U.S. patent application number 15/458234 was filed with the patent office on 2017-12-28 for small molecule therapeutic compounds targeting thioesterase deficiency disorders and methods of using the same.
The applicant listed for this patent is The USA, as represented by the Secretary, Dept. of Health and Human Services, The USA, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Anil Baran MUKHERJEE, Chinmoy SARKAR, Zhongjian ZHANG.
Application Number | 20170367998 15/458234 |
Document ID | / |
Family ID | 46001782 |
Filed Date | 2017-12-28 |
United States Patent
Application |
20170367998 |
Kind Code |
A1 |
MUKHERJEE; Anil Baran ; et
al. |
December 28, 2017 |
SMALL MOLECULE THERAPEUTIC COMPOUNDS TARGETING THIOESTERASE
DEFICIENCY DISORDERS AND METHODS OF USING THE SAME
Abstract
The invention provides methods of inhibiting the development or
progression of a thioesterase deficiency disorder in a mammal by
the administration of a compound that functionally mimics the
enzymatic activity of all thioesterases in mammals. Such
thioesterase deficiency disorders include cancers and adult- or
infant-neuronal ceroid lipofuscinoses (NCLs). The invention also
provides small molecule mimics of thioesterases useful in the
methods of the invention and pharmaceutical compositions containing
the therapeutically effective compounds and methods of using the
same.
Inventors: |
MUKHERJEE; Anil Baran;
(Brookeville, MD) ; SARKAR; Chinmoy; (Rockville,
MD) ; ZHANG; Zhongjian; (Rockville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The USA, as represented by the Secretary, Dept. of Health and Human
Services |
Bethesda |
MD |
US |
|
|
Family ID: |
46001782 |
Appl. No.: |
15/458234 |
Filed: |
March 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14110393 |
Feb 5, 2014 |
9629816 |
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PCT/US12/32772 |
Apr 9, 2012 |
|
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15458234 |
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61473692 |
Apr 8, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/137 20130101;
A61K 31/00 20130101; A61K 45/06 20130101; A61K 31/133 20130101;
A61P 43/00 20180101; A61K 31/136 20130101 |
International
Class: |
A61K 31/133 20060101
A61K031/133; A61K 31/137 20060101 A61K031/137; A61K 31/136 20060101
A61K031/136; A61K 45/06 20060101 A61K045/06; A61K 31/00 20060101
A61K031/00 |
Claims
1. A method of preventing, treating or ameliorating a thioesterase
deficiency disorder, comprising administering to a mammal in need
of such treatment, a therapeutically effective amount of a compound
that functionally mimics a thioesterase enzymatic activity.
2. The method of claim 1, wherein the compound mimics palmitoyl
protein thioesterase-1 enzymatic activity.
3. The method of claim 1, wherein the compound inhibits the
progression or development of a neurodegenerative disease
associated with a thioesterase deficiency in humans.
4. The method of claim 1, wherein the compound is at least one
compound selected from the group consisting of:
N-Methylhydroxylamine, N,N-Dimethylhydroxylamine,
N,N-Diethylhydroxylamine, N-Cyclohexylhydroxylamine,
N-(tert-Butyl)hydroxylamine, N,O-Bis(trimethylsilyl) hydroxylamine,
N-Benzylhydroxylamine, N-Benzyloxycarbonyl),
N,O-Di-Boc-hydroxylamine, N-Benzoyl-N-phenyl hydroxylamine,
N,N-Dibenzylhydroxylamine, N-tert-Butyl-O-[1-[4-(chloromethyl)
phenyl]ethyl]-N-(2-methyl-1-phenylpropyl) hydroxylamine, and
pharmaceutically-acceptable salts thereof.
5. The method of claim 1, wherein the compound is N-t-butyl
hydroxylamine or a pharmaceutically-acceptable salt thereof.
6. (canceled)
7. (canceled)
8. The method of claim 1, wherein the compound is administered to
the mammal in a pharmaceutical composition.
9. The method of claim 8, wherein the pharmaceutical composition is
a mono-phasic pharmaceutical composition suitable for parenteral or
oral administration consisting essentially of a
therapeutically-effective amount of the compound, and a
pharmaceutically acceptable carrier.
10-16. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/110,393, filed Feb. 5, 2014, and issued as U.S. Pat.
No. 9,629,816 on Apr. 25, 2017; which is a national stage
application under 35 U.S.C. 371 and claims the benefit of PCT
Application No. PCT/US2012/032772 having an international filing
date of 9 Apr. 2012, which designated the United States; and which
PCT application claimed the benefit of U.S. Provisional Application
No. 61/473,692 filed on 8 Apr. 2011; the entire disclosure of each
is incorporated by reference herein.
TECHNICAL FIELD
[0002] The invention relates to therapeutic compounds,
pharmaceutical compositions containing these compounds, and their
use in the prevention or treatment of diseases resulting from
thioesterase deficiency disorders.
BACKGROUND OF INVENTION
[0003] Lysosomal storage disorders represent a group of at least 50
genetically distinct, biochemically related, inherited diseases.
Individually, these disorders are considered rare, although high
prevalence values have been reported in some populations. These
disorders are devastating for individuals and their families and
result in considerable use of resources from health care systems,
however, the magnitude of the problem is not well defined. Included
amongst these lysosomal storage disorders are mutations or other
disruptions of thioesterases, which result in the accumulation of
posttranslationally lipid-modified proteins in lysosomes.
[0004] Thioesterases are enzymes in the esterase family (members of
E.C.3.1.2), which split an ester (specifically at a thiol group)
into an acid and alcohol, in the presence of water. Examples of
human thioesterases include acetyl-coA hydrolase, palmitoyl-CoA
hydrolase, succinyl-CoA hydrolase, formyl-CoA hydrolase, acyl-CoA
hydrolase, and ubiquitin thiolesterase and human genes encoding
thiol esterases include: ACOT1, ACOT2, ACOT4, ACOT6, ACOT7, ACOT8,
ACOT9, ACOT11(STARD14), ACOT12 (STARD15), OLAH, APT1, APT2, PPT1,
PPT2, THEM2 (ACOT13), THEM4, THEM4P1, and THEM5.
[0005] An example of a lysosomal storage disorder resulting from
one or more mutations in a thioesterase is infantile neuronal
ceroid lipofuscinosis (INCL), which is a lethal childhood
neurodegenerative storage disorder caused by palmitoyl protein
thioesterase-1 (PPT1) gene mutations. Palmitoylation is a
posttranslational modification in which a 16-carbon fatty acid,
palmitate, is attached to specific cysteine residues in
polypeptides via thioester linkage. PPT1 cleaves thioester linkages
in S-acylated proteins facilitating degradation or recycling and
its deficiency leads to lysosomal storage of these proteins causing
INCL pathogenesis, as depicted in FIG. 1. Currently, there is no
effective treatment for INCL.
[0006] As an example of cancers associated with thioesterase
deficiencies, Ras is mutated in cancer more frequently than any
other oncogene. Hence, Ras has been a focus for the development of
rationally designed anti-cancer drugs, yet to date none have been
successfully developed. Posttranslational lipid-modification of Ras
proteins is essential for Ras membrane association and
transformation. The differences in the four Ras isoforms, N-Ras,
H-Ras, K-Ras4A and KRas4B reside in the C-terminal region referred
to as the hypervariable region (HVR), which is modified by
posttranslational lipid-modifications. H-Ras is modified by two
cysteine palmitoylations and one cysteine farnesylation, whereas
N-Ras and K-Ras(A) are modified by one cysteine palmitoylation and
one cysteine farnesylation. In contrast, K-Ras(B) is not
palmitoylated. Reversible palmitoylations of H- and N-Ras GTPases
control their membrane attachment and specific localization on the
plasma membrane and the Golgi. Proper steady state localization
requires a dynamic cycle of palmitoylation on the Golgi, which
redirects Ras to the plasma membrane, and ubiquitous
depalmitoylation to counteract spontaneous nonspecific distribution
over cellular endo membranes. Disruption of this dynamic cycle
results in a reduction of Ras localization on the Golgi and the
plasma membrane, due to random redistribution to endo membranes,
indicating that inhibitors of palmitoylation as well as enzymatic
depalmitoylation alter the steady state localization of Ras GTPases
and thus Ras signaling.
[0007] Thus, thioesterases present a compelling therapeutic target
for the prevention and treatment of thioesterase deficiency
disorders such as lysosomal storage disorders including INCL, and
cancers associated with Ras localization and activation, and there
exists a need for effective methods of inhibiting Ras GTPases for
the treatment and prevention of these thioesterase deficiency
disorders.
SUMMARY OF INVENTION
[0008] The present invention provides small molecules that
functionally mimic thioesterase activity, as well as therapeutic
uses of these molecules to prevent or treat neurodegenerative
disorders or slow the growth and metastasis of cancers resulting
from a thioesterase deficiency in a mammal.
[0009] Despite the fact that hydroxylamine (HA) specifically
cleaves thioester linkages in S-acylated proteins, HA-mediated
methemoglobin production causes toxicity because methemoglobin
cannot carry oxygen like hemoglobin does. Thus, toxicity of HA
precludes its clinical use. However, HA-derivatives may be
non-toxic. The present inventors have screened 12 HA-derivatives
for their ability to cleave thioester linkages like the parent
compound, HA and discovered that they do indeed have this property.
(See Table 1 for a listing of these HA-derivatives). They found
that one of these HA-derivatives, N-t-butyl hydroxylamine (NtBuHA),
is non-toxic to both cultured cells from INCL patients and to
Ppt1-Knockout (Ppt1-KO) mice, which recapitulate virtually all
clinical and pathological features of INCL. It also does not cause
elevated levels of methemoglobin. (See Table 3). Moreover, it has
potent antioxidant properties, cleaves thioester linkage in
S-acylated proteins and reduces intracellular ceroid load in
cultured INCL cells. Additional animal studies showed that dietary
NtBuHA showed no toxicity, depleted lysosomal ceroid, reduced
endoplasmic reticulum- and oxidative-stresses, suppressed
apoptosis, improved neurological function, and markedly extended
lifespan in Ppt1-KO mice, which mimic INCL, as compared to their
untreated littermates, demonstrating that NtBuHA may be used in the
treatment of thioesterase deficiency disorders such as INCL.
