U.S. patent application number 15/296247 was filed with the patent office on 2017-02-23 for methods of treating disorders associated with protein polymerization.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Tunde Hidvegi, George Konstantine Michalopoulos, David Hirsch Perlmutter.
Application Number | 20170049783 15/296247 |
Document ID | / |
Family ID | 43544626 |
Filed Date | 2017-02-23 |
United States Patent
Application |
20170049783 |
Kind Code |
A1 |
Perlmutter; David Hirsch ;
et al. |
February 23, 2017 |
METHODS OF TREATING DISORDERS ASSOCIATED WITH PROTEIN
POLYMERIZATION
Abstract
The present invention relates to methods of treatment of
clinical disorders associated with protein polymerization
comprising administering, to a subject, an effective amount of
carbamazepine, oxcarbazepine or another carbamazepine-like
compound. It is based, at least in part, on the discovery that, in
cells having a genetic defect in .alpha.1-antitrypsin,
carbamazepine was able to decrease levels of the mutant protein.
Furthermore, carbamazepine reduced the hepatic load of mutant
.alpha.1-antitrypsin and the toxic effect of that mutant protein
accumulation, hepatic fibrosis, in vivo using a mouse model of the
disease. As patients having this defect in .alpha.1-antitrypsin
exhibit toxic accumulations of the protein, treatment according to
the invention may be used to ameliorate symptoms and signs of
disease.
Inventors: |
Perlmutter; David Hirsch;
(Pittsburgh, PA) ; Michalopoulos; George Konstantine;
(Pittsburgh, PA) ; Hidvegi; Tunde; (Gibsonia,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
43544626 |
Appl. No.: |
15/296247 |
Filed: |
October 18, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14535210 |
Nov 6, 2014 |
9511074 |
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15296247 |
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13362606 |
Jan 31, 2012 |
8906905 |
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14535210 |
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PCT/US2010/044243 |
Aug 3, 2010 |
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13362606 |
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61230921 |
Aug 3, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/28 20180101;
A61P 25/16 20180101; A61K 31/155 20130101; A61K 31/55 20130101;
A61P 43/00 20180101 |
International
Class: |
A61K 31/55 20060101
A61K031/55 |
Goverment Interests
GRANT INFORMATION
[0002] This invention was made with government support under grants
HL037784 and DK076918 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1-5. (canceled)
6. A method of treating Parkinson's Disease consisting essentially
of administering, to a subject in need of such treatment, a
therapeutically effective amount of carbamazepine or a
carbamazepine-like compound selected from the group consisting of
carbamazepine-10,11-epoxide, iminostilbene, dihydrocarbamazepine,
ethyl urea, phenyl urea, diphenylurea, dicyclohexylurea, phenytoin,
substituted and unsubstituted iminobenzyl compounds, imipramine,
(S)-(-)-10-acetoxy-10,11-dihydro-5Hdibenz[b,f]azepine-5-carboxamide
(BIA 2-093), and
10,11-dihydro-10-hydroxyimino-5Hdibenz[b,f]azepine-5-carboxamide
(BIA 2-024).
7. The method of claim 6 where the carbamazepine-like compound is
carbamazepine.
8. The method of claim 6 where the carbamazepine-like compound is
oxcarbazepine.
9. The method of claim 6 where the carbamazepine-like compound is
metabolized, in the subject, to form
10,11-dihydro-10-hydroxy-carbamazepine.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/535,210 filed Nov. 6, 2014, which is a
continuation of U.S. patent application Ser. No. 13/362,606 filed
Jan. 31, 2012, now U.S. Pat. No. 8,906,905, which is a
continuation-in-part of International Patent Application No.
PCT/US2010/044243 filed Aug. 3, 2010 which claims priority to U.S.
Provisional Patent Application No. 61/230,921 filed Aug. 3, 2009,
to each of which priority is claimed and the contents of each of
which are incorporated by reference in their entireties herein.
1. INTRODUCTION
[0003] The present invention relates to methods of treatment of
clinical disorders associated with protein polymerization
comprising administering, to a subject, an effective amount of
carbamazepine ("CBZ"), oxcarbazepine ("OBZ") or another
carbamazepine-like compound.
2. BACKGROUND OF THE INVENTION
[0004] The classical form of .alpha.1-antitrypsin ("AT") deficiency
is an autosomal co-dominant disorder that affects approximately 1
in 2000 live births (25). It is caused by a point mutation that
alters the folding of an abundant liver-derived plasma glycoprotein
during biogenesis and also renders it prone to polymerization (43).
In addition to the formation of insoluble aggregates in the ER of
liver cells, there is an 85-90% reduction in circulating levels of
AT, the pre-dominant physiologic inhibitor of neutrophil elastase.
Individuals who are homozygous for the mutant allele are
susceptible to premature development of chronic obstructive
pulmonary disease. Pulmonary involvement is believed to be caused
by a loss-of-function mechanism, as lack of AT in the lung permits
elastase to slowly destroy the pulmonary connective tissue matrix
(44).
[0005] AT deficiency is the most common genetic cause of liver
disease in children and also causes liver disease and
hepatocellular carcinoma in adults. In contrast to pulmonary
involvement, liver inflammation and carcinogenesis are believed to
be caused by a gain-of-toxic function mechanism. This is most
clearly demonstrated by introducing the mutant human ATZ allele as
transgene into genetically engineered mice (45, 11). Insoluble
aggregates in hepatocytes, hepatic inflammation and carcinogenesis
evolve even though the endogenous anti-elastases of the transgenic
mouse are intact.
[0006] Cohort studies from an unbiased Swedish newborn screening
program have shown that only 8-10% of the affected homozygous
population develop clinically significant liver disease through the
first 30 years of life (26). This has led to the concept that
genetic and/or environmental modifiers determine whether an
affected homozygote is susceptible to, or protected from, liver
disease. Furthermore, it has led to consideration of two general
explanations for the effects of such modifiers: variation in the
function of intracellular degradative mechanisms and/or variation
in the signal transduction pathways that are activated to protect
the cell from protein mislocalization and/or aggregation.
[0007] Studies in this area have so far indicated that the
proteasome is responsible for degrading soluble forms of ATZ (29,
46) and that macroautophagy is specialized for disposal of the
insoluble polymers/aggregates that accumulate in the ER (30, 47).
In terms of cellular response pathways, it is thought that
accumulation of ATZ activates NF.kappa.B and autophagy but not the
unfolded protein response (1, 16).
[0008] Polymerization of protein is associated with a number of
other disorders. Among these is Alzheimer's Disease ("AD"), a
disorder which affects four million people in the United States and
has an incidence estimated at 1 in 68 individuals. As such, AD is
the most common form of age-dependent neurodegeneration. Most cases
are recognized by the sporadic onset of dementia during the seventh
decade of life while the less common, mutation-linked familial
cases cause dementia that is recognized by the fifth decade. AD is
associated with the accumulation of aggregation-prone peptides in
the brain, especially amyloid-.beta. ("A.beta.") peptides, but
hyperphosphorylated tau proteins also contribute to the tangles and
plaques that constitute the histological hallmarks of the
disease.
