrity of
cells. As a lipid-soluble molecule, vitamin E is believed to insert
into the cell membrane for its function. The plasma concentration
of vitamin E is one per 2000-3000 lipids and is easily depleted if
it is not regenerated or is deficient in diet. The deficiency of
vitamin E presents symptoms primarily in neuronal system including
ataxia, dysarthria, hyporeflexia and reduced vibration sense as
well as cardiomyopathy and retinitis pigmentosa. On other hand,
high doses of vitamin E have been used and propose for the
treatment of neurological disorders. The potential link of vitamin
E deficiency with cancer and inflammation has been reported with
regard to the antioxidant scavenging activity of vitamin E.
[0043] Lysosomal storage diseases include many disorders such as
mucopolysaccharidosis, mucolipidosis, spingolipidosis, GM2
gangliosidosis, GM2 activator protein deficiency, Niemann-Pick
disease, Gaucher's disease, Fabry's disease, Farber's disease,
Metachromatic leukodystrophy, mucosulfatidosis, Krabbe's disease,
Sphingolipid activator protein deficiencies, Lysosomal acid lipase
deficiency, Wolman's disease, Cholesterol ester storage disease,
Cerebrotendious xanthomatosis, Neuronal ceroid lipofuscinosis,
oligosaccharidosis and related disorders, and lysosomal transport
defects.
[0044] Blood cholesterol level of cholesterol is well regulated
wherein .about.70% cholesterol is synthesized by the body and
.about.30% of it is absorbed/reabsorbed from the intestine.
Cholesterol is a vital structural component in cell membrane, as
cellular cholesterol homeostasis is important to maintain a variety
of cellular functions. Cholesterol is usually bound to lipid
binding proteins in blood and delivered to cells by LDL. The
endocytosis formed coated vesicles containing LDL reach the late
endosome/lysosome and the free cholesterols are released from LDL
by acid lipase. The free cholesterol is then delivered to NPC2 and
NPC1 proteins located in late endosome/lysosome. Free cholesterol
is then moved to the trans-Golgi network (TNG) for efflux through
cell membrane and/or to ER for esterification for normal
intracellular cholesterol storage. Mutations in NPC1/2 proteins
result in the malfunction in this cholesterol trafficking pathway
in cells with the consequence of free cholesterol accumulation in
late endosome/lysosome. This cholesterol recycling malfunction
subsequently results in the accumulation of other membrane lipids
in the NPC cells. In addition, it is well known that cholesterol
and sphingolipids accumulation induce oxidative stress. Thus, in
the case of NPC oxysterols accumulation has been link with the
apoptotic triggering. In brain, loss of Purkinje neurons in the
cerebellar cortex due to neuronal degeneration is most predominant
in NPCD. Purkinje neurons rely on the cholesterols synthesized in
astrocytes which are delivered to them. Deficiency in NPC1 or NPC2
proteins may block the cholesterol movement from late
endosome/lysosome to other compartments in cells which impairs the
critical lipid recycling/movement (including many other lipids) and
eventually results in cell death. We disclose a correction of
cholesterol movement malfunction in NPC cells with an
activation/enhancement of an alternative cholesterol recycling
pathway as a new therapeutic strategy to treat the deadly NPCD. In
addition, the antioxidative effect of .delta.-tocopherol should
have an impact in reducing the oxysterols levels and therefore
decreasing the apoptotic signal as part of the overall proposed NPC
treatment. Similar effects in reducing the oxidative stress
observed in other LSD should being considered.
[0045] We disclose tocopherols in NPC cells as well as in other LSD
cells. In normal conditions, cholesterol recycling/movement in the
membrane mainly relies on the NPC1/NPC2 protein dependent pathway.
The cholesterol influx and efflux is balanced maintaining normal
cellular functions. These functions include the rapid
endocytic/exocytic processes and lipid movement/recycling in the
membrane system. The continuous lipid movements in the plasma
membrane and in intracellular compartments are critical for
maintaining the proper functions of cellular signaling pathways.
After the treatment with .delta.-tocopherols inserted into the
membranes, including the plasma membrane as well as the membranes
in intracellular organelles, the membrane lipid movement is
slightly enhanced accounting for the slight reduction of free
cholesterol in wild type cells caused by an increase in cholesterol
efflux. The resultant reduction in cholesterol esters is probably
due to the increasing need for free cholesterol. In NPC cells, the
mutations in NPC1 and NPC2 proteins result in the cessation of
cholesterol movement in the membrane system as well as the other
lipids. The cholesterol efflux is significantly reduced and free
cholesterol accumulates in lysosome. With .delta.-tocopherol
treatment, the accumulated free cholesterols in late
endosome/lysosome are reduced which are mediated by an alternative
NPC1/NPC2-independent pathway. This alternative lipid movement
pathway is stimulated/enhanced by insertion of .delta.-tocopherols
into the membrane that led to an increase in cholesterol efflux.