[0010] Therefore, the present invention provides compounds that
functionally mimic thioesterase activity in mammals having a
thioesterase deficiency, and pharmaceutically-acceptable salts and
prodrugs thereof. The invention also provides pharmaceutical
compositions containing these compounds. The invention also
provides methods of using these compounds and pharmaceutical
compositions to treat or prevent thioesterase deficiency disorders
in a mammal in need of, or suspected of needing, such
treatment.
[0011] One embodiment of the invention is a method of treating a
thioesterase deficiency disorder by administering to a mammal in
need of such treatment, a therapeutically effective amount of a
compound that functionally mimics thioesterase enzymatic activity.
In one aspect of this embodiment, the compound mimics
palmitoyl-protein thioesterase-1 (PPT1), thereby treating
PPT1-deficiency or preventing abnormal lysosomal ceroid
accumulation responsible for PPT1-deficiency. In another aspect of
the invention, the compound mimics all thioesterases thereby
treating cancers associated with the deficiencies of these
thioesterases and preventing the growth and metastasis of these
tumors.
[0012] In a preferred embodiment of these aspects of the invention,
the compound is at least one of the compounds of Table 1.
[0013] In a preferred embodiment, the compound is administered to
the mammal in a pharmaceutical composition of the invention. In a
particularly preferred embodiment of the invention, the compound
administered is N-t-butyl hydroxylamine (NtBUHA).
[0014] Additionally, the invention provides pharmaceutical
compositions containing one or more of the compounds of Table 1, or
pharmaceutically-acceptable salts thereof, with at least one
pharmaceutically-acceptable carrier. Also provided herein is a
pharmaceutical composition comprising at least one prodrug of at
least one compound of the invention, with at least one
pharmaceutically acceptable carrier.
[0015] Another embodiment of the invention is a method of
preventing or treating cancers associated with thioesterase
deficiencies, by administering a therapeutically effective amount
of one of the compounds of the invention, or a pharmaceutically
acceptable salt thereof, or prodrug thereof, to a mammal in need of
such treatment or suspected of having a thioesterase deficiency
related cancer or a metastasis of a thioesterase deficiency related
cancer.
[0016] Another embodiment of the invention is a method of
preventing or treating neurodegenerative disorders associated with
thioesterase deficiencies, by administering a therapeutically
effective amount of one of the compounds of the invention, or a
pharmaceutically acceptable salt thereof, or prodrug thereof, to a
mammal in need of such treatment or suspected of having a
neurodegenerative disorder associated with a thioesterase
deficiency disorder. In a related embodiment, the invention is a
method of preventing or treating infantile NCL (INCL) caused by
palmitoyl-protein thioesterase-1 (PPT1)-deficiency.
[0017] Another embodiment of this invention is a method of treating
cancer by administering a therapeutically effective combination of
at least one of the compounds of Table 1 and one or more other
known anti-cancer or anti-inflammatory treatments. For example,
other anti-cancer treatments may include surgery, chemotherapy,
radiation, immunotherapy, or combinations thereof.
[0018] Also provided herein are methods for the prevention,
treatment or prophylaxis of neurodegeneration in a mammal secondary
to lysosomal storage disorders including, but not limited to,
neuronal ceroid lipofuscinoses (NCLs; also known as Batten disease)
and infantile NCL (INCL; also known as infantile Batten disease).
These methods comprise administering to a mammal in need of such
treatment, therapeutically-effective amounts of any of the
compounds of Table 1, pharmaceutical compositions of the invention
containing at least one of the compounds of Table 1, and/or
pharmaceutical compositions comprising at least one prodrug of the
compounds of Table 1.
[0019] Also provided herein are methods for preventing at least one
symptom of lysosomal storage disorders in a mammal including, but
not limited to, psychomotor retardation, retinal blindness,
myoclonus, seizures, intracellular autofluorescent storage material
and reduced lifespan. These methods comprise administering to a
mammal suspected of having a lysosomal storage disorder
therapeutically-effective amounts of any of the compounds of Table
1, pharmaceutical compositions of the invention containing at least
one of the compounds of Table 1, and/or pharmaceutical compositions
comprising at least one prodrug of the compounds of Table 1, prior
to the development of a symptom of a lysosomal storage
disorder.
[0020] Also provided herein are pharmaceutical packages comprising
a pharmaceutical composition comprising therapeutically-effective
amounts of at least one compound of Table 1, optionally together
with at least one pharmaceutically acceptable carrier. The
pharmaceutical compositions may be administered separately,
simultaneously or sequentially, with other compounds or therapies
used in the prevention, treatment or amelioration of thioesterase
deficiency-related cancers and/or neurodegenerative disorders.
These packages may also include prescribing information and/or a
container. If present, the prescribing information may describe the
administration, and/or use of these pharmaceutical compositions
alone or in combination with other therapies used in the
prevention, treatment or amelioration of thioesterase
deficiency-related cancers and/or neurodegenerative disorders.
[0021] Also provided herein are pharmaceutical packages containing
a pharmaceutical composition of at least one prodrug of a compound
of Table 1, optionally together with at least one
pharmaceutically-acceptable carrier. These packages may also
include prescribing information and/or a container. If present, the
prescribing information may describe the administration, and/or use
of these pharmaceutical compositions alone or in combination with
other therapies used in the prevention, treatment or amelioration
of thioesterase deficiency-related cancers and/or neurodegenerative
disorders.
[0022] Another embodiment of this invention is a method of testing
the susceptibility of a mammal having a thioesterase
deficiency-related cancer and/or neurodegenerative disorder to
treatment with a putative mimic of thioesterase activity by testing
the mammal for a response to the putative mimic, wherein the
response is indicative of growth inhibition or reduction in cancer
cell number or neurodegenerative disorders in the mammal.
[0023] Other aspects of the invention will be set forth in the
accompanying description of embodiments, which follows and will be
apparent from the description or may be learnt by the practice of
the invention. However, it should be understood that the following
description of embodiments is given by way of illustration only
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
and are encompassed within the scope of this invention.
BRIEF DESCRIPTION OF DRAWINGS
[0024] FIG. 1 models the suggested mechanism of lysosomal storage
in PPT1-deficient cells versus normal cells.
[0025] FIG. 2 depicts the screening method utilized to test
HA-derivatives for their ability to cleave thioester linkage in
[.sup.14C]palmitoyl .about.CoA (a model thioester substrate of
PPT1).
[0026] FIG. 3 depicts the densitometric analysis of the free
[.sup.14C]-palmitate bands for twelve HA-derivatives that were
screened for their ability to cleave thioester linkage in
[.sup.14C]palmitoyl .about.CoA.
[0027] FIGS. 4A-4C chart the results of an MTT assay determining
the viability, as a percentage of untreated control, of INCL
fibroblasts following 48 hours of treatment with ten
HA-derivatives: N-t-butyl hydroxylamine (NtBuHA),
N-benzylhydroxylamine, N-Methyl hydroxylamine, N-cyclohexyl
hydroxylamine, N-benzyloxycarbonyl hydroxylamine, N,N-dimethyl
hydroxylamine, N,N-diethyl hydroxylamine, N-benzoyl-N-phenyl
hydroxylamine, (i) N,O-bis(trimethylsilyl) hydroxylamine, and
N,O-Di-BOC hydroxylamine.
[0028] FIGS. 5A-5B depict the thioester linkages of NtBuHA-treated
INCL cells in a dose-dependent (FIG. 5A) and time-dependent (FIG.
5B) manner.
[0029] FIG. 6 charts the viability, as a percentage of untreated
control, of INCL fibroblasts following treatment with different
amounts of three HA-derivatives.
[0030] FIG. 7 depicts the densitometric analysis of
[.sup.35S]cysteine-thioester compounds of NtBuHA-treated INCL cells
(labeled "+") and untreated control INCL cells (labeled "-"), at 12
hours, 24 hours, and 48 hours after testing.
[0031] FIG. 8 depicts the densitometric analysis of
[.sup.35S]cysteine-labeled lipid bands for both untreated--(labeled
"-") and NtBuHA-treated (labeled "+") lympohblasts in the cell
lines of nine NCL patients.
[0032] FIGS. 9A-9D show TEM analysis (and magnified insets) of
GRODs in untreated control INCL lymphoblasts (FIG. 9A),
NtBuHA-treated INCL lymphoblasts (FIGS. 9B and 9C), and in
NtBuHA-treated lymphoblasts three weeks after treatment was
withdrawn.
[0033] FIGS. 10A-10B show TEM analysis (and magnified insets) of
GRODs in untreated control INCL fibroblasts (FIG. 10A) and in
NtBuHA-treated INCL fibroblasts (FIG. 10B).
[0034] FIG. 11 charts the quantitative analysis of GRODs in
untreated control INCL lymphoblasts, NtBuHA-treated INCL
lymphoblasts, and in NtBuHA-treated lymphoblasts three weeks after
treatment was withdrawn.
[0035] FIGS. 12A-12C show TEM analysis of GRODs in the brain cells
of wild-type mice (FIG. 12A), untreated PPT1-KO mice (FIG. 12B),
and NtBuHA-treated PPT1-KO mice (FIG. 12C).
[0036] FIGS. 13A-13C show autofluorescent analysis of the brain
tissues of wild-type mice (FIG. 13A), untreated PPT1-KO mice (FIG.
13B), and NtBuHA-treated PPT1-KO mice (FIG. 13C).
[0037] FIGS. 14A-14B show Western blot analysis of the brain
lysates from wild-type mice (labeled "WT"), untreated PPT1-KO mice
(labeled "KO"), and ntBuHA-treated PPT1-KO mice (labeled "ntBuHA")
for ER-stress markers Grp-78, Grp-94, and ATF6 (FIG. 14A) as well
SOD2 and catalase (FIG. 14B).