[0009] AD is thought to be caused by a gain-of-toxic function
mechanism that is triggered by the accumulation of aggregated
A.beta. and tau and worsened by aging (36). Recent studies have
shown that the prevalence of autophagosomes is increased in
dystrophic neurons of the AD brain, a finding that is recapitulated
in mouse models of the disease (37). Most of the evidence suggests
that autophagy plays a role in disposal of aggregated proteins that
might have toxic effects on neurons (38, 39). In fact, the
neuropathological effects of A.beta. in a mouse model of AD were
ameliorated by enhancing autophagy via overexpression of the
autophagy protein beclin 1 (39). In a study by Cohen et al.,
breeding of a mouse model of AD to a mouse model with targeted
disruption of the IGF-1 receptor demonstrated that reduced IGF-1
signaling blunted and delayed the toxic effect of A.beta.
accumulation (40). Although this could be attributed in part to
sequestration of soluble A.beta. oligomers into dense aggregates of
lower toxicity, it is well established that IGF-1 signaling
inhibits autophagy and therefore that these mice would likely have
enhanced autophagy. Thus, based on the current literature,
autophagy may be increased in AD, but the load of oligomers may be
too great to avoid toxic A.beta. accumulation.
[0010] Other disorders associated with increased protein aggregates
include Parkinson's Disease and Huntington's Chorea. Parkinson's
Disease is associated with the presence of protein aggregates in
the form of "Lewy Bodies", which contain a number of proteins
including one or more of alpha-synuclein, ubiquitin, neurofilament
protein, alpha B crystallin and tau protein. Interestingly, a
number of other disorders manifested as dementia are also
associated with the presence of Lewy Bodies in neurons--these
include Alzheimer's Disease, Pick's Disease, corticobasal atrophy,
multiple system atrophy, and so-called "dementia with Lewy Bodies"
or "DLB". Huntington's Chorea is associated with aggregates of
huntingtin protein containing a mutation that results in long
tracts of polyglutamine ("polyQ") which result in improper protein
processing and aggregate formation.
[0011] Carbamazepine ("CBZ"; also known as Tegretol.RTM.,
Carbatrol, and Equetro), is a drug that has been used for many
years as an anticonvulsant in the treatment of epilepsy and as a
specific analgesic for treatment of trigeminal neuralgia. It is
believed to act by reducing post-synaptic responses and blocking
post-tetanic potentiation in the nervous system. CBZ is known to
increase hepatic cytochrome P450 activity and thereby affect the
clearance of other pharmaceuticals eliminated through that system.
It is metabolized in the liver (see Prescribing Information from
Novartis Pharmaceuticals).
[0012] Oxcarbazepine ("OBZ", also known as Trileptal.RTM.) is, like
CBZ, a drug used in the treatment of seizures and trigeminal
neuralgia; in addition, it is used as a mood stabilizer. Unlike
CBZ, neither OBZ nor its monohydroxy derivative induce hepatic
oxidative metabolism (with the possible exception of P450IIIA
isozyme (58).
3. SUMMARY OF THE INVENTION
[0013] The present invention relates to methods of treatment of
clinical disorders associated with protein polymerization
comprising administering, to a subject, an effective amount of
carbamazepine or a carbamazepine-like compound. It is based, at
least in part, on the discoveries that CBZ could decrease steady
state levels of ATZ protein in cells and animals manifesting the
ATZ mutation, and was observed to decrease the amount of ATZ
accumulated in the liver in a mouse model of AT deficiency, and
that OBZ was able to decrease the cellular ATZ load at lower doses
than CBZ. According to the invention, treatment with CBZ, OBZ or a
similar compound may therefore be used to ameliorate the symptoms
and signs of AT deficiency as well as other disorders marked by
protein polymerization, including, but not limited to, Alzheimer's
Disease, Parkinson's Disease, and Huntington's Disease.
[0014] Without being bound by any particular theory, it appears
that CBZ lowers ATZ levels by not only increasing autophagy, but
also by increasing proteasomal degradation of ATZ as well as by
another mechanism outside the lysosomal and proteosomal
systems.
4. BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1A-D. (A) Effect of CBZ on steady state levels of ATZ
in the HTO/Z cell line. Immunoblot analysis of HTO/Z cells treated
with different concentrations of CBZ, separated into soluble and
insoluble fractions and then probed with antibodies to AT (top) and
GAPDH (bottom). (B) Effect of CBZ and rapamycin (RAP) on steady
state levels of ATZ in the HTO/Z cell line. After a 48-hour
incubation in CBZ or RAP in the concentrations indicated at the
bottom of the figure, cells were homogenized, cell homogenates
separated into insoluble and soluble fractions and these fractions
were then subjected to immunoblot analysis for AT (top) and GAPDH
(bottom). (C) Effect of different concentrations of CBZ on steady
state levels of ATZ in the HTO/Z cell line. Densitometric results
from 8 different experiments are plotted on the vertical axis and
concentration of CBZ on the horizontal axis. The number of samples
at each concentration is indicated just above the horizontal axis.
Results for the insoluble fraction are shown on the left and for
the soluble fraction on the right. Results are expressed as
mean+/-SD. (D) Effect of CBZ on steady state levels of wild type AT
in the HTO/M cell line and on steady state levels of BiP in the
HTO/Z cell line. This was carried out as in FIG. 1B.
[0016] FIG. 2A-D. Effect of CBZ on synthesis (A) and kinetics of
secretion (B, C) of ATZ in the HTO/Z cell line. (A) Cell lysates
after pulse labeling were immunoprecipitated with anti-AT; (B) Cell
lysates (IC) and extracellular fluid (EC) were immunoprecipitated
with anti-AT after pulse-chase labeling. (C) Kinetics of
disappearance from IC was determined by densitometric scanning of
fluorograms from 5 separate experiments. Data is shown as
mean+/-standard error. Dashed lines show the half-time for
disappearance. (D) Effect of CBZ on the fate of ATZ in pulse-chase
analysis. Fluorographic images from 5 separate pulse-chase
experiments described in FIGS. 2B and 2C were subjected to
densitometric scanning. In contrast to FIG. 2C the data from both
intracellular and extracellular contents are displayed in a
histogram. The relative densitometric intensity of the AT band at
T0 IC is set at 100% and every other band is compared to that. The
relative amount IC is shown in white and EC as hatched. The results
for control are shown on the left and CBZ on the right. Using this
display it can be seen that in CBZ-treated cells there is an
increase in the rate of disappearance of ATZ from the IC, a
decrease in the amount of ATZ that appears in the EC, and a
decrease in the amount of ATZ recovered from IC and EC together.
Together, this data demonstrates that the effect of CBZ is solely
an enhancement of ATZ degradation--i.e. CBZ does not affect
secretion.
[0017] FIG. 3A-F. (A) Effect of CBZ on LC3 conversion in the HTO/Z
cell line by immunoblot. Densitometric values are shown at the
bottom. (B and C) Effect of CBZ on ATZ in autophagy-deficient
(Atg5-/-) (B) versus wild-type (Atg5+/+) (C) cell lines. (D) Effect
of CBZ on levels of the AT Saar variant in the HTO/Saar cell line
compared to ATZ in the HTO/Z line. (E) Effect of CBZ on ATZ levels
in the presence of proteasomal inhibitors. For the last 6 hours of
incubation with CBZ (30 mM) or control, proteasomal inhibitors were
added to some of the monolayers. The experiments were done as in
FIG. 1A. For loading control, immunoblots for GAPDH are shown in
the lower panels. Similar results were obtained in three separate
experiments. (F) Effect of inducing expression of AT on LC3
conversion in HTO/Z (Z, left panel) and HTO/Saar (Saar, right
panel) cell lines. Each cell line was incubated in the absence or
presence of doxycycline (DOX) for 4 weeks. Separate monolayers that
were incubated in the absence or presence of dox were incubated
with lysosomal protease inhibitors (Lys. inh.), E64d (20 .mu.g/ml)
and pepstatin A (20 .mu.g/ml), for the last 4 hours prior to
harvesting and homogenization. These homogenates were subjected to
immunoblot analysis for LC3. Densitometric values for the
LC3-II/LC3-I ratio are also shown, with the relative densitometric
value in the presence of DOX but not Lys inh arbitrarily set as
1.0. The results show that there is an increase in the LC3-II to
LC3-I ratio when dox is removed in the Z cell line (compare lanes 3
and 4 to lanes 1 and 2) and this is further increased in the
presence of lysosomal inhibitors (compare lanes 7 and 8 to 5 and 6;
it will also help to compare lanes 5-8 to lanes 1-4). This LC3
conversion is specific for Z as shown by the results of inducing
Saar. There is no increase in LC3 II when dox is removed in the
Saar cell line (compare lane 10 to 9) and no change when lysosomal
inhibitors are added (lane 11). These results are representative of
3 separate experiments.
[0018] FIG. 4A-E. In vivo effect of CBZ on (A) hepatic AT load, (B
and C) globules, (D) autophagosomes, and (E) hepaticfibrosisin
PiZ.times.GFP-LC3 mice. Male mice at 5 months of age were treated
for 2 weeks with CBZ (250 mg kg-1) or solvent (dimethyl sulfoxide)
by gavage. Samples from two control and two CBZ-treated mice are
shown. (A) Immunoblot; (B) histochemical staining with periodic
acid-Schiff and diastase; (C) immunostaining with anti-AT; (D)
immunostaining with anti-GFP; (E) histochemical staining with
Sirius red. Globules are purple in (B). Globules are red and nuclei
blue in (C). Autophagosomes are green in (D). Scale bars, 100
mm.
[0019] FIG. 5A-C. (A) Green fluorescent autophagosomes in the liver
of PiZ.times.GFP-LC3 and GFP-LC3 mice. At 5 months of age, male
mice were sacrificed after 24 hours of regular feeding or
starvation. Liver sections were stained with anti-GFP to enhance
the detection of green fluorescent autophagosomes. The results
indicate that hepatic autophagy is activated in the PiZ mouse
without stimulation by starvation whereas hepatic autophagy is only
activated in the GFP-LC3 mouse after starvation. (B) Relationship
between green fluorescent autophagosomes and ATZ-containing
globules in liver cells of untreated PiZ.times.GFP-LC3 mice. Liver
from 5-month old males was doublestained with anti-AT (with
secondary antibody for red fluorescence) and anti-GFP. The arrows
point to globule-containing hepatocytes and the arrowhead points to
a globule-devoid hepatocyte. The result demonstrate the
autophagosomes are predominantly located in globule-containing
hepatocytes in the liver of PiZ mice that have not been treated
with CBZ. (C) Effect of rapamycin on hepatic AT accumulation in PiZ
mice. Male PiZ mice at 5 months of age were treated for 2 weeks
with rapamycin 2 mg/kg/day by intraperitoneal injection every other
day. The control group received the solvent DMSO in the identical
volume. Livers were harvested and AT levels determined by
immunoblot as described above.
[0020] FIG. 6. Effect of CBZ on serum levels of human ATZ in PiZ
mice. Serum levels were determined by ELISA specific for human AT.
Sufficient amount of serum was available from 11 control PiZ mice
and 18 PiZ mice treated with CBZ 250 mg/kg/day for 2 weeks. Results
are shown as mean+/-SD.
[0021] FIG. 7. Hepatic hydroxyproline content in FVB/N, PiZ and
PiZ.times.IKK.beta..hep mice in the absence or presence of CBZ
treatment. The results are shown as % control with the hepatic
hydroxyproline content in the untreated PiZ mouse set at 100%.
Mouse strain, dose of CBZ and duration of CBZ treatment are shown
at the bottom. The absolute figure for % control is shown at the
top of each bar.
[0022] FIG. 8. Levels of smooth muscle actin, collagen IA and
TGF.beta. RNA reported as mean+/-SD. As determined by Q-PCR.
[0023] FIG. 9. HTO/Z cells were incubated for 48 hrs in the absence
or presence of OBZ. Insoluble and soluble fractions from cell
homogenates were subjected to western blot for ATZ and GAPDH.
[0024] FIG. 10. Plaque load detected by staining of brain sections
with x-34, anti-A.beta.1-40 or anti-A.beta.1-42.
[0025] FIG. 11. Effect of CBZ on pulmonary fibrosis in
PiZ.times.GFP-LC3 mice. Sirius Red staining (% area) was measured.
3 month-old mice were treated for 3 weeks, 5 days per week, by oral
gavage. Doses are mg/kg/day. * denotes significant difference from
the DMSO control.
5. DETAILED DESCRIPTION OF THE INVENTION
[0026] For clarity of description and not by way of limitation, the
detailed description of the invention is divided into the following
subsections:
[0027] (i) treatment agents;
[0028] (ii) disorders of protein polymerization; and
[0029] (iii) methods of treatment.
5.1 Treatment Agents
[0030] Treatment agents which may be used according to the
invention include carbamazepine ("CBZ"), oxcarbazepine ("OBZ") and
other CBZ-like compounds.
[0031] CBZ is 5H-dibenz[b,f]azepine-5-carboxamide. The chemical
structure of CBZ is shown in Formula I:
##STR00001##
[0032] Oxcarbazepine, also known by the trade name Trileptal.RTM.
is 10,11-dihydro-10-oxo-5H-dibenz[b,f]azepine-5-carboxamide, and
its structural formula is:
##STR00002##
[0033] Other CBZ-like compounds include CBZ metabolites, including
but not limited to carbamazepine-10,11-epoxide and iminostilbene,
as well as structurally related compounds, and oxcarbazepine
metabolites, such as but not limited to
10,11-dihydro-10-hydroxy-carbamazepine (also known as "MHD").
Non-limiting examples of compounds structurally related to CBZ and
OBZ include dihydro-CBZ, ethyl urea, phenyl urea, diphenylurea,
dicyclohexylurea, phenytoin, substituted and unsubstituted
iminobenzyl compounds, imipramine,
(S)-(-)-10-acetoxy-10,11-dihydro-5H-dibenz[b,f]azepine-5-carboxamide
(BIA 2-093), and
10,11-dihydro-10-hydroxyimino-5H-dibenz[b,f]azepine-5-carboxamide
(BIA 2-024) (22, 23, 24). CBZ and CBZ-like compounds that are able
to cross the blood brain barrier offer advantages for the treatment
of disorders of protein polymerization in the central nervous
system. The ability of compounds structurally related to CBZ to
treat disorders of protein polymerization may be confirmed to have
activity in decreasing protein polymers (aggregates), for example,
using the HTO/Z cell line or its equivalent, or the
PiZ.times.GFP-LC3 transgenic mouse or its equivalent, or the model
system developed in Caenorhabditis elegans, as described in U.S.
Provisional Application No. 61/258,384, filed Nov. 5, 2009.
5.2 Disorders of Protein Polymerization
[0034] Disorders of protein polymerization (also sometimes referred
to in the art as disorders of protein aggregation or accumulation)
that may be treated according to the invention include, but are not
limited to, .alpha.1-antitrypsin deficiency, hepatic fibrosis,
pulmonary fibrosis, Alzheimer's Disease, Parkinson's Disease,
Pick's Disease, corticobasal atrophy, multiple system atrophy, Lewy
Body Disease, familial encephalopathy with neuroserpin inclusion
bodies (FENIB), Huntington's Disease, amyloidosis (e.g., primary,
secondary, familial, senile), prion-associated diseases (e.g.,
Creuzfeld-Jacob disease, mad cow's disease), protein polymerization
resulting from ischemic or traumatic brain injury (for example
dementia pugilistica (chronic traumatic encephalopathy)),
progressive supranuclear palsy, Lytico-Bodig disease (Parkinson
dementia complex of Guam), ganglioma, subsacute sclerosing
panencephalitis, certain forms of congenital diabetes, certain
forms of retinitis pigmentosa, certain forms of long QT syndrome,
hereditary hypofibrinogenemia, certain forms of osteogenesis
imperfecta, certain forms of hereditary angioedema,
Charcot-Marie-Tooth disease and Pelizaeus-Merzbacher
leukodystrophy.
5.3 Methods of Treatment
[0035] The present invention relates to methods of treating
clinical disorders associated with protein polymerization
comprising administering, to a subject in need of such treatment,
an effective amount of CBZ or a CBZ-like compound.
[0036] A subject in need of such treatment may be a human or a
non-human subject, and may be suffering from a disorder associated
with protein polymerization or be at risk of developing such a
disorder due to age, family history, or exposure to a toxic
agent.
[0037] An effective amount, as that term is used herein, is an
amount that (i) reduces one or more sign and/or symptom of the
disorder; and/or (ii) inhibits progression of the disorder; and/or
(iii) prolongs survival of the subject. It is this reduction in a
sign and/or symptom, inhibition of progression, or prolongation of
survival which constitutes treatment of the disorder.
[0038] Signs and symptoms of a disorder associated with protein
polymerization depend upon the particular disorder and are known to
the person skilled in the art. For all disorders treated according
to the invention, one sign that may be "reduced" may be the
accumulation of polymerized protein, in which either the rate of
accumulation may be slowed or (but not necessarily) the amount of
polymerized protein accumulated may stabilize or decrease.
[0039] For example, but not by way of limitation, where the
disorder is AT-deficiency, signs or symptoms that may be reduced or
otherwise ameliorated according to the invention include hepatitis,
hepatic enlargement, hepatic fibrosis, hepatocarcinoma, impaired
liver function, abdominal distension from ascites, jaundice, edema,
enlarged spleen, hypersplenism, gastrointestinal bleeding,
encephalopathy, renal failure, prolonged bleeding from injuries,
shortness of breath, wheezing, cough, decreased serum oxygen,
increased serum carbon dioxide, increased total lung capacity,
decreased FEV1/FVC ratio, increased incidence of pulmonary
infection, pulmonary infection, weight loss and fatigue. Although
the working example below addresses effects of ATZ accumulation on
the liver additional evidence is consistent with a similar toxic
function of ATZ in the lung, such that signs or symptoms of
pulmonary dysfunction may be treated according to the
invention.
[0040] As a further non-limiting example, where the disorder is
Alzheimers Disease, signs or symptoms that may be reduced or
otherwise ameliorated according to the invention include impairment
of short term memory, impairment of abstract thinking, impairment
of judgment, impairment of language skills, and mood changes.
[0041] As a further non-limiting example, where the disorder is
Parkinson's Disease, signs or symptoms that may be reduced or
otherwise ameliorated according to the invention include tremor,
bradykinesia, rigidity, impaired speech, and dementia.
[0042] As a further non-limiting example, where the disorder is
Huntington's Disease, signs or symptoms that may be reduced or
otherwise ameliorated according to the invention include dementia
and choreoform movements.
[0043] As a further non-limiting example, where the disorder is
amyloidosis, signs or symptoms that may be reduced or otherwise
ameliorated according to the invention include thickening of the
skin, rash, cardiomyopathy, congestive heart failure, cardiac
arrhythmias and/or conduction defects, shortness of breath,
fatigue, impaired renal function, hyothyroidism, anemia, bone
damage/fracture, impaired liver function, impaired immunity, and
glossitis.
[0044] As a further non-limiting example, where the disorder is a
prion disease, signs or symptoms that may be reduced or otherwise
ameliorated include dementia and choreoform movements.
[0045] In additional non-limiting embodiment, the present invention
provides for a method of decreasing the amount of polymerized
protein in a cell comprising exposing the cell to an effective
amount of CBZ or a CBZ-related compound. The cell may be a cell
affected by a disorder of protein polymerization, as set forth
above, for example, but not by way of limitation, a liver cell or a
lung cell from a subject suffering from AT deficiency, a neuron
from a subject suffering from Alzheimer's Disease, Parkinson's
Disease, Huntington's disease, a prion disease, or a cell from a
subject suffering from any of the other aforelisted disorders
associated with protein polymerization.
[0046] CBZ or a CBZ-related compound may be administered by any
route of administration, including oral, intravenous,
intramuscular, subcutaneous, intrathecal, intraperitorneal,
intrahepatic, by inhalation, e.g., pulmonary inhalation, etc. In a
preferred non-limiting embodiment of the invention, CBZ or a
CBZ-related compound may be administered orally.
[0047] In preferred non-limiting embodiments of the invention, CBZ
may be administered at a dose of 400 mg/day in 2-4 divided doses.
Said dose may optionally be increased weekly by 200 mg until a
therapeutically effective dose, or a dose of up to 1000 mg/day (for
children 12-15 years of age) or a dose of up to 1200 mg/day (for
persons greater than 15 years of age), is reached. Children between
the ages of 6-12 may desirably be treated with an initial dose of
200 mg/day which is then increased weekly by 100 mg/day until a
dose of 800 mg/day is reached. Children under 6 years of age may
desirably be treated with CBZ at a dose of 10 mg/kg/day, which dose
may be increased weekly by 5 mg/kg/day until a dose of 20 mg/kg/day
is reached.
[0048] In certain non-limiting embodiments of the invention, the
dose of CBZ administered produces a serum concentration or
cerebrospinal fluid concentration of at least about 0.1 micromolar
and preferably at least about 3 micromolar or at least about 1
microgram per milliliter. To determine the lower dosage limit of a
CBZ-related compound, said related compound may be tested in an
assay system as described in the example section below and the
concentration of related compound which creates approximately the
same inhibitory effect on ATZ accumulation as 3 mM CBZ may be
determined.
[0049] In certain non-limiting embodiments of the invention, CBZ
may be administered at a total dose of at least about 100 mg/day,
which may optionally be administered as a divided dose.
[0050] In certain non-limiting embodiments of the invention, CBZ
may be administered at a total dose of between about 25 and 1500
mg/day, or between about 100 and 1200 mg/day, or between about 400
and 1200 mg/day, or between about 100 and less than about 400
mg/day, any of which doses may optionally be administered as a
divided dose.
[0051] Where a CBZ-like compound is used, the dose of compound may
be determined based on the above doses for CBZ and a comparison of
the related compound's potency to that of CBZ in reducing AZT
accumulation in vitro or in vivo, for example as determined using
one or more assay described in the example below.
[0052] For example, and not by limitation, the present invention
provides for a method of treating clinical disorders associated
with protein polymerization comprising administering, to a subject
in need of such treatment, an effective amount of OBZ.
[0053] In certain non-limiting embodiments of the invention, OBZ
may be administered at a total dose of between about 5 and 1500
mg/day, or between about 50 and 1000 mg/day, or between about 50
and 600 mg/day, or between about 50 and 300 mg/day, or between
about 50 and 200 mg/day, or between 50 and less than about 300
mg/day, any of which doses may optionally be administered as a
divided dose.
[0054] In certain non-limiting embodiments of the invention, the
dose of OBZ administered produces a serum concentration or
cerebrospinal fluid concentration of at least about 0.01 micromolar
and preferably at least about 0.1 micromolar or at least about 1
microgram per milliliter.
[0055] In certain non-limiting embodiments of the invention, OBZ
may be administered at a total dose of at least about 100 mg, which
may optionally be administered as a divided dose.
[0056] In certain non-limiting embodiments, the dose may be
administered daily, about every other day, about twice a week, or
about once a week.
[0057] Treatment may be administered continuously or for intervals
interrupted by breaks.
[0058] Prior to treatment with CBZ, it is desirable to test whether
a subject carries the HLA-B* 1502 allele, as subjects carrying this
allele may have a severe skin reaction to CBZ, which may include
toxic epidermal necrolysis or Stevens Johnson Syndrome.
6. EXAMPLE
An Autophagy-Enhancing Drug Promotes Degradation of Mutant
.alpha.1-Antitrypsin Z and Reduces Hepatic Fibrosis
6.1 Materials and Methods
[0059] Materials Rabbit anti-human AT antibody was purchased from
DAKO (Santa Barbara, Calif.) and goat anti-human AT from Diasorin
(Stillwater, Minn.). Antibody to GAPDH was purchased from US
Biochemical and antibody to LC3 was from Axora LLC (San Diego,
Calif.). Antibody to murine BiP was purchased from Santa Cruz
Biotechnology (Santa Cruz, Calif.). Rapamycin (RAP) was purchased
from Sigma and prepared as a stock solution of 2 mgs/ml in DMSO.
Carbamazepine (CBZ) was purchased from Sigma and prepared in a
stock solution of 25 mg/ml DMSO. Doxycycline was purchased from
Sigma and prepared 1 mg/ml in water. MG132 was purchased from
Calbiochem (stock solution 10 mM in DMSO), lactacystin from Boston
Biochem (stock solution 10 mM in DMSO), E64D from Peptide
International (stock solution 20 mg/ml in DMSO) and pepstatin A
from Sigma (stock solution 20 mg/ml in DMSO).
[0060] Cell Lines
[0061] The human epidermal HeLa cell line with
doxycycline-regulated expression of ATZ (HTO/Z) has been described
previously (1). HTO/M and HTO/Saar are HeLa cell lines with
doxycycline-regulated expression of wild type AT and the AT Saar
variant, respectively (1). A murine embryonic fibroblast cell line
(MEF) with targeted disruption of Atg5 (2) was engineered for
stable expression of ATZ using the previously described pRc/RSV-ATZ
expression plasmid (3). A wild type MEF cell line was also
engineered for stable expression of ATZ in the same way to serve as
control. For experiments with CBZ, the inducible cell lines were
cultured in the absence of doxycycline for at least 4 weeks for
maximal expression of AT. The cells were then subcultured into
separate monolayers in fresh complete growth medium and incubated
for 48 hours in the absence or presence of CBZ or rapamycin (RAP).
CBZ or RAP were added to the growth medium. The duration of
incubation with CBZ was determined to be optimal at 48 hours based
on experiments in which the duration was varied from 12 to 72
hours. Doses of CBZ were based on previous studies of its effects
in cell lines (5,6). Doses of RAP were based on previous positive
effects on autophagic disposal of polyglutamine-repeat proteins
(7). After the incubation cells were homogenized and cell
homogenates separated into insoluble and soluble fractions
according to our previously established technique (8). Samples of
20 .mu.gs each were subjected to immunoblot analysis for AT, BiP
and GAPDH.
For experiments in which proteasomal inhibitors were used, MG132
was used at 30 .mu.M and lactacystin at 10 .mu.M for the last 6
hours of the incubation with CBZ or control. Cells that were
incubated with MG132 or lactacystin alone served as control to
validate that the proteasome was inhibited. For investigation of
LC3 conversion, lysosomal inhibitors (E64D and pepstatin A at 20
.mu.g/ml) were added to the medium for the last 4 hours of the
incubation with CBZ or control. This has been shown to inhibit the
lysosomal degradation of LC3-II and when compared to the LC3-II
levels in the absence of lysosomal inhibitors to provide a true
reflection of autophagic flux (9). If the number of separate
experiments done in the cell line models is not specifically
indicated in the text or figure legend, at least 3 separate
experiments were done in each case.
[0062] Transgenic Mice
[0063] PiZ mice that have been bred into the C57/BL6 background
have been described previously (1). For the second and third series
of experiments with CBZ we used PiZ mice that were re-derived onto
the FVB/N background for a move into a new animal facility. The
transgene that was used to generate the PiZ mouse is a genomic
fragment of DNA that contains the coding regions of the ATZ gene
together with introns and .about.2 kilobases of upstream and
downstream flanking regions (10). It is important to point out that
the endogenous murine ortholog of AT is not knocked out in this
mouse so it does not have deficient serum levels of AT. In this
perspective it is not an exact phenocopy of the classical form of
AT deficiency. In particular it cannot be a model for the
loss-of-function mechanisms associated with the classical form of
AT deficiency. It is known to have abundant expression of ATZ in
hepatocytes and other cell types that express ATZ in humans (11).
In the liver there are abundant ATZ-containing intrahepatocytic
globules and inflammation that is characteristic of what is seen in
the human liver (11,12). It was found that the liver of the PiZ
mouse resembles that in humans with the classical form of AT
deficiency in terms of regenerative activity, steatosis, dysplasia,
mitochondrial injury, activation of autophagy, NF.kappa..B and
genes associated with fibrosis (1, 12-16). In this study Sirius Red
staining and quantification of hydroxyproline in the liver of these
mice was used for the first time and it was found that there is
also significant hepatic fibrosis, the most important hepatic
histological marker of hepatic injury that occurs in the human
disease. FIG. 7 shows that the hepatic hydroxyproline content is
more than 2-fold higher than that in the background FVB/N strain.
Taken together, these observations indicate that the PiZ mouse is
an appropriate model for the gain-of-toxic function mechanism that
is responsible for liver damage in the classical form of AT
deficiency. Indeed, the fact that these mice are endowed with
endogenous AT function and therein normal levels of AT in the serum
and body fluids make them an even more `pure` model for liver
damage by gain-of-toxic function. PiZ.times.GFP-LC3 mice, which
generated green fluorescent autophagosomes, have been described
previously (2). PiZ.times.IKK.beta..hep were generated by mating of
PiZ to IKK.beta..hep, which has conditional hepatocyte-specific
disruption of NF.B activation (M. Karin, 17). There is increased
injury in the liver of these mice as evidenced by the hepatic
hydroxyproline content, 153.3% of that in the PiZ mouse (FIG.
7).
[0064] Histology
[0065] Sections of liver tissue were stained with hematoxylin and
eosin, PAS, PAS after diastase treatment, TUNEL, PCNA, Ki67 and
Sirius Red using standard techniques (18). Previous methods for
staining with antibody to smooth muscle actin were used (19). Each
was examined by the pathologist (GM) who was completely blinded to
the experimental protocol. Sections of liver tissue were also
stained with goat anti-human AT followed by donkey anti-goat Cy3 to
detect AT-containing intracellular globules. Finally sections of
liver tissue were stained with anti-GFP to optimize the detection
of green fluorescent autophagosomes. The number of inflammatory
nodules, AT-containing globules and autophagosomes were each
quantified blindly by counting cells in 6 microscopic fields of 10
different sections for each liver. The number of nuclei, as
determined by Hoechst staining, was used to exclude the possibility
that different numbers of cells were counted in liver sections from
mice treated with CBZ as compared to controls. Hepatic
hydroxyproline concentration was determined by a well-established
protocol (20,21).
[0066] Therapeutic Regimens
[0067] For the initial series of experiments in mice in vivo, the
dose of 250 mg/kg/day for CBZ was based on previous studies of its
biological effects in mice (22,23). The duration of 2 weeks was
more effective in reducing the hepatic ATZ load than 7 days or 10
days. CBZ was administered in DMSO by gavage once per day. Control
mice were given an equivalent volume of DMSO by gavage. In a second
series of experiments, doses of 200, 100 and 50 mg/kg/day of CBZ
were administered for 6 weeks. The dose and route of administration
of RAP, 2 mg/kg/day by intraperitoneal injection for 2 weeks, was
based on previous studies that have shown activation of hepatic
autophagy (24).
[0068] Quantitative PCR (Q-PCR)
[0069] Levels of mRNA from smooth muscle actin, collagen IA and
TGF.beta. in liver of PiZ mice were determined by Q-PCR using
primers from ABI and previous described conditions (19).
[0070] Radioimmunoprecipitation, SDS-PAGE and Immunoblot
Analysis
[0071] Biosynthetic labeling, pulse-chase labeling,
immunoprecipitation and SDS-PAGE/fluorography for AT followed
previously published protocols (1). Radioactivity measured in TCA
precipitates, using previous methods (1), did not show any effects
of CBZ on total protein synthesis or secretion. For the pulse
labeling experiments, HTO/Z cells were incubated for 48 hours in
the absence or presence of CBZ in several different concentrations
and then subjected to labeling for 30 mins. The cell lysates were
then examined by immunoprecipitation and the immunoprecipitates
analyzed by SDS-PAGE/fluorography. For the pulse-chase experiments,
HTO/Z cells were incubated for 48 hours in the absence of presence
of CBZ 30 .mu.M and then pulse labeled for 30 mins. The cells were
then washed and incubated in growth medium without tracer for
several different time intervals to constitute the chase. CBZ 30
.mu.M was included during the pulse and chase periods. The
extracellular fluid and cell lysate samples were subjected to
immunoprecipitation and the immunoprecipitates analyzed by
SDS-PAGE/fluorography. All fluorograms were subjected to
densitometry. The relative densitometric value of T0 is set at 100%
and the remainder of the data set expressed as % of this control.
The data are shown as mean+/-SE and the mean value at each time
point is shown at the bottom of the figure.
[0072] For immunoblot analysis to detect AT, GAPDH or LC3, cells
were lysed in 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, pH 8.0.
Protein levels were quantified using the BCA protein assay (Pierce
Biotechnology, Rockford, Ill.). 10-50 .mu.g samples were loaded
onto 7.5% precast gels. PVDF membranes were blocked in TBS, 0.5%
Tween 20 (TBST), 5% milk and then incubated with primary antibody
in 5% milk TBST solution. Horseradish peroxidase anti-goat Ig or
anti-mouse Ig (Jackson Labs, Bar Harbor, Me.) were used as
secondary antibodies in TBST. Blots were visualized with Super
Signal West Dura or West Femto from Pierce.
[0073] For immunoblot on liver, the liver was snap frozen in liquid
nitrogen and stored at -80.degree. C. Liver was homogenized in 50
mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM KCl, 2 mM MgCl2, 0.5% Triton
X-100, 0.5% deoxycholic acid containing 0.1 mM phenylmethylsulfonic
acid and complete protease inhibitor cocktail from Roche. Total
protein concentration was measured by BCA assay (Pierce). Soluble
and insoluble fractions were separated by centrifugation (14,000
rpm, 10 min, 4.degree. C.). The insoluble pellet was washed twice
in 50 mM Tris-HCl (pH7.4, 150 mM NaCl) and resuspended in 50 mM
Tris-HCl (pH6.8, 5% SDS, 10% glycerol). Equal amounts of total
protein (1 ug) were loaded on 8% SDS-PAGE. After transfer to PVDF
membrane, the blots were blocked in PBS-Tween20 containing 5%
non-fat milk for 1 hr at RT, then goat-anti human AT antiserum
(Diasorin, 1:2500) was applied followed by three washes. Donkey
anti-goat IgG-HRP (Santa Cruz, 1:1,000,000) and West Dura (Pierce)
was used for detection of AT. The blots were stripped (Pierce) and
after the blocking step anti-mouse GAPDH (US Biologicals, 1:10,000)
and rabbit anti-mouse IgG-HRP (Jackson Labs, 1:5000) were used to
detect GAPDH.
[0074] For ELISA on mouse serum specimens, Nunc Maxisorp plates
were first coated with goat anti-human AT (Bethyl), then blocked in
PBS-Tween20 containing 5% nonfat milk. Serum samples were loaded
into the wells in 1:20,000 dilution using purified human AT serial
dilutions (1.56 to 100 ng/ml) as a standard. Rabbit-anti human AT
(Dako) was used as capturing antibody, and goat anti-rabbit IgG-HRP
(Dako) a secondary antibody. Protein levels were detected with OPD
(Sigma).
[0075] Statistical Analysis
[0076] Students t-test was used for most comparisons but the
Welch-modified t-test was used to compare experimental groups that
were not paired and did not assume equal variances. Kinetic curves
were analyzed by two-way ANOVA with the Bonferroni post-test using
the Prism software application.
6.2 RESULTS AND DISCUSSION
[0077] The classical form of .alpha.1-antitrypsin (AT) deficiency
is caused by a point mutation (lysine for glutamate 342) that
alters the folding of an abundant liver-derived plasma glycoprotein
during biogenesis and also renders it prone to polymerization (25).
In addition to the formation of insoluble aggregates in the
endoplasmic reticulum (ER) of liver cells, there is an 85-90%
reduction in circulating levels of AT, the pre-dominant physiologic
inhibitor of neutrophil elastase. Liver fibrosis and carcinogenesis
are caused by a gain-oftoxic function mechanism. Indeed, AT
deficiency is the most common genetic cause of liver disease in
childhood but can also present for the first time with cirrhosis
and/or hepatocellular carcinoma in adulthood (25).
[0078] Genetic and/or environmental modifiers determine whether an
affected homozygote is susceptible to liver disease (26). Two
general explanations for the effects of such modifiers have been
postulated:variation in the function of intracellular degradative
mechanisms (27,28) and/or variation in the signal transduction
pathways that are activated to protect the cell from protein
mislocalization and/or aggregation. As for degradation, the
proteasome is responsible for degrading soluble forms of
.alpha.1-antitrypsin Z (ATZ) (29), and macro-autophagy is
specialized for disposal of the insoluble polymers and aggregates
(30, 31). However, disposal of ATZ may involve other degradative
mechanisms, as yet not well defined (32, 1). In terms of cellular
response pathways, accumulation of ATZ activates nuclear factor kB
(NF-kB) and autophagy but not the unfolded protein response (30, 1,
16).
[0079] Because the autophagic response participates in both
degradation of ATZ and in the cellular response to accumulation of
ATZ in the ER, we examined whether a drug that enhances autophagy
could ameliorate hepatotoxicity in this disorder. From a list of
drugs that have been recently shown to enhance autophagic
degradation of aggregation-prone proteins with polyglutamine
repeats (5, 33, 34), we selected carbamazepine (CBZ) for detailed
studies of its effect on ATZ because it has the most extensive
safety profile in humans.
[0080] First, we found that CBZ mediated a marked decrease in
steady-state levels of ATZ in both the insoluble and soluble
fractions in the HeLa inducible cell line HTO/Z (FIG. 1A). The
effect of CBZ was also specific because rapamycin, a drug that
activates autophagy by inhibiting target of rapamycin (TOR) kinase,
had no effect on ATZ levels (FIG. 1B). CBZ was dose dependent in
the range of 1 to 60 mM (FIG. 1C) and did not affect wild-type AT
levels in the HTO/M cell line or BiP levels in the HTO/Z line (FIG.
1D).
[0081] To further characterize the effect of CBZ on ATZ, we carried
out pulse labeling and pulse-chase labeling experiments in the
HTO/Z line. CBZ did not affect synthesis of ATZ (FIG. 2A), and
disappearance of ATZ from the intracellular compartment was more
rapid in cells treated with CBZ than in the untreated cells (FIGS.
2, B and C). A statistically significant increase in disappearance
of ATZ from the intracellular compartment was mediated by CBZ
(P=0.0007 by two-way analysis of variance with Bonferroni
adjustment), with a half-time of 130 min compared to 200 min in
untreated cells. The increase in intracellular disappearance of ATZ
mediated by CBZ could not be attributed to enhanced secretion
(FIGS. 2B and 2D). Thus, CBZ appears exclusively to change the rate
of intracellular degradation.
[0082] To determine whether CBZ enhances autophagy in the HTO/Z
line, we examined its effect on isoform conversion of
autophagosomal membrane-specific protein LC3, an indicator of
autophagosome formation (FIG. 3A). The LC3-II to LC3-I ratio
increased in a dose-dependent manner and was greater in the
presence of lysosomal enzyme inhibitors, indicating that CBZ
elicits increased autophagic flux. This effect of CBZ on autophagic
flux exceeded the increase that results from intracellular
accumulation of ATZ (FIG. 3F). Thus, CBZ stimulates autophagy in
cells that have already activated the autophagic pathway in
response to ER accumulation of ATZ.
[0083] To determine whether the effect of CBZ on ATZ degradation
involved enhanced autophagy, we examined its effect on ATZ levels
in an autophagy (Atg5)-deficient cell line (FIGS. 3B and 3C). CBZ
mediated a decrease in levels of insoluble ATZ in the wild-type
mouse embryonic fibroblast (MEF) cell line but not in the Atg5
deficient cell line. CBZ also mediated a decrease in levels of
soluble ATZ in both wild-type and Atg5-deficient cells. Thus, CBZ
enhances the disposal of insoluble ATZ by autophagy and has an
independent action on the disposal of soluble ATZ by mechanism(s)
that do not involve the conventional autophagic pathway.
[0084] To determine whether the effects of CBZ were specific for
the Z variant of AT, we investigated its effect on disposal of AT
Saar, a variant of AT that accumulates in the ER but does not
aggregate and is predominantly degraded by a proteasomal mechanism
(1). AT Saar was present only in the soluble fraction, but it was
degraded by CBZ in a manner almost identical to that of ATZ (FIG.
3D), suggesting an effect of CBZ also on the proteasome.
[0085] Thus, we examined the effect of CBZ on steady-state levels
of ATZ in the presence of proteasomal inhibitors (FIG. 3E).
Although they had no effect on levels of insoluble ATZ, lactacystin
and MG132 partially reversed the effect of CBZ on levels of soluble
ATZ [lactacystin: reversal of 23.1.+-.14.0% (mean T SD), n=3
experiments; MG132: reversal of 12.3, average of n=two
experiments]. Increased levels of ATZ in the presence of
lactacystin and MG132 alone provided validation for proteasome
inhibitory activity under the conditions of these experiments.
Thus, CBZ mildly enhances proteasomal degradation of ATZ and has an
independent action on nonproteasomal mechanisms for disposal of
soluble ATZ.
[0086] Next, we examined the effect of CBZ on hepatic load of ATZ
in vivo using PiZ.times.GFP-LC3 mice. The PiZ mouse was created
with the human ATZ gene as transgene. Although it differs from the
human disorder in having normal circulating levels of the
endogenous murine ortholog of AT, the PiZ mouse is a robust model
of liver disease associated with AT deficiency, as characterized by
intrahepatocytic ATZ-containing globules, inflammation, and
increased regenerative activity, dysplasia, and fibrosis (12). It
has been bred onto the GFP-LC3 background to monitor autophagy
(30). When administered at 250 mg kg-1 day-1 for 2 weeks by gavage,
CBZ mediated a marked decrease in total, insoluble, and soluble ATZ
in the liver (FIG. 4A). The treatment was also associated with a
marked decrease in intrahepatocytic ATZ-containing globules (FIGS.
4B and 4C). Quantitative morphometry showed a decrease in
globule-containing hepatocytes by a factor of 3.36 (P<0.001 by
Mann-Whitney rank sum test). Serum concentrations of human AT were
not significantly affected by CBZ treatment (FIG. 6), arguing
against any effect on secretion of ATZ in vivo.
[0087] Using indirect immunofluorescence, an increase in number of
hepatic green fluorescent autophagosomes was detected in areas of
liver that lacked AT-stained globules after CBZ treatment (FIG.
4D), and this was confirmed by quantitative morphometry
(mean.+-.SD: 565.7.+-.185.7 mm2 in control versus 1055.3.+-.139.7
mm2 in CBZ; P=0.049 by t test). The increase in autophagosomes
mediated by CBZ superseded the increase that occurs predominantly
in globule-containing hepatocytes from ATZ expression alone (30)
(FIGS. 5A and 5B). The effect of CBZ in vivo was specific in that
rapamycin had no effect on hepatic ATZ levels (FIG. 5C). Next, we
examined the effect of CBZ on hepatic fibrosis because it is a key
feature of the liver disease associated with AT deficiency (12).
CBZ mediated a marked decrease in fibrosis (FIG. 4E). Furthermore,
there was a marked and statistically significant reduction in
hepatic hydroxyproline concentration in PiZ mice treated with CBZ
(mean.+-.SD: 1.21.+-.0.7 in CBZ versus 2.27.+-.1.02 mg per
milligram of dry weight in control, P=0.0074 by t test with Welch
modification). Hepatic hydroxyproline content was decreased 46.7%
by CBZ, reaching a level that was indistinguishable from that of
the background FVB/N strain (FIG. 7). CBZ also mediated a decrease
in hepatic hydroxyproline concentration in the PiZ.times.IKKbDhep
mouse model (FIG. 7). On this hepatocyte-specific NFkB-deficient
background, there is more severe liver damage as reflected by
hydroxyproline concentrations that are >150% of the levels in
the PiZ mouse on the FVB/N background (FIG. 7). CBZ treatment
decreased levels of stellate cell activation markers, including
smooth muscle actin, collagen 1A, and transforming growth factor b,
but only the decrease in actin reached statistical significance
(FIG. 8).
[0088] To determine whether lower doses of CBZ for more prolonged
time intervals could reduce hepatic fibrosis, we examined the
effect of CBZ at lower doses for 6 weeks. Hepatic hydroxyproline
concentrations decreased at the dose of 200 mg kg-1 day-1 but not
at doses of 50 and 100 mg kg-1 day-1 (FIG. 7). Although the lowest
effective dose of CBZ (200 mg kg-1 day-1) was considerably higher
than the doses used in humans (10 to 20 mg kg-1 day-1), effective
doses of drugs can be 10 to 20 times as high in mice because of the
higher ratio of surface area to body weight when compared to
humans.
[0089] Thus, CBZ reduces the hepatic load of mutant ATZ and hepatic
fibrosis in the PiZ mouse. Mechanistic studies indicate that CBZ
increases both autophagic and proteasomal degradation of ATZ. That
rapamycin does not enhance autophagic disposal of ATZ may mean that
a TOR-independent pathway is involved in the effect of CBZ. The
effect of CBZ on ATZ disposal cannot be fully accounted for by the
proteasomal and conventional macroautophagic pathways. The capacity
to enhance disposal of both insoluble and soluble ATZ could
represent an important characteristic of CBZ as a potential
therapeutic in that it might provide for elimination of the
putative hepatotoxic form of ATZ, whether it is soluble monomeric,
soluble oligomeric, and/or insoluble polymeric ATZ species.
[0090] Because it is theorized that clinically significant liver
damage occurs only in AT-deficient patients who also have a
"second" defect in quality control and that these second defects
are heterogeneous among the affected population, one might conclude
that CBZ would be effective only in individuals in whom the
"second" defect is related to the specific mechanism of CBZ action.
However, our results suggest that CBZ can enhance autophagy beyond
the extent to which it has already been activated by the
pathological state. CBZ also appears to affect several mechanisms
of intracellular disposal and therefore may not require mechanistic
specificity for a beneficial effect. It is also encouraging that
CBZ reduced hepatic fibrosis in the PiZ.times.IKKbDhep mouse model,
which could be viewed as a mouse with a type of "second" defect--in
this case, reduced functioning of the hepatocyte NF-kB signaling
pathway.
[0091] In addition to its potential for the treatment of liver
disease due to AT deficiency, CBZ should be considered for its
ability to enhance intracellular disposal pathways for the
treatment of other diseases in which tissue damage involves
gain-of-toxic function mechanisms caused by misfolded or
aggregation-prone proteins (34). Our results also provide further
evidence for the concept that the endogenous protein homeostasis
machinery can be used to prevent tissue damage from mutant proteins
(35).
7. EXAMPLE
Oxcarbazepine Decreases Cellular ATZ Load at Lower Doses than
CBZ
[0092] OBZ is a structural derivative of CBZ and has been used
extensively with an exceptional safety profile. Like CBZ it
permeates the blood-brain barrier but it has several advantages
over CBZ: it does not induce the liver microsomal membranes and
cytochrome P450 activities; it does not cause the serious side
effects of anemia and agranulocytosis that occasionally develop
from CBZ administration. The effect of OBZ on steady state levels
of ATZ in the HTO/Z cell line (FIG. 9) was evaluated. It was found
that OBZ mediated a marked decrease in insoluble ATZ. The effect
was dose-dependent with an effect evident at doses as low as 0.1
uM. This means that OBZ is effective at significantly lower doses
than CBZ which has a minimal effective dose of 3 .mu.M. OBZ is also
different than CBZ in that it appears to have a minimal effect on
soluble ATZ levels, suggesting that it only stimulates
autophagy.
8. EXAMPLE
CBZ Reduces Plaque Load in a Mouse Model of AD
[0093] The APP-PS1 mouse model of AD is associated with accelerated
amyloid deposition with plaques resembling AD in humans starting to
appear at 12 weeks of age (12) and progressive behavioral changes
starting at 6 months of age (55, 56). Only a limited number of mice
were available at the ideal age, 9 wks, at the time of the study,
so the pilot study was very small. 9-12 wks of age was selected as
the ideal age because it represents the age with the earliest
consistent appearance of A.beta. deposition in APP-PS1 mice. Mice
were treated by orogastric gavage with CBZ 200 mg/kg/day, 5 doses
per wk, for 3 wks; n=2 for CBZ and n=4 for vehicle (DMSO). Brain
sections were stained with X-34, antibodies to A.beta.1-40 and
A.beta.1-42 to determine plaque load using techniques as described
previously (57). FIG. 10 shows that CBZ mediated a marked reduction
in plaque load. ELISA for soluble and insoluble A.beta.1-40 and
A.beta.1-42 using previously described methods (57) also showed a
trend toward lower values in the CBZ-treated mice but with a much
greater degree of variation.
9. EXAMPLE
CBZ Decreases Pulmonary Fibrosis in PiZ Mice
[0094] Experiments were performed to determine if CBZ can mediate a
decrease in lung fibrosis in the PiZ mouse in vivo. 3-month-old PiZ
mice were treated 5 days per week for 3 weeks with diluent DMSO or
CBZ 200 mg/kg/day (n=3-8 mice per group). In this series of
experiments lung fibrosis was assayed by Sirius Red staining with
quantitative morphometry. The results, depicted in FIG. 11, show a
statistically significant reduction in Sirius red staining (and
hence pulmonary fibrosis) when PiZ mice were given CBZ. Of note,
the drug treatment reduces lung fibrosis to levels that are
comparable to what is found in wild type mice at this age.
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[0153] Various publications are cited herein, the contents of which
are hereby incorporated by reference in their entireties.
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