U.S. patent application number 13/195552 was filed with the patent office on 2011-12-08 for use of pramipexole to treat amyotrophic lateral sclerosis.
This patent application is currently assigned to University of Virginia Patent Foundation. Invention is credited to James P. Bennett, JR..
Application Number | 20110301210 13/195552 |
Document ID | / |
Family ID | 26991607 |
Filed Date | 2011-12-08 |
United States Patent
Application |
20110301210 |
Kind Code |
A1 |
Bennett, JR.; James P. |
December 8, 2011 |
USE OF PRAMIPEXOLE TO TREAT AMYOTROPHIC LATERAL SCLEROSIS
Abstract
The present invention is directed to compositions comprising
pramipexole and the use of such compositions to treat
neurodegenerative diseases such as amyotrophic lateral sclerosis
(ALS). As shown in FIG. 6B the mean +/-SEM serum 2,3-DHBA levels
for the 12 ALS participants decreased significantly after
pramipexole treatment.
Inventors: |
Bennett, JR.; James P.;
(Crozet, VA) |
Assignee: |
University of Virginia Patent
Foundation
Charlottesville
VA
|
Family ID: |
26991607 |
Appl. No.: |
13/195552 |
Filed: |
August 1, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11614528 |
Dec 21, 2006 |
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13195552 |
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10496714 |
May 26, 2004 |
7157480 |
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PCT/US02/39970 |
Dec 2, 2002 |
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11614528 |
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60339383 |
Dec 11, 2001 |
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60347371 |
Jan 11, 2002 |
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Current U.S.
Class: |
514/367 ;
435/375 |
Current CPC
Class: |
Y02A 50/30 20180101;
A61P 9/10 20180101; A61P 25/28 20180101; A61P 21/00 20180101; A61P
39/06 20180101; A61P 25/16 20180101; A61K 31/428 20130101; A61P
25/00 20180101; A61P 25/14 20180101; A61P 43/00 20180101 |
Class at
Publication: |
514/367 ;
435/375 |
International
Class: |
A61K 31/428 20060101
A61K031/428; A61P 25/00 20060101 A61P025/00; C12N 5/02 20060101
C12N005/02 |
Goverment Interests
US GOVERNMENT RIGHTS
[0002] This invention was made with government support under Grant
Nos. NS35325, AG14373, NS39788 and NS39005 awarded by National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of treating ALS patients, said method comprising the
step of administering to said patient a composition comprising a
tetrahydrobenthiazole having the general structure: ##STR00006##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently
selected from the group consisting of H and C.sub.1-C.sub.3
alkyl.
2. The method of claim 1 wherein R.sub.1, R.sub.2, and R.sub.4, are
H and R.sub.3 is C.sub.1-C.sub.3 alkyl.
3. The method of claim 1 wherein said tetrahydrobenthiazole is
pramipexole.
4. The method of claim 3 wherein greater than 90% of the
pramipexole in said composition is R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole
5. A method for enhancing the bioelectric potential (.DELTA..PSI.)
across mitochondrial membranes of cells with impaired mitochondrial
energy production, said method comprising the step of contacting
said cells with a composition comprising a tetrahydrobenthiazole
having the general structure ##STR00007## wherein R.sub.1, R.sub.2,
R.sub.3, and R.sub.4, are independently selected from the group
consisting of H and C.sub.1-C.sub.3 alkyl.
6. The method of claim 5 wherein said tetrahydrobenthiazole is
pramipexole.
7. The method of claim 6 wherein greater than 90% of the
pramipexole compound is R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole.
8. A method of reducing oxidative stress in an ALS patient, said
method comprising the step of administering to said patient a
composition comprising a tetrahydrobenthiazole having the general
structure: ##STR00008## wherein R.sub.1, R.sub.2, and R.sub.3 are H
and R.sub.4 is C.sub.1-C.sub.3 alkyl.
9. The method of claim 8 wherein said tetrahydrobenthiazole is
pramipexole.
10. The method of claim 9 wherein said pramipexole is R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/614,528, filed on Dec. 21, 2006, which is a
continuation of U.S. patent application Ser. No. 10/496,714, filed
on May 26, 2004, which is a .sctn.371 filing of PCT/US02/39970,
filed on Dec. 2, 2002, which claims priority under 35 U.S.C.
.sctn.119(e) to provisional patent application Nos. 60/339,383
filed Dec. 11, 2001 and 60/347,371 filed Jan. 11, 2002, the
disclosures of which are incorporated herein.
FIELD OF THE INVENTION
[0003] The present invention relates to the use of pramipexole
(2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole) to treat
neurodegenerative diseases. More particularly the invention is
directed to the use of the substantially pure sterioisomer R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole and the
pharmacologically acceptable salts thereof as a neuroprotective
agent to treat neurodegenerative diseases.
BACKGROUND OF THE INVENTION
[0004] Neurodegenerative diseases (NDD) such as Alzheimer's disease
(AD) and Parkinson's disease (PD) arise from the accelerated loss
of certain populations of neurons in the brain. Parkinson's (PD)
and Alzheimer's (AD) diseases usually appear sporadically without
any obvious Mendelian inheritance patterns, but may show maternal
biases. Although rare or uncommon inherited forms of adult NDD
exist, the relevance of pathogenesis in these autosomal genetic
variants to the much more commonly occurring sporadic forms is a
subject of intense debate.
[0005] Accumulating evidence provides compelling support that a
primary etiologic component of sporadic adult NDD relates to
mitochondrial dysfunction and the resulting increased cellular
oxidative stress. PD and AD brains and non-CNS tissues show
reductions in mitochondrial electron transport chain (ETC)
activity. When selectively amplified in cytoplasmic hybrid
("cybrid") cell models, mitochondrial genes from PD (Swerdlow, et
al, Exp Neurol 153:135-42, 1998) and AD (Swerdlow, et al, Exp
Neurol 153:135-42 1997) subjects recapitulate the ETC deficits,
produce increased oxidative stress and a variety of other important
mitochondrial and cellular dysfunctions. The combined weight of
evidence from tissue studies and cybrid models of sporadic PD and
AD suggests that relief of oxidative stress, by agents capable of
scavenging oxygen free radicals and protecting cells from
mitochondrially generated cell death, be considered as a primary
characteristic of compounds developed as neuroprotective agents for
these diseases (Beal, Exp Neurol 153:135-42, 2000).
[0006] Oxidative stress has also been associated with the fatal
neurodegenerative disorder amyotrophic lateral sclerosis (ALS).
ALS, also known as Lou Gehrig's disease, is a progressive, fatal
neurodegenerative disorder involving the motor neurons of the
cortex, brain stem, and spinal cord. It is a degenerative disease
of upper and lower motor neurons that produces progressive weakness
of voluntary muscles, with eventual death. The onset of disease is
usually in the fourth or fifth decade of life, and affected
individuals succumb within 2 to 5 years of disease onset. ALS
occurs in both sporadic and familial forms.