[0038] FIG. 15 charts the number of apoptotic cells (by percentage)
after FACS analysis of H.sub.2O.sub.2 incubated (labeled "+") and
non-H.sub.2O.sub.2 incubated (labeled "-") untreated INCL
lymphoblasts (labeled "control"), NtBuHA-pretreated INCL
lymphoblasts, and NtBuHA pre- and co-treated INCL lymphoblasts.
[0039] FIGS. 16A-16C depict (TUNEL) assays conducted using cortical
tissue sections from wild-type mice (FIG. 16A), untreated PPT1-KO
mice, and NtBuHA-treated PPT1-KO mice (FIG. 16C) to determine
apoptosis, with arrows indicating apoptotic cells. FIG. 16D charts
the quantitative analysis of this TUNEL assay of apoptotic cells,
wherein cortical tissues of wild-type mice are labeled "1,"
untreated PPT1-KO mice are labeled "2," and NtBuHA-treated PPT1-KO
mice are labeled "3."
[0040] FIG. 17 shows Western blot analysis of caspase-9 and cleaved
PARP-1 cortical tissue of wild-type, untreated and NtBuHA-treated
PPT1-KO mice showed a marked reduction in level
[0041] FIGS. 18A-18D depict an immunohistochemical analyses of
cortical tissue sections from wild-type (FIG. 18A),
untreated-PPT1-KO (FIG. 18B), and NtBuHA-treated PPT1-KO (FIG. 18C)
mice was performed using antibodies against a neuronal marker,
NeuN. FIG. 18D charts the quantitative results of this analysis by
number of NeuN-positive cells, wherein "1" represents cortical
tissues from wild-type mice, "2" represents cortical tissues from
untreated-PPT1-KO mice, and "3" represents cortical tissues
NtBuHA-treated PPT1-KO mice.
[0042] FIGS. 19A-19F chart the motor coordination of 6-month and
8-month old wild-type ("WT"), untreated-PPT1-KO mice ("KO") and
NtBuHA-treated PPT1-KO mice ("NtBuHA") on the Rotarod Performance
Test for motor coordination (FIGS. 19A-19D) and the open field test
exploratory behavior (FIGS. 19E-19F).
[0043] FIG. 20 compares the survival rates of untreated PPT1-KO
mice and NtBuHA-treated PPT1-KO mice by percentage surviving over a
given course of time.
DETAILED DESCRIPTION OF EMBODIMENTS
[0044] Based on their compelling clinical significance in cancer
and neurodegeneration, the present inventors have identified and
used thioesterases as molecular targets. The thioester linkage is
labile and nucleophilic attack readily disrupts this linkage. The
present inventors therefore reasoned that pharmacological agents
with nucleophilic properties might have a therapeutic potential for
the treatment of thioesterase deficiency disorders by mimicking
thioesterase activity. To develop a therapeutic strategy for such
thioesterase deficiency disorders with small molecules, the present
inventors rationalized that for a compound to be a potential drug
candidate it must satisfy at least three criteria:
[0045] 1) It must be non-toxic,
[0046] 2) It must be a compound with nucleophilic properties,
and
[0047] 3) It must cross the blood-brain barrier.
[0048] Hydroxylamine (HA) is a small molecule with nucleophilic
activity and it cleaves thioester linkages with high specificity
and thus, functionally mimics thioesterase activity, especially
PPT1 activity. Unfortunately, HA toxicity precludes its clinical
use for any disorder. For this reason, the present inventors
examined some non-toxic derivatives of HA for indications of
anti-oxidant properties and the ability of these compounds to
cleave thioester linkages in s-acylated proteins and to facilitate
the depletion of ceroid in cell and animal models. The
HA-derivatives of Table 1 were determined to be non-toxic and able
to cleave thioester linkages in [.sup.14C]-palmitoyl.about.CoA, a
model high-energy thioester substrate of PPT1. Additionally, the
present inventors demonstrated that each of the HA-derivatives of
Table 1 hydrolyze the thioester linkage in
[.sup.14C]-palmitoyl.about.CoA as well as in s-acylated proteins
from cultured INCL lymphoblasts in a dose- and time-dependent
manner.
[0049] In one aspect, the invention is a method of inhibiting the
development of neurodegenerative disorders or the growth and/or
metastasis of cancer in a mammal by administering at least one
compound of Table 1, or pharmaceutically-acceptable salts and/or
prodrugs thereof to the mammal.
TABLE-US-00001 TABLE 1 Compounds screened for thioesterase-like
activity in the present invention Chemical Formula Description 1.
NH.sub.2OH Hydroxylamine 2. CH.sub.5NO N-Methylhydroxylamine 3.
C.sub.2H.sub.7NO N,N-Dimethylhydroxylamine 4. C.sub.4H.sub.11NO
N,N-Diethylhydroxylamine 5. C.sub.6H.sub.13NO
N-Cyclohexylhydroxylamine 6. C.sub.6H.sub.15NO.sub.3
N-(tert-Butyl)hydroxylamine 7. C.sub.6H.sub.19NOSi.sub.2
N,O-Bis(trimethylsilyl)hydroxylamine 8. C.sub.7H.sub.9NO
N-Benzylhydroxylamine 9. C.sub.8H.sub.9NO.sub.3
N-Benzyloxycarbonyl) 10. C.sub.10H.sub.19NO.sub.5
N,O-Di-Boc-hydroxylamine 11. C.sub.13H.sub.11NO.sub.2
N-Benzoyl-N-phenyl hydroxylamine 12. C.sub.14H.sub.15NO
N,N-Dibenzylhydroxylamine 13. C.sub.23H.sub.32ClNO
N-tert-Butyl-O-[1-[4-(chloromethyl)phenyl]ethyl]-
N-(2-methyl-1-phenylpropyl)hydroxylamine
[0050] As used herein, the term "compound" means a chemical or
biological molecule such as a simple or complex organic molecule, a
peptide, a protein or an oligonucleotide. Exemplary therapeutic
compounds of the invention are those compounds listed in Table 1 of
this disclosure.
[0051] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problems or complications commensurate with a reasonable
benefit/risk ratio.
[0052] "Pharmaceutically-acceptable salts" refer to derivatives of
the disclosed compounds wherein the parent compound is modified by
making acid or base salts thereof. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines, or alkali or
organic salts of acidic residues such as carboxylic acids.
Pharmaceutically-acceptable salts include the conventional
non-toxic salts or the quaternary ammonium salts of the parent
compound formed, for example, from non-toxic inorganic or organic
acids. Such conventional nontoxic salts include those derived from
inorganic acids such as hydrochloric, hydrobromic, sulfuric,
sulfamic, phosphoric, nitric and the like; and the salts prepared
from organic acids such as acetic, propionic, succinic, glycolic,
stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic,
hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,
sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, and the
like. Pharmaceutically acceptable salts are those forms of
compounds, suitable for use in contact with the tissues of human
beings and animals without excessive toxicity, irritation, allergic
response, or other problem or complication, commensurate with a
reasonable benefit/risk ratio.
[0053] Pharmaceutically-acceptable salt forms of compounds provided
herein are synthesized from the compounds of Table 1 that contain a
basic or acidic moiety by conventional chemical methods. Generally,
such salts are, for example, prepared by reacting the free acid or
base forms of these compounds with a stoichiometric amount of the
appropriate base or acid in water or in an organic solvent, or in a
mixture of the two; generally, nonaqueous media like ether, ethyl
acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists
of suitable salts are found in at page 1418 of Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton,
Pa., 1985.
[0054] "Prodrugs" are intended to include any covalently bonded
carriers that release an active parent drug of the present
invention in vivo when such prodrug is administered to a mammalian
subject. Since prodrugs are known to enhance numerous desirable
qualities of pharmaceuticals (i.e., solubility, bioavailability,
half life, manufacturing, etc.) the compounds of the present
invention may be delivered in prodrug form. Thus, the present
invention is intended to cover prodrugs of the presently claimed
compounds, methods of delivering the same, and compositions
containing the same. Prodrugs of the present invention are prepared
by modifying functional groups present in the compound in such a
way that the modifications are cleaved, either in routine
manipulation or in vivo, to a compound of the invention. Prodrugs
include compounds of the present invention wherein an acyl,
hydroxy, amino, or sulfhydryl group is bonded to any group that,
when the prodrug of the present invention is administered to a
mammalian subject, is cleaved to form a free acetyl, hydroxyl, free
amino, or free sulfhydryl group, respectively. Examples of prodrugs
include, but are not limited to, acetate, formate, and benzoate
derivatives of alcohol and amine functional groups in the compounds
of the present invention.
[0055] The term "therapeutically-effective amount" of a compound of
this invention means an amount effective to inhibit the formation
or progression of a thioesterase deficiency disorder following
administration to a mammal having a thioesterase deficiency
disorder.
[0056] It will be appreciated by those skilled in the art that
compounds of the invention having a chiral center may exist in, and
may be isolated in, optically active and racemic forms. It is to be
understood that the compounds of the present invention encompasses
any racemic, optically-active, regioisomeric or stereoisomeric
form, or mixtures thereof, which possess the therapeutically useful
properties described herein. It is well known in the art how to
prepare optically active forms (for example, by resolution of the
racemic form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase). It is
also to be understood that the scope of this invention encompasses
not only the various isomers, which may exist but also the various
mixtures of isomers, which may be formed. For example, if the
compound of the present invention contains one or more chiral
centers, the compound can be synthesized enantioselectively or a
mixture of enantiomers and/or diastereomers can be prepared and
separated. The resolution of the compounds of the present
invention, their starting materials and/or the intermediates may be
carried out by known procedures, e.g., as described in the four
volume compendium Optical Resolution Procedures for Chemical
Compounds: Optical Resolution Information Center, Manhattan
College, Riverdale, N.Y., and in Enantiomers, Racemates and
Resolutions, Jean Jacques, Andre Collet and Samuel H. Wilen; John
Wiley & Sons, Inc., New York, 1981, which is incorporated in
its entirety by this reference. Basically, the resolution of the
compounds is based on the differences in the physical properties of
diastereomers by attachment, either chemically or enzymatically, of
an enantiomerically pure moiety resulting in forms that are
separable by fractional crystallization, distillation or
chromatography.