Cholesterol efflux via lipid rafts and/or other vesicle are
candidates for this alternative lipid movement pathway. This model
explains the effect of .delta.-tocopherols on the reduction of
enlarged lysosome size in four types of LSDs as the enhanced lipid
movement may clean the accumulated lipids in late endosome and
lysosome.
[0046] Free cholesterol accumulation in cells is a hallmark of NPC
disease. The Amplex red cholesterol assay was used to screen an
approved drug library in the NPC patient-derived skin fibroblasts
that have free cholesterol accumulation in late endosome and
lysosome. Surprisingly, we disclose .delta.-tocopherol.
.delta.-tocopherol significantly reduced the cholesterol amount in
the NPCD fibroblasts. This cholesterol reduction effect was
concentration dependent and proportional to the time of compound
treatment (2 to 5 days) to the cells (FIG. 1a). We then show
.delta.-tocopherol in the filipin cholesterol staining assay, a
method used clinically for NPCD diagnosis. We disclose the
cholesterol reduction effect of .delta.-tocopherol and free
cholesterol accumulation in NPCD patient cells was significantly
reduced (FIG. 1b). Lysosome size is usually enlarged in the NPCD
cells using a lysotracker staining assay. As shown in FIG. 1c, the
enlarged lysosome size in NPCD fibroblasts was decreased and
shrunken to the size similar to the wild type cells. These effects
on free cholesterol amount, filipin cholesterol staining, and
lysotracker staining observed for .delta.-tocopherol were more
significant and potent than any of the other vitamin E isoforms.
The effects of cholesterol reduction and lysosome size decrease by
.delta.-tocopherol in the NPC fibroblasts required a compound
treatment for 2 to 5 days with a significant effect after the 4-5
day treatment. .delta.-tocopherol at 40 .mu.M was more potent than
.alpha.-tocopherol at 80 .mu.M.
[0047] We disclose .delta.-tocopherol in 9 additional fibroblast
primary cells derived from different NPCD patients using above
assays to examine the potential variability in the cells derived
from different NPCD patients. No significant difference was found
in these cells and the effects on cholesterol reduction and
lysosome size decrease were confirmed in all of these patient
cells. Similar to the initial experimental results, the effect of
.delta.-tocopherol was more potent than that of .alpha.-tocopherol
in all these cells.
[0048] In order to examine if the cholesterol reduction effect of
tocopherols was related to an antioxidant effect, we tested a
potent antioxidant agent N-acetyl cysteine (NAc) in the same set of
assays. We found that NAc did not have any effect on the
cholesterol reduction nor lysosome size decrease (FIG. 2c),
indicating that antioxidant effect of vitamin E may not account for
the effects we observed on these NPCD cells. We also found that the
other lipid soluble and water soluble vitamins including vitamin D,
vitamin K, and vitamin C were not active in these assays further
teaching that the effects of .delta.-tocopherol on cholesterol
reduction and lysosome size decrease were specific and were not
generalized in other vitamins (FIG. 2c). We disclose that the
compound's cytotoxicity could interfere with these cell-based
assays that often appeared as the false positives for cholesterol
reduction and lysosome size decrease. We show that the cytotoxicity
of compounds with the cell/nuclear dye co-staining methods and an
ATP content assay in parallel with the cholesterol and lysotracker
assays. Cytotoxicity was not present at the effective concentration
of these .delta.-tocopherol (FIG. 2d). .delta.-tocopherol, at a
concentration of 50 .mu.M or less did not show significant
cytotoxicity after up to a 5-day compound treatment. We found that
the impure compound preparation in combination with too high of a
concentration of tocopherols could lead to the significant
cytotoxicity in both wild type and NPCD fibroblasts. Although the
tocotrienol effect on these NPCD cells were observed, they were
more toxic in our assays and the additional compound purification
(to 99%).