[0007] About 10% of all ALS patients are familial cases, of which
20% have mutations in the superoxide dismutase 1 (SOD 1) gene
(formerly known as Cu,Zn-SOD), suggesting that an abnormally
functioning Cu,Zn-SOD enzyme may play a pivotal role in the
pathogenesis and progression of familial amyotrophic lateral
sclerosis (FALS). It is believed that the increased generation of
oxygen free radicals, especially hydroxyl radicals, by mutant SOD1,
to be the initiating factor that results in the sequence of events
leading to motor neuron death in FALS. This hypothesis is supported
by recent reports that transfection of neuronal precursor cells
with mutant SOD1 results in increased production of hydroxyl
radicals and enhanced rate of cell death by apoptosis. Furthermore,
applicants believe that oxidative stress is responsible motor
neuron death in sporadic forms of ALS as well.
[0008] Recent research has revealed that a likely inciting event in
the premature neuronal death that is associated with ALS is the
presence of mutated mitochondrial genes (mitochondrial DNA, mtDNA).
These mtDNA mutations lead to abnormalities in functioning of
energy production pathways in mitochondria, resulting in an
excessive generation of damaging oxygen derivatives known as
"reactive oxygen species" (ROS), including entities called "oxygen
free radicals." When ROS production exceeds the capacity of
cellular mechanisms to remove/inactivate ROS, the condition known
as "oxidative stress" exists.
[0009] Oxidative stress can damage many cellular components. Work
by the inventor has shown that a critical cell component damaged by
oxidative stress in cell models of AD and PD is a particular
mitochondrial protein complex known as the "mitochondrial
transition pore complex" (MTPC). Normal activity of the MTPC is
essential for the maintenance of a bioelectric potential (
).THETA.) across mitochondrial membranes, which in turn is used for
mitochondrial synthesis of energy storage chemicals such as ATP.
Loss of ).THETA. results in depolarization of mitochondria and
initiates a cascade of biochemical reactions which ultimately
result in cell death by a mechanism known as "programmed cell
death" or "apoptosis." Apoptosis mechanisms have been observed not
only in AD and PD, but also in other NDD such as amyotrophic
lateral sclerosis (ALS) and Huntington's disease.
[0010] Accordingly, one strategy for treating these various
neurodegenerative diseases involves the administration of a
neuroprotective agent. Effective neuroprotective agents for these
debilitating and fatal illnesses should not only be effective in
cell culture and animal models of these diseases, but must be
tolerated chronically in high enough doses to achieve therapeutic
levels in nervous tissues. Ideally such agents would also target
cellular components involved in control of cell death pathways and
interrupt disease pathophysiology.
[0011] In accordance with one embodiment of the present invention a
method is provided for treating a neurodegenerative disease such as
ALS. The method comprises the steps of administering pramipexole to
the individual in an amount effective to reduce oxidative stress in
that individual.
[0012] Pramipexole (PPX,
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole)
##STR00001##
exists as two stereoisomers: The S(-) enantiomer is a potent
agonist at D2 family dopamine receptors and is extensively used in
the symptomatic management of PD. The synthesis, formulation and
administration of pramipexole is described in U.S. Pat. Nos.
4,843,086, 4,886,812 and 5,112,842, the disclosures of which are
incorporated herein. S (-) PPX has been shown by several groups to
be neuroprotective in cellular and animal models of increased
oxidative stress, including MPTP toxicity to dopamine neurons (see
U.S. Pat. Nos. 560,420 and 6,156,777, the disclosures of which are
incorporated herein). S(-) PPX reduces oxidative stress produced by
the parkinsonian neurotoxin and ETC complex I inhibitor
methylpyridinium (MPP+) both in vitro and in vivo and can block
opening of the mitochondrial transition pore (MTP) induced by MPP+
and other stimuli (Cassarino, et al, 1998). The lipophilic cationic
structure of PPX is suggestive of the possibility that
concentration into mitochondria across ).THETA..sub.9, in
combination with its low reduction potential (320 mV), may account
for these desirable neuroprotective properties.
[0013] Dosing with S(-) PPX is limited in humans by its potent
dopamine agonist properties and will restrict achievable brain drug
levels. Because the R(+) enantiomer of PPX has very little dopamine
agonist activity (Schneider and Mierau, J Med Chem 30:494-498,
1987) but may retain the desirable molecular/antioxidant properties
of S(-) PPX, this compound is suggested herein as having utility as
an effective inhibitor of the activation of cell death cascades and
loss of viability that occurs in neurodegenerative diseases.
SUMMARY OF THE INVENTION
[0014] The present invention provides methods for preventing and/or
delaying symptoms, or alleviating symptoms relating to a wide
variety of neurodegenerative diseases. More particularly, the
invention is directed to compositions comprising pramipexole and
methods of using such compositions to treat amyotrophic lateral
sclerosis (ALS).
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1A represents a time course of MPP+-induced release of
cytochrome C and FIG. 1B represents a time course of MPP+-induced
activation of caspase 3. SH-SY5Y cells were incubated with 5 mM
MPP+ for varying times and harvested. In FIG. 1A, cells were
homogenized in isotonic sucrose, centrifuged, and 100:g of
supernatant protein electrophoresed using SDS-PAGE, transferred to
nylon membranes and immunostained for cytochrome C with enhanced
chemiluminescence detection. The most lefthand band (designated
"Ohr" on the x axis) corresponds to mitochondrial cytochrome C at
time "0", and the other bands represent electrophoresed cytoplasmic
protein immunostained for cytochrome C. Positions of MW markers are
shown on the y axis. Other batches of cells were assayed for
caspase 3 using a commercial system, according to manufacturer's
instructions (Biomol). Caspase assays shown in FIG. 1B are the
results of 3-4 independent experiments. *p<0.05 compared to
activity at 0.25 hrs.
[0016] FIG. 2 represents data showing the inhibition of
MPP+-induced caspase 3 activation by PPX, bongkreckic acid and
aristolochic acid. SH-SY5Y cells were incubated with 5 mM MPP+ for
24 hours in the absence of or presence of 1 mM S(-) PPX, 1 mM R(+)
PPX, 250:M bongkreckic acid (BKA) or 25:M aristolochic acid (ARA)
or combinations thereof. Cells were then assayed for caspase 3
activity. Shown in FIG. 2 are the mean +/-SEM for 4-5 independent
experiments. Lanes A-F represent caspase 3 activation by cells
incubated with the following compounds: A, control (no compounds
added); B, MPP+; C, MPP+/BKA; D, MPP+/S(-) PPX; E, MPP+/R(+) PPX;
F, MPP+/ARA. For lanes B, p<0.05 compared to control; and for
lanes C--F, p<0.05 compared to MPP+ treated cells (lane B).