[0057] The compounds used in making the pharmaceutical compositions
of the present invention may be purchased commercially. The
compounds of the present invention, including the salts and
prodrugs of these compounds, may also be prepared in ways well
known to those skilled in the art of organic synthesis. The
compounds of the invention may be prepared using the reactions
performed in solvents appropriate to the reagents and materials
employed and suitable for the transformation being effected. It is
understood by one skilled in the art of organic synthesis that the
functionality present on various portions of the molecule must be
compatible with the reagents and reactions proposed. Such
restrictions to the substituents, which are compatible with the
reaction conditions, will be readily apparent to one skilled in the
art and alternate methods must then be used.
[0058] Also provided herein are pharmaceutical compositions
containing compounds of the invention and a
pharmaceutically-acceptable carrier, which are media generally
accepted in the art for the delivery of biologically active agents
to animals, in particular, mammals. Pharmaceutically-acceptable
carriers are formulated according to a number of factors well
within the purview of those of ordinary skill in the art to
determine and accommodate. These include, without limitation: the
type and nature of the active agent being formulated; the subject
to which the agent-containing composition is to be administered;
the intended route of administration of the composition; and, the
therapeutic indication being targeted. Pharmaceutically-acceptable
carriers include both aqueous and non-aqueous liquid media, as well
as a variety of solid and semi-solid dosage forms. Such carriers
can include a number of different ingredients and additives in
addition to the active agent, such additional ingredients being
included in the formulation for a variety of reasons, e.g.,
stabilization of the active agent, well known to those of ordinary
skill in the art. Descriptions of suitable
pharmaceutically-acceptable carriers, and factors involved in their
selection, are found in a variety of readily available sources,
such as Remington's Pharmaceutical Sciences, 17th ed., Mack
Publishing Company, Easton, Pa., 1985.
[0059] This invention further provides a method of treating a
mammal afflicted with a thioesterase deficiency disorder or
preventing the development of such thioesterase deficiency disorder
in a mammal, which includes administering to the mammal a
pharmaceutical composition provided herein. Such compositions
generally comprise a therapeutically effective amount of a compound
of Table 1 in an amount effective to prevent, ameliorate, lessen or
inhibit the thioesterase deficiency disorder. Such amounts
typically comprise from about 0.1 to about 100 mg of the compound
per kilogram of body weight of the mammal to which the composition
is administered. Therapeutically effective amounts can be
administered according to any dosing regimen satisfactory to those
of ordinary skill in the art.
[0060] Administration may be, for example, by various parenteral
means. Pharmaceutical compositions suitable for parenteral
administration include various aqueous media such as aqueous
dextrose and saline solutions; glycol solutions are also useful
carriers, and preferably contain a water soluble salt of the active
ingredient, suitable stabilizing agents, and if necessary,
buffering agents. Antioxidizing agents, such as sodium bisulfite,
sodium sulfite, or ascorbic acid, either alone or in combination,
are suitable stabilizing agents; also used are citric acid and its
salts, and EDTA. In addition, parenteral solutions can contain
preservatives such as benzalkonium chloride, methyl- or
propyl-paraben, and chlorobutanol.
[0061] Alternatively, compositions can be administered orally in
solid dosage forms, such as capsules, tablets and powders; or in
liquid forms such as elixirs, syrups, and/or suspensions. Gelatin
capsules can be used to contain the active ingredient and a
suitable carrier such as, but not limited to, lactose, starch,
magnesium stearate, stearic acid, or cellulose derivatives. Similar
diluents can be used to make compressed tablets. Both tablets and
capsules can be manufactured as sustained release products to
provide for continuous release of medication over a period of time.
Compressed tablets can be sugar-coated or film-coated to mask any
unpleasant taste, or used to protect the active ingredients from
the atmosphere, or to allow selective disintegration of the tablet
in the gastrointestinal tract.
[0062] A preferred formulation of the invention is a mono-phasic
pharmaceutical composition suitable for parenteral or oral
administration for the prevention, treatment or prophylaxis of a
thioesterase deficiency disorder, consisting essentially of a
therapeutically-effective amount of a compound of the invention,
and a pharmaceutically acceptable carrier.
[0063] Another preferred formulation of the invention is a
mono-phasic pharmaceutical composition suitable for the prevention,
treatment or prophylaxis of a thioesterase deficiency disorder,
consisting essentially of a therapeutically-effective amount of a
prodrug of a compound of the invention, and a pharmaceutically
acceptable carrier.
[0064] Examples of suitable aqueous and nonaqueous carriers that
may be employed in pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0065] These compositions may also contain adjuvants such as
wetting agents, emulsifying agents and dispersing agents. It may
also be desirable to include isotonic agents, such as sugars,
sodium chloride, and the like in the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents that delay absorption such
as aluminum monosterate and gelatin.
[0066] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution, which in turn may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug is accomplished by
dissolving or suspending the drug in an oil vehicle.
[0067] Injectable depot forms are made by forming microencapsulated
matrices of the drug in biodegradable polymers such as
polylactide-polyglycolide. Depending on the ratio of drug to
polymer, and the nature of the particular polymer employed, the
rate of drug release can be controlled. Examples of other
biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the drug in liposomes or microemulsions that are
compatible with body tissue. The injectable materials can be
sterilized for example, by filtration through a bacterial-retaining
filter.
[0068] For preparing solid compositions such as tablets, the
principal active ingredient is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a compound of the present invention. When
referring to these preformulation compositions as homogeneous, it
is meant that the active ingredient is dispersed evenly throughout
the composition so that the composition may be readily subdivided
into equally effective unit dosage forms such as tablets, pills and
capsules. This solid preformulation is then subdivided into unit
dosage forms of the type described above containing from, for
example, 0.1 to about 500 mg of the therapeutic compounds of the
present invention.
[0069] Formulations of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, powders, granules or as a solution or a suspension in an
aqueous or non-aqueous liquid, or an oil-in-water or water-in-oil
liquid emulsions, or as an elixir or syrup, or as pastilles (using
an inert base, such as gelatin and glycerin, or sucrose and
acacia), and the like, each containing a predetermined amount of a
compound or compounds of the present invention as an active
ingredient. A compound or compounds of the present invention may
also be administered as bolus, electuary or paste.
[0070] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, cetyl alcohol and glycerol
monosterate; (8) absorbents, such as kaolin and bentonite clay; (9)
lubricants, such as talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0071] A tablet may be made by compression or molding optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid
diluent.
[0072] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient only, or preferentially, in
a certain portion of the gastrointestinal tract, optionally, in a
delayed manner. Examples of embedding compositions that can be used
include polymeric substances and waxes. The active ingredient can
also be in microencapsulated form.
[0073] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by an enteric layer that serves to
resist disintegration in the stomach and permit the inner component
to pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0074] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically-acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0075] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0076] Suspensions, in addition to the active compounds, may
contain suspending agents as, for example, ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, and mixtures thereof.
[0077] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more
compounds of the invention with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or salicylate, and which is
solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active compound. Formulations of the present invention which
are suitable for vaginal administration also include pessaries,
tampons, creams, gels, pastes, foams or spray formulations
containing such carriers as are known in the art to be
appropriate.
[0078] Dosage forms for the topical or transdermal administration
of compounds of this invention include powders, sprays, ointments,
pastes, creams, lotions, gels, solutions, patches, drops and
inhalants. The active ingredient may be mixed under sterile
conditions with a pharmaceutically-acceptable carrier, and with any
buffers, or propellants that may be required.
[0079] The ointments, pastes, creams and gels may contain, in
addition to an active ingredient, excipients, such as animal and
vegetable fats, oils, waxes, paraffins, starch, tragacanth,
cellulose derivatives, polyethylene glycols, silicones, bentonites,
silicic acid, talc and zinc oxide, or mixtures thereof.
[0080] Powders and sprays can contain, in addition to an active
ingredient, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder or
mixtures of these substances. Sprays can additionally contain
customary propellants such as chlorofluorohydrocarbons and volatile
unsubstituted hydrocarbons, such as butane and propane.
[0081] Transdermal patches have the added advantage of providing
controlled delivery of compounds of the invention to the body. Such
dosage forms can be made by dissolving, dispersing or otherwise
incorporating one or more compounds of the invention in a proper
medium, such as an elastomeric matrix material. Absorption
enhancers can also be used to increase the flux of the compound
across the skin. The rate of such flux can be controlled by either
providing a rate-controlling membrane or dispersing the compound in
a polymer matrix or gel.
[0082] Pharmaceutical formulations include those suitable for
administration by inhalation or insufflation or for nasal or
intraocular administration. For administration to the upper (nasal)
or lower respiratory tract by inhalation, the compounds of the
invention are conveniently delivered from an insufflator, nebulizer
or a pressurized pack or other convenient means of delivering an
aerosol spray. Pressurized packs may comprise a suitable propellant
such as dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
In the case of a pressurized aerosol, the dosage unit may be
determined by providing a valve to deliver a metered amount.
[0083] Alternatively, for administration by inhalation or
insufflation, the composition may take the form of a dry powder,
for example, a powder mix of one or more of the anti-cancer
compounds of the invention and a suitable powder base, such as
lactose or starch. The powder composition may be presented in unit
dosage form in, for example, capsules or cartridges, or, e.g.,
gelatin or blister packs from which the powder may be administered
with the aid of an inhalator, insufflator or a metered-dose
inhaler.
[0084] For intranasal administration, compounds of the invention
may be administered by means of nose drops or a liquid spray, such
as by means of a plastic bottle atomizer or metered-dose inhaler.
Typical of atomizers are the Mistometer (Wintrop) and Medihaler
(Riker).