[0049] It has been reported that the inhibition of cholesterol de
novo synthesis and increase in NPC protein expression might correct
the phenotype in these NPCD cells. In addition, the over expression
of Rab9 or acid sphingomyelinase has been reported to reduce the
cholesterol accumulation in the NPCD cells. We found that neither
the expression of HMG-CoA reductase, a key enzyme in the
cholesterol synthesis pathway, nor that of NPC-1 protein was
changed by the .delta.-tocopherol treatment (FIG. 2d). The activity
of HMG-CoA reductase or acid sphingomyelinase was not changed as
well. The Rab9 and acid sphingomyelinase protein levels remained
same in these cells after the treatment with the .delta.-tocopherol
(FIG. 2d). We therefore disclose that these proteins may not be
directly involved in the mechanism of action of the
.delta.-tocopherol treatment as it relates to cholesterol reduction
and lysosome size decrease. Thus, we further disclose a pathway for
lipid recycling/membrane movement in the membrane system which is
enhanced by the .delta.-tocopherol treatment. Through this enhanced
lipid recycling pathway, the clearance of the accumulated free
cholesterols in lysosome occurs resulting in the decrease in
lysosome size in these NPCD cells.
[0050] Skin fibroblasts derived from patients with lysosomal
storage diseases are commonly used for clinic diagnosis. They are
also readily available from Coriell Cell Repository and can be
passaged up to 20-30 times in cell culture. Although not all the
patient derived fibroblasts exhibit lysosomal storage of
macromolecules, the lipid storage and enlargement of lysosome size
are showed in fibroblasts from patients with Farber, Niemann-Pick
type A, Wolman, and Tay Sachs disease. We found that the
enlargement of lysosome size in these four types of fibroblasts can
be measured by the lysotracker dye staining method, similarly to
that used in the cells obtained from patients with Niemann-Pick
type C disease. These patient derived fibroblasts can be used as
the disease models for drug testing. Farber, Niemann-Pick Type A,
Wolman and Tay Sachs diseases caused accumulation of ceramide,
sphingomyelin, cholesterol-ester and GM2 gangliosides accumulation
in lysosomes, respectively. (See FIGS. 8-23).
[0051] Four other types of patient derived skin fibroblasts were
tested in the lysotracker assay that visualizing the acidic
compartments inside cells including late endosome and lysosome. We
disclose that .delta.-tocopherol significantly reduced the size of
enlarged lysosomes in all four cell types including these of
Niemann Pick type A, Wolman, Farber and Tay-Sachs diseases (FIG. 3,
see also FIGS. 8-23). These cell types a distinct lysosomal storage
disorders with the enlarged lysosomes in cells. Cholesterol esters
instead of free cholesterols are accumulated in lysosomes of Wolman
disease due to the mutations of acid lipase. Niemann Pick type A
disease is caused by deficiency in acid sphigomyelinase (ASM)
activity which results in accumulation of sphingomyelin in
lysosomes. Ceramides, produced by ASM from sphingomyelin,
accumulated in lysosomes as a result of ceramidase deficiency in
Farber disease. Tay-Sachs is caused by the hexosaminidase A
deficiency and accumulation of GM2 lipids in lysosomes. The
structures of these lipids involving these four additional
lysosomal storage diseases are different from free cholesterol. We
disclose the use of .delta.-tocopherol on the lysosome size
decrease in all of these diseases. We further disclose in an
alternate embodiment, that .delta.-tocopherol inserts into the
membrane and facilitates the movement of accumulated lipids out of
lysosome resulting in a decrease in the enlarged lysosome size in
these cells.
[0052] The direct measurements using a GC-MS method revealed that
the free cholesterol was reduced in the NPC fibroblasts after
.delta.-tocopherol treatment in NPCD fibroblasts (FIG. 4a). The
effect of .delta.-tocopherol was profound in the free cholesterol
level in NPCD cells was nearly recovered to the normal level as in
the wild type cells (FIG. 4a), while the effect of
.alpha.-tocopherol was not statistically different due to the high
variation among the three test groups. The free cholesterol level
in normal cells was also reduced slightly after the treatment. The
amount of cholesterol ester in both NPCD and wild cells after the
treatments of both .alpha.- and .delta.-tocopherol was
significantly reduced (FIG. 4b). The sphingomyelin amount in both
cell types was not significantly changed after the treatment (FIG.
4c). We also show that the cholesterol efflux significantly
increased after the treatments with .delta.-tocopherol in the baby
hamster kidney cells (FIG. 4d), indicating the free cholesterols
removed from late endosomes and lysosomes were transferred out of
cells via the increased cholesterol efflux. Thus, we disclose
.delta.-tocopherol facilitates the lipid movement in membrane. The
reduction of intracellular free cholesterol as the result of
increased cholesterol efflux by .delta.-tocopherol mobilizes the
storage cholesterol or the utilization of storage cholesterols is
also enhanced by .delta.-tocopherol.