[0017] FIG. 3 represents data showing the inhibition of
MPP+-induced caspase 9 activation by PPX, bongkreckic acid and
aristolochic acid. SH-SY5Y cells were incubated with 5 mM MPP+ for
4 or 8 hours in the absence of or presence of 1 mM S(-) PPX, 1 mM
R(+) PPX, 250:M bongkreckic acid (BKA) or 25:M aristolochic acid
(ARA). Cells were then assayed for caspase 9 activity. Shown in
FIG. 3 are the mean +/-SEM for 3-4 independent experiments. Lanes
A-I represent caspase 9 activation by cells incubated with the
following compounds: A, control (no compounds added); B, 15 min
MPP+; C, 4 hr MPP+; D, 4 hr MPP+/PPX; E, 8 hr MPP+; F, 8 hr
MPP+/BKA; G, 8 hr MPP+/S(-) PPX; H, 8 hr MPP+/R(+) PPX; I, 8 hr
MPP+/ARA. For lanes C and E, p<0.01 compared to control; and for
lanes F, G, H, and I p<0.05 compared to control.
[0018] FIGS. 4A & 4B. Inhibition of MPP+-induced cell death by
PPX enantiomers. SH-SY5Y cells were incubated with 5 mM MPP+ for 24
hours in the absence of or presence of increasing concentrations of
S(-) PPX (FIG. 4A) or R(+) PPX (FIG. 4B). Lanes A-F represent cells
treated with 0 nM, 3 nM, 30 nM, 300 nM, 3 uM, and 30 um of PPX,
respectively. The cells were then incubated with calcein-AM, washed
and read on a fluorescent plate reader. The number of independent
experiments is indicated on each bar, and the P values by t-test
for comparison to no PPX (MPP+ only) are shown above each bar. In
the presence of MPP+, 3 nM, R(+) PPX caused greater calcein
retention than S(-) PPX (p=0.035), and 3:M, S(-) PPX caused greater
calcein retention than R(+) PPX (p=0.027).
[0019] FIG. 5 is a graphic representation of the time course of
changes in serum 2,3-DHBA levels in ALS and control (CTL) subjects
after administration of 1.3 grams of aspirin. FIG. 5A shows the
means +/-SEM for changes in serum 2,3-DHBA concentration; FIG. 5B
shows the ratio of serum 2,3-DHBA concentration to salicylate
concentration; and FIG. 5C shows the serum salicylate concentration
over the time course.
[0020] FIG. 6 is a graphic representation of the effect of
pramipexole on serum 2,3-DHBA concentration. FIG. 6A indicates the
DHBA concentrations in individual subjects both pre and post
pramipexole treatment. Subjects 2, 3, 7 and 12 were non-ambulatory.
Subjects 3 and 7 were ventilator dependent. FIG. 6B provides the
mean +/-SEM serum levels of 2,3-DHBA pre and post pramipexole
treatment. FIG. 6C provides the mean +/-SEM levels of serum of
2,3-DHBA concentration/salicylate pre and post pramipexole
treatment.
[0021] FIG. 7. Mice were adminstered R(+) PPX in their drinking
water and then treated with a neurotoxin
(N-methyl-4-pheny-1,2,3,6-tetrahydropyridine, MPTP) which increases
oxidative stress in the brain and forebrain 2,3-DHBA levels were
determined.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0022] In describing and claiming the invention, the following
terminology will be used in accordance with the definitions set
forth below.
[0023] As used herein, the term "purified" and like terms relate to
the isolation of a molecule or compound in a form that is
substantially free of contaminants normally associated with the
molecule or compound in a native or natural environment.
[0024] As used herein, the term "treating" includes prophylaxis of
the specific disorder or condition, or alleviation of the symptoms
associated with a specific disorder or condition and/or preventing
or eliminating said symptoms.
[0025] As used herein, an "effective amount" means an amount
sufficient to produce a selected effect. For example, effective
amount of pramipexole or derivative thereof encompasses an amount
that will inhibit the generation of or decrease the levels of
reactive oxygen species present in an individual.
[0026] As used herein, the term "halogen" means Cl, Br, F, and I.
Especially preferred halogens include Cl, Br, and F. The term
"haloalkyl" as used herein refers to a C.sub.1-C.sub.4 alkyl
radical bearing at least one halogen substituent, for example,
chloromethyl, fluoroethyl or trifluoromethyl and the like.
[0027] The term "C.sub.1-C.sub.n alkyl" wherein n is an integer, as
used herein, represents a branched or linear alkyl group having
from one to the specified number of carbon atoms. Typically
C.sub.1-C.sub.6 alkyl groups include, but are not limited to,
methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl, sec-butyl,
tert-butyl, pentyl, hexyl and the like.
[0028] The term "C.sub.2-C.sub.n alkenyl" wherein n is an integer,
as used herein, represents an olefinically unsaturated branched or
linear group having from 2 to the specified number of carbon atoms
and at least one double bond. Examples of such groups include, but
are not limited to, 1-propenyl, 2-propenyl, 1,3-butadienyl,
1-butenyl, hexenyl, pentenyl, and the like.
[0029] The term "C.sub.2-C.sub.n alkynyl" wherein n is an integer
refers to an unsaturated branched or linear group having from 2 to
the specified number of carbon atoms and at least one triple bond.
Examples of such groups include, but are not limited to,
1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the
like.
[0030] The term "C.sub.3-C.sub.n cycloalkyl" wherein n=8,
represents cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl, and cyclooctyl.
[0031] As used herein the term "aryl" refers to a mono- or bicyclic
carbocyclic ring system having one or two aromatic rings including,
but not limited to, phenyl, benzyl, naphthyl, tetrahydronaphthyl,
indanyl, indenyl, and the like. The term (C.sub.5-C.sub.8
alkyl)aryl refers to any aryl group which is attached to the parent
moiety via the alkyl group.
[0032] The term "heterocyclic group" refers to a mono- or bicyclic
carbocyclic ring system containing from one to three heteroatoms
wherein the heteroatoms are selected from the group consisting of
oxygen, sulfur, and nitrogen.
[0033] As used herein the term "heteroaryl" refers to a mono- or
bicyclic carbocyclic ring system having one or two aromatic rings
containing from one to three heteroatoms and includes, but is not
limited to, furyl, thienyl, pyridyl and the like.
[0034] The term "bicyclic" represents either an unsaturated or
saturated stable 7- to 12-membered bridged or fused bicyclic carbon
ring. The bicyclic ring may be attached at any carbon atom which
affords a stable structure. The term includes, but is not limited
to, naphthyl, dicyclohexyl, dicyclohexenyl, and the like.
[0035] The compounds encompassed by the present invention include
compounds that contain one or more asymmetric centers in the
molecule. In accordance with the present invention a structure that
does not designate the stereochemistry is to be understood as
embracing all the various optical isomers, as well as racemic
mixtures thereof.
[0036] As used herein, the term neuroprotective agent refers to an
agent that prevents or slows the progression of neuronal
degeneration and/or prevents neuronal cell death.
[0037] The Invention
[0038] The present invention is directed to the use of
tetrahydrobenthiazoles to treat neurodegenerative diseases,
including ALS. More particularly, the tetrahydrobenthiazoles of the
present invention have the general structure:
##STR00002##
wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4, are independently
selected from the group consisting of H, C.sub.1-C.sub.3 alkyl and
C.sub.1-C.sub.3 alkene. In one preferred embodiment the
NR.sub.3R.sub.4 group is in the 6-position. In another embodiment
R.sub.1, R.sub.2 and R.sub.4, are H, R.sub.3 is C.sub.1-C.sub.3
alkyl, and the NR.sub.3R.sub.4 group is in the 6-position. In one
embodiment the compound has the general structure of formula I,
wherein R.sub.1 and R.sub.2 are each H and R.sub.3, and R.sub.4 are
H or C.sub.1-C.sub.3 alkyl.