[0085] Drops, such as eye drops or nose drops, may be formulated
with an aqueous or nonaqueous base also comprising one or more
dispersing agents, solubilizing agents or suspending agents. Liquid
sprays are conveniently delivered from pressurized packs. Drops can
be delivered by means of a simple eye dropper-capped bottle or by
means of a plastic bottle adapted to deliver liquid contents by
drop by means of a specially shaped closure.
[0086] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the type described above.
[0087] The dosage formulations provided by this invention may
contain the therapeutic compounds of the invention, either alone or
in combination with other therapeutically active ingredients, and
pharmaceutically acceptable inert excipients. The term
`pharmaceutically acceptable inert excipients` includes at least
one of diluents, binders, lubricants/glidants, coloring agents and
release modifying polymers.
[0088] Suitable antioxidants may be selected from amongst one or
more pharmaceutically acceptable antioxidants known in the art.
Examples of pharmaceutically acceptable antioxidants include
butylated hydroxyanisole (BHA), sodium ascorbate, butylated
hydroxytoluene (BHT), sodium sulfite, citric acid, malic acid and
ascorbic acid. The antioxidants may be present in the dosage
formulations of the present invention at a concentration between
about 0.001% to about 5%, by weight, of the dosage formulation.
[0089] Suitable chelating agents may be selected from amongst one
or more chelating agents known in the art. Examples of suitable
chelating agents include ethylenediaminetetraacetic acid (EDTA),
edetic acid, citric acid and combinations thereof. The chelating
agents may be present in a concentration between about 0.001% and
about 5%, by weight, of the dosage formulation.
[0090] The dosage form may include one or more diluents such as
lactose, sugar, cornstarch, modified cornstarch, mannitol,
sorbitol, and/or cellulose derivatives such as wood cellulose and
microcrystalline cellulose, typically in an amount within the range
of from about 20% to about 80%, by weight.
[0091] The dosage form may include one or more binders in an amount
of up to about 60% w/w. Examples of suitable binders include methyl
cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose,
polyvinyl pyrrolidone, eudragits, ethyl cellulose, gelatin, gum
arabic, polyvinyl alcohol, pullulan, carbomer, pregelatinized
starch, agar, tragacanth, sodium alginate, microcrystalline
cellulose and the like.
[0092] Examples of suitable disintegrants include sodium starch
glycolate, croscarmellose sodium, crospovidone, low substituted
hydroxypropyl cellulose, and the like. The concentration may vary
from 0.1% to 15%, by weight, of the dosage form.
[0093] Examples of lubricants/glidants include colloidal silicon
dioxide, stearic acid, magnesium stearate, calcium stearate, talc,
hydrogenated castor oil, sucrose esters of fatty acid,
microcrystalline wax, yellow beeswax, white beeswax, and the like.
The concentration may vary from 0.1% to 15%, by weight, of the
dosage form.
[0094] Release modifying polymers may be used to form extended
release formulations containing the therapeutic compounds of the
invention. The release modifying polymers may be either
water-soluble polymers, or water insoluble polymers. Examples of
water-soluble polymers include polyvinylpyrrolidone, hydroxy
propylcellulose, hydroxypropyl methylcellulose, vinyl acetate
copolymers, polyethylene oxide, polysaccharides (such as alginate,
xanthan gum, etc.), methylcellulose and mixtures thereof. Examples
of water-insoluble polymers include acrylates such as
methacrylates, acrylic acid copolymers; cellulose derivatives such
as ethylcellulose or cellulose acetate; polyethylene, and high
molecular weight polyvinyl alcohols.
[0095] Another embodiment of the invention relates to the use of
any of the prodrug compounds or compositions described herein in
the preparation of a medicament for the treatment of a thioesterase
deficiency disorder.
[0096] Also encompassed by the present invention are methods for
screening potential therapeutic agents that may prevent, treat or
inhibit the formation of a thioesterase deficiency disorder, by
functionally mimicking a thioesterase enzymatic activity
comprising: (a) combining an s-acylated protein having a thioester
linkage and a potential therapeutic compound under conditions in
which they interact, and; (b) monitoring the cleavage of the
thioester linkage; wherein a potential therapeutic compound is
selected for further study when it mimics the thioesterase
enzymatic activity compared to a control sample to which no
potential therapeutic compound has been added. In one embodiment,
the potential therapeutic compound is selected from the group
consisting of a pharmaceutical agent, a cytokine, a small molecule
drug, a cell-permeable small molecule drug, a hormone, a
combination of interleukins, a lectin, a bispecific antibody, and a
peptide mimetic. In another embodiment, the potential therapeutic
compound is a compound of Table 1 or an analog or derivative
thereof.
[0097] Another embodiment of the invention relates to the use of
any of the compounds or compositions of the invention in the
preparation of a medicament for the inhibition of a thioesterase
deficiency disorder in a mammal.
[0098] Each publication or patent cited herein is incorporated
herein by reference in its entirety.
[0099] Although the invention has been described in conjunction
with specific embodiments thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention.
Other aspects, advantages, and modifications within the scope of
the invention will be apparent to those skilled in the art to which
the invention pertains.
EXAMPLES
[0100] Unless otherwise indicated, the practice of the present
invention can employ conventional techniques known to those of
skill of the arts of the pharmaceutical industry and the like. To
the extent such techniques are not described fully herein, one can
find ample reference to them in the scientific literature.
Materials and Methodology
Cell Culture
[0101] PPT1-deficient immortalized INCL lymphoblast-cultures were
obtained from the laboratory of the late Dr. Krystina E.
Wisniewski. PPT1-deficient fibroblasts were derived from skin
biopsy sample from an INCL patient admitted to a clinical protocol
(#01-CH-0086), approved by the Institutional Review Board (IRB) of
the NICHD, NIH. This patient was homozygous for one of the most
lethal PPT1 gene mutations (R122W). A list of cell lines used and
the PPT1-mutations they carried have been provided in Table 2,
below. Fibroblasts were cultured in DMEM supplemented with 10% heat
inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml
penicillin and streptomycin at 37.degree. C. in humidified
atmosphere with 5% CO.sub.2. INCL and normal lymphoblasts were
cultured in RPMI supplemented with 16% FBS at 37.degree. C. in
humidified atmosphere with 5% CO.sub.2.
TABLE-US-00002 TABLE 2 PPT1 deficient cells Cell Line PPT1-Mutation
Lymphoblasts 1. C11568 Missense A364T (R122W) Missense G541A
(V181M) 2. C12275 Missense G353A (G118D) Nonsense C451T (R151X) 3.
C11796 Nonsense C451T (R151X) Nonsense C451T (R151X) 4. C10949
Nonsense C490T (R161X) Nonsense C490T (R161X) 5. C1045LT Nonsense
C451T (R151X) Nonsense C451T (R151X) 6. C7142L Nonsense C451T
(R151X) Missense G541A (V181M) 7. C10320LT Nonsense C451T (R151X)
Missense G749T (G250V) 8. C11560 Missense A223C (T75P) Nonsense
C451T (R151X) 9. C12488 Missense A223C (T75P) Missense A223C (T75P)
10. C9955 Normal Lymphoblasts Fibroblasts INCL Fibroblasts Missense
A364T (R122W) Missense A364T (R122W)
Animals
[0102] All animal experiments were performed according to the
institutional guidelines after an animal protocol was approved by
the Animal Care and Use Committee of the National Institute of
Child Health and Development, National Institutes of Health.
PPT1-KO mice (a gift from Dr. S. L. Hofmann, University of Texas
Southwestern Medical Center at Dallas, Dallas, Tex., USA) were
generated by targeted disruption of the last exon in the PPT1 gene
in embryonic stem (ES) cells. These mice were subsequently
backcrossed for 10 generations with wild type C57BL/6 mice in order
to obtain congenic C57 background and a breeding pair was provided
by Dr. Mark Sands to start the inventors' colony. Animals were
housed and maintained in a pathogen-free facility. Three-month old
PPT-KO mice were given NtBuHA in their drinking water (1 mM NtBuHA
and 1 mM NaCl).
Transmission Electron Microscopy
[0103] Transmission electron microscopy was performed using
standard methodology.
[0104] Briefly, after treatment with PTC124 for 7 days, cells were
fixed with 2.5% glutaraldehyde in sodium phosphate buffer and then
washed with Millonig's phosphate buffer once and kept in the same
buffer at 4.degree. C. until final processing. Ultra-thin sections
were then prepared and then stained with lead citrate and uranyl
acetate and examined with a LEO 912 electron microscope (JFE
Enterprises).
Western Blot Analysis
[0105] Western blot analysis was performed as using standard
methodology. Briefly 30 .mu.g of protein was resolved in 4-12% Bis
tris gel (Invitrogen) and electro-transferred in PVDF membrane. The
membranes were blocked with 5% non fat dried milk for 1 hour at
room temperature and were then probed overnight with primary
antibody at 4 C. After washing the blots were incubated with
horseradish peroxidase conjugated secondary antibody (Santacruz
Biotechnology) for 1 hour at room temperature and developed using
enhanced chemiluminescences detection reagents (Pierce). Primary
antibodies used in this study were caspase-3, caspase-9 (cell
signaling) SOD2, catalase, (3-Actin (US Biologicals).
Immunohistochemistry
[0106] The NtBuHA treated and untreated mouse brain tissues were
fixed in 3.7% paraformaldehyde, embedded in paraffin and processed
for histological analyses. Briefly, after being treated with xylene
and then successively with different concentrations of ethanol in
phosphate buffered saline (PBS) (100% to 0%), tissue sections were
blocked with 5% BSA in PBS. Sections were then probed overnight
with anti-synaptophysin (Abcam) and anti-GFAP followed by
incubation with alexa fluor 594 conjugated anti-rabbit and
anti-mouse secondary antibodies (Invitrogen). Sections were mounted
with DAPI containing mounting medium and imaged using Zeiss LSM 510
Inverted Meta confocal microscope (Carl Zeiss).
