U.S. patent application number 16/247880 was filed with the patent office on 2019-07-04 for methods and compositions for treatment of lipid storage disorders.
The applicant listed for this patent is Icahn School of Medicine at Mount Sinai, Neurotrope BioScience, Inc.. Invention is credited to Lawrence ALTSTIEL, David R. CROCKFORD, Yiannis A. IOANNOU, Sathapana KONGSAMUT.
Application Number | 20190201377 16/247880 |
Document ID | / |
Family ID | 54321042 |
Filed Date | 2019-07-04 |
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United States Patent
Application |
20190201377 |
Kind Code |
A1 |
IOANNOU; Yiannis A. ; et
al. |
July 4, 2019 |
METHODS AND COMPOSITIONS FOR TREATMENT OF LIPID STORAGE
DISORDERS
Abstract
Treating subjects having a lipid storage disorder with a
composition comprising a PKC activator, such as bryostatins,
bryologs, and polyunsaturated fatty acids. Accordingly, the present
disclosure provides methods for treating human subjects suffering
from lipid storage disorders, such as Niemann-Pick disease, by
administering PKC activators. The present disclosure provides,
according to certain embodiments, methods comprising administering
to a subject with Niemann-Pick Type C disease a pharmaceutically
effective amount of bryostatin 1.
Inventors: |
IOANNOU; Yiannis A.; (New
York, NY) ; ALTSTIEL; Lawrence; (Stonington, CT)
; CROCKFORD; David R.; (Newburyport, MA) ;
KONGSAMUT; Sathapana; (Madison, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Neurotrope BioScience, Inc.
Icahn School of Medicine at Mount Sinai |
Newark
New York |
NJ
NY |
US
US |
|
|
Family ID: |
54321042 |
Appl. No.: |
16/247880 |
Filed: |
January 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15304838 |
Oct 17, 2016 |
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PCT/US2015/025821 |
Apr 14, 2015 |
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16247880 |
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61987360 |
May 1, 2014 |
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61981473 |
Apr 18, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/20 20130101;
A61P 3/00 20180101; A61K 31/365 20130101; A61K 31/35 20130101; A61K
31/366 20130101; A61P 25/00 20180101; A61K 31/215 20130101; A61K
31/00 20130101 |
International
Class: |
A61K 31/366 20060101
A61K031/366; A61K 31/35 20060101 A61K031/35; A61K 31/20 20060101
A61K031/20; A61K 31/365 20060101 A61K031/365; A61K 31/215 20060101
A61K031/215 |
Claims
1-5. (canceled)
6. A method of treating Niemann-Pick Type C disease in a patient in
need thereof comprising: administering to the patient with
Niemann-Pick Type C disease a pharmaceutically effective amount of
bryostatin 1, a bryolog, or a pharmaceutically acceptable salt or
solvate thereof.
7. The method of claim 6, wherein the bryostatin 1 or bryolog is
administered from 1 to 4 times per day, twice a week, once a week,
once every two weeks, once every three weeks, once every four
weeks, once every six weeks, once every eight weeks, or less
frequently.
8. The method of claim 6, wherein the bryostatin 1 or bryolog is
administered orally, intraperitoneally, subcutaneously,
intranasally, intramuscularly, buccally, trans-dermally, or
intravenously.
9. The method of claim 6, wherein the pharmaceutically effective
amount of bryostatin 1 or bryolog is from about 0.0000001 mg/kg to
about 250 mg/kg per dose.
10. The method of claim 6, wherein the pharmaceutically effective
amount of bryostatin 1 or bryolog is from about 0.00001 mg/kg to
about 5.0 mg/kg per dose.
11. The method of claim 6, wherein the bryostatin 1 or bryolog is
administered at a dose from 0.01-25 .mu.g/m.sup.2
intravenously.
12. A method of treating Niemann-Pick Type C disease in a patient
in need thereof comprising: administering to the patient with
Niemann-Pick Type C disease a pharmaceutically effective amount of
a bryolog, wherein the bryolog is selected from the group
consisting of: ##STR00001## ##STR00002## ##STR00003## ##STR00004##
##STR00005## ##STR00006## ##STR00007## ##STR00008## wherein one
R.sup.D is hydrogen and the other R.sup.D is --CO.sub.2CH.sub.3,
##STR00009## where OR.sup.A and R.sup.B are selected from:
TABLE-US-00004 OR.sup.A R.sup.B --O.sub.2C--CH.sub.3
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3 --OH
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3
--O.sub.2C--C(CH.sub.3C).sub.3 --O.sub.2C--CH.sub.2--CH.sub.3
--O.sub.2C--C(CH.sub.3C).sub.3 --O.sub.2C--CH.sub.3
--O.sub.2C--CH.sub.2--CH.sub.3 --O.sub.2C--CH.sub.3
--O.sub.2C--CH.sub.3 --O.sub.2C--CH.sub.3
--O.sub.2C--CH.sub.2--CH.sub.3 --O.sub.2C--CH.sub.2--CH.sub.3
--O.sub.2C--CH.sub.3 --O.sub.2C--CH.sub.2--CH.sub.3
--O.sub.2C--C(CH.sub.3C).sub.3 --H --O.sub.2C--CH.sub.3 --H
--O.sub.2C--CH.sub.2--CH.sub.3
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3
--O.sub.2C--CH.sub.2--CH.sub.3 --H --O.sub.2C--C(CH.sub.3C).sub.3
--OH --O.sub.2C--CH.sub.3
--O.sub.2C--CH.dbd.CH--CH.dbd.CH--CH.sub.2--CH.sub.2--CH.sub.3
--O.sub.2C--C(CH.sub.3C).sub.3 --H;
##STR00010## or a pharmaceutically acceptable salt or solvate
thereof.
13. The method of claim 12, wherein the bryolog is administered
from 1 to 4 times per day, twice a week, once a week, once every
two weeks, once every three weeks, once every four weeks, once
every six weeks, once every eight weeks, or less frequently.
14. The method of claim 12, wherein the bryolog is administered
orally, intraperitoneally, subcutaneously, intranasally,
intramuscularly, buccally, trans-dermally, or intravenously.
15. The method of claim 12, wherein the pharmaceutically effective
amount of the bryolog is from about 0.0000001 mg/kg to about 250
mg/kg per dose.
16. The method of claim 12, wherein the pharmaceutically effective
amount of the bryolog is from about 0.00001 mg/kg to about 5.0
mg/kg per dose.
17. The method of claim 12, wherein the bryolog is administered at
a dose from 0.01-25 .mu.g/m.sup.2 intravenously.
Description
PRIORITY CLAIM
[0001] This application claims priority to U.S. provisional patent
application No. 61/981,473 filed Apr. 18, 2014 and U.S. provisional
patent application No. 61/987,360 filed May 1, 2014, the contents
of which are expressly incorporated by reference.
BACKGROUND
[0002] All references cited herein are expressly incorporated by
reference.
[0003] Lipid storage disorders (or lipidoses) are a group of
inherited metabolic disorders in which harmful amounts of lipids
accumulate in some of the body's cells and tissues. People with
these disorders generally either do not produce enough of one of
the enzymes needed to metabolize lipids or they produce enzymes
that do not work properly. Over time, this excessive storage of
fats can cause permanent cellular and tissue damage.
[0004] Many lipid storage disorders lack adequate therapeutics for
treatment. These disorders include, for example, Niemann-Pick
disease types A, Band C, Gaucher disease Type II, Fabry disease
(note that an enzyme replacement is available), gangliosidoses
including Tay-Sachs disease, Sandhoff disease, Krabbe disease,
Metachromatic leukodystrophy, and cholesteryl ester storage disease
(Wolman's disease).