[0053] The lethality of NPCD is primarily caused by the neuronal
degeneration though the cholesterol accumulation is observed in
many cell types. We show the cholesterol reduction effect of
.delta.-tocopherol is present in human NPC neuronal cells, using
the neuron-like cells differentiated from the mesenchymal stem
cells (MSC) derived that were obtained from two NPCD patients. Both
the NPCD patient derived MSC cell lines showed the similar
cholesterol accumulation as that in the NPCD fibroblasts. The
phenotype of cholesterol accumulation was preserved in these
neuron-like cells after the differentiation as well as in the MSC
cells (FIG. 5a). A set of neuronal markers were used to examine
both wild type and NPCD cells after the differentiation including
GAD65 (glutamic acid decarboxylase), galactocerebroside,
neurofilament M, NeuN (neuronal nuclear antigen) and
neuron-specific beta III tubulin. We show that only neuron-specific
beta III tubulin was positive in both cells after neuronal
differentiation whereas none of matured markers were found,
indicating that these cells were neuron-like cells but not matured
neurons. In addition, only a small portion of cells were
differentiated similarly between wild type and mutant cells.
.delta.-tocopherol treatment showed significant reduction in
cholesterol accumulation as well as a decrease in lysosome size in
these neuron-like NPCD cells that were co-localized by the filipin
staining with a neuronal marker (FIG. 5b). .delta.-tocopherol
effects on the reduction of accumulated free cholesterol and
decrease in lysosome size in late endosomes and lysosomes exist in
the neuron-like NPCD cells and the NPCD MSC cells, as it was
observed in the NPCD fibroblasts.
[0054] The gene expression pattern of NPCD cells after the
treatment with .delta.-tocopherol is disclosed in comparison with
the wild type fibroblasts. (Table 1)
[0055] Among the genes identified in Table 1, the expressions of
LDLR and ABCA1 genes significantly reduced in NPC cells after the
.delta.-tocopherol treatment. NPC1 mutant cells were noted for an
upregulation of (1) cholesterol homeostasis (transport) genes, (2)
genes involved in membrane trafficking, (3) genes associated with
calcium regulation, (4) genes involved in oxidative stress and (5)
genes associated with Alzheimer's disease.
[0056] NPCD patient-derived fibroblasts have an altered expression
of many genes that regulate lipid and cholesterol homeostasis,
including those that affect lipid and protein transport, and de
novo synthesis of saturated lipids and cholesterol (Table 1).
[0057] In the NPCD fibroblasts, LDLR and ABCA1 were upregulated
inducing more transport of cholesterol into the cell. The
upregulation of LDLR in NPC cells is not the result of reduced
available free cholesterols in cells although they are accumulated
in late endosomes and lysosomes. ABCA1 transport was reported as
one of cholesterol efflux mechanisms. It may also transport
cholesterols into cells dependent on the balance of cholesterols in
and out of cells. The treatment with .delta.-tocopherol in NPCD
cells decreased expression of these two genes. In addition, the
expression of several lipid and cholesterol synthetic genes, such
as SCD and HMGCR, were also decreased after the .delta.-tocopherol
treatment in NPCD cells which may be due to a direct interaction
with or decreased expression of sterol sensors in the cell like
SREBPs and INSIG1, reflecting an overall reduction in endoplasmic
reticulum stress.
[0058] Beta-cyclodextrin (cyclo) has been reported to reduce
cholesterol accumulation in NPC mouse and cat models as well as
increase in life span in these models. Cyclodextrin is also a
pharmaceutical excipient that is used for formulation of
hydrophobic drugs. The mechanism of action for cyclo to treat NPC
is still unclear. It has been implicated that the cyclo induced
endocytosis and/or exocytosis may contribute to its effect on the
cholesterol reduction in NPC cells and animal models. However, high
concentration (0.3 to 10 mM in vitro) and large dose (4g/Kg body
weight) are need for the effect of cyclo on the NPC that may cause
the side and toxic effects. We disclose the combination therapy of
.delta.-tocopherol and cyclodextrin in reduced concentrations.
[0059] While the invention has been particularly shown and
described with reference to a number of embodiments, it would be
understood by those skilled in the art that changes in the form and
details may be made to the various embodiments disclosed herein
without departing from the spirit and scope of the invention and
that the various embodiments disclosed herein are not intended to
act as limitations on the scope of the claims.