[0039] In accordance with one embodiment, the present invention is
directed to a method of treating ALS. The method comprises
administering to a patient a compound having the general
structure:
##STR00003##
wherein R.sub.1 and R.sub.2 are H, and R.sub.3, and R.sub.4, are H,
or C.sub.1-C.sub.3 alkyl. In one preferred embodiment R.sub.1,
R.sub.2 and R.sub.4, are H, and R.sub.3 is propyl. In another
embodiment, the composition comprises pramipexole wherein
pramipexole component consists essentially of one of the two
sterioisomers of pramipexole (either
R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or
S(-)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole). In one
embodiment the active agent of the composition consists of the
pramipexole sterioisomer,
R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole
dihydrochloride, or other pharmacologically acceptable salts,
substantially free of its S(-) enantiomer. In one embodiment a
composition is provided wherein greater than 80% of the pramipexole
compounds present in the composition are in the R(+) conformation,
and more preferably greater than 90% or greater than 95% of the
pramipexole compounds are in the R(+) conformation. In one
embodiment a composition comprising pramipexole is provided wherein
greater than 99% of the pramipexole compounds are in the R(+)
conformation.
[0040] In one embodiment a composition is provided that comprises
an active agent consisting essentially of
R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole
dihydrochloride, or other pharmacologically acceptable salts
thereof, and a pharmaceutically acceptable carrier. This
composition can be administered orally on a chronic basis for
preventing neural cell loss in NDD (and more particularly reducing
oxidative stress in ALS patients), or it can be formulated and
administered intravenously for prevention of neural cell loss in
acute brain injury.
[0041] The neuroprotective effect of the compositions of present
invention derives at least in part from the active compound's
ability to prevent neural cell death by at least one of three
mechanisms. First, the present tetrahydrobenzthiazoles are capable
of reducing the formation of ROS (both in vivo in rat brain and in
vitro in cells with impaired mitochondrial energy production)
induced with neurotoxins that can mimic PD. In this manner the
tetrahydrobenzthiazoles function as "free radical scavengers."
Second, the tetrahydrobenzthiazoles can partially restore the
reduced ).THETA. that is correlated with AD and PD mitochondria.
Third, tetrahydrobenzthiazoles can block the apoptotic cell death
pathways which are produced by pharmacological models of AD and PD
mitochondrial impairment.
[0042] In accordance with one embodiment a method is provided for
reducing oxidative stress in an ALS patient. A reduction in
oxidative stress, for the purposes of the present invention, is
intended to include any reduction in the level of reactive oxygen
species present in the patient, including for example, decreased
serum ROS levels as detected by the conversion of salicylate to 2,3
DHBA. The method comprises administering an effective amount of a
tetrahydrobenzthiazole having the general formula:
##STR00004##
wherein R.sub.1, R.sub.2 and R.sub.4, are H, and R.sub.3 is
C.sub.1-C.sub.3 alkyl. In one embodiment, the
tetrahydrobenzthiazole is
R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or
S(-)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or a
racemic mixture of the R(+) and S(-) sterioisomers. The amount of
tetrahydrobenzthiazole administered to treat ALS will vary based on
route of administration, as well as additional factors including
the age, weight, general state of health, severity of the symptoms,
frequency of the treatment and whether additional pharmaceuticals
accompany the treatment. The dosage amount of the active compound
to be administered is easily determined by routine procedures known
to those of ordinary skill in the art.
[0043] Alternatively, the present invention provides a method for
enhancing the bioelectric potential ( ).THETA.) across
mitochondrial membranes of cells with impaired mitochondrial energy
production. The method comprises the steps of contacting cells
having impaired mitochondrial energy production with a composition
comprising a pramipexole active agent and a pharmaceutically
acceptable carrier. In one embodiment the pramipexole active agent
consists essentially of the R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole sterioisomer
and the pharmacologically acceptable salts thereof.
[0044] A number of central nervous system diseases and conditions
result in neuronal damage and each of these conditions can be
treated with the tetrahydrobenzthiazole compositions of the present
invention. Conditions which can lead to nerve damage include:
Primary neurogenerative disease; Huntington's Chorea; Stroke and
other hypoxic or ischemic processes; neurotrauma; metabolically
induced neurological damage; sequelae from cerebral seizures;
hemorrhagic stroke; secondary neurodegenerative disease (metabolic
or toxic); Parkinson's disease, Alzheimer's disease, Senile
Dementia of Alzheimer's Type (SDAT); age associated cognitive
dysfunctions; or vascular dementia, multi-infarct dementia, Lewy
body dementia, or neurogenerative dementia.
[0045] The invention is also safe to administer to humans. S(-)
pramipexole, which is a potent dopamine agonist approved for the
treatment of PD symptoms is the enantiomer of R(+) pramipexole.
However, R(+) pramipexole lacks pharmacological dopamine activity.
Accordingly, R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole and the
pharmacologically acceptable salts thereof can be administered in
much larger doses than S(-) pramipexole and can achieve brain
levels capable of providing neuroprotection. In accordance with one
embodiment ALS is treated by administering either R(+) or R(-)
pramipexole, however the administration of R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole is preferred
because much higher doses can be given. As indicated in Example 1,
the S(-) and R(+) isomers are approximately equipotent in reducing
oxidative stress. However the use of the R(+) isomer allows one to
administer higher doses and thus achieve greater reduction in toxic
oxygen free radicals. Accordingly, in one embodiment a method of
reducing neural cell death in patients with amyotrophic lateral
sclerosis (ALS) is provided, wherein the patient is administered a
pharmaceutical composition comprising a compound
of the general structure:
##STR00005##
The synthesis of Pramipexole is described in European Patent 186
087 and its counterpart, U.S. Pat. No. 4,886,812, the disclosure of
which is incorporated herein.
[0046] In one embodiment, pramipexole, and more preferably
R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole is
compounded with binding agents to yield tablets for oral
administration, or with substances known to the art to yield a
transdermal patch ("skin patch") for continuous delivery.
Alternatively, pramipexole can be formulated with the necessary
stabilizing agents to produce a solution that can be administered
parenterally (ie, intravenously, intramuscularly, subcutaneously).
The oral and/or transdermal preparations of the invention are used
to reduce neural cell death in patients with NDD (ie Alzheimer's
disease, Parkinson's disease, Huntington's disease, amyotrophic
lateral sclerosis). The parenteral formulations of the invention
are used to reduce neural cell death in patients with acute brain
injury (ie. stroke, sub-arachnoid hemorrhage, hypoxic-ischemic
brain injury, status epilepticus, traumatic brain injury,
hypoglycemic brain injury).
[0047] The use of R(+) pramipexole to treat NDD, by virtue of its
being a relatively inactive stereoisomer of the dopamine agonist
S(-) pramipexole (Mirapex, Pharmacia and Upjohn), solves an
important problem associated with the use of S(-) pramipexole as a
dopamine agonist. Dosing of Mirapex is limited by dopaminergic side
effects on blood pressure and mentation.