[0005] Niemann-Pick disease is an inherited autosomal recessive
lipid storage disorder characterized by excessive accumulation of
sphingomyelin in the lysosomes of cells such as macrophages and
neurons, which impairs normal cellular function. Niemann-Pick Type
A results from a deficiency of acid sphingomyelinase and is a
rapidly progressive neurodegenerative disease. It typically results
in death within two to three years of age. Niemann-Pick Type B is a
milder form that results in the enlargement of the liver and
spleen, and respiratory distress with death generally ensuing by
early adulthood. These two forms of Niemann-Pick disease which are
both associated with acid sphingomyelinase (ASM) deficiencies are
referred to collectively herein as Niemann-Pick disease, or ASM
deficiency (ASMD). Other types of Niemann-Pick disease, e.g., Type
C, do not involve mutations in the ASM gene and are not directly
attributable to the function of ASM. The nature of the biochemical
and molecular defects that underlie the remarkable clinical
heterogeneity of the A and B subtypes remains unknown. Although
patients with both subtypes have residual ASM activity (about 1 to
10% of normal), biochemical analysis cannot reliably distinguish
the two phenotypes. Moreover, the clinical course of Type B NPD is
highly variable, and it is not presently possible to correlate
disease severity with the level of residual ASM activity.
[0006] Niemann-Pick Type C is results from mutations in NPC1 and
NPC2 genes. In Niemann-Pick Type C, the protein product of the
major mutated gene NPC1 is not an enzyme but appears to function as
a transporter in the endosomal-lysosomal system, which moves large
water-insoluble molecules through the cell. The protein coded by
the NPC2 gene has been shown to be a small cholesterol-binding
protein that resides in the lysosome lumen. The disruption of this
transport system results in the accumulation of cholesterol and
glycolipids in lysosomes.
[0007] Niemann Pick disease, as well as other lipid storage
disorders, is a disorder for which there remains an overwhelming
need for therapeutics for treatment. Presently there is no FDA
approved therapy for Neimann Pick disease in the United States; and
treatments for this disease are limited with most people afflicted
with Type A dying by age 18 months, while those with Type B or Type
C, frequently live into their teenage years.
SUMMARY
[0008] The protein kinase C (PKC) family of enzymes is responsible
for a multitude of cellular processes through the enzymes' ability
to regulate proteins via signal transduction cascades. The members
of this kinase family are structurally and functionally similar and
are categorized into conventional (.alpha., .beta.I, .beta.II and
y), novel (.delta., .epsilon., .eta., and .theta.), and atypical
isoforms (.zeta., and .lamda.). These isoforms have been implicated
in a variety of diseases and pathological conditions.
[0009] The present disclosure is based in part on the previously
unappreciated role for PKCs in lipid storage disorders such as
Niemann-Pick Type C (NPC1) disease. We observed that the
intermediate filament, vimentin, is hypophosphorylated in NPC1
cells compared to wild-type (WT) cells and that this
hypophosphorylation results from reduced activity [5]. Vimentin is
involved in a variety of cellular processes, including vesicular
membrane transport [6, 7], signal transduction [8, 9] and cell
motility [10]. Similar to NPC1 cells, cells lacking vimentin are
unable to transport LDL-derived cholesterol from their lysosomes to
the endoplasmic reticulum for esterification [11]. The decreased
vimentin phosphorylation in NPC1 cells reduces the pool of soluble
vimentin, likely disrupting the vimentin cycle, which is necessary
for transport to take place [12, 13]. Vimentin has been shown to be
phosphorylated by several proteins, including the PKCs [14] and in
particular the a [15], .epsilon. [10] and .beta.II [16,17]
isoforms.
[0010] Accordingly, the present disclosure provides methods for
treating human subjects suffering from lipid storage disorders,
such as Niemann-Pick disease, by administering PKC activators.
[0011] The present disclosure provides, according to certain
embodiments, methods comprising administering to a subject with a
lipid storage disorder a pharmaceutically effective amount of a PKC
activator.
[0012] The present disclosure provides, according to certain
embodiments, methods comprising administering to a subject with
Niemann-Pick Type C disease a pharmaceutically effective amount of
bryostatin 1.
[0013] The features and advantages of the present disclosure will
be readily apparent to those skilled in the art upon a reading of
the description of the embodiments that follows.
DRAWINGS
[0014] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0015] Some specific embodiments of the disclosure may be
understood by referring, in part, to the following description and
the accompanying drawings.
[0016] FIG. 1A and FIG. 1B are Western blots showing the effects of
transient PKC expression on vimentin solubilization in human NPC1
cells. Representative Western blot analyses of soluble and
insoluble vimentin levels in human NPC1 3123 (A) and human null
NPC1o (B) cells transfected with PKC .epsilon., .beta. or a show
that the three isoforms increase levels of soluble vimentin and
Rab9 with a concurrent decrease of insoluble vimentin relative to
untransfected cells(-). The levels of vimentin solubilized are
similar to that seen in cells expressing Rab9 (Rab9). The blots
shown are representative of at least 3 independent experiments.
[0017] FIG. 2 is a Western blot showing Rab9 release from insoluble
vimentin fraction of NPC1 cell lysates. The insoluble vimentin
fraction from NPC1 cell lysates was incubated with various PKC
isoforms. All isoforms tested can affect Rab9 release to some
degree from the insoluble vimentin fraction, with PKC.alpha. being
the most effective and PKC.GAMMA. being the least effective. The
blots shown are representative of at least 3 independent
experiments.
[0018] FIG. 3 is a graph showing the effects of PKCs and fatty
acids on cholesterol esterification in M12 NPC1 CHO cells. M12
cells were treated with 50 .mu.g/ml fatty acids for 2 days and then
transfected with the indicated PKC isoforms. Following
transfection, cholesterol transport was assessed by esterification
assay. Both free fatty acids and PKCs alleviate the cholesterol
transport defect of NPC1 cells and their effects appear to be
additive.
[0019] FIG. 4 are images showing the effects of transient PKC
expression on the NPC1 phenotype. M12 cells were transfected with
PKC isoforms or Rab9 for 48 hrs and then analyzed by filipin
staining for cholesterol storage. Cells positive for transfection
stain positive for GFP (left panel) and show decreased filipin
staining (outlined cells, right panel) compared to surrounding
untransfected cells, confirming the role of PKCs in mobilizing
stored cholesterol from the NPC1 endosomes. Bar, 20 .mu.m.
[0020] FIG. 5A, FIG. 5B and FIG. 5C show the effects of fatty acids
on vimentin solubilization and the NPC1 phenotype. (A) Human NPC1
3123 cells were treated with 50 .mu.g/ml linoleic or oleic acid for
24 hrs, after which the levels of soluble vimentin were analyzed by
Western blotting. Cholesterol storage in NPC1 CHO (B) or human 3123
(C) cells was analyzed by filipin staining. Fluorescence intensity
was quantitated in at least 150 cells for each sample. The bar
graph represents average values .+-.SEM from 3 independent
experiments. * and * * * denote statistically significant
differences between treated and untreated cells with P<0.05 and
P<0.0001, respectively, as determined by Student's t-test.
[0021] FIG. 6 shows the effects of PKC activation on the NPC1
phenotype. NPC1 CHO cells (B through F) were treated with 100 .mu.M
DCP-LA (C), 10 .mu.M DHA (D), or 100 .mu.M diazoxide (E) and
cholesterol storage was quantified by filipin fluorescence. WT CHO
cells are shown in (A). Filipin intensity was quantitated in at
least 150 cells for each sample. The bar graph represents average
values .+-.SEM from 3 independent experiments. *, * *, and * * *
denote statistically significant differences between treated and
untreated cells with P<0.05, P<0.01 and P<0.0001,
respectively, as determined by Student's t-test.