EXAMPLES
[0060] The following example is provided for illustrative purposes
only and is not intended to limit the scope of the invention.
Example 1
[0061] Human skin fibroblast cell lines GM5657, GM56599 and GM3123
were purchased from the Coriell Cell Repository (Caden, N.J.).
Other human skin fibroblast cell lines NPC19, NPC20, and NPC25 were
established by Forbes D. Porter. Cell growth media (DMEM),
L-glutamine, FBS, penicillin/streptomycin, Hoechst and LysoTracker
Red were purchased from Invitrogen (Carlsbad, Calif.). Filipin,
gamma-tocopherol (T1782, >96% purity) were obtained from
Sigma-Aldrich (St. Louis, Mo.). Delta-tocopherol was purchased from
Sigma-Aldrich and purified to 99% by AAA using a protocol
co-developed by AAA and NCGC. Alpha- (cat#100008377),
gamma-(cat#10008494), delta- (cat#10008513) tocotrienols were
obtained from Cayman Chemical (Ann Arbor, Mich.) in ethanol
solutions. They were lyophilized and dissolved in DMSO.
Beta-tocopherol (99% purity, cat #ASB-00020319-050) was purchased
from ChromaDex (Irvine, Calif.).
[0062] The primary human skin fibroblasts were cultured in DMEM
media (Invitrogen, cat#11995-040) supplemented with 10% FBS, 100
unit/ml penicillin and 100 .mu.g/ml streptomycin in a humidified
incubator (5% CO2) at 37.degree. C.
[0063] The adipose mesenchymal stem cells (HMSC.AD-100, HMSC-NPC22
and HMSC-NPC23) were isolated from two NPC patients as well as one
heath individual and provided by Celleng-tech (Coralville, Iowa).
These cells were maintained in MSC expansion media (Celleng-tech,
cat# HMSC.E.Media-450) supplemented with 10% FBS 100 unit/ml
penicillin and 100 .mu.g/ml streptomycin at 5% CO.sub.2, 37.degree.
C. For neural differentiation, cells were initially plated at 30%
confluency (.about.2500 cells/cm.sup.2) using MSC expansion media.
After 24 hours, MSC expansion media were withdrew. Cells were
washed twice with Dulbecco's phosphate Buffer saline (Invitrogen)
and incubated with neural differentiation media (Celleng-tech, cat#
NEU.D.Media-450) supplemented with 10% FBS, 100 unit/ml penicillin
and 100 .mu.g/ml streptomycin at 5% CO.sub.2, 37.degree. C. for
additional 3 days.
[0064] A collection of 2,800 compounds enriched in approved drugs
were set up internally at NIH Chemical Genomics Center and
dissolved in DMSO solution as 10 mM stock solution. The compounds
were serially diluted at 1:2.236 ratio in DMSO in 384-well plates
to yield fifteen concentrations and then formatted into 1,536-well
plates at 5 .mu.l/well as the compound source plates. The
concentrations of diluted compounds in the source plates ranged
from 0.28 .mu.M to 10 mM. In the screen experiments, 23 nl of
compounds in DMSO solution was transferred to assay plates
containing 5 .mu.l/well cell culture medium. The final
concentrations of compounds in the screening assay plates ranged
from 1.32 nM to 46 .mu.M.
[0065] The free cholesterol level in these cells was then measured
by a cholesterol oxidase coupled HRP/amplex red detection method
with a commercial available kit (Invitrogen). The cells were seeded
to black, tissue culture-treated 1536-well plates (Greiner Bio-One,
Monroe, N.C.) at 800 cells/well in 5 .mu.l medium by a Multidrop
Combi dispenser (Thermo Scientific, Waltham, Mass.) and cultured
for 24 hours. The assay plates were added with 23 nl/well of
compound DMSO solution using a pintool station (Kalypsys, San
Diego, Calif.) and cultured for 3 days. The 1536-well assay plate
was washed by a plate-inverted centrifugation method that removing
medium by centrifuging plates upside down against a stack of paper
towel at 1000 rpm for 1 min followed by addition of 7 .mu.l/well
PBS buffer, repeating for two times. The PBS buffer was added with
a 45 degree angled dispenser (Kalypsys) or a Multi-Drop Combi
dispenser set at medium speed to reduce disturbance to the cells.