R(+)-2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole has 1%
or less of the potency to produce the side effects that result from
the use of S(-) pramipexole. Thus, the present invention can be
administered more safely to AD patients, who are typically
intolerant of even small doses of dopamine agonist medication. Also
the present invention can be administered intravenously in much
larger doses than S(-) pramipexole. Thus, it can safely be used in
conditions such as stroke, where lowering of blood pressure can be
detrimental.
[0048] In one embodiment a method for treating a patient having a
neurodegenerative disease is provided, that simultaneously reduces
the risk of dopaminergic side effects. The method comprises the
step of administering a composition comprising a pramipexole active
agent and a pharmaceutically acceptable carrier, wherein the
pramipexole active agent consists essentially of the R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole sterioisomer
and the pharmacologically acceptable salts thereof. In one
embodiment the neurodegenerative disease to be treated is selected
from the group consisting of ALS, Alzheimer's disease and
Parkinson's disease, and the composition is administered at a
dosage of about 10 mg to about 500 mg per day of R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or the
pharmacologically acceptable salts thereof.
[0049] The dosage to be used is, of course, dependent on the
specific disorder to be treated, as well as additional factors
including the age, weight, general state of health, severity of the
symptoms, frequency of the treatment and whether additional
pharmaceuticals accompany the treatment. The amounts of the
individual active compounds are easily determined by routine
procedures known to those of ordinary skill in the art. For
example, the tetrahydrobenzthiazoles of the present invention can
be administered orally to humans with NDD in daily total doses
between 10 mg and 500 mg. Alternatively, the
tetrahydrobenzthiazoles can be administered parenterally to humans
with acute brain injury in single doses between 10 mg and 100 mg,
and/or by continuous intravenous infusions between 10 mg/day and
500 mg/day.
[0050] In one preferred embodiment, R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole (or a
pharmacologically acceptable salts thereof) is administered to a
patient suffering from a neurogenerative disease such as ALS to
treat the disease. As used herein, the term "treating" includes
alleviation of the symptoms associated with a specific disorder or
condition and/or preventing or eliminating said symptoms. More
particularly, a composition comprising a pramipexole active agent
and a pharmaceutically acceptable carrier is administered to the
patient to prevent or substantially reduce neural cell death,
wherein the pramipexole active agent consists essentially of the
R(+) 2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole (and the
pharmacologically acceptable salts thereof) sterioisomer of
pramipexole. The synthesis, formulation and administration of
pramipexole is described in U.S. Pat. Nos. 4,843,086; 4,886,812;
and 5,112,842; which are incorporated by reference herein.
[0051] Hydroxyl radical generation in the tissues of an individual
results in an increase in the conversion of salicylate to 2,3
dihydroxybenzoic acid (DHBA) (Floyd et al. (1984) J. Biochem.
Biophys. Methods 10:221-235; Hall et al. (1993) J. Neurochem.
60:588-594). Thus the accumulation of 2,3 DHBA in the serum of
individuals can be used as an indicator of level of oxidative
stress suffered by that individual. The effect of R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole or S(-)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole on 2,3
dihydroxybenzoic acid (DHBA) serum levels was investigated. As is
shown in FIGS. 5 and 6, individual serum levels of 2,3 DHBA
decreased in ALS patients after treatment with S(-)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole. These data
demonstrate that treatment with pramipexole lowers oxidative stress
in vivo in ALS patients.
[0052] Furthermore, as shown in FIG. 7 additional studies were
conducted on mice and demonstrate the effectiveness of pramipexole
in reducing oxidative stress in vivo. In this study mice were
administered R(+) PPX in their drinking water for 8 weeks at 3
different daily doses, and then were given a neurotoxin
(N-methyl-4-pheny-1,2,3,6-tetrahydropyridine, MPTP) which increases
oxidative stress in the brain. Brain tissue was then analyzed for
oxygen free radical production. The data show that the 30 mg/kg/day
and 100 mg/kg/day doses resulted in significant reduced free
radical levels in the brain.
[0053] Toxicology studies have also been conducted and no evidence
of adverse effects were detected. In particular, an 8 week
toxicology study was performed in the mice given R(+) PPX in their
drinking water. All their major organs were examined pathologically
and no lesions were found. While this does not address the issue of
effectiveness of R(+) PPX as a neuroprotectant, it does demonstrate
the potential safety (and thus feasibility) of administering very
high doses of the drug to humans chronically.
[0054] The present invention is also directed to pharmaceutical
compositions comprising the tetrahydrobenzthiazole compounds of the
present invention. More particularly, the tetrahydrobenzthiazole
compounds can be formulated as pharmaceutical compositions using
standard pharmaceutically acceptable carriers, fillers, solublizing
agents and stabilizers known to those skilled in the art.
Pharmaceutical compositions comprising the tetrahydrobenzthiazoles
are administered to an individual in need thereof by any number of
routes including, but not limited to, topical, oral, intravenous,
intramuscular, intra-arterial, intramedullary, intrathecal,
intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, enteral, topical, sublingual, or rectal means.
[0055] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention. In
accordance with one embodiment a kit is provided for treating an
ALS patient. In this embodiment the kit comprises one or more of
the tetrahydrobenzthiazoles of the present invention, and more
particularly the R(+)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole sterioisomer.
These pharmaceuticals can be packaged in a variety of containers,
e.g., vials, tubes, microtiter well plates, bottles, and the like.
Preferably, the kit will also include instructions for use.
Example 1
R(+) and S(-) PPX are Effective Inhibitors of Activation of Cell
Death Cascades
Materials and Methods.
[0056] Cell Culture.
[0057] SH-SY5Y human neuroblastoma cells were obtained from the
American Tissue Culture Collection (www.atcc.org) and maintained in
culture in a replicating state. For caspase assays and cytochrome C
release studies they were grown in T75 flasks with DMEM/high
glucose containing 10% fetal bovine serum, antibiotic/antimicotic
(100 IU/ml of penicillin, 100:g/ml of streptomycin sulfate,
0.25:g/ml of amphotericin B) and 50:g/ml of uridine and 100:g/ml of
pyruvate in a 5% CO.sub.2 atmosphere at 37.degree. C. to
approximately full confluence (2.times.10.sup.7 cell/flask). They
were then incubated with 5 mM methylpyridinium iodide (MPP+; Sigma;
www.sigma-aldrich.com) or 100:M 25-35 or 35-25 beta amyioid
peptides (Bachem; www.bachem.com) for varying times, then
harvested. For cell death studies, the cells were plated into 96
well black-bottom plates and grown for 24 hours in DMEM media
before being exposed to toxin.
[0058] Caspase Assays.