Immunofluorescence Analysis
[0107] Immunofluoresence analysis was performed according to the
method of Tanida et al. Briefly cells were grown in 2-chamber slide
(Lab-Tek) and fixed with 4% paraformaldehyde for 10 min at room
temperature. Cells were blocked with 2% BSA and 5% serum in PBS for
1 h at room temperature and then probed overnight with primary
antibody at 4 C, followed by incubation with secondary antibodies
at room temperature for 1 h. Primary antibodies used were
anti-Saposin A and D. Secondary antibodies used are Alexa fluor 488
conjugated anti-rabbit, alexa fluor conjugated 594 anti-mouse
(Invitrogen). Nuclei were stained with DAPI (Sigma-Aldrich).
Fluorescence was visualized using Zeiss LSM 510 Inverted Meta
confocal microscope (Carl Zeiss), and the image obtained was
processed and analyzed with the LSM image software (Carl
Zeiss).
Cellular Apoptosis Detection
[0108] To evaluate whether NtBuHA protects against oxidative
stress-induced apoptosis in INCL lymphoblasts the cells were
treated with 1 mM NtBuHA for 12 hours and then incubated with 500
.mu.M H.sub.2O.sub.2 for 3 hours in presence or absence of NtBuHA
(1 mM). Untreated INCL lymphoblasts were also treated with
H.sub.2O.sub.2 for 3 hours. For the detection of apoptosis the
cells were stained with annexin V using Apoptosis detection kit
(Biovision) and analyzed by FACS (Guava EasyCyte Mini System,
Millipore).
Palmitoylated Protein Isolation
[0109] Palmitoylated proteins were identified by acyl-biotinyl
exchange protocol as described by Roth et al (2006) with minor
modifications. Briefly cells were lysed with RIPA buffer (Pierce)
and incubated overnight with 10 mM N-ethylmalemide (NEM, Pierce)
and 1.times. protease inhibitor cocktail (PI, Pierce) at 4.degree.
C. with gentle mixing. The next day, NEM was removed by three
sequential precipitation by the chloroform-methanol (CM) method
described previously (Wessel and Flugge, 1984. Following third
precipitation, the protein was divided into two parts. One part was
treated with 1 M hydroxylamine (Sigma) pH 7.4 (freshly prepared), 1
mM BMCC-biotin (Pierce), 0.2% Triton X-100 (Sigma), and 1.times.PI
and the other part was treated with similar mixture without
hydroxylamine for 1 h at room temperature. The protein was then
precipitated by CM method and treated with 200 .mu.M BMCC-biotin,
0.2% Triton X-100, and 1.times.PI at room temperature 25.degree. C.
for 1 hour. HPDP-biotin was then removed by three sequential CM
precipitations. Following the third precipitation, protein was
dissolved in SDS-PAGE loading buffer and boiled for 5 min. The
sample was then subjected to Western blot analysis with avidin-HRP
conjugate.
Transmission Electron Microsocopy
[0110] Transmission electron microsocopy (TEM) of INCL lymphoblasts
and fibroblasts was performed. Briefly, cells were first treated
with 250 .mu.M NtBuHA for 3 weeks. Medium was replaced with fresh
250 .mu.M of NtBuHA at every 72 hours. After treatment, cells were
fixed with 2.5% glutaraldehyde in sodium phosphate buffer, washed
three times with Millonig's phosphate buffer and stained with 2.5%
uranyl acetate. Ultra-thin sections were then prepared using AO
Reichert Ultracut ultramicrotome, stained with lead citrate and
examined by using Zeiss EM10 CA.
[0111] Cortical tissue sections were prepared from wild-type mice
and untreated--as well as NtBuHA-treated PPT1-KO littermates.
Cortical tissues (approximately 1 mm.sup.3) were dissected from
those animals, fixed in 2.5% glutaradlehyde in 0.1M sodium
cacodylate buffer, pH 7.4 followed by three washing in 0.1 M sodium
cacodylate buffer at room temperature. Tissues were then post-fixed
with 1% osmium tetroxide, dehydrated by sequential treatment of
50%, 70%, 90% and 100% ethanol and treated with Spurr's
resin/ethanol using a variable wattage Pelco BioWave Pro microwave
oven. Cortical tissues were embedded and polymerized in 100% resin
for 18 hours at 70.degree. C. Tissue sections (50 nm thick) were
prepared using Reichert-Jung Ultracut-E ultramicrotome and
collected on LuxFilm grids (Ted Pella, Inc.) of 30 nm film
thickness. The grids were then post-stained with uranyl acetate and
lead citrate and examined in a FEI Tecnai G2 transmission electron
microscope operating at 80 kV.
Real Time Polymerase Chain Reaction
[0112] Real time polymerase chain reaction (RT PCR) was performed
using standard protocols.
Motor Coordination Test
[0113] Motor coordination of the untreated- and NtBuHA-treated
PPT1-KO mice was assessed using Rotarod) (UGO Basile, Italy) at
three different speeds (4, 8 and 12 rpm). In all these speeds the
direction of rotation was reversed every 15 seconds. Animals were
trained twice at all speed settings for 1 minute each for 3 days.
Animals were then given a rest for 1 minute between two trials.
Rotarod experiments were performed for at least 60 seconds on day 4
and the amount of time a mouse was on the rotarod before falling
from the rotating rod was recorded.
Statistical Analysis
[0114] All data were expressed as the mean of at least three
experiments .+-.SD. Results were analyzed using one way ANOVA and
p<0.05 was considered significant.
Example 1
[0115] This Example demonstrates that derivatives of hydroxylamine
(HA) are able to cleave the thioester linkage in
[.sup.14C]palmitoyl-CoA in vitro.
[0116] It has been reported that thioester linkages in s-acylated
proteins are labile, and that compounds having nucleophilic
properties are able to disrupt such linkages. Thus, the ability of
derivatives of hydroxylamine to disrupt thioester linkages in
s-acylated proteins was tested. Twelve HA-derivatives, described in
Table 1, were screened for their ability to cleave thioester
linkage in [.sup.14C]palmitoyl .about.CoA (a model thioester
substrate of PPT1), mediating the release of free
[.sup.14C]palmitic acid, a process depicted in FIG. 2. The parent
compound, HA, as well as recombinant human PPT1, served as positive
controls. [.sup.14C]-palmitoyl-CoA was incubated for 1 hour with
each of the HA derivatives and the reaction mixture was resolved by
thin layer chromatography (TLC) followed by autoradiography. The
free [.sup.14C]-palmitate bands were visible and quantitated by
densitometric analysis of the bands. The results of this study
demonstrated that all twelve hydroxylamine derivatives hydrolyze
the thioester linkage in [.sup.14C]palmitoyl-CoA, as shown in FIG.
3. These results suggest that HA-derivatives are capable of
efficiently cleaving the thioester linkage in a model substrate of
PPT1.
Example 2
[0117] This example demonstrates that ten HA-derivatives did not
adversely affect the viability of cultured cells from INCL
patients.
[0118] Ten out of the twelve HA-derivative compounds listed in
Table 1 were tested to determine whether they adversely affected
the viability of cultured cells from INCL patients. Two derivatives
from Table 1, namely, N, N-dibenzylhydroxylamine and
N-tert-Butyl-O-[1-[4-(chloromethyl)phenyl]ethyl]-N-(2-methyl-1-phenylprop-
yl) hydroxylamine, were not tested because they were insoluble in
tissue culture media. Briefly, following treatment with
HA-derivatives INCL cells were incubated with Thiazolyl Blue
Tetrazolium Blue (MTT) for 4 hours at 37.degree. C. Formazan
crystals thus formed were dissolved in acidified isopropanol and
absorption was measured at 570 nm. Viability was expressed as
percent of untreated control. As illustrated in FIG. 4A-C, the
results showed that all ten compounds except N-Benzoyl-N-phenyl
hydroxylamine are non-toxic up to a concentration of 1 mM as there
was no evidence for any adverse effect that decreased the viability
of these cells.
Example 3
[0119] This Example demonstrates that three derivatives of
hydroxylamine (HA) cleave the thioester linkage in
[.sup.14C]palmitoyl-CoA in a time- and dose-dependent manner.
[0120] To determine whether hydrolysis of the thioester linkage by
hydroxylamine derivatives, namely N-t-butyl hydroxylamine (NtBuHA),
benzyl-HA, and methyl-HA, is dose dependent, a fixed amount of
[.sup.14C]palmitoyl-CoA was incubated with varying concentrations
of each of these three derivatives, and the production of
[.sup.14C]palmitate measured. To determine whether hydrolysis of
the thioester linkage by the hydroxylamine derivatives is also
time-dependent, a fixed amount of [.sup.14C]palmitoyl-CoA was
incubated with each of the three derivatives, and the production of
[.sup.14C]palmitate measured at various times during incubation.
The results of this study demonstrated that all three of the
hydroxylamine derivatives tested cleaved the thioester linkage in a
dose-dependent and time-dependent manner. FIGS. 5 A and B shows the
results obtained using NtBuHA.
Example 4
[0121] This Example demonstrates that derivatives of hydroxylamine
(HA) are non-toxic with respect to cultured INCL fibroblasts.
[0122] Before further testing the ability of hydroxylamine
derivatives to cleave thioester bonds in s-acylated proteins, the
potential toxicity of these derivatives was tested in cultured
cells. Briefly, normal fibroblasts (GM 00498) were obtained from
the Coriell Institute of Medical Research, USA. PPT1-deficient INCL
fibroblasts were isolated from skin biopsy samples of INCL patients
admitted to an ongoing clinical protocol (#01-CH-0086) approved by
the Institutional Review Board (IRB) of the National Institute of
Child health and Human Development (NICHD) at the National
Institute of Health (NIH). The fibroblasts were cultured in
Dulbecco's Modified eagle medium (DMEM) (GIBCO.RTM.), supplemented
with 10% heat inactivated fetal bovine serum (FBS), 2 mM glutamine,
and 100 U/ml penicillin and streptomycin, at 37 C in humidified
atmosphere containing 5% CO.sub.2. Semi-confluent cells were
harvested with 0.025% trypsin and 0.52 mM
ethylenediaminetetraacetic acid (EDTA) and re-plated at a density
of 5.times.10.sup.5 cells/well in a 6-well plate. The fibroblasts
were cultured in the presence or absence of varying concentrations
of each of the HA-derivatives for 48 hours, after which the
viability of the cells was determined using the MTT-Cell Viability
Assay by T. Mossman, (Rapid colorimetric assay for cellular growth
and survival. J. Immunol. Method. 65 (1983) 55-63). The results of
this study, which are shown in FIG. 6, demonstrated that the three
hydroxylamine derivatives are non-toxic to cultured
fibroblasts.