After the cell wash, 2.5 .mu.l/well of detection mixture from the
kit was added followed by 1 hour incubation at 37.degree. C. With
the addition of the detection mixture, the cells were lysed by the
detergent containing buffer and free cholesterols in cell lysate
were oxidized by cholesterol oxidase to produce H.sub.2O.sub.2
which reacting with amplex red dye in the presence of horseradish
peroxidase to yield red fluorescence signal. A ViewLux plate reader
(PerkinElmer, Boston, Mass.) was used to measure the fluorescent
signal in assay plates with the excitation of 573 nm and emissions
of 610 nm.
[0066] Filipin dye staining was used to determine the level of free
cholesterol accumulation in cells (27, 28). Cells were seeded at
1000 cells/well in 100 .mu.l medium in the black/clear bottom,
tissue culture treated 96-well plates (Greiner Bio-One, Monroe,
N.C.) for culture overnight or in a designated time. The cells were
washed twice with the HBBS buffer and fixed with 100 .mu.l/well
3.2% formaldehyde at room temperature for 30 minutes. After three
times of plate wash with PBS buffer, cells were stained with 50
.mu.g/ml filipin, which was freshly-dissolved in DMSO at 10 mg/ml
and then diluted in PBS buffer. Samples were then washed twice with
PBS buffer and stored in 4.degree. C. On the day of imaging, cells
were stained with 2 .mu.M of CellMask Red (Invitrogen, H32711) in
PBS at room temperature for 1 h. Samples were viewed and
photographed using a Nikon Eclipse Ti microscope with a 20.times.
objective equipped with CCD camera. DAPI filter and TRITC filter
were used to visualize filipin and CellMask Red, respectively. The
images were processed by Adobe Photoshop.
[0067] Lysotracker is fluorescent dye that stains acidic
compartments in live cells and is well-retained after aldehyde
fixation. We have optimized a lysotracker staining assay to
visualize the enlarged lysosome size in the NPC skin fibroblasts by
applying appropriate concentration of lysotracker dye in comparison
with these normal fibroblast cells. Cells were seeded at 8000
cells/well in 100 .mu.l medium in a black/clear bottom, tissue
culture treated plate and cultured overnight or in a dedicated
time. The cells were live-stained with 100 u/well 50 nM Lysotracker
Red-99 dye (Invitrogen) dissolved medium at 37.degree. C. for 1 hr
followed by two-times plate wash with PBS buffer using the
plate-inverted centrifugation method. The plate was then added with
100 .mu.l/well 3.2% formaldehyde and 1 .mu.g/ml Hoechst 33342
(Invitrogen) in PBS and incubated at room temperature for 30
minutes. After three times of plate wash with PBS buffer, samples
were stored at 4.degree. C. until imaging. Samples were
photographed using an IN Cell Analyzer 1000 (GE Healthcare) with a
20.times. objective equipped with CCD camera. DAPI filter and TRITC
filter were used to visualize Hoechst and Lysotracker red,
respectively. The images were processed by Adobe Photoshop.
[0068] To measure the cytotoxicity of compounds, an ATP content
assay was used with a commercial assay kit (ATP-Lite, PerkinElmer).
Cells were seeded in white solid 1536-well plates and treated with
compounds same as in the cholesterol oxidase assay describe above.
After 3-day compound treatment, 3 .mu.l/well detection mixture
(prepared according to the manufacturer's instructions) was added
to each well and the plates were incubated for 10 min at room
temperature followed by a measurement using a Viewlux plate reader
in a luminescence mode. The buffer in the detection mixture lyses
cells and releases cellular ATP which reacts with luciferin in the
presence of luciferase to produce light. The luminescence signal
reduces if the cells are killed by the compound during the 3-day
treatment.
[0069] Cell pellets were collected from 100-mm dishes and washed
once with 10 ml PBS. Proteins were assayed using the BCA kit
(Sigma-Aldrich) and lipids were extracted by modified Bligh and
Dyer procedure (29) from the cells. Internal standards were added
based on protein concentration that included N12:0 sphingomyelin,
D7-cholesterol, 17:0 cholesterol ester (Avanti Polar Lipids, Inc).
A portion of the lipid extract was used for detection of
sphingomyelins using direct infusion mass spectrometric assay with
lithium hydroxide for facilitating ionization and the rest was used
to convert cholesterol and its ester into acetate by
derivatization. The derivatization was performed by treating the
dried crude lipid extract with solutions of 1M acetic acid with 1M
DMAP in chloroform and 1M EDC in chloroform at 50.degree. C. for 2
hours. The derivatized product was extracted with hexane and
analyzed by direct infusion mass spectrometric assay with 5%
ammonium hydroxide for facilitating ionization.