[0022] FIG. 7 are images showing the effects of PKC activation on
sphingolipid transport. Human NPC1 3123 cells were treated with (B)
20 .mu.M DCP-LA, (C) 2 .mu.g/ml oleic acid, (D) 2 .mu.g/ml linoleic
acid, or (F) 100 nM PMA for 48 hours before BODIPYLacCer staining
was performed. In untreated cells (A), transport of the lipid to
the trans-Golgi network (TGN) is inhibited and staining is visible
only in punctate endocytic vesicles. In contrast, in treated cells
the lipid can be seen in the TGN (arrows) in treated cells,
indicating release of the transport block that characterizes
NPC1.
[0023] FIG. 8 is a graph showing the ability of DCPLA, diazoxide
and bryostatin 1 to decrease stored cholesterol levels in NPC3-SV
cells at 48 h.
[0024] FIG. 9 is a graph showing the ability of DCPLA, and
bryostatin 1 to decrease stored cholesterol levels in NPC3-SV cells
at 72 h.
[0025] FIG. 10 is a graph showing the ability of DCPLA, and
bryostatin 1 to decrease stored glycosphingolipid levels in NPC3-SV
cells at 72 h using verotoxin B (VTB) as a probe stain.
[0026] FIG. 11 is a graph showing the ability of DCPLA and
bryostatin 1 to decrease filipin accumulation using an NPC24-SV
cell line.
[0027] FIG. 12 are photomicrographs showing the ability of DCPLA,
and bryostatin 1 to release the ganglioside transport block in
NPC3SV cells.
[0028] FIG. 13 is a graph showing the ability of bryostatin 1
(0.1-100 nM) to decrease cholesterol accumulation in human NPC24-SV
cells.
[0029] FIG. 14 is a graph showing the ability of bryostatin 1
(0.1-100 nM) to decrease glycosphingolipid accumulation in human
NPC24-SV cells.
[0030] FIG. 15 are images showing representative fields of treated
cells used in the analysis shown FIGS. 13-14.
[0031] FIG. 16 is a graphic representation of an NPC1 cell showing
the itineraries of various lipids stored.
[0032] FIG. 17 is a graph showing untreated C57 NPC1 mice. Weight
gain is observed up to around day 70-72 of life after which a rapid
weight loss is observed.
[0033] While the present disclosure is susceptible to various
modifications and alternative forms, specific example embodiments
have been shown in the figures and are herein described in more
detail. It should be understood, however, that the description of
specific example embodiments is not intended to limit the invention
to the particular forms disclosed, but on the contrary, this
disclosure is to cover all modifications and equivalents as
illustrated, in part, by the appended claims.
DESCRIPTION
[0034] In general, the present disclosure provides methods for
treating lipid storage disorders using PKC activators. As used
herein, "protein kinase C activator" or "PKC activator" refers to a
substance that increases the rate of the reaction catalyzed by
protein kinase C, upregulates the expression of PKC (e.g.,
upregulates the expression of PKC.alpha., PKC .beta.II, PKC .gamma.
and/or PKC .epsilon.), or otherwise facilitates the activation of
PKC.
[0035] In certain embodiments, the present disclosure provides
methods comprising administering to a human subject with a lipid
storage disorder a pharmaceutically effective amount of a PKC
activator. The PKC activator may be administered as part of a
composition suitable for administration to a human subject.
[0036] In certain embodiments, the PKC activator may be any of
bryostatin 1-20, .alpha. bryolog, neristatin, a polyunsaturated
fatty acid, or combinations thereof.
[0037] Bryostatins may be used in the methods of the present
disclosure. The bryostatins are a family of naturally occurring
macrocyclic compounds originally isolated from marine bryozoa.
Currently, there are about 20 known natural bryostatins which share
three six-membered rings designated A, Band C, and which differ
mainly in the nature of their substituents at C7 (OR.sup.A) and C20
(R.sup.B). Bryostatin 1 and derivatives of bryostatin 1 are
described in U.S. Pat. No. 4,560,774 (incorporated herein by
reference). Examples of suitable bryostatins that may be used with
the methods of the present disclosure include, bryostatin 1,
bryostatin 2, bryostatin 3, bryostatin 4, bryostatin 5, bryostatin
6, bryostatin 7, bryostatin 8, bryostatin 9, bryostatin 10,
bryostatin 11, bryostatin 12, bryostatin 13, bryostatin 14,
bryostatin 15, bryostatin 16, bryostatin 17 bryostatin 18,
bryostatin 19, and bryostatin 20.
[0038] Analogs of bryostatins, commonly referred to as bryologs,
also may be used in the methods of the present disclosure. Bryologs
are structural analogues of bryostatin. While bryostatin has two
pyran rings and one 6-membered cyclic acetal, in most bryologs one
of the pyrans of bryostatin is replaced with a second 6-membered
acetal ring. This modification reduces the stability of bryologs,
relative to bryostatin, for example, in both strong acid or base,
but has little significance at physiological pH. Bryologs also have
a lower molecular weight (ranging from about 600 to 755), as
compared to bryostatin (988), a property which may facilitate
transport across the blood-brain barrier. Examples of suitable
bryologs include, but are not limited to analogs and derivatives of
bryostatins such as those disclosed in U.S. Pat. Nos. 6,624,189,
7,256,286 and 8,497,385 (the disclosures of which are incorporated
herein by reference).
[0039] In certain embodiments, polyunsaturated fatty acid esters
(PUFAs or polyenoic fatty acids)) may be used in the methods of the
present disclosure for treating lipid storage disorders. A PUFA is
a fatty acid containing more than one double bond. There are three
classes of PUFAs, omega-3 PUFAs, omega-6 PUFAs, and omega-9 PUFAS.
In omega-3 PUFAs, the first double bond is found 3 carbons away
from the last carbon in the chain (the omega carbon). In omega-6
PUFAs the first double bond is found 6 carbons away from the chain
and in omega-9 PUFAs the first double bond is 9 carbons from the
omega carbon. As used herein, the term PUFA includes both
naturally-occurring and synthetic fatty acids. A major source for
PUFAs is from marine fish and vegetable oils derived from oil seed
crops. Examples of PUFA's suitable for use in the methods of the
present disclosure include, but are not limited to, esters of
8-[2-(2-pentylcyclopropylmethyl) cyclopropyl]-octanoic acid
(DCPLA), as well as those described in U.S. Pat. No. 8,163,800 and
in PCT publication WO 2010014585 A1.
[0040] Another example of suitable PKC activators include potassium
channel activators such as, for example, diazoxide.
[0041] In certain embodiments, neristatins, such as neristatin 1,
may be used in the methods of the present disclosure for treating
lipid storage disorders.
[0042] Other suitable PKC activators include, but are not limited
to, phorbol-12- myristate-13-acetate (PMA), okadaic acid,
1.alpha.,25-dihydroxyvitamin D3, 12- deoxyphorbol-13-acetate
(prostratin), 1,2-dioctanoyl-sn-glycerol (DOG), 1-oleoyl-2-
acetyl-sn-glycerol (OAG),
(2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)b
enzolactam(a-amyloid precursor protein modulator), cis-9-
octadecenoic acid (oleic acid), ingenol 3-angelate,
resiniferatoxin,
L-.alpha.-Phosphatidyl-D-myo-inositol-4,5-bisphosphate, triammonium
salt (PIP2), phorbol-12, 13-dibutyrate, 8(S-hydroxy-(5Z, 9E, 11Z,
14Z)-eicosatetraenoic acid (8(S)-HETE),
12.beta.-[(E,E)-5-Phenyl-2,4-pentadienoyloxy]daphnetoxin
(merzerein), clomiphene citrate, sodium oleate, phorbol 12,
13-diacetate, phorbol-12, 13-didecanoate,
1,2-dipalmitoyl-sn-glycerol, 1-Stearoyl-2-linoleoyl-sn-glycerol,
phorbol-12, 13-didecanoate, 1,2-dipalmitoyl-sn-glycerol,
1-stearoyl-2-linoleoyl-sn-glycerol, phorbol 12, 13-dihexanoate,
prostratin and its analogs, resiniferonol
9,13,14-ortho-phenylacetate, C-8 ceramide,
1,6-bis(Cyclohexyloximinocarbonylamino)hexane;
1,6-Di(0-(carbamoyl)cyclohexanone oxime)hexane (RHC-80267),
(+/-)-1-oleoyl-2-acetylglycerol, 5(S), 6(R), 15(S)-TriHETE (Lipoxin
A4), (-)-Indolactam V, SC-9, SC-10, zoledronic acid monohydrate,
12-deoxyphorbo 13-angelate 20-acetate,
6-(N-decylamino)-4-hydroxymethylindole, 4aphorbol 12,
13-dibutyrate, 1,2-dihexanoyl-sn-glycerol, zoledronic acid disodium
salt tetrahydrate, arachidonic acid methyl ester, arachidonic
acid-d8.