[0059] After exposure to MPP+ or beta amyloid peptide, cells were
collected in PBS and centrifuged at 450.times.g for 6 minutes at
4.degree. C. Cell pellets were resuspended in a hypotonic cell
lysis buffer [25 mM HEPES, 5 mM MgCl.sub.2, 5 mM EDTA, 1M DTT and
protease inhibitor cocktail (Sigma Chemical)] at a concentration of
2.times.10.sup.7 cells/100:1 of lysis buffer. Lysates were
subjected to four cycles of freezing and thawing. Cell lysates were
then centrifuged at 16,000.times.g for 30 minutes at 4.degree. C.
The supernatant fractions were collected, and protein content was
measured by Lowry assay (BioRad). 100:g of protein was used to
measure caspase activity in 96 well plates and was assayed in
quadruplicate. The activity was measured using the assay buffer and
the protocol provided by the manufacturers. (Biomol, caspase 3;
Promega, caspases 3 and 9). Caspase activity is based on cleavage
of synthetic peptide substrates resulting in liberation of colored
(p-nitroaniline (p-NA), Biomol) or fluorescent (aminomethylcoumarin
(AMC), Promega) chromogens. Only activated caspases are capable of
cleaving these substrates, and chromogen generation is completely
inhibited when the caspase inhibitor provided with each assay kit
is included in the assay. Under the indicated assay conditions
linear rates of generation of chromogen were observed over 2 hours.
Chromogen absorbance (p-NA) was measured on an OptiMax plate reader
or chromogen fluorescence (AMC) on a SpectraMax Gemini plate reader
at zero time and after 30 minutes of incubation at 37 degrees to
estimate relative caspase activities. Chromogen signal at zero time
was subtracted from the readings at 30 minutes.
[0060] Cell Death.
[0061] Cell death was estimated by measuring loss of calcein
retention with the "Live-Dead" assay (Molecular Probes;
www.molecularprobes.com) in cells grown in 96 well plates and
incubated with calcein-AM, according to manufacturer's
instructions. Calcein signals were assayed in a SpectraMax Gemini
adjustable fluorescent plate reader (Molecular Devices). Calcein
fluorescence from cells preincubated with methanol was subtracted
from all readings as background. Each assay was performed with 8
wells/condition, which were averaged. 3-8 independent experiments
were performed to evaluate a broad range of concentrations of S(-)
and R(+) PPX in this paradigm.
[0062] Cytochrome C Western Blot.
[0063] Cytochrome C was detected by Western blot following
polyacrylamide electrophoresis of 100:g of cell supernatant protein
and transfer to nylon membrane. The primary antibody was a mouse
monoclonal anti-cytochrome C, obtained from Pharmingen and used at
1:10,000 dilution. Detection was performed with enhanced
chemiluminescence (Pierce) and imaged on a BioRad FluorS imaging
station.
[0064] Drugs
[0065] R(+) and S(-) PPX (gifts of Pharmacia Corporation) were
obtained as their dihydrochloride salts and dissolved directly into
culture media. Bongkreckic acid, an antagonist of the ATP binding
site on the adenine nucleotide translocator, was provided as a
solution in 1M NH.sub.4OH. Aristolochic acid (sodium salt), a
phospholipase A2 inhibitor, was obtained from Sigma Chemical Co. In
the caspase experiments, drugs were added 1 hour before MPP+ or
beta amyloid peptide. In the calcein/cell death experiments, drugs
were added 4 hours before MPP+.
Results
[0066] Activation of Caspases by MPP+ and BA 25-35.
[0067] FIG. 1B shows the time course of caspase 3 activity during
incubation of SH SY5Y cells with 5 mM MPP+. Increased activity was
detectable by 4 hours and had increased about 2-fold by 24 hours.
FIG. 1A shows the Western blot result for cytochrome C protein
released into cytoplasm. Similar to the biochemical activity curve,
cytoplasmic cytochrome C is detectable in small amounts by 4 hours
and increases substantially by 12 hours.
[0068] FIG. 2 shows that both R(+) and S(-) PPX enantiomers
suppressed caspase 3 activation during MPP+ exposure. MPP+-induced
increases in caspase 3 activity were also blocked by bongkreckic
acid, a specific antagonist of the ATP binding site on the inner
membrane site of the adenine nucleotide translocator. Aristolochic
acid, a phospholipase A2 inhibitor, previously shown to block
MPP+-induced apoptosis of SH-SY5Y cells (Fall and Bennett, 1998),
also prevented increases in caspase 3 activity. Activation of
caspases by MPP+ and BA 25-35 peptide was blocked by PPX
enantiomers and agents active at the mitochondrial transition pore.
S(-) PPX reduced by about 70% the increases in caspase 3 activity
following incubation with BA 25-35 peptide and had no suppressive
effect by itself. MPP+ exposure also increased activity of caspase
9 with a time course similar to activation of caspase 3 (see FIG.
3). The increases in caspase 9 activity were also blocked by S(-)
and R(+) PPX, bongkreckic acid and aristolochic acid.
[0069] FIG. 4 shows the effects on cellular calcein retention of
incubating SH-SY5Y cells with varying concentrations of R(+) or
S(-) PPX prior to exposure to 5 mM MPP+ for 24 hours. Calcein is a
fluorescent dye that is retained inside cells as a function of
their ability to maintain a plasma membrane potential. MPP+ alone
reduced calcein uptake by about 60%. Both PPX enantiomers
substantially restored calcein uptake at 30 nM levels, and this
protective effect was retained through 30:M PPX's.
Discussion.
[0070] This study focused on the use of the neurotoxins MPP+ and
25-35 beta amyloid peptide added to replicating SH-SY5Y
neuroblastoma cells as cell culture models for studying potential
neuroprotective compounds useful for Parkinson's and Alzheimer's
diseases, respectively. SH-SY5Y cells are neoplastic, dividing
cells of neuroectodermal origin, not primary neurons. They are
mitotic as a result of a Ras mutation, leading to chronic
activation of MAPK/ERK signaling. SH-SY5Y cells are relatively
insensitive in short-term incubations to MPP+, compared to primary
neurons. Applicants have found that 2.5 and 5 mM, but not 1 mM,
MPP+ produced apoptotic morphology and DNA pyknotic fragments
within 18-24 hours. However, longer incubations with lower
concentrations of MPP+ may more closely approximate the in vivo
MPTP model of PD in animals, and it has been reported that longer
term exposure to lower MPP+ levels still can activate the
mitochondrial cell death cascade.
[0071] SH-SY5Y cells are also sensitive to beta amyloid peptides.
Li, et al (1996) found that serum-starved SH-SY5Y showed extensive
DNA nicked end labeling after 3 days exposure to 100:M beta amyloid
2S-3S and exhibited a concentration-dependent increase in DNA
laddering. Beta amyloid peptide-induced activation of caspases has
not apparently been described in SH-SY5Y, but several reports have
shown caspase activations from exposure to beta amyloid peptides in
various primary neuron lines. These include activation of
caspases-2, -3 and -6 by the 25-35 analogue in cerebellar granule
cells, and caspase-3 in rat primary cortical neurons. Thus, it is
not surprising that caspase-3 activity (DEVDase) was observed in
SH(-)SY5Y exposed to 100:M beta amyloid 25-35 but no caspase
activation was observed after exposure to the reverse 35-25
sequence.