[0070] A triple-quadrupole mass spectrometer equipped with an
electrospray was used for analysis of lipids in positive mode.
Sphingomyelins, cholesterol and cholesterol esters were detected by
neutral loss scan of 213 (collision energy: 50 V), neutral loss
scan of 77 (collision energy: 14 V) and precursor ion scan of m/z
369 (collision energy: 14 V) respectively. Data processing of mass
spectrometric analyses including ion peak selection, data
transferring, peak intensity comparison and quantitation was
conducted using self-programmed Microsoft Excel macros (30).
[0071] MSC cells were seeded on coverslips on day 0 in MSC
expansion medium. On day 1, cells were washed twice with warm PBS
buffer and then incubated with MSC neuronal differentiation medium.
On day 4, media were replaced with fresh neuronal differentiation
medium. On day 6, cells were fixed with 3.2% paraformaldehyde in
HBSS for 15 min. Immunocytochemistry for neuronal beta III tubulin
and unesterified cholesterol (filipin staining) was performed as
described. The coverslips were examined y a XXX microscope. The
following filter sets were used: for filipin, excitation 360/40 nM,
emission filter 460/50 nM.
[0072] Five NPC fibroblast cell lines as well as five normal
fibroblast cell lines were treated with 40 .mu.M delta-tocopherol
for 5 days with a medium change and sully of fresh compound on
3.sup.rd day. The total RNAs was prepared as the standard method
described previously ( ). The microarray gene expression experiment
was carried out in a core microarray facility at NHGRI with a set
of AAAAA chips (Affymetrix).
[0073] Partek Genomics Software Suite (Copyright, Partek Inc.) was
used to perform analysis of microarray data. Affymetrix cell files
were loaded into the software using the RMA algorithm, which
involved background correction, quantile normalization, log-2
transformation, and median polish summarization. PCA and
hierarchical clustering identified one sample as an outlier. This
sample (sample number 12) was not considered for further
analysis.
[0074] ANOVA was performed to identify the differentially expressed
genes for three comparisons: 1) NPC_DMSO vs WT_DMSO 2)
NPC_tocopherol-treated vs NPC_DMS0-treated and 3)
WT_tocopherol-treated vs WT_DMSO-treated. An FDR cut-off of 0.05
failed to identify any genes with differential expression. So, a
p-value cut-off of 0.01 was applied with an aim to validate the
true positives using RT-PCR.
[0075] Cell lysates were prepared as previously described (31) and
proteins in lysates were quantified using Bicinchoninic Acid (BCA)
Protein Assay Kit (Sigma-Aldrich). Samples were heated at 65 C for
10 min before resolved by SDS-PAGE under reducing conditions.
Proteins were transferred to PVDF membranes (Bio-Rad Laboratory)
using either Criterion Blotter (Bio-Rad Laboratory) or iBlot dry
blotting devices (Invitrogen). For NPC1 and HMG-CoA reductase
detection, rabbit polyclonal antibodies against NPC1 (Abcam, cat #
ab36983, 1:2000) or HMG-CoA reductase (Millipore, cat #07-457,
1:1000) were used, respectively. A peroxidase-conjugated donkey
anti-rabbit IgG (Santa Cruz) was used as secondary antibody at a
1:500 dilution.
[0076] The compound library screen data was analyzed using software
developed internally at NIH Chemical Genomics Center (32).
Concentration-response curves analyzed and EC50/IC50s were
calculated with the Prism.RTM. software (GraphPad, San Diego,
Calif.).
Example 2
[0077] Patient derived primary skin fibroblasts have been used as
disease models for testing the effect of alpha- and
delta-tocopherols. Fibroblasts from patients with Farber disease,
Niemann Pick Type A disease, Wolman diseases and Say Tachs disease
were obtained from Coriell Cell repository (Camden, N.J.) that
accumulating ceramide, sphingomyelin, cholesteryl ester and GM2
gangliosides in lysosomes, respectively.
[0078] (1) Amplex-red cholesterol assay. This biochemical assay
(Invitrogen) uses cholesterol oxidase and a HRP-Amplex-re reporting
system to measure the free cholesterol level in cells. With the
addition of acid lipase to this assay, that hydrolyzes cholesterol
ester to free cholesterol, the level of total cholesterol in cells
including the cholesterol ester can be measured. This assay was
used for detecting the total cholesterol level of in fibroblasts
from patients with Wolman disease.