[0043] As used herein, "a pharmaceutically effective amount" is an
amount of a pharmaceutical compound or composition having a
therapeutically relevant effect on a lipid storage disorder. A
therapeutically relevant effect relates to some improvement in a
biomechanical process (e.g., gait, use of limbs, and the like) or a
change in the cellular, physiological or biochemical parameters
associated with any of the causes of a particular lipid transport
disorder (e.g., vimentin solubility, cholesterol esterification,
cholesterol accumulation and transport, glycosphingolipid
accumulation and transport).
[0044] In certain embodiments, a pharmaceutically effective amount
for bryostatins and bryologs may be from about 0.0000001 to about
500 mg per kg host body weight per day, which can be administered
in single or multiple doses. In some embodiments, the dosage level
may be: from about 0.0000001 mg/kg to about 250 mg/kg per day; from
about 0.0000005 mg/kg to about 100 mg/kg per day; from at least
about 0.0000001 mg/kg to about 250 mg/kg per day; from at least
about 0.00000005 mg/kg to about 100 mg/kg per day; from at least
about 0.000001 mg/kg to about 50 mg/kg per day; or from about
0.00001 mg/kg to about 5.0 mg/kg per dose. In other embodiments,
the dosage may be about 0.00000001 mg/kg to about 0.00005 mg/kg;
0.00005 mg/kg to about 0.05 mg/kg; about 0.0005 mg/kg to about 5.0
mg/kg per day; about 0.0001 mg/kg to about 0.5 mg/kg per dose; or
0.001 to 0.25 mg/kg per dose.
[0045] In certain specific embodiments, in which the lipid storage
disorder is Niemann-Pick disease, a pharmaceutically effective
amount of a PKC activator may be an amount sufficient to solubilize
vimentin and/or release trapped Rab9.
[0046] In certain embodiments, the dosing is from about 1 .mu.g/kg
(3-25 .mu.g/m.sup.2) to 120 .mu.g/kg (360-3000 .mu.g/m.sup.2). In
other embodiments, the dosing is from about 0.04-0.3 .mu.g/kg (1
.mu.g/m.sup.2) to about 1-10 .mu.g/kg (25 .mu.g/m.sup.2). In other
embodiments, the dosing is from about 0.01 .mu.g/m.sup.2 to about
25 .mu.g/m.sup.2. In other embodiments, the dosing is from about
0.0002-0.0004 .mu.g/kg to about 0.05-1 .mu.g/kg.
[0047] In certain embodiments, the PKC activator is a PUFA
administered at a dosage of about 0.001 to 100 mg/kg; 0.01 to about
50 mg/kg; about 0.1 to about 10 mg/kg.
[0048] In certain embodiments, the PKC activator present in the
compositions used in the methods of the present disclosure is a
bryostatin or bryolog, and the bryostatin or bryolog is used in an
amount from about 0.0001 to about 1000 milligrams. In some
embodiments, the bryostatin or bryolog is used in an amount from at
least about 0.0001, 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005,
0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06,
0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,
5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0,
250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, or about
1000.0 milligrams.
[0049] The compositions used in the methods of the present
disclosure may be administered via any suitable route; for example,
orally, intraperitoneally, subcutaneously, intranasally, buccally,
trans-dermally intramuscularly, intrarectally, intravenously, and
by oral inhalation.
[0050] The compositions used in the methods of the present
disclosure may be administered on a regimen of 1 to 4 times per
day, and in some embodiments, the compositions are administered
twice a week, once a week, once every two weeks, once every three
weeks, once every four weeks, once every six weeks, once every
eight weeks or even less frequently depending on the needs of the
patient.
[0051] The compositions used in the methods of the present
disclosure may be administered as part of a course of treatment
lasting for about 1 to about 30 days; about 1 to about 90 days;
about 1 to about 120 days; about 1 to about 180 days; about 1 to
365 days; one year; two years; three years; or for the patient's
lifetime.
[0052] It will be understood, however, that the specific dose level
and frequency of dosage for any particular host may be varied and
will depend upon a variety of factors including the activity of the
specific compound employed, the metabolic stability and length of
action of that compound, the age, body weight, general health, sex,
diet, mode and time of administration, rate of excretion, drug
combination, the nature of the disorder, the severity of the
particular disorder, and the host undergoing therapy.
[0053] To facilitate a better understanding of the present
invention, the following examples of specific embodiments are
given. In no way should the following examples be read to limit or
define the entire scope of the invention
EXAMPLES
Example 1
[0054] Materials and Methods
[0055] Dulbecco's Modified Eagle Medium (DMEM), trypsin,
L-glutamine, gentamicin, and NuPage gels and buffers were obtained
from Invitrogen (Carlsbad, Calif.) while FBS was from Hyclone,
Thermo Scientific (Rockford, Ill.). The monoclonal anti-vimentin
(V9), conjugated anti-mouse-IgG and anti-rabbit-IgG antibodies were
from Santa Cruz Biotechnologies, Inc. (Santa Cruz, Calif.). The
anti-GAPDH antibody was from Millipore (Billerica, Mass.) and the
anti-Rab9 polyclonal antibody has been described elsewhere [27].
Filipin was from Polysciences, Inc. (Warrington, Pa.). Lumilight
Plus substrate and FuGENE.TM. 6 transfection reagent were both from
Roche Diagnostics (Indianapolis, Ind.). [9,10-3H(N)] oleic acid (15
Ci/mmol) was obtained from NEN Life Science Products (Boston,
Mass.) and LDL was from EMD Biosciences Inc. (La Jolla, Calif.).
All other chemicals were acquired from Sigma-Aldrich (St. Louis,
Mo.).
[0056] Cell Culture and Transfection
[0057] The human wild-type fibroblast (GM05387), NPC1o fibroblast
(GM09341), and NPC1 fibroblast (GM03123) cell lines were obtained
from Coriell Cell Repositories (Camden, N.J.). The M12 Chinese
hamster ovary (CHO) cell line and its wild-type parental line were
obtained and cultured. Fibroblast cell lines were cultured in DMEM,
and CHO cells were cultured in DMEM/F12 (50:50) medium,
supplemented with 10% FBS, 2 mM L-glutamine, and 50 .mu.g/ml
gentamicin in a humidified incubator at 37.degree. C. with 5%
CO2.
[0058] The cDNA for PKC a was cloned into the bicistronic vector
pIRES (Stratagene), which contains GFP for monitoring successful
transfection. The cDNAs for PKC .beta.II, and PKC .epsilon. (ATCC)
were cloned into vector pYDual, which expresses a nuclear-targeted
RFP (Ioannou, unpublished). A Rab9-YFP fusion construct (described
in [5]) was used for Rab9 expression. Transient overexpression was
achieved by transfecting cells at 70% confluency using the
FuGENE.TM. 6 reagent (Roche Diagnostics) according to the
manufacturer's suggestions.