[0072] The focus of the present experiments was to determine if PPX
enantiomers can prevent activation of caspases and can promote
calcein retention as a marker of cell survival in acute toxin
exposure, cell culture models of AD and PD. Activation of both the
"initiator" caspase 9 and "executioner" caspase 3 was blocked by
both PPX enantiomers in the MPP+ model for PD, and activation of
caspase 3 was blocked by S(-) PPX in the BA 25-3S model for AD.
Both PPX enantiomers at nanomolar levels could promote cell
survival in the MPP+ model for PD. Thus, the present findings add
to the growing body of work describing the neuroprotective actions
of PPX and suggest potential clinical utility of this family of
compounds in neurodegenerative diseases.
[0073] While this study did not examine the most proximate site of
action of PPX, several finding implicate the mitochondrial
transition pore complex (MTPC) in these cell models. First,
bongkreckic acid, a selective adenine nucleotide translocator
antagonist and inhibitor of MTP opening, blocked MPP+ induced
activation of both caspases 9 and 3. This would be consistent with
MPP+ bringing about MTP opening either directly or through
mechanisms that involve oxidative stress. In isolated liver
mitochondria MPP+ can bring about a classical MTP opening that is
incompletely blocked by free radical scavenging enzymes and is more
completely blocked by S(-) PPX. Neurotoxic 25-35 BA peptide can
also stimulate MTP opening in isolated mitochondria. Both MPP+ and
2S-35 BA peptide increase oxidative stress in brain microdialysis
studies in vivo and in neural cell culture in vitro, and S(-) PPX
has been shown to reduce MPP+-induced oxidative stress in vitro and
in vivo.
Example 2
Use of Pramipexole to Treat ALS
[0074] Oxidative abnormalities have been identified both in
familial amyotrophic lateral sclerosis (FALS) and the more
prevalent sporadic ALS (SALS). 2,3-DHBA is a hydroxylated
salicylate byproduct that has been shown to be a reliable in vivo
marker of increased free radical activity and is reliably assayed
by HPLC. Following the administration of an oral salicylate load,
elevated serum levels of 2,3-dihydroxybenzoic acid (2,3-DHBA) and
DHBA/salicylate were observed in SALS subjects. As described herein
12 SALS patients were studied to determine the levels of 2,3-DHBA
both before and after treatment with pramipexole.
Methods
Participant Preparation
[0075] The present study was conducted in two phases. In the first
phase eleven definite SALS subjects who met Airlie House criteria,
and 7 controls were studied. These participants underwent aspirin
loading with subsequent 2,3-DHBA analysis. After receiving 1.3
grams of aspirin po, blood was drawn 2, 3, and 4 hours later. Serum
was separated, frozen and stored at -80 degrees. Aliquots of serum
were subsequently coded and blinded for 2,3-DHBA and salicylate
assays.
[0076] In the second phase, 17 subjects with definite SALS were
randomly selected from a clinic population. The subjects received
1.3 grams of aspirin po, and blood was drawn 3 hours later.
Following acquisition of these baseline samples, SALS participants
began pramipexole therapy. Dosage escalation was performed similar
to PD patients with an attempt made to reach 1.5 mg t.i.d.-q.i.d.
as the final dose, following a 7 week titration. After each
participant was on his/her highest pramipexole dose for three
weeks, the aspirin loading study was performed once again. Twelve
of the original seventeen SALS subjects could complete the
pramipexole escalation phase. Of these all but two were able to
reach a pramipexole dosage of 6 mg per day, and all patients
obtained at least a dosage of 3 mg per day. Each participant was
then offered the opportunity to continue with pramipexole
treatment.
Specimen Preparation
[0077] Preparation of serum: 0.9 ml of serum at 4.degree. C. was
mixed with 0.2 ml of 1 M perchloric acid and centrifuged at 15,000
rpm for 10 minutes in a refrigerated microcentrifuge.
[0078] Assay for 2,3-DHBA: 20 uL of supernatant were injected in
duplicate onto a C18 "Catecholamine" Adsorbosphere column (Alltech)
perfused at 0.6 ml/min with buffer consisting of 125 ml/L
acetonitrile, 1.5 gm/L of sodium heptane sulfonate, 3 ml/L
triethylamine, 100 mg/L of Na.sub.2 EDTA with final pH adjusted to
2.8 with phosphoric acid. Detection utilized a CouloChem II
flow-through electrochemical detector (ESA, with the following
settings: guard cell=+600 mV; E1=-100 mV; E2=+400 mV). 2,3-DHBA
eluted at 10-10.5 minutes under these conditions.
[0079] Assay for salicylate: 50 uL of supernatant were injected in
duplicate onto a C8 Kromosil HPLC column (Alltech) perfused at
0.8-1.0 ml/min with 70% methanol/30% water/0.5% trifluoroacetic
acid. Salicylate eluted at 6-7 minutes under these conditions and
was detected by ultraviolet absorption at 315 nM.
Results
[0080] FIG. 5A shows the time course of increase in 2,3-DHBA serum
levels in the 11 SALS (59.2 .A-inverted. 12.3 yr) and 7 age matched
control (56.7 .A-inverted. 10.7 yr) subjects studied in the first
phase. The SALS patients exhibited a range from symptom onset of 10
to 156 months, representing both acute and chronic stages of this
disease. Maximum 2,3 DHBA levels were found in SALS subjects at 3
hours after aspirin dosing, and this time point was chosen for the
second phase of the study. In addition, 2-way ANOVA revealed a
difference in production of 2,3-DHBA when SALS and control groups
were compared across time. This difference was significant at the
p=0.033 level for the two populations; post-hoc testing (Tukey
test) did not reveal any significant differences between individual
time points.
[0081] As an additional comparison, ratios of 2,3-DHBA/salicylate
were compared across time for the two populations. The ALS group
showed an approximate 1.5-fold increase in this normalized marker
of 2,3-DHBA production at 3 hours after administration of aspirin.
2-way ANOVA showed a difference significant at the p=0.06 level
(FIG. 5B). Serum salicylate levels were not significantly different
over time between the ALS and CTL populations (FIG. 5C). Of the
original 17 patients who entered the second phase of the study, 5
dropped out due to complications from the disease or inability to
tolerate the medication. The remaining participants consisted of 8
males and 4 females. The average age was 63.2 years. Pramipexole
therapy was well tolerated by these ALS subjects. These
participants did not exhibit any evidence of clinical dementia, nor
did they have significant cardiovascular instability.
[0082] The patients enrolled in this study represented various
clinical stages of disease progression. Four participants were
non-ambulatory, two of which were ventilator-dependant. The other
eight were ambulatory at the onset of the study. The average time
from the entry into the study and the final blood draw was 76.6
days with a range of 49-105 days.