[0079] (2) Lysotracker dye (Invitrogen) staining assay. Lysotracker
dye stains the acidic compartment in cells. We use an optimal
concentration of lysotracker dye that shows the enlarged lysosomes
due to the storage of lipids in fibroblasts from patients with
lysosomal storage diseases including Farber, Wolman, Tay Sachs and
Niemann Pick Type A diseases. Drug treatment can reduce the size of
the enlarged lysosome as the lipid storage is decreased.
[0080] (3) Nile-red dye staining assay. Nile red dye (Invitrogen)
stains two groups of lipids. At excitation1=460-480 nm and
emission1=535 nm, it stains the non-polar lipids such as
cholesterol ester and triglycerides. At excitation2=500-540 nm and
emission2 590-615 nm it stains the total lipids including the polar
lipids such as phospholipids.
[0081] (4). Intracellular calcium assay. We used a fluorescence
calcium dye (Fluo-8, AAT Bioquest, CA) to measure the transit
increase of intracellular calcium stimulated by alpha-tocopherol
and delta-tocopherol. Cytosal calcium increase is a second
messenger which mediates a variety of cellular functions including
exocytosis.
[0082] .delta.-tocopherol has significant effect on reduction of
enlarged lysosome size and storage of macromolecules in patient
derived fibroblasts of five lysosomal storage diseases. The
mechanism of action for .delta.-tocopherol is direct insertion into
the lipid membrane in cells that facilitates lipid movement in
membrane and discharge of accumulated lysosomal macromolecules out
of cells. Thus, .delta.-tocopherol can be used to treat all types
of lysosomal storage diseases. We teach the combination therapy
using vitamin E and cyclodextrin has synergetic or additive effect
that can reduce dosage and adverse effect as well as increase the
efficacy due to different mechanism of action for two drugs.
Example 3
[0083] It is envisioned that .delta.-tocopherol will be used either
alone or in combination with inhibitors of CYP4F2 for the treatment
of lysosomal storage disorders. Purified Delta-tocopherol purified
to pharmacologically acceptable levels for the treatment of humans
would be performed. Dosage curves would be examined for both
cytotoxicity and efficacy, as would be understood by one with skill
in the art. Patients could have multiple dose treatments, for
multiple days, depending on efficacy, toxicity and half life with
regard to delta tocopherol.
TABLE-US-00001 TABLE 1 Genes differentially expressed in NPC
fibroblasts, or with tocopherol treatment NPC vs WT Toco Toco Gene
Class untreated treat NPC treat WT LDLR Cholesterol trafficking 1.3
-2.0 -2.0 ABCA1 Cholesterol trafficking 1.3 -2.0 -1.4 CPE
Intracellular transport 2.7 NC NC SCD Lipid and cholesterol 2.1
-3.9 -3.5 synthesis FADS2 Lipid and cholesterol 1.2 -2.5 -2.0
synthesis ACACA Lipid and cholesterol 1.1 -1.5 -1.5 synthesis HMGCR
Lipid and cholesterol 1.8 -1.7 -1.5 synthesis HMGCS1 Lipid and
cholesterol 1.6 -1.9 -1.6 synthesis SC4MOL Lipid and cholesterol
2.3 -1.4 -1.7 synthesis DHCR7 Lipid and cholesterol 1.3 -1.7 -1.6
synthesis INSIG1 Cholesterol homeostasis 1.6 -1.8 -1.6 SREBF1
Cholesterol homeostasis -1.1 -1.4 -1.2 SREBF2 Cholesterol
homeostasis 1.1 -1.6 -1.6 MMP1 Inflammation 4.9 2.7 5.0 MMP3
Inflammation 5.1 1.7 2.0 TNFAIP6 Inflammation 2.9 NC NC FRRS1
Ferric Iron reductase 2.9 1.1 -1.1 NOX4 Generation of ROS 2.7 -1.1
-1.7
[0084] The description of the present invention has been presented
for purposes of illustration and description, but is not intended
to be exhaustive or limiting of the invention to the form
disclosed. The scope of the present invention is limited only by
the scope of the following claims. Many modifications and
variations will be apparent to those of ordinary skill in the art.
The embodiment described and shown in the figures was chosen and
described in order to best explain the principles of the invention,
the practical application, and to enable others of ordinary skill
in the art to understand the invention for various embodiments with
various modifications as are suited to the particular use
contemplated.
* * * * *