[0059] Protein Analyses
[0060] Transfected cells were harvested with PBS containing 2 mM
EDTA at 2 days post-transfection. Soluble and insoluble cell
fractions were prepared as described previously [5]. Briefly, to
obtain the soluble/cytoplasmic fraction, cells were incubated on
ice for 30 min in cold "phospho" buffer [150 mM NaCl, 20 mM NaF,
100 .mu.M Na.sub.3VO.sub.4, 20 mM Hepes, pH 7.5), 1% (v/v) Igepal,
10% (v/v) glycerol, and 1 .mu.L/20 mg tissue of protease inhibitor
cocktail] and then centrifuged for 20 min at 14,000 rpm at
4.degree. C.; the clear supernatant was frozen in aliquots at a
concentration of 1 .mu.g/.mu.L. The pellet (insoluble fraction) was
washed 3 times in ice-cold PBS containing 2 mM EDTA and then
resuspended in a volume of "Triton" buffer [PBS, 1% (w/v) SDS, and
0.1% (v/v) Triton X-100] equal to the "phospho" buffer. This
solution was boiled for 10 min and sonicated until the solution
became clear. The protein concentration of this fraction was
adjusted to 1 .mu.g/.mu.l according to the protein concentrations
determined for the soluble/cytoplasmic fraction. Protein
concentrations were determined using the fluorescamine method as we
have described [36]. 4-12% Bis-Tris precast gel (Invitrogen,
Carlsbad, Calif.) and then transferred onto a Protran membrane
using an XCell II apparatus (Invitrogen, Carlsbad, Calif.)
according to the manufacturer's instructions. Blots were processed
as described previously [27].
[0061] For Rab9 dissociation studies, 1.0.times.107 NPC1 3123 cells
were collected in ice-cold PBS and lysed by sonication 4 times for
lOs each. The lysate was centrifuged at 14,000 rpm for 10 minutes
to separate the soluble from the insoluble fractions and the total
protein concentration was determined using a modified Bradford
assay (Bio-Rad, Hercules, Calif.). An equal amount of each
insoluble fraction was mixed with each purified PKC isoform from a
PKC isozyme panel (Sigma, St. Louis, Mo.) and incubated for 60 min
at 37.degree. C. An equal volume of each sample was resolved
through a 4-12% Bis-Tris precast gel, transferred to a membrane,
and processed as described above.
[0062] Cholesterol Esterification
[0063] The preparation of [3H] oleate substrate and esterification
assays were performed as previously described [5]. Cells were
treated with 50 .mu.g/ml fatty acid for 2 days and then transfected
with PKE.alpha. or PKC.epsilon. for 24 hrs before esterification
assays. All values were generated in triplicate and normalized for
total cell protein.
[0064] Immunofluorescence Microscopy
[0065] For filipin staining in transfected cells, cells were
transfected with PKC or Rab9 using Fugene 6 according to the
manufacturer's recommendations. After 48 hrs, cells were stained
with filipin as we have previously described [3 7]. Cells were
mounted onto slides using Fluoromount-G (Southern Biotech,
Birmingham, Ala.) and analyzed on a Nikon Eclipse microscope fitted
with a charge-coupled-device camera (Nikon, Melville, N.Y.). Images
were acquired with MetaVue software and then deconvoluted using
AutoDeblur software from AutoQuant Imaging, Inc (Troy, N.Y.). For
quantitation of filipin fluorescence, cells were seeded at
3.times.10.sup.5 cells/well in 6-well dishes and allowed to settle
overnight, after which the medium was replaced with medium
containing 10% lipoprotein deficient serum (LPDS) for 4 days. Cells
were incubated with oleic/linoleic acids for 48 hours, DCPLA/DHA
for 24 hrs, or diazoxide for 72 hrs before fixing and staining with
filipin as we have previously described. Images were acquired using
the same exposure time for all samples. Fluorescence intensity was
determined using the integrated intensity function of MetaVue
software; at least 150 cells were quantitated for each sample and
each experiment was repeated 3 times. For analysis of sphingolipid
transport, cells were incubated with oleic/linoleic acids, DCP-LA,
or PMA for 48 hours before BODIPY-LacCer staining was performed as
previously described [30].
[0066] PKC Expression Increases the Levels of Soluble Vimentin in
NPC1 Cells
[0067] NPC1 cells with missense or null (NPC1o) mutations contain
decreased or virtually undetectable levels of soluble
phosphorylated vimentin relative to WT cells, respectively [5].
Furthermore, the vimentin present in NPC1 cells exists as large
disorganized filaments (dephosphorylated state) near the plasma
membrane. Thus, NPC1 cells behave essentially as vimentin-null
cells, which, similar to NPC1 cells, are unable to esterify
LDL-derived cholesterol [11]. In extending those studies, it was
hypothesized that decreased vimentin phosphorylation was the result
of protein kinase C (PKC) inhibition in NPC1 cells. In support of
this, it was observed in that study that treatment of NPC cells
with the PKC activator phorbol-12-myristate-13-acetate (PMA)
increased levels of soluble vimentin and ameliorated the NPC lipid
storage phenotype, whereas conversely, treatment of WT cells with
PKC inhibitors resulted in the disappearance of soluble vimentin in
those cells. These results strongly implicate PKC in the
maintenance of the soluble vimentin pool in cells and by extension
normal lysosomal cholesterol efflux. Extending those studies by
evaluating different PKC isoforms and their effects on soluble
vimentin levels in NPC cells, it can be shown that the PKC isoforms
.alpha., .beta.II, and .epsilon. have been implicated in vimentin
phosphorylation [10, 17, 18]; therefore, the focus on these
isoforms. They were transiently expressed in human NPC1 cells and
their effects on soluble vimentin levels were characterized.
Expression of PKC .beta.II caused a significant increase in soluble
vimentin levels (.about.38-fold higher than untransfected NPC1
cells), which was higher than the levels seen in WT cells
(.about.20-fold higher than NPC1 cells), whereas expression of PKCs
.alpha. or .epsilon. caused smaller but still significant increases
(.about.3-fold and .about.7-fold, respectively) in soluble vimentin
levels (FIG. 1A, and FIG. 1B). As a control, expression of Rab9 in
these cells also led to a significant increase (-30-fold) in
soluble vimentin, consistent with what was previously reported [5].
As noted all three isoforms resulted in an increase of soluble Rab9
levels to a similar degree (.about.2500-fold higher than
untransfected NPC1 cells). Furthermore, insoluble vimentin levels
decreased as soluble vimentin levels increased in PKCexpressing
cells, suggesting that the increase in soluble vimentin was due to
solubilization (phosphorylation) of insoluble vimentin (FIG.
1A).
[0068] Similarly, in the severely affected NPC1o cells, which
normally have almost no detectable soluble vimentin, expression of
any of the three PKC isoforms resulted in increased levels of
soluble vimentin (FIG. 1B). With respect to vimentin
solubilization, all three isoforms work equally well in the NPC1o
cells, in contrast to the NPC1 cells, in which the .beta.II isoform
seemed to be the most effective in solubilizing vimentin.
Furthermore, soluble Rab9 levels also increased to similar levels
as a result of PKC Expression (FIG. 1B), a result also seen in
PKC-expressing NPC1 cells (FIG. 1A).
[0069] PKC Expression Induces Rab9 Dissociation from Vimentin
[0070] The observations that the small GTPase Rab9 is trapped in
vimentin filaments in NPC1 cells [5] and that Rab9 overexpression
corrects the NPC1 phenotype [19] strongly suggest that Rab9
availability is reduced in NPC1 cells. The disease cells may
attempt to compensate for this deficit by upregulating Rab9 protein
expression. This idea is supported by the fact that NPC1 cells do
contain more Rab9 protein than WT cells (FIG. 1A and [19]). As
described above, PKC expression increased not only soluble vimentin
levels but also soluble Rab9 levels significantly (FIG. 1A and FIG.