Example 5
[0123] This Example demonstrates the ability of NtBuHA to hydrolyze
thioester linkages in S-acylated proteins in cultured INCL
lymphoblasts, and to mediate depletion of lipid-extractable
[.sup.35S]cysteine thioesters from these cells.
[0124] It has been demonstrated that not only is NtBuHA non-toxic,
but that it is also a potent antioxidant. Thus, this derivative was
chosen for more extensive study of its ability to hydrolyze
S-acetylated proteins, and to deplete lipid-extractable
[.sup.35S]cysteine thioesters from fibroblasts and lymphoblasts.
INCL fibroblasts were obtained and cultured as described in Example
4. The cells were then labeled with [.sup.35S]cysteine. Briefly,
following incubation for six hours in medium lacking serum and
cysteine/cystine, the cells were washed and then re-cultured for
varying lengths of time in medium containing NtBuHA, with a change
of medium every 12 hours. As controls, [.sup.35S]cysteine-labeled
cells were cultured in drug-free medium as well as medium
containing 1M HA. Following incubation, cells were washed twice
with cold PBS and then centrifuged at 2,250 g at 4.degree. C. for 5
minutes. Lipid thioesters were then extracted from the cells and
subject to analysis by high-performance thin layer chromatography.
Briefly, the extracted fatty acyl esters were dissolved in
chloroform:methanol (1:1) and an aliquot applied to a TLC plate and
resolved using a mixture of chloroform:methanol:water (65:25:4).
The plates were then dried and autoradiographs obtained.
[0125] The results of this study demonstrated that compared with
untreated control cells, treatment with NtBuHA for 24 and 48 hours
yielded [.sup.35S]cysteine-labeled lipid extractable thioester
compounds of significantly reduced intensity, as shown in FIG.
7.
[0126] The ability of NtBuHA to deplete
[.sup.35S]cysteine-thioester containing compounds was tested using
[.sup.35S]cysteine-labeled, immortalized lymphoblasts. Although the
cells were obtained from patients who were PPT-deficient due to
inactivating mutations in PPT1, they had been clinically diagnosed
to have either infantile (INCL; n=3), late infantile (LINCL; n=3)
or juvenile (JNCL; n=3) forms of NCL. The cells were labeled and
then incubated in medium containing NtBuHA for 48 hours. Following
incubation, the lipid esters were extracted and resolved. As shown
in FIG. 8, the results demonstrated a clear reduction in the
intensity of the [.sup.35S]cysteine-labeled lipid bands in every
cell line treated with NtBuHA, when compared with lipid bands from
untreated cells. As expected, the normal control cells did not show
abnormal accumulation of [.sup.35S]cysteine-labeled lipid bands.
These results demonstrate that treatment of cells with NtBuHA
results in hydrolysis of thioester linkages in S-acylated
proteins.
Example 6
[0127] This Example demonstrates that NtBuHA treatment depletes
granular osmiophilic deposits (GRODs) in cultured PPT1-deficient
cells.
[0128] It has been demonstrated that there is a physical
correlation between ceroid accumulation and the presence of high
concentrations of GRODs in the cells of INCL patients. Since
experiments revealed that NtBuHA treatment of PPT1-deficient cells
from INCL patients results in the depletion of ceroids, it was
hypothesized that NtBuHA treatment of such cells would also result
in depletion of GRODs, which are detectable by TEM. Accordingly,
untreated- and NtBuHA-treated cultured INCL lymphoblasts were
analyzed using TEM. The results showed that compared with the
untreated controls, FIG. 9A, the NtBuHA-treated cells contained
either no GRODs, FIG. 9B, or a substantially reduced number of
GRODs that were considerably smaller in size, FIG. 9C. Similar
experiments were conducted using cultured INCL patient fibroblasts,
which showed that while the untreated fibroblasts contained
numerous large highly dense GRODs, as shown in FIG. 10A, the
NtBuHA-treated cells had significantly lower number of GRODs with
markedly reduced size and density, as in FIG. 10B. These results
from two different cell types from INCL patients demonstrated that
NtBuHA is effective in depleting GRODs, which are
characteristically found in INCL.
Example 7
[0129] This example shows that withdrawal of NtBuHA treatment
promotes GROD reaccumulation in cultured INCL cells.
[0130] For a therapeutic agent to be effective for INCL it must not
only mediate depletion of ceroid by cleaving thioester linkage in
S-acylated proteins but it must also prevent reaccumulation of
ceroid. To determine whether withdrawal of NtBuHA causes
reaccumulation, immortalized INCL lymphoblasts that were cultured
in medium containing NtBuHA for three weeks, which showed depletion
of GRODs. Some of these cultures were washed with PBS and then
cultured for an additional 3 weeks in medium without NtBuHA before
being analyzed by TEM. The GRODS in 15-20 informative cells were
counted and presented graphically, as shown in FIG. 11. The results
showed that while NtBuHA-treatment reduced GRODs as expected (FIGS.
9B and 9C), its withdrawal from the culture medium caused
reaccumulation (FIG. 9D).
Example 8
[0131] This experiment demonstrates the depletion of GRODs in brain
tissues of NtBuHA-treated PPT1-KO mice.
[0132] Since the results from in vitro experiments are not always
be replicable in vivo, PPT1-KO mice were obtained from Dr. S. L.
Hofman at the University of Texas Southwestern Medical Center at
Dallas, in Dallas, Tex. for in vivo testing. Both the knockout mice
and their wild-type litter-mates (the control mice) were housed and
maintained in a pathogen-free facility. To determine whether
treatment with NtBuHA mediates the depletion of GRODs in vivo as
well as in vitro, 3-month old PPT1-KO mice were treated with NtBuHA
until they were 6 months old. Untreated age- and sex-matched
PPT1-KO and wild-type mice were kept as controls. 6-month old
animals were chosen because PPT1-KO mice at this age manifest signs
of neurological impairment as well as pathological features of INCL
including ER- and oxidative-stress, accumulation of GRODs,
increased apoptosis and decreased brain volume compared with their
wild-type litter-mates. At the end of this treatment period, the
brain cells of the mice were examined by TEM. The results showed
that while the wild-type mice had no GRODs (FIG. 12A) high levels
of GRODs were clearly visible in the brain cells of untreated
PPT1-KO mice (FIG. 12B). The NtBuHA-treated PPT1-KO mice showed
markedly reduced number of GRODs (FIG. 12C). Interestingly, when an
infrequent GROD was detected in the brain cells of an
NtBuHA-treated PPT1-KO mouse, the GRODs appeared remarkably smaller
in size than those found in untreated animals (FIG. 12C). These
results demonstrate that NtBuHA-treatment mediates depletion of
GRODs in PPT1-KO brain cells and suggests, albeit indirectly, that
this compound or its active metabolite(s) crosses the blood-brain
barrier to effect this positive change. These results, taken
together with those detailed in Examples 6 and 7, demonstrated that
NtBuHA facilitates reduction of total ceroid load and consequent
reduction in GROD levels in cultured cells from INCL patients as
well as those in brain cells of PPT1-KO mice.
Example 9
[0133] This example shows that autofluorescence is diminished in
the brain cells of NtBuHA-treated PPT1-KO mice.
[0134] Accumulation of intracellular autofluorescent lipopigment
material in the brain and other tissues is a characteristic
pathological finding in INCL patients and in PPT1-KO mice. Given
this, it was hypothesized that NtBuHA-mediated depletion of GRODs
may also reduce the intensity of autofluorescence in brain tissues
of NtBuHA-treated PPT1-KO mice. Accordingly, a comparison of the
autofluorescence of brain tissues from NtBuHA-treated PPT1-KO mice,
untreated PPT1-KO mice, and wild-type litter-mates was performed.
Results showed that while the brain tissues from wild-type mice had
no autofluorescence (FIG. 13A) those of the untreated PPT1-KO mice
manifested intense autofluorescence (FIG. 13B). NtBuHA-treated
Ppt1-KO mice had markedly reduced autofluorescence (FIG. 13C)
compared with those of the untreated PPT1-KO mice. These results
further confirmed that NtBuHA mediated depletion of intracellular
ceroid and consequently, diminished the intensity of
autofluorescence.
Example 10
[0135] This Example demonstrates that treatment of mice with NtBuHA
promotes degradation of long-lived proteins.
[0136] It has been reported previously that in mice lacking Atg7,
the degradation of long-lived proteins is impaired under normal and
starvation conditions (Komatsu et al., 2005). PPT1-KO mice lack
Atg7, which facilitates autophagosome formation. Since it appears
that impairment of autophagosome-lysosome fusion in PPT1-deficient
cells is due to failure of dynamic palmitoylation of Rab7, an
experiment was conducted to determine whether NtBuHA treatment
facilitating dynamic palmitoylation would also promote degradation
of long-lived proteins in the cells of PPT1-KO mice.
[0137] PPT1-KO mice were given drinking water containing NtBuHA.
Wild-type controls were given regular water. Some mice were
subjected to one day of fasting prior to harvest of cells. At the
end of the treatment period, fibroblasts were obtained from both
the PPT1-KO and wild-type mice and incubated with long-lived and
short-lived proteins. At the end of the experiment, the percentage
of protein degradation was calculated. The results showed that the
amount of total proteins decreased following one day of fasting in
the fibroblasts of the control mice. In contrast, fasting did not
significantly decrease the amount of total cellular proteins in the
cells of the PPT1-KO mice. The amount of total proteins in the
cells of the PPT1-KO mice was higher compared to normal control
cells. These results indicated that the decrease of total proteins
is dependent on autophagosome-lysosome fusion and that it was
impaired in the cells of PPT1-KO mice.
Example 11
[0138] This example shows that NtBuHA suppresses endoplasmic
reticulum and oxidative-stress in the brain cells of PPT1-KO
mice.
[0139] It has previously reported that endoplasmic reticulum (ER-)
and oxidative-stress contribute to neuropathology in INCL patients.
Recently, it has been demonstrated also that mutations in the CLN3
gene that underlie juvenile neuronal ceroid lipofuscinosis (JNCL or
Batten disease) also cause oxidative stress leading to
neurodegeneration in Drosophila flies. Therefore, the inventors
sought to determine whether NtBuHA-treatment of PPT1-KO mice
ameliorate the ER- and oxidative-stress in brain tissues of these
mice. Accordingly, the levels of ER-stress markers Grp-78, Grp-94
and ATF6 were determined by Western blot analysis of brain lysates.
The results showed that the levels of all three ER-stress markers
were reduced in brain tissues of NtBuHA-treated PPT1-KO mice (FIG.
14A). Western blot analysis of brain lysates from NtBuHA-treated
and untreated PPT1-KO mice was also performed to determine the
levels of superoxide dismutase-2 (SOD2) and catalase, which
increases with elevated oxidative-stress. Results showed that
compared with untreated PPT1-KO mice, NtBuHA-treatment
significantly reduced the levels of both SOD2 and catalase (FIG.
14B). Taken together, these results demonstrated that
NtBuHA-treatment reduces both ER- and oxidative stress levels in
the brain tissues of PPT1-KO mice.
Example 12
[0140] This example shows suppression of apoptosis in
NtBuHA-treated cultured cells from INCL patients
[0141] Reports have indicated that PPT1 plays critical roles in
protecting neuroblastoma cells against apoptosis. Moreover, in
these cells antisense-mediated inhibition of PPT1 leads to
increased apoptosis. Consistent with these results, an increased
rate of apoptosis in the brain biopsy material and in cultured
immortalized lymphoblasts from INCL patients have been reported. In
addition, increased neuronal apoptosis in the brain of PPT1-KO mice
has also been reported. Together, these results at least in part
provided an explanation for the rapidly progressive cerebral
atrophy in INCL patients. Thus, the inventors sought to determine
whether the NtBuHA-treatment protects cultured immortalized INCL
lymphoblasts from oxidative-stress mediated apoptosis. Cultured
lymphoblasts from INCL patients were chosen because PPT1 is
expressed in all tissues and PPT1-deficiency in cultured
lymphoblasts and fibroblasts from INCL patients show virtually
identical pathological changes (accumulation of ceroid, GRODs and
increased apoptosis) as are found in postmortem neuronal tissues
from such patients. Accordingly, INCL lymphoblasts were pretreated
with NtBuHA for 12 hours and then incubated with hydrogen peroxide
in the presence or absence of NtBuHA for 3 hours. Untreated INCL
cells were also exposed, to the same concentration of hydrogen
peroxide for same amount of time. Apoptosis of the
H.sub.2O.sub.2-treated cells was then quantitated by florescence
activated cell sorting (FACS) analysis. The results, charted in
FIG. 15, showed that while treatment of INCL lymphoblasts with
H.sub.2O.sub.2 in the absence of NtBuHA increased apoptosis the
H.sub.2O.sub.2-induced oxidative-stress in INCL lymphoblasts
pretreated with NtBuHA failed to induce apoptosis above the base
level. Similarly cells that were pretreated with NtBuHA and
incubated with H.sub.2O.sub.2 in presence of NtBuHA (co-treated)
also showed no alteration in apoptosis above the base level in
these cells. These results showed that NtBuHA protects cultured
INCL lymphoblasts from oxidative-stress mediated apoptosis.
Example 13
[0142] This example shows that NtBuHA reduces the level of
apoptosis in Ppt1-KO mouse brain cells
[0143] It has been reported that postmortem brain tissues from INCL
patients as well as those from PPT1-KO mice manifest high levels of
oxidative stress, which induces neuronal apoptosis contributing to
neuropathology. Since the results of the testing described in
Example 12 showed that NtBuHA reduces the level of apoptosis in
cultured cells from INCL patients, the inventors sought to
determine whether this treatment also reduces apoptosis in the
brain of PPT1-KO mice. Accordingly, a terminal deoxynucleotidyl
transferase dUTP nick end labeling (TUNEL) assay was performed
using cortical tissue sections from wild-type, untreated PPT1-KO
and NtBuHA-treated PPT1-KO mice to determine the level of
apoptosis. A visual assessment (FIGS. 16A-C) and quantitative
analysis (D) showed that the wild-type mouse brain cells had
virtually no TUNEL-positive apoptotic cells, the brain cells of
untreated PPT1-KO mice contained numerous apoptotic cells (FIG.
16B), and the brain tissues from NtBuHA-treated PPT1-KO mice showed
appreciably lower levels of TUNEL-positive apoptotic cells than in
the brain cells of the untreated PPT1-KO mice. Furthermore, Western
blot analysis of cortical tissue homogenates of wild-type,
untreated and NtBuHA-treated PPT1-KO mice showed a marked reduction
in caspase-9 and cleaved PARP-1 level (FIG. 17). Since elevated
levels of apoptosis in the brain of PPT1-KO mice and INCL patients
mediate progressive decline in number of cortical neurons, the
inventors also sought to further confirm whether decreased
apoptosis in NtBuHA-treated PPT1-KO mice prevented neuronal loss.
Accordingly, an immunohistochemical analyses of cortical tissue
sections from wild-type, untreated-PPT1-KO, and NtBuHA-treated
PPT1-KO mice was performed using antibodies against a neuronal
marker, NeuN. Results showed that compared with the brain of WT
mice (FIG. 18A) that of the untreated-PPT1-KO mice had an
appreciably reduced number of NeuN-positive cells (FIG. 18B).
Remarkably, the brain sections from NtBuHA-treated PPT1-KO mice
showed a modestly higher level of NeuN-positive cells (FIG. 18C).
Quantitative analysis of the NeuN-positive cells in brain tissues
from wild-type, untreated and NtBuHA-treated PPT1-KO mice (FIG.
18D) confirmed the visual assessment. Taken together, these results
provided supporting evidence that NtBuHA-treatment had a
neuroprotective effect on PPT1-KO mice.
Example 14
[0144] The ability of the HA-derivative NtBuHA to increase
methemoglobin levels was tested by placing PPT1-KO mice on a diet
containing NtBuHA. After three months on the diet, the level of
methemoglobin in the animal's blood was measured. The results of
this study are shown below in Table 3.
TABLE-US-00003 TABLE 3 Methemoglobin wild-type, untreated PPT1-KO,
NtBuHA-treated PPT1-KO mice % Genotype Methemoglobin Mean .+-. SD
WT-1 1.6 1.5 0.310913 WT-2 1.0 WT-3 1.1 WT-4 0.9 KO-1 1.1 1.025
0.221736 KO-2 1.3 KO-3 0.8 KO-4 0.9 BuHA-1 0 0.45 0.310913 BuHA-2
0.5 BuHA-3 0.7 BuHA-4 0.6
[0145] The results demonstrate that NtBuHA treatment up to three
months does not increase methemoglobin levels in PPT1-KO mice.
Example 15
[0146] This example shows that NtBuHA-treated PPT1-KO mice retain
near normal motor coordination and exploratory behavior at 6- and
8-months old
[0147] The natural history of INCL shows that psychomotor
deterioration is one of the first signs to be recognized in
patients with INCL. It has been recently demonstrated that
transplantation of human neuroprogenitor stem cells into the brain
of PPT1-KO mice delayed loss of motor coordination, most likely by
producing PPT1. Since NtBuHA functionally mimics PPT1, the
inventors tested 6-month and 8-month old wild-type, untreated- and
NtBuHA-treated PPT1-KO mice for motor coordination and exploratory
behavior. The Rotarod Performance Test, which measures parameters
such as riding time (seconds) and/or endurance was used to measure
motor coordination. The open field test was utilized to evaluate
the exploratory behavior. The results showed that NtBuHA-treated
PPT1-KO mice retained near normal motor function (FIG. 19A-D) as
well as exploratory behavior (FIG. 19E-F). These results suggest
that NtBuHA-treatment of PPT1-KO mice prevents deterioration of
motor coordination and helps maintain exploratory behavior in
PPT1-KO mice.
Example 16
[0148] This example shows that NtBuHA extends lifespan in PPT1-KO
mice.
[0149] For this study, 3-month old PPT1-KO mice were divided into
two groups. In the first group, 55 mice received no treatment
(control). In the second group, 59 mice were treated with NtBuHA.
The status of all mice was monitored on a daily basis. At 5.5
months after the initiation of the experiment the results showed
that while 52 out of 59 mice (88%) NtBuHA-treated mice were still
alive and apparently healthy whereas only 10% of the untreated mice
were alive (FIG. 20). This experiment shows that NtBuHA-treatment
expands lifespan in PPT1-KO mice.
[0150] The foregoing examples of the present invention have been
presented for purposes of illustration and description.
Furthermore, these examples are not intended to limit the invention
to the form disclosed herein. Consequently, variations and
modifications commensurate with the teachings of the description of
the invention, and the skill or knowledge of the relevant art, are
within the scope of the present invention. The specific embodiments
described in the examples provided herein are intended to further
explain the best mode known for practicing the invention and to
enable others skilled in the art to utilize the invention in such,
or other, embodiments and with various modifications required by
the particular applications or uses of the present invention. It is
intended that the appended claims be construed to include
alternative embodiments to the extent permitted by the prior
art.
* * * * *