[0083] Serum levels of 2,3-DHBA were compared pre- and post
pramipexole treatment. Without exception, the individual serum
levels of 2,3-DHBA were reduced (FIG. 6A). FIG. 6B shows the mean
+/-SEM for serum 2,3-DHBA concentration in the ALS subjects before
and after achieving stable pramipexole dosing. The mean reduction
was about 45% and was significant at the p=0.015 level. Serum
salicylate levels (.mu.M) were unchanged both before (18.8+/-7.2,
S.D.) and during (20.2+/-5.5, S.D.) pramipexole treatment. FIG. 6C
shows that serum levels of 2,3-DHBA normalized to salicylate levels
for each subject were reduced an average of 59% by pramipexole
treatment; t-test showed this difference to be significant at the
p=0.010 level.
Discussion.
[0084] This study revealed that an approximate two-fold increase of
in vivo oxidative stress was observed in ALS patients after oral
aspirin loading, based on an increased production of 2,3-DHBA. The
increase in 2,3-DHBA was observed at various stages of clinical
disease progression, suggesting that this metabolite may serve as a
reliable marker of increased oxidative stress throughout this
disease process, but particularly in the early stages, when the
diagnosis often remains uncertain. Because of the small population
size, no attempt was made to correlate the level of 2,3-DHBA
production with disease stage.
[0085] The present study also revealed that pramipexole therapy, at
doses typically tolerated in Parkinson's disease, reduced in vivo
oxidative stress in ALS subjects. Serum from 12 patients before and
several weeks after reaching maximum tolerated doses of pramipexole
revealed a reduction of about 45% of basal 2,3-DHBA and about 59%
of basal 2,3-DHBA normalized to salicylate level. It is likely that
pramipexole brought about this reduction in ROS production as a
result of its free-radical scavenging/antioxidant properties.
Pramipexole has been shown capable of reducing ROS production in
both SY5Y neuroblastoma cells in vitro and rat striatum in vivo
acutely exposed to complex I inhibition with methylpyridinium
(Cassarino et al., J. Neurochem 1998; 71:295-301). Pramipexole also
reduces brain ROS production in vivo following infusion of the
neurotoxin 6-hydroxydopamine (Ferger et al., Brain Res 2000;
883:216-23), inhibits cytochrome C release and reduces brain lipid
oxidation in vivo following treatment with the pro-neurotoxin MPTP
[16].
[0086] Since approximately equivalent neuroprotective actions of
pramipexole are observed in the R(+) and S(-) enantiomers, and
since dopamine agonist actions reside primarily in the S(-)
enantiomer, the ROS-scavenging actions likely have no relationship
to dopamine agonist properties. If this is true, then the R(+)
enantiomer of pramipexole should be tolerated in much higher doses
than the S(-) enantiomer used in the present study, with the
potential for increased antioxidative activity in vivo.
[0087] Oxidative stressors are well established in sporadic ALS,
however the etiology of increased oxidative stress remains obscure.
Markers of CNS oxidative stress damage in ALS include increased
immunohistochemical staining in lumbar spinal cord for lipid
peroxidation product, and increased spinal fluid levels of
nitrotyrosine and nitrosylated manganese superoxide dismutase.
These results are consistent with increased damage to ALS tissue by
ROS that include nitric oxide derivatives.
[0088] Animal and cell models of ALS also provide insight into
oxidative stress and motor neuronal vulnerability. ALS spinal cord
motor neurons have reduced activity of cytochrome C oxidase,
(studied histochemically) and such reduced electron transport chain
function may serve to increase oxygen free radical production.
Spinal cord microdialysis of mice carrying a mutant human SOD1 FALS
gene revealed increased production of ROS and levels of
malondialdehyde, consistent with that concept. Furthermore, it has
been reported that a CuZn-SOD FALS mutation increased the
vulnerability of spinal motor neurons to death from excitotoxicity,
and the mechanism involved increased oxidative stress. Finally,
increased oxidative stress in ALS may be responsible for increased
markers of apoptotic cell death processes observed in ALS spinal
cord, and for involvement of caspases in motor neuron death in a
FALS mouse model.
[0089] Mitochondrial pathology occurs early in the course of
experimental motor neuron disease. These structures are not only
sensitive to oxidative injury, but their dysfunction leads to
accelerated free radical production and possible damage to
mitochondrial DNA. Visualization of mitochondrial function in ALS
muscle biopsies revealed reduced activity of complex I, and
ultrastructural analysis of ALS anterior horn cell dendrites
revealed aggregated, dark mitochondria. Selective loss of glial
excitatory amino acid transporter-2 (EAAT2) around degenerating
anterior horn cells in ALS may reflect additional protein damage
that could contribute to increased excitotoxicity.
[0090] Oxidative damage in ALS may therefore represent a primary
neurodegenerative process, a secondary epiphenomena of motor neuron
mitochondrial pathology, or a combination of both. Cybrid studies
with amplification of SALS mitochondrial genes have shown that
increased oxidative stress in SALS can be understood in terms of a
primary mitochondrial genetic etiology. How mitochondrial DNA
becomes defective in SALS is not known; both maternally inherited
and sporadically acquired mechanisms are possible.
[0091] The use of salicylate loading and 2,3-DHBA measurements as
an estimate of relative in vivo oxidative stress levels has been
applied to adult-onset diabetes, liver dysfunction in alcoholics
and arthritis. Multiple oxidizing species can contribute to the
production of 2,3-DHBA, including hydroxyl radical and
peroxynitrite anion. Thus, a particular ROS source cannot be
assigned for the observed 2,3-DHBA levels, nor can the tissue
source(s) of increased salicylate hydroxylation be defined.
[0092] In summary, basal serum levels of 2,3-DHBA, a marker of
oxidative stress, were found to be increased in a cohort of SALS
subjects, and these levels were reduced following treatment with
pramipexole. As is shown in FIG. 6 individual serum levels of 2,3
DHBA decreased in ALS patients after treatment with S(-)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole. In
particular, the patients were administered orally a daily dose of
3-6 mg of S(-)
2-amino-4,5,6,7-tetrahydro-6-propylaminobenzathiazole for seven
weeks. At the end of seven weeks serum samples were taken and the
concentration of 2,3 DHBA was measured and compared to 2,3 DHBA
levels in serum samples taken prior to treatment. These data
demonstrate that treatment with pramipexole lowers oxidative stress
in vivo in ALS patients.
Example 3
Effectiveness of Pramipexole in Reducing Mptp Induced Oxidative
Stress In Vivo
Methods
[0093] Male C57BL/6 mice received daily R(+) pramipexole
dihydrochloride in drinking water for 8 weeks at doses calculated
to provide 0, 10, 30 or 100 mg/kg/day. On the day of the study the
mice were injected with 30 mg/kg s.c. of the neurotoxin
N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to increase
oxidative stress in the brain. One hour later the mice were
injected with 100 mg/kg i.p. of sodium salicylate. One hour after
salicylate injection the mice were killed and forebrains analyzed
for 2,3-dihydroxybenzoic acid (2,3-DHBA) content.
[0094] As shown in FIG. 7 the results show that 30 and 100
mg/kg/day treatment with R(+) pramipexole significantly reduced
forebrain oxidative stress produced by MPTP. Toxicology studies
have also been conducted and no evidence of adverse effects were
detected. In particular, an 8 week toxicology study was performed
in the mice given R(+) PPX in their drinking water. All their major
organs were examined pathologically and no lesions were found.
* * * * *
References