1B). To determine whether the increased Rab9 levels in
PKC-expressing cells was a result of Rab9 release from insoluble
vimentin after it was phosphorylated, PKC assays were performed in
vitro with nine purified PKC isoforms using the insoluble vimentin
fraction of NPC1 cell lysates as the PKC substrate. All isoforms
were able to effect Rab9 release from insoluble vimentin to varying
degrees (FIG. 2), suggesting that, at least in vitro, most PKC
isoforms can catalyze vimentin phosphorylation and Rab9 release.
Interestingly, PKC .alpha. caused the greatest increase in Rab9
release, while PKC .beta.II and .epsilon. were less effective and
PKC y was almost ineffective. These results differ from the results
of transiently transfected PKC-expressing cells, which suggest that
PKC .beta.II is more effective in increasing soluble vimentin
levels than PKC .alpha. and .epsilon. (FIG. 1A). The discrepancies
in isoform effectiveness may be due to the nature of the assays,
the inherent activity of each isoform, or the in vivo subcellular
location of the different isoforms and their access to vimentin
[10, 16, 17, 20]; however, it is clear that PKC is able to
solubilize vimentin and in doing so release entrapped Rab9.
[0071] Overexpression of PKCs Induces a Partial Correction of the
NPC1 Phenotype
[0072] Based on the data above and observations that Rab9
overexpression results in increased soluble vimentin and correction
of the NPC1 phenotype [5], it was determined whether increased
vimentin solubility caused by PKC overexpression would also result
in correction of the NPC1 phenotype. NPC1 CHO (M12) cells
containing a deletion of the NPC1 locus [21] were transfected with
PKC .alpha. or PKC .epsilon. and the amount of LDL-derived free
cholesterol transported from the E/L system to the ER for
esterification by acyl-CoA: cholesterol acyltransferase (ACAT) [22]
was measured. Esterification levels for M12 cells were less than
10% of the esterification activity of the parental WT CHO cells
(FIG. 3), which is consistent with a block in cholesterol transport
out of the E/L system. Expression of PKC .alpha. or PKC .epsilon.
ameliorated the cholesterol transport block, increasing the level
of M12 cell esterification by approximately 4-and 6.5-fold,
respectively, over that of untransfected M12 cells (FIG. 3,
+PKC.alpha., +PK.epsilon.). These results indicate that
solubilization of vimentin mediated by expression of PKCs can
partially release the NPC1 lipid transport block.
[0073] Cholesterol storage in PKC-transfected cells was also
determined qualitatively by staining with filipin, a fluorescent
probe that binds to free cholesterol [23]. This analysis yielded
similar results to the esterification studies shown in FIG. 3.
Cells that were positive (as determined by GFP co-expression) for
PKC.alpha., PK.epsilon., or PKC.beta.II showed significantly less
filipin staining than surrounding untransfected cells (FIG. 4).
These results were similar to those seen in cells over-expressing
Rab9 (FIG. 4, Rab9), which has previously been shown to correct the
NPC1 cholesterol storage phenotype [19].
[0074] Exposure to Fatty Acids Increases Soluble Vimentin Levels in
NPC1 Cells
[0075] Fatty acids and in particular oleic acid have been shown to
induce PKC activity [24], whereas a downstream metabolite of
linoleic acid, DCP-LA
(8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid), has
been shown to potently activate PKC .epsilon. [25, 26].
Furthermore, it was shown previously that NPC1 endosomes store
large amounts of fatty acids [27], which could potentially limit
the amount of free fatty acids available to the cell for PKC
activation and other processes.
[0076] To determine if exogenously added fatty acids can increase
vimentin solubilization in NPC1 cells, human NPC1 fibroblasts were
treated with oleic or linoleic acid for 48 hours and the levels of
soluble vimentin in cell lysates were analyzed. NPC1 fibroblasts
contain very little soluble vimentin (FIG. 5A and [5]). Treatment
with either oleic or linoleic acid significantly increases the
amount of soluble vimentin, with oleic acid being slightly more
effective. These results suggest that exogenously added fatty acids
can effect vimentin solubilization in NPC1 cells, presumably by
activating PKCs.
[0077] Exposure to Fatty Acids Induces Correction of the NPC1
Phenotype
[0078] Since fatty acids increase solubilization of vimentin in
NPC1 cells, they might also improve the NPC1 phenotype. M12 CHO
cells (FIG. 5B) and human NPC1 fibroblasts (FIG. 5C) were treated
with each fatty acid and then stained with filipin. WT cells (FIGS.
5B/C, 1) stain very weakly with filipin, whereas NPC1 (FIGS. 5B/C,
2) cells contain bright, punctate staining that is indicative of
free cholesterol in endocytic vesicles. Filipin fluorescence in
NPC1 cells from both species was significantly decreased after
exposure to either fatty acid (FIGS. 5B/C, 3 and 4). Following
quantitation of filipin fluorescence levels by integrated
morphometry, both fatty acids were found to dramatically reduce the
levels of cholesterol accumulation in both NPC1 cell lines,
reducing filipin fluorescence to .about.75% of levels in untreated
cells (FIGS. 5B/C, graphs). Human fibroblasts exhibit more
heterogeneous filipin staining patterns than either CHO or mouse
NPC1 cell lines, which is reflected in the higher standard
deviation in untreated 3123 cells (FIG. 5C, graph). The effect of
fatty acids on cholesterol esterification in M12 cells was also
evaluated. Linoleic acid increased cholesterol esterification by
greater than 2-fold compared to untreated M12 cells (FIG. 3, +Lin).
In M12 cells treated with oleic or linoleic acids followed by
expression of PKC E, the correction of the NPC1 phenotype was more
pronounced, with esterification levels that were .about.3.5-fold
over untreated cells (FIG. 3, +PKCE/oleic and
+PKC.epsilon./linoleic). These results suggest that fatty acids and
PKC expression have an additive effect on correction of the NPC1
cholesterol transport block.
[0079] To further characterize the effects of fatty acids on the
NPC phenotype, the ability of docosahexanoic acid (DHA) and a
metabolite of linoleic acid, DCP-LA, were tested for their ability
to decrease cholesterol storage in M12 CHO cells. Both fatty acids
have been shown to potently activate PKC .epsilon. [24, 28].
Treatment of M12 CHO cells with these compounds overnight resulted
in decreased cholesterol storage (FIG. 6C and FIG. 6D) relative to
untreated cells (FIG. 6B). DCP-LA (FIG. 6C) was slightly more
effective than DHA (FIG. 6D). Quantitation of filipin intensity in
these cells revealed that both compounds reduced cholesterol
storage in M12 CHO cells by .about.50% (FIG. 6G), lending further
support to the positive effect of free fatty acids on the NPC
disease phenotype, possibly through activation of PKC
.epsilon..
[0080] To provide further support for the role of PKCs in NPC
rescue, M12 cells were treated with diazoxide, which has been shown
to activate PKC .epsilon. [29]. This treatment resulted in reduced
cholesterol accumulation in M12 CHO cells (FIG. 6F) relative to
untreated cells (FIG. 6E). Quantitation of the filipin intensity in
these cells indicated that diazoxide reduced cholesterol storage by
.about.50%, similar to the results seen with the free fatty acids
(FIG. 6G).
[0081] To further confirm the positive effects of DCP-LA and fatty
acids on the NPC1 phenotype, human NPC1 cells were treated with
DCP-LA, fatty acids, or PMA to activate PKCs. Cells were then
labeled with BODIPY-LacCer, which has previously been shown to
provide a dynamic view of the endocytic pathway [30]. Following
absorption to the plasma membrane, the LacCer sphingolipid enters
normal cells via endocytosis and eventually reaches the trans-Golgi
network (TGN; [30]). Due to the lipid transport block in NPC1 cells
however, this sphingolipid is trapped in endosomes and its
targeting to the TGN is dramatically inhibited [30]. Human NPC1
cells treated as indicated in FIG. 7 show a dramatic improvement in
lipid transport with the BODIPY-LacCer effectively reaching the TGN
(FIG. 7; arrows) compared to untreated cells that show only
punctate, endosomal fluorescence (FIG. 7A). These results provide
further support that these agents are able to release the NPC1
lipid block.
[0082] Taken together, these results indicate that exposure to free
fatty acids, which may act by activating PKC.epsilon. has a
positive effect on the NPC cholesterol storage phenotype.
[0083] Discussion
[0084] Rab9 expression in NPC1 cells restored lipid transport from
the E/L system and normalized cholesterol esterification [19] and
subsequently showed that Rab9 was entrapped in insoluble vimentin
filaments in NPC1 cells [5]. Consequently, accumulated lipids, such
as sphingosine [31, 32] in NPC1 cells, might exert an inhibitory
effect on various PKC isoforms, resulting in a disruption of the
vimentin phosphorylation/dephosphorylation cycle [19].
[0085] To characterize the nature of PKC inhibition and vimentin
hyposphorylation in NPC1 cells, a number of PKC isoforms (.alpha.,
.beta.II and .epsilon.) in NPC1 cells were expressed and their
effect characterized on vimentin solubilization and correction of
the NPC1 phenotype. All three isoforms had a positive effect on
vimentin solubilization to varying degrees (FIG. 1A and FIG.
1B).
[0086] Furthermore, PKC-induced vimentin solubilization was
accompanied by the release of the entrapped Rab9 (FIG. 1A and FIG.
1B). To further determine which PKC isoforms might be more
effective in vimentin phosphorylation and release of Rab9, eight
different PKC isoforms were tested in an in vitro assay. Most
isoforms were able to release Rab9 from vimentin to varying degrees
(FIG. 2), which may not be in case in vivo. This discrepancy is
likely due to the different in vivo subcellular locations of PKC
isoforms and their access to vimentin filaments [16, 20]. However,
it is also possible that vimentin may not be a direct substrate for
certain PKCs, as has been shown with regards to PKC .delta.
controlled phosphorylation of vimentin [10]. In that study, PKC
.epsilon. mediated vimentin phosphorylation, which was shown to be
critical for proper integrin recycling through the cell. These
studies indicate that expression of PKC isoforms in NPC1 cells
results in the partial correction of the NPC1 disease phenotype,
i.e., cholesterol accumulation in the endosomal/lysosomal
system.
[0087] Also, many studies have shown that long chain fatty acids
such as oleic and linoleic, along with downstream metabolites such
as DCP-LA, are able to activate PKC .epsilon. [24, 25], an isoform
shown to phosphorylate vimentin filaments [10]. Since it has been
previously reported that the availability of free fatty acids may
be limited in NPC1 cells [27, 33], exogenously added fatty acids
were considered to have a positive effect on the NPC1 phenotype,
presumably by activating PKC .epsilon. and leading to
phosphorylation of vimentin and release of Rab9. As predicted,
addition of oleic acid, linoleic acid, or DCPLA resulted in an
increase of soluble vimentin in NPC1 cells (FIG. 5 A, FIG. 5B and
FIG. 5C). Furthermore, fatty acid addition resulted in a
significant improvement in cholesterol esterification by NPC1 cells
(FIG. 3), indicating that lipid transport from the E/L system was
restored. The ability of diazoxide, a known activator of PKC [29],
was tested to correct the NPC1 phenotype, providing further support
for the involvement of insufficient PKC phosphorylation of vimentin
in contributing to NPC1 pathogenesis. In agreement with the results
presented here, diazoxide was able to reduce cholesterol
accumulation in NPC1 cells by 50% (FIG. 6). Although results with
multiple PKC .epsilon. activators strongly suggest that PKC
.epsilon. is mediating these changes within the NPC cells, the
possibility that these agents may be acting through some other
pathway or protein besides PKC cannot be excluded.
[0088] These data are consistent with previous observations of
aberrant PKC expression in NPC mouse liver [34]. In those studies
the expression of PKC .alpha.a, .delta., .epsilon., and .zeta. were
evaluated by immunoblot. Whereas PKC .alpha. and 6 were about
3-fold higher in NPC1 livers compared to WT livers, PKC .epsilon.
was not significantly increased and PKC was higher only in
heterozygous livers. It is interesting to postulate that PKC
.epsilon. does not render itself amenable to upregulation but can
be activated, via fatty acids for example, and such activation can
yield beneficial results in NPC1 cells. There is strong evidence
that PKC .epsilon. is responsible for phosphorylating vimentin,
which in turn controls the vesicular transport of various ligands
such as integrins [10]. Considering the difficulties of delivering
proteins as therapeutics, which are significantly amplified in
diseases with neuropathology such as NPC1, a small lipid activator
of a key regulator such as PKC 6 would be greatly advantageous.
These results suggest that identification of the PKC isoform(s)
responsible for vimentin phosphorylation may provide new
therapeutic targets for the treatment of Niemann-Pick type C
disease, as well as other lysosomal storage disorders that lead to
E/L lipid accumulation [35].
Example 2
[0089] We also studied the effect of the PKC activators bryostatin
1 and DCPLA on the phenotype of Niemann-Pick C disease using probes
for free cholesterol (Filipin), glycosphingolipid levels (VTB) and
gangliosides movement (CTB), following treatment of human NPC1
cells for 48-72 hrs.
[0090] Materials and Methods
TABLE-US-00001 TABLE 1 Human NPC1 Cell Lines NPC3-SV: SV 40 large T
immortalized Species: Human Source: Mount Sinai-Proprietary NPC
genotype: V1165M and 3741-44delACTC NPC24 SV 40 large T
immortalized Species: Human Source: Mount Sinai-Proprietary NPC
genotype: p.I1061T/p.I1061T
TABLE-US-00002 TABLE 2 Reagents PKC Activators: Bryostatin 1,
DCPLA, Diazoxide Source: Santa Cruz Biotech, Sigma-Aldrich Staining
Reagents: Filipin, Cholera toxin B, Verotoxin B Source:
Polysciences, Sigma-Aldrich, MS Proprietary
[0091] General Methodology
[0092] Cells are plated in 6-well dishes and treated with the
appropriate compound (dissolved in DMSO) at the indicated dose
daily for 48 hrs. Control cells receive DMSO. After 48 hrs cells
are transferred to cover slips with fresh compound and grown for
another 24 hrs. Cover slips are collected and processed for
microscopy.
[0093] Assay Protocol
[0094] For detection of unesterified cholesterol in lysosomes,
cells were fixed in formalin for 30 min at 4.degree. C., washed
2.times., 5 min in 0.9% NaCl, incubated for 45 min with 0.01%
filipin in PBS at room temperature, and then washed 2.times., 5
min. Fluorescence was observed using a Nikon Eclipse fluorescence
microscope (Nikon, Melville, N.Y.) equipped with a CCD camera.
Fluorescence signals were quantified using Nikon's imaging and
quantitation software package NIS Elements, v.3.22.
[0095] Verotoxin B (VTB): For VTB staining (glycosphingolipid
detection) cells are washed with PBS and fixed in formalin, 30 min,
4.degree. C. Following a wash with 0.9% sodium chloride, 2.times.,
5 min at room temperature cells are permeabilized with digitonin 50
.mu.g/well in 1.5 ml PBS. Cells are washed with PBS, and
alexa-labeled VTB is added at 0.5 m/well in 1 ml PBS. Cells are
incubate 45 min at RT on a shaker in the dark, washed with 0.9%
sodium chloride, 2.times., 5 min at RT, and mounted for
viewing.
[0096] Cholera toxin B (CTB): For CTB labeling (ganglioside
movement) cells are washed with PBS, and 0.5 .mu.g/well CTB is
added in 1.5 ml Opti-MEM per well. Cells are incubated in a
37.degree. C. incubator for 1 hr. Complete DMEM with FBS media is
added and incubation is continued for 4 hr at 37.degree. C. Cells
are washed with PBS and fixed with formalin for 15 min at 4.degree.
C. Cells are washed with 0.9% sodium chloride, 2.times., 5 min at
RT and mounted for viewing.
[0097] Analysis, Results and Conclusions
[0098] Niemann-Pick Cl (NPC1) cells have a defective NPC1 protein
and are characterized by the extreme accumulation of a number of
lipids such as cholesterol, sphingolipids and gangliosides in
various endosomal vesicles (FIG. 2). In the characterization of
NPC1 cells from various patients and evaluation of potential
compounds that may be beneficial in treating this disease a number
of different assays that measure the clearance and/or movement of
various NPC1-specific lipids were performed. For example filipin
was used to detect the level of stored cholesterol (Assay protocol)
whereas verotoxin B (VTB) allows the assessment of the levels of
glycosphingolipids which are sometimes stored in endosomes distinct
from those that contain cholesterol (Assay protocol). In addition,
movement of gangliosides from the plasma membrane by using a
different probe, cholera toxin can be monitored (Assay
protocol).
[0099] These studies indicate that all three PKC activators,
bryostatin 1, DCPLA and diazoxide are able to induce clearance of
cholesterol from NPC1 cells to varying degrees and at different
drug concentrations (FIG. 8). However, bryostatin 1 appears to be
the most active compound showing activity in the nanomolar
concentration range (FIG. 9). At 10 nM bryostatin 1 also shows
statistically significant effectiveness in decreasing the
glycosphingolipid accumulation of NPC1 cells (FIG. 10).
[0100] To determine whether the effects of bryostatin 1 is
dependent on the genotype of the NPC1 cells used in the above
experiments (i.e. the specific NPC1 gene mutations carried by a
particular patient) a different patient cell line with a completely
distinct NPC1 genotype was used. Bryostatin 1 was still able to
effect cholesterol 2 clearance from these cells at 10 nM
concentration indicating that the activity of bryostatin 1 is
independent of the NPC1 genotype (FIG. 11).
[0101] Bryostatin 1 also was effective in a kinetic assay that
monitors the movement of lipids from the plasma membrane to the
Golgi network through endosomes. At 10 mM bryostatin 1 was able to
clear the endosome-trapped cholera toxin (CTB) from NPC1 cells
(FIG. 12).
[0102] To confirm these results bryostatin 1 was used at a range of
0.1 nM to 100 nM and evaluated both in cholesterol and sphingolipid
assays (FIG. 13). At 0.1 nM bryostatin has no effect in both
assays. The effect becomes statistically significant at 1 nM and
increases with increasing bryostatin 1 concentration.
[0103] Accordingly, bryostatin 1 showed a positive therapeutic
effect for human NPC1 disease cells at a concentration of 10-100
nM.
Example 3
[0104] Niemann-Pick C disease is a severe inherited lipidosis that
leads to neurodegeneration and early childhood death. The
biochemical and cellular events that lead to neurodegeneration are
currently poorly understood. However, it has been shown that
PKC.epsilon., activation can restore the blocked lipid transport
pathway and lead to a reduction of stored lipid material in the NPC
endosomes/lysosomes. Thus, treatment of NPC1 mice with bryostatin
1, a natural product activator of PKC , should lead to an
improvement of disease progression in this animal model.
[0105] A total of 30 C57B16 NPC1 mice, mixed sex, were used. These
mice were separated into 5 groups of 5 mice each, Groups 1-5.
[0106] Study Drug
[0107] Bryostatin 1 (purity .gtoreq.:95%) from Aphios (Woburn,
Mass.), 1 vial 2 mg, was solubilized in a 5% DMSO, 20% Solutol and
75% Saline solution and used as the study drug. The negative
(vehicle) control is a 5% DMSO, 20% Solutol and 75% Saline
solution. DCP-LA from Sigma-Aldrich: 5 mg oil/vial was used as an
additional in vitro active compound. Bryostatin 1 (API) was stored
at or below -20.degree. C. and formulated as needed. The formulated
bryostatin 1 was stored at 2.degree. to 8.degree. C. for less than
24 hour(s). The DCP-LA and the vehicle controls were stored at
2.degree.-8.degree. C.
[0108] The stock solution of bryostatin 1 is 1 Omg/ml in DMSO,
which is kept at -80.degree. C. in aliquots. The dosing formulation
is made by diluting the stock first in DMSO, then adding solutol,
and lastly saline-with more insoluble compounds, adding the drug
stock to complete vehicle will cause the compound to come out of
solution, so we make formulations stepwise. The dosing volume is
always 100 .mu.l. Mice weigh approximately 20 g.
[0109] Dosing and Frequency
[0110] The study agents are dosed intraperitoneally (IP) as shown
in Table 3. The mice are dosed twice weekly (Mon and Thu) starting
at 30 days for up to 150 days.
TABLE-US-00003 TABLE 3 Dosing regimen Group Agent Dosing Group 1:
Vehicle control (mL) Group 2: Bryostatin-1 [BHD-1] 40 .mu.g/kg (120
.mu.g/m.sup.2) Group 3: Bryostatin-1 [BHD-2] 30 .mu.g/kg (90
.mu.g/m.sup.2) Group 4: Bryostatin-1 [BHD-3] 20 .mu.g/kg (60
.mu.g/m.sup.2) Group 5: Bryostatin-1 [BHD-3] 10 .mu.g/kg (30
.mu.g/m.sup.2) Group 6: Bryostatin-1 [BHD-3] 5 .mu.g/kg (15
.mu.g/m.sup.2) Group 7: Active (DCP-LA mg oil/kg)
[0111] Study Duration
[0112] C57 NPC1 mice have an average life span of .about.110 days.
By Day 70 mice reach an average weight of 18-22 grams. Untreated
NPC1 mice develop ataxia and will begin to lose weight at .about.70
days (see FIG. 17). Ataxia (or lack of voluntary muscle control) is
a clinical feature of NPC1 mice, which may be described as shaking.
Treatment will be initiated at .about.30 days of age, average NPC
mouse weight of 10 g. If the drug is efficacious injections will
continue past day 70-80. An extension of life of about 20-30% will
mean that animals will need to be treated up to day 130-150.
[0113] Data Collection
[0114] Initially mice will be weighed and weights will be recorded
prior to each injection. Mice may periodically be tested in a
rotarod and time on rotarod will be recorded.
[0115] Upon euthanasia blood will be collected and the brains will
be frozen and stored for evaluation of cholesterol storage and
Purkinje cell survival. In addition, the livers and spleens from
these mice will be extracted, prepared for histology and stored
frozen for the option of determining the effects of bryostatin 1
treatment on peripheral organ lipid accumulation.
[0116] Data Analysis
[0117] Primary outcome will be weights collected on dosing days
(2.times./week) over the course of the study. Differences will be
examined between vehicle control and different doses of bryostatin
1, particularly after age 70 days, when animals have historically
experienced a weight loss.
[0118] Secondary outcomes include: (1) lipid accumulation in liver
and spleen, and (2) cerebellar Purkinje cell survival and
cholesterol storage (which are to be conducted only if the primary
outcome is positive).
[0119] Results
[0120] Mice in dose groups 30, 20 and 10 .mu.g/kg lived past the
age of 100 days.
[0121] One of skill in the art will appreciate that the examples
herein are not intended to be limiting and that one of skill in the
art will readily be able to apply the teachings herein to treating
lipid storage disorders. Therefore, the present invention is well
adapted to attain the ends and advantages mentioned as well as
those that are inherent therein. While numerous changes may be made
by those skilled in the art, such changes are encompassed within
the spirit of this invention as illustrated, in part, by the
appended claims